Transport Phenomena & Membrane Digestion in Small Intestinal Mucosa: An Electrophysiological Approach

Transport phenomena and membrane digestion in small intestinal mucosa An electrophysiological approach

Transport phenomena and membrane digestion in small intestinal mucosa An electrophysiological approach by Sergey T. Metelsky

Sofia–Moscow 2011

Transport phenomena and membrane digestion in small intestinal mucosa An electrophysiological approach by Sergey T. Metelsky

Linguistic editor: Anne Devismes

First published 2011 ISBN 978-954-642-592-8 (paperback) ISBN 978-954-642-593-5 (e-book)

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Printed in Bulgaria, June 2011

Contents€€€€€5

To the memory of A. Ugolev

6€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

Contents€€€€€7

Contents Preface by the author..............................................................................................14 Preface by Dr. D.D.F. Loo......................................................................................... 17

Chapter 1. Transport processes in epithelial tissues

19

1.1. Introduction....................................................................................................19 1.2. The structure of an epithelial sheet............................................................... 21 1.3. Link between digestive and transport processes..........................................22 1.4. Absorption...................................................................................................... 25

1.4.1. Passive transport............................................................................... 26 1.4.1.1. Passive transport of substances through a brush border membrane......................................................................................... 26 1.4.1.2. Facilitated diffusion, Transport through transporters and by means of carriers...............................................................................27 1.4.1.3. Transepithelial transport of water and solvent drag......................27 1.4.1.4. An endocytosis and an exocytosis, a transcytosis........................... 29 1.4.1.5. Paracellular transport and persorption............................................ 31 1.4.2. Active, ion-dependent, or coupled transport processes.................32 1.4.2.1. The coupled transport of water-soluble substances...................... 34

1.5. Transport characteristic of epithelium and epitheliocytes.......................... 35

1.5.1. 1.5.2. 1.5.3. 1.5.4.

Transport of nutrients through a brush border............................. 38 Transport of other compounds through a brush border............... 38 Transport processes in intestinal basolateral membrane............... 40 Interaction between transport processes in apical and basolateral membranes....................................................................................... 44 1.5.5. Sodium transport through the apical membrane and through the epitheliocyte..................................................................................... 46

8€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

1.6. Transport of water......................................................................................... 48 1.7. Final remarks...................................................................................................51

Chapter 2. Investigative techniques of sodium transport in the epitheliocyte 53 2.1. Isotope studies............................................................................................... 53 2.2. Methods of molecular biology...................................................................... 54 2.3. Optical studies................................................................................................ 56 2.2. Electrophysiological studies.......................................................................... 57

2.2.1. Short circuit current method and the rate limiting step for transepithelial transport of Na2+...................................................... 57 2.2.2. Transient characteristics measurement technique......................... 60 2.2.3. Analysis of noise................................................................................61 2.2.4. Patch-clamp technique......................................................................61

2.3. Non polarized preparation............................................................................ 62 2.4. Fractionation techniques............................................................................... 62 2.5. Pharmacological analysis............................................................................... 63

2.5.1. Inhibitors........................................................................................... 63 2.5.2. Stimulants......................................................................................... 64

2.6. Limitations of the used realizations of the short circuit current technique. ........................................................................................................................ 65 2.7. Final remarks.................................................................................................. 66

Chapter 3. Some aspects€ of an adequate short circuit current techinque

67

3.1. The chamber construction.............................................................................68 3.2. Rate of perfusion...........................................................................................68 3.3. Effect of subepithelial tissues on the results obtained................................. 71 3.4. Updating the SCC techinque for clinic study................................................. 71 3.5. Final remarks...................................................................................................72

Contents€€€€€9

Chapter 4. Osmotic phenomena and water fluxes

77

4.1. Effects of osmotic pressure gradient through two types of epithelia.........77

4.1.1.

Influence of the gradient of osmotic pressure upon electric characteristics.....................................................................................77 4.1.1.1. Streaming potential......................................................................... 78 4.1.1.2. Opening of tight cell junctions........................................................ 79

4.2. Osmotic effects in small intestine.................................................................80 4.3. Influence of water fluxes on absorption...................................................... 82 4.4. Final remarks..................................................................................................86

Chapter 5. Transport of monosaccharides and the contribution to its study made by electrophysiological techniques 89 5.1. Transport of sugars through a brush border and a basolateral membrane. ........................................................................................................................89 5.2. Data on sugar transport obtained by the SCC techinque........................... 91

5.2.1. SCC responses to glucose................................................................. 92 5.2.2. The one-sideness of the glucose response...................................... 94 5.2.3. Time-dependence of SCC responses to glucose in experiment..... 95 5.2.3.1. Increase in SCC responses to glucose on the background of fast initial decline of basal SCC............................................................... 95 5.2.3.2. Increase in SCC responses to glucose on the background of quasistationary reduction of the basal SCC............................................. 95 5.2.4. Dependence of stimulating glucose effect on its concentration...... ........................................................................................................... 96 5.2.5. Dependence of the parameters of the SCC response to glucose from rate of perfusion..................................................................... 98 5.2.6. Influence of some physiological factors on stimulating effect of glucose.............................................................................................100 5.2.6.1. A proximo-distal gradient of stimulating effect of glucose.........100 5.2.6.2. Influence of thermal stress on transport of sugars and on stimulating effect of glucose..........................................................100

5.3. Final remarks................................................................................................. 101

10€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

Chapter 6. The single response method

105

6.1. The principles of the method.......................................................................105 6.2. Dependence of kinetic parameters of coupled sodium and glucose transport on the rate of perfusion..............................................................106 6.3. Concentration dependence of the constant of binding of a nutrient with the transporter..............................................................................................108 6.4. Time dependence of SCC responses on glucose.......................................... 110 6.5. Final remarks..................................................................................................112

Chapter 7. Unstirred layers of the fluid at the mucosa surface

115

7.1. A phenomenon of unstirred layer.............................................................. 115 7.2. Measurement of the thickness of unstirred layer....................................... 116 7.3. Dependence of the thickness of an unstirred layer on the rate of perfusion....................................................................................................... 119 7.4. Does the thickness of the unstirred layer change during experiment?....120 7.5. Dependence on temperature of the unstirred layer thickness...................121 7.6. Final remarks................................................................................................. 122

Chapter 8. Regulation of sodium transport in a small intestine

125

8.1. Active sodium transport in the absence of nutrients (basal SCC).............. 125 8.2. Control of active sodium transport in tight epithelia................................ 125

8.2.1. Control by intracellular sodium......................................................126 8.2.2. Control by intracellular calcium......................................................126 8.2.3. Control of the interaction of sodium with the surface of an apical membrane........................................................................................126 8.2.4. Control of permeability of the single channel.............................. 127 8.2.5. Control of the number of channels................................................ 127 8.2.6. Control by change of permeability for counterion....................... 127 8.2.7. Dependence of sodium transport on cell volume......................... 127

Contents€€€€€11

8.3. Control of ion absorption in an enterocyte................................................128

8.3.1. Neuro-endocrine control.................................................................129 8.3.2. Hormones and drugs.......................................................................130 8.3.3. Inhibitors of transport..................................................................... 133 8.3.3.1. The ouabain..................................................................................... 133 8.3.3.2. Amiloride......................................................................................... 135 8.3.3.3. Phlorizin...........................................................................................138 8.3.4. Link with energetics........................................................................140 8.3.4.1. Aerobic metabolism. Influence of oxygen access..........................140 8.3.4.2. Anaerobic metabolism....................................................................142 8.3.5. Neuro-endocrine control, Theophillin, hormones......................... 143 8.3.6. Influence of some other agents......................................................145 8.3.6.1. Influence of ethylene diamine tetraacetate (EDTA) and calcium..... ..........................................................................................................145 8.3.6.2. Carbodiimides..................................................................................149 8.3.6.3. Copper and p-hydroxymercuribenzoate........................................150 8.3.6.4. Guanidine and urea.........................................................................150 8.3.6.5. Papaverine—the first chemical uncoupler of the coupled sodium and glucose transport in the rat small intestine?.......................... 151 8.3.7. Effects of some other drugs............................................................ 151

8.4. Final remarks................................................................................................. 152

Chapter 9. Molecular mechanisms of the coupled transport of glucose

155

9.1. Mechanisms of facilitated glucose transport through plasmatic membranes....................................................................................................155 9.2. Molecular mechanisms (models) of Na+-dependent transport..................158 9.3. The predictions of serial transporter models (the common carrier and channel).........................................................................................................160 9.4. The parallel multipathway€ cotransporter model....................................... 161

9.4.1.

The influence of temperature on sodium and glucose transport..... ..........................................................................................................162 9.4.2. Evidence for a multi-pathway model..............................................171 9.4.3. Structure of a multi-pathway cotransporter.................................. 174 9.4.3.1. The gate mechanism....................................................................... 174 9.4.3.2. The sodium channel........................................................................ 177

9.5. Final remarks.................................................................................................183

12€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

CHAPTER 10. Hydrolysis-dependent disaccharide transport

185

10.1. The mechanisms offered for an explanation of the phenomenon...........185 10.2. The dependence on sodium of transport of the glucose liberated as a result of hydrolysis....................................................................................... 188 10.3. The enzyme-transport ensemble.................................................................190 10.4. Final remarks.................................................................................................198

CHAPTER 11. Transport systems for amino acids

199

11.1. Attempts to classify the transporters of amino acids.................................199 11.2. Mutual inhibition between sugars and amino acids in their transport......... ...................................................................................................................... 202 11.3. The two-pathway transporter for glycine.................................................. 203

11.3.1. The electrophysiological effects of glycine................................... 205 11.3.2. Link between stimulating effects of glycine and glucose on the SCC................................................................................................... 206 11.3.3. Hypothesis about the parallel multi-pathway cotransporter for nutrients...........................................................................................210

11.4. Final remarks................................................................................................. 213

CHAPTER 12. Mechanisms of peptide transport in the small intestine

215

12.1. The pH-dependence of peptide effects on the SCC....................................216 12.2. Final remarks.................................................................................................218

CHAPTER 13. Gerontological aspects of absorption and membrane digestion in the small intestine 221 13.1. Kinetic parameters of Na+-dependent absorption of nutrients in young and old animals............................................................................................. 221 1 3.2. Final remarks.................................................................................................224

Contents€€€€€13

Chapter 14. intestinal Absorption at Satiety, fasting and refeeding

227

14.1. Final remarks................................................................................................ 230

CHAPTER 15. Clinical study of absorption and membrane digestion

233

15.1. Absence of an influence of essential amino acids on the SCC...................234 15.2. Absorption of nutrients, coupled with sodium, in the small intestine of patients with irritable bowel syndrome and celiac disease.......................234 15.3. Use of SCC technique on ischemic diseases of the digestive tract............ 236 15.4. New experimental opportunities for the development of clinical nutrition and nutritional supplements....................................................................... 238 15.5. Final remarks and prospects........................................................................ 239

Chapter 16. Conclusions

241

16.1. Osmotic responses and transtissue transport of water.............................. 241 16.2. Intestinal absorption: a stage of the approach of nutrients from bulk to a mucosa surface..............................................................................................242 16.3. Application of the single response method for a wide spectrum of nutrients and inhibitors of transport. The underestimated approach 16.4. Parallel multi-pathway cotransporter —transporters with gate mechanisms...................................................................................................243 16.5. The SCC technique is the only one that can enable online estimations of vector properties of mucosa biopsies from the human gastrointestinal tract............................................................................................................... 248

Acknowledgements.............................................................................................. 250 Abbreviations and designations........................................................................... 251 References..............................................................................................................253

14€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

Preface by the author This book gives a modern and, to some extent, nontraditional view on fundamental physiological problems of small intestinal transport and membrane digestion. Experts in the physiology of digestion and absorption, and other adjacent areas, as well as experts in short circuit current technique will be slightly puzzled after opening this book. For many years, we have obtained rather important results in these areas, allowing us to resolve many theoretical and applied issues, sometimes in new ways, but these results as a whole remained inaccessible to the scientific community. One can point to two objective reasons for this situation: “perestroika” (it was not the best times for science) and the death of my mentor A.M. Ugolev (1991). I tried to work, no matter the circumstances. My book “Transport Processes and Membrane Digestion in Small Intestinal Mucosa” published in 2007 (in Russian) has received a lot of positive comments from most leading experts in the physiology of intestinal digestion and absorption and in gastroenterology. Only after that was I inspired to prepare two books to be published in English, based on the original Russian book: this book and another one called “The Short Circuit Current Technique”, which will be published later. This fragmentation is caused by two circumstances. First, these two books will have two rather different circles of readers. Second, the account of this book is more consistent now and is not interrupted by technical aspects and theoretical analysis of the short circuit current technique. One can find all these special issues in the second book. Only a very brief account of our updates of classic short circuit current technique in this book is given here for a better understanding of the data presented in this book. More than 25 years ago, we essentially improved the technique mentioned above, and on its basis, a principally new powerful SCC single response method was developed. By using this method, new results regarding unstirred layers, osmotic phenomena, and the regulation of transport processes in the small intestine have been obtained. Briefly (Chapter 6), three parameters of the short circuit current response to nutrient, the usual one (amplitude) and two others suggested by us (relative initial rates of development and washing out of the response), are enough to describe the short circuit current response dynamic to the addition of nutrient in a first approximation. That dynamic contains information about kinetic parameters of hydrolysis and transport processes and about the unstirred layer thickness.

Preface by the author€€€€€15

Data concerning the application of our express-method for studies of animal and human gastrointestinal transport physiology and pathological physiology in terms of molecular mechanism of absorption and digestion are described. In all chapters of this book, not only the latest literature data on the discussed problems are summarized, but our own results obtained by using the above-mentioned method are presented as well. The book begins (Chapter 1) with a brief account of the structure and transport processes in epithelial tissues as a whole and in small intestinal mucosa in particular. Passive (simple and facilitated diffusion) and active transport of ions, nutrients, and water are among them. Chapter 2 briefly treats the method for investigation of sodium transport in epitheliocytes, the usual one and our specially developed to study flat epithelial tissues. Electrophysiological approaches (in particular the short circuit current technique) have engaged our attention. Conclusions were reached that the known short circuit current technique has some limitations. Chapter 3 briefly describes the adequate short circuit current technique in which the observed limitations are eliminated. Intestinal epithelium proves to be extremely sensitive to osmotic pressure gradient through it. The role of water flows and its influence on intestinal absorption are discussed in Chapter 4. In Chapter 5, the transport of monosaccharides and its contribution to studying electrophysiological techniques are discussed. The conclusion was drawn that short circuit current responses to glucose reflect the physiological reality and contain information on the mechanism of coupling of sodium and glucose transport. In this book, topics such as transport of water and osmotic phenomena, as well as new data concerning the measurement of unstirred layer thickness near the mucosa surface under different conditions, are analyzed. Chapter 7 is, in our opinion, one of the more dramatic chapters in this book. It was shown that the thickness of unstirred layer can be determined both with the response of electric parameters (in particular the short circuit current) upon addition of sugar (mannitol) and with the nutrients transported in a Na+-dependent manner. Values of layer thickness determined with mannitol and with glucose or with glycine are close. Traditional issues of sodium transport process control are also considered (Chapter 8). It turns out that neuro-endocrine control of both types of sodium transporters (amilorid-sensitive and nutrient-dependent) is absent in vitro. It is important to keep in mind that by means of the short circuit current technique, one can test not only the activity of transcellular processes but also the state of cell tight junctions as well. Data about the possibility of dissociation of active transport and induced effects of glucose have allowed us to propose the two-pathway model of the coupled cotransporter for glucose and sodium (Chapter 9). The model is characterized by the presence of two interacting transporters located side by side (for glucose and for sodium) and of a superficial gate mechanism binding glucose on input in transport system, result-

16€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

ing in the opening of the sodium transporter. The multi-pathway model is proved in additional experiments. It was revealed (Chapter 10) that the more easily disaccharide is hydrolyzed by intestinal enzymes, the larger its stimulating effect on the SCC. When summarizing all experimental data, we conclude that separate independent transport systems for sodium and glucose, at least in rats, do not exist, and glucose is transported through Na+-dependent hydrolysis-transport ensemble. The resolution of the issue of hydrolysis-dependent transport within the framework of the parallel multi-pathway cotransporter carrying out transport of monomers and dimers allows us to explain a number of well-known facts that have not yet obtained a satisfactory explanation. It turns out that glycine and glucose transporters may be united in one quaternary structure with the same sodium transporter (Chapter 11). After that, the model of a multi-pathway parallel cotransporter has been proposed. In accordance with developed views, the transporter composition of a multi-pathway cotransporter can vary both in quantity of subunits and in the type of the formed separate transporters for nutrients. At neutral рН, the proportion between efficiency of absorption from a solution with dipeptide and from a solution containing a mixture of the corresponding amino acids can take any value (Chapter 12). Hence, the widespread approach for the determination of dipeptide transport efficiency by comparison (at neutral рН) of rates of absorption from a solution with a mixture of amino acids and from a solution containing the corresponding dipeptide only is incorrect. The adequacy of the used methods and approaches is illustrated by data on nutrient-absorption investigation in rats under fasting and satiety conditions (Chapter 14) and on aging (Chapter 13) as well. Each fundamental concept discussed here, even the rather theoretical ones such as unstirred liquid layer, is illustrated as far as possible throughout the book by its clinical significance. Incentive examples of using our modification of the short circuit current technique to determine the kinetic constants of coupled sodium and glucose transport in intestinal mucosa in real-time operation mode in clinic investigations are given (Chapter 15). Finally, Chapter 16 summarizes all essential points of discussions given in this book. This preface gives a brief account of what the reader will find in this book. This monograph is intended for specialists in gastroenterology, physiology of nutrition, biophysics, biochemistry, pharmacology, and pathology of the digestive system. It may be of interest for general practitioners and for postgraduate students as well.

Preface by Dr. D.D.F. Loo€€€€€17

Preface by Dr. D.D.F. Loo The small intestine is the major site for the absorption of water, salt, and nutrients (sugars, amino acids, peptides) into the body. This book discusses the transport processes involved (ion channels, pumps, transporters), and the experimental methods used for their study. In recent years, molecular biological, biochemical, biophysical and protein crystallography approaches have resulted in great advances in our understanding of the mechanisms. The epithelial Na+ channel, Na+/K+ pump, facilitative glucose transporter, and ion-coupled cotransporters (Na+/glucose, Na+/phosphate, and H+/dipeptide) have been cloned and sequenced, and structural models on the atomic level have been proposed for some of these proteins. Many techniques are available for transport studies at different levels of integration. With the short circuit current technique, current across an epithelium is measured under conditions of zero trans-epithelial voltage. Multiple transport systems may contribute to the short circuit current. Concurrent measurements of short circuit current and transepithelial flux of radioactive substances (such as ions, sugars, amino acids) allow identification of the transported substances underlying the short circuit current. Fluctuation or noise analysis involves measurements of the fluctuations in short circuit current under stationary conditions. The current fluctuations are due to the opening and closing of ion channels, and the power spectrum yields information on channel density and occupancy probabilities in the open and closed states of the channel. Patch clamp methods offer the highest resolution, with measurements of single channel currents in cell-attached or excised patches, and whole cell recordings of currents from channels and electrogenic cotransporters. Each technique has strengths and weaknesses. For example, patch clamp studies enable characterizations of channels and transporters, but data on a single population of proteins need to be integrated into the higher level function of the whole tissue. On the other hand, complex tissue geometry could make interpretation of the short circuit current problematic. Since voltages across the apical and basolateral membranes are not controlled, it is difficult to draw conclusions on the properties of the underlying transport systems. In addition, in transport studies on whole tissues, there is an unstirred region adjacent to the apical cell membrane where concentrations of the transported substances differ from that of the bulk phase. Consequently, kinetic

18€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

properties - such as substrate affinities and maximal transport rates, depend on the thickness of the unstirred layer. The major focus of the book is on studies using the short circuit current (SCC) technique. The technique was developed in the 1940’s, and it remains the method of choice for whole tissue studies, as well as studies of substances across monolayer’s of cultured cells. Different aspects of the procedure, such as experimental chamber design and tissue complexity are discussed. A method, called the single response SCC, is described for estimating the thickness of the unstirred layer, and results from SCC studies are compensated for unstirred layer effects. Following the overview of transepithelial transport and description of SCC, the subsequent chapters are concerned with the molecular mechanisms of the transport of water, sugars (monosaccharides and disaccharides), amino acids and dipeptides. Finally, the later chapters review the studies on topics such as the effects of aging, intestinal absorption at satiety, fasting refeeding, clinical study of absorption and membrane digestion, and use of SCC on ischemic diseases of the digestive tract. Dr. D. D. F. Loo

Chapter 1. Transport processes in epithelial tissues€€€€€19

Chapter 1. Transport processes in epithelial tissues 1.1. Introduction To remain alive, any living organism must ensure itself an adequate nutrient supply from the immediate environment. For vertebrates, the assimilation of nutrients by the digestive system takes place mainly in the small intestine. This organ makes the selection on which food component will enter the internal environment. The most important products of digestion (splitting of food polymers into oligo- and monomers), or nutrients, enter the enterocytes through active transport. The mechanism of active transport of nutrients, which has long been known to be carried out against an electrochemical potential gradient, assumes coupling with any exergonic reaction serving as the driving force for active transport. Transport systems of membranes are subdivided into primary-energized transport systems (pumps) and secondary-energized transport systems (cotransporters). If free energy is not used, the transmembrane fluxes are passive. For instance, Na+-K+ ATPase is an example of a pump working through splitting of ATP molecules. An example of an exergonic-coupled reaction of the second type, which has been found in recent years to be abundant and important, is the movement of some monovalent ions, in particular sodium, down the electrochemical gradient through a membrane. The important progress in the physiology of transport processes observed over the past few years results in several essentially new lines of investigation. Indeed, some researchers suggest making no distinction between biologically active substances, bioactive€additions, and food because the differences between them are rather conditional; so far, there are no strict criteria for their discrimination. In addition, many substances, such as glycoproteins, which are xenobiotics, organize their own transport. The method of short circuit current (SCC) and the model of transcellular sodium transport, both developed by H. Ussing (Holtug et al., 1996), are among the main achievements in the physiology of transport processes over the last few decades. They will both be considered in more details below. These achievements have resulted in rapid increase of the number of publications on the electrophysiology of epithelial tissues.

20€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

Over the past 20 years only, about 3500 studies were carried out based on use of the SCC technique; among these studies, more than 60 were on biopsies of human intestine. Below, we shall try to consider and evaluate the results of this revolution. One of the most significant achievements in the treatment of diseases of the digestive system organs and correction of morphofunctional impairments of the gastrointestinal (GI) tract was the use of nutrients for pharmacotherapy. In this case, nutrients can directly affect the GI tract or increase the response to pharmacotherapy. In the future, such pharmacotherapy may be a powerful addition to traditional approaches used for the treatment of the corresponding groups of patients (Rothstein, Rombeau, 1998). The topics under consideration, that is, absorption mechanisms and membrane digestion, are quite complex. On the one hand, they are at the interface of many sciences, such as gastroenterology, physiology, biophysics, biochemistry, molecular biology, pharmacology, and so forth. On the other hand, these results are obtained in studies of various groups of organisms, from protozoans to humans. Furthermore, it is necessary to take into account that whole organisms, single organs, isolated cells, and macromolecules (enzymes, etc.) are used in these studies, which are carried out both in vivo and in vitro). It has long been known that one can study the coupled transport of ions (Fig. 1) and nutrients, happening in any animal gut, from two equivalent viewpoints: biochemistry and biophysics (for example, electrophysiology). In biochemical studies, one can determine the absorption of nutrients and the effects of ions (e.g., sodium) during this process, and in electrophysiological studies, one can determine the currents generated while ions are moving through membranes and the effects of the corresponding nutrients on this process. A. M. Ugolev was maybe the first to unify these two trends (biochemical and electrophysiological) in studying intestinal transport processes within one Lab in 1979 (Laboratories of Nutrition Physiology, Institute of Physiology, USSR Academy of Sciences). It should be pointed out that the author of the present book worked in laboratories headed by А. M. Ugolev from the end 1979 to 1991, and a part of the data presented here were obtained during this time. To help the reader have a more complete picture of the considered phenomena, we shall analyze the results obtained not only on intestine mucosa, but also on other more studied biological objects and structures. The theoretical basis of such an approach is the concept formulated by A. M. Ugolev in 1985 (Ugolev, 1985; Ivashkin et al., 1990). According to these authors, all variety of wildlife is caused by a combination of a rather restricted number of universal functional blocks, such as enzymes, multi-enzyme complexes, channels, transporters, carriers, molecules, and so forth. So, for example, such a concept, in parallel with a set of other facts, explains why the structure and properties of functional blocks such as Na+-K+ ATPases, isolated from an enterocyte, a neuron, or an erythrocyte, are only slightly distinguishable.

Chapter 1. Transport processes in epithelial tissues€€€€€21

1.2. The structure of an epithelial sheet Columnar cells of the small intestine (enterocytes) are derivatives of a population of crypt stem cells. The diversity of epitheliocytes (columnar, goblet, endocrinocytes, and Paneth cells) are derived from cambial cells of crypts, from where they migrate on intestinal villi (except for Paneth cells). The process of migration takes from 3 to 6 days, during which time their differentiation occurs. It has long been known that intestinal epithelial cells are characterized by expressed polarity: sides of plasmatic membranes turn into a lumen of organ, and in the internal environment of an organism, they have essential morphological distinctions. The zone of the junction between epithelial cells represents a highly specialized area, which not only provides mechanical stability to an epithelium but also serves as a barrier on the pathway for the migration of plasmatic membrane proteins from one side of the cell the other. Because solutes and water are transported from an intestinal lumen through an epithelial sheet in one direction, the cells should be polarized so that substances may enter through an apical plasmalemma and leave through a basolateral plasmalemma (Fig. 1). As a consequence, the transport properties of both parts of membranes are distinguished. Transport proteins, being synthesized inside a cell, should be integrated into various parts of a plasmatic membrane. When delivered to the corresponding parts of the plasmalemma, the proteins should stay there. A tight junction is a barrier limiting free lateral diffusion of protein molecules along a plasmatic membrane. Such a barrier is essential because it divides the membrane of an epithelial cell into two parts, characterized by their own set of membrane proteins (luminal or apical membrane), and the basolateral membrane turns to lamina propria of a mucosa containing numerous microvessels. Association of cells into the cellular ensembles forming tissues occurs in no accidental manner. Cells form highly ordered structures. Every tissue is formed because of specific adhesion of cellular ensembles, the cytoskeleton, and interactions with an extracellular matrix. During tissue formation, separate cells occupy certain positions that are appropriate for interactions and concerted functioning of cells and supracellular structures. The specific functions of a tissue are determined by complex interactions between the tissue and its environment. Adhesion of cells as the tissue forming factor is coupled with a set of signaling processes controlling information transfer between neighboring cells. So, the epithelial cells lining a digestive tract are attached to each other through tight junctions. These cells are arranged on a thin basal membrane (Fuller, Shields, 2006). In an epithelium, there is a special cell junction which first provides a link and interaction between cells and second gives cells some stability against physical influences because of the presence of filaments. A cell junction is a structure within a tissue of a multicellular organism. Cell junctions are especially abundant in epithelial tissues. They consist of protein complexes

22€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

and provide contact between neighboring cells and between a cell and the extracellular matrix, or they built up the paracellular barrier of epithelia and control the paracellular transport. Junctions through which epithelial cells are connected with each other and with an extracellular matrix can be divided into three groups. 1. € Tight junctions: they form an impenetrable barrier between neighboring cells and impair the penetration of even the smallest molecules (or for example, ions) from one side of an epithelial sheet to the other. 2. € Anchoring junctions: they attach cells to each other and to an extracellular matrix. 3. € Gap junctional intercellular communication: such junctions convey small molecules and electric signals between neighboring cells. Molecules entering into a cell through an apical plasmalemma leave it through a basolateral plasmalemma and enter into blood. Hence, in polarized epitheliocytes, many cellular proteins (for example, transporters of carbohydrates or amino acids and ionic pumps) are situated only on an apical or only on a basolateral membrane. In studies of interactions of epithelial cells with various viruses, for example, flu or vesicular stomatitis, it has been found that various proteins of viruses selectively bound either with the apical or with the basolateral parts of a plasmatic membrane. New virus particles go through only one surface of the infected cells. For example, flu viruses leave through the apical surface, and vesicular stomatitis viruses leave through the basolateral surface of epithelial cells. Gap junctions are the most widespread cell junctions. Under an electronic microscope, they look as strips separating plasmatic membranes of neighboring cells by a narrow gap with a width of 2–4 nm (Fuller, Shields, 2006). The gap junction is built up of protein subunits named connexines (26000–54000 Da). Six identical subunits form the annular structure named connexone. During alignment of connexones of two neighboring cells, the water channel along which small molecules (<1000 Da) can pass is formed.

1.3. Link between digestive and transport processes Food enters into an organism mainly in the form of polymers; however, carbohydrates, proteins, and fats appear in blood in the form of their components (monomers). The process of hydrolysis of food substances into monomers is referred to as digestion. The process of assimilation takes place because of cavital, intracellular digestion (Kushak, 1983; Ugolev, 1967, 1990, 1972; De Jesus, Smith, 1974) and membrane digestion (Smirnov, Ugolev, 1981; Ugolev, 1960, 1967, 1972; Korot’ko, 1987). The digested food can now pass into the blood vessels in the wall of the intestine through the process known as absorption. The small intestine is the site where most of

Chapter 1. Transport processes in epithelial tissues€€€€€23

the nutrients from ingested food are absorbed. The end product of amylase digestion of starch and glycogen is mainly a disaccharide (maltose). After peptic and pancreatic proteolysis, average food proteins are converted into a mix consisting mostly of peptides; the content of free amino acids in the mix represents approximately 20%. Thus, as a result of digestion under physiological conditions, carbohydrates and proteins enter transport systems of brush border membranes of the small intestine mostly in the form of maltose, amino acids, and small peptides (Parsons, 1978; Ernst, Mills, 1980). The latter are near the same cellular surface, where digestive enzymes are located because the work of enzyme-transport ensembles is especially effective (Ugolev, 1972, 1977, 1985; Ugolev, Smirnova, 1977; Ugolev, Kuzmina, 1993). The majority of hypotheses explaining the cohesiveness of hydrolytic and transport processes in intestines is based on the recognition that a membrane digestion is the basic mechanism providing transition from hydrolytic processes to transport. However, in some cases, in particular to account for the mechanism of absorption of oligopeptides, the transport of intact compounds through a brush border membrane has to be taken into account. Epithelia can transport solutes in two directions: absorption (or reabsorption in kidneys) and secretion. Transport of a fluid can be either isotonic (in comparison with plasma) or hypertonic (Hill, Shachar-Hill, 2006). The two-membrane model of transepithelial transport of solutes, which at present is the basic concept, assumes the vector nature of transporting epithelia when substances enter into a cell through one membrane and leave it through the other one. Ions enter into a cell through two principal pathways: ion channels or transporters and possibly carriers (Fig. 2). Energy for the entrance of ions into a cell is usually provided by the electrochemical gradient of Na+ through the plasmatic membrane, generated and maintained by the Na+-K+ ATPase pump. As a rule, this pump is localized on a membrane turning to blood. Owing to the work of this pump, the intracellular concentration of Na+ is equal to 10–20 mM and that of K+ to 90–120 mM. The energy which is released from the electrochemical potential of Na+ (difference of ionic concentration + difference of electric potentials on a membrane) while Na+ is entering through a plasmatic membrane can be used by other transport systems. Hence, the Na+-K+ ATPase pump carries out two main important functions: it pumps out Na+ from cells and generates the electrochemical gradient providing energy for the mechanisms allowing solute entrance. So, the prominent feature of substance transport in an epithelium of the GI tract lies in the fact that it is carried out through a monolayer of cells. Such transport is named transepithelial transport. Intestinal epitheliocytes (enterocytes) are asymmetric or polarized: apical and basal membranes are distinguished from each other by permeability, by a set of enzymes, by the value of the potential difference, and they carry out different transport functions (Fig. 1).

24€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

Mucosa

Epithelium

Na+ - channel: - number-50 1/μm2 - current -0.3-0.5 pA - lifetime- 60 h - open - 1 s, - closed -6-10 s

Serosa

[Na] = 145-160 mM [K] = 6 mM [Cl ] = 145 mM

Na+ [Na]= 10-20 mM [K] = 90-120 mM [Cl] = 55 mM

Nutrient-dependent

Basal

Electrogenic Na+ transport (Short circuit current technique)

- 40- 50 mV 1.0

3.8-4.1 4.2

GLUT2 ATP-dependent Ca2+ pump Na+/Ca2+ exchanger Na+-pump of baso-lateral membrane (rheogenic):

Na+ 3 Na+ 2K+

- transport rate 4-6 µmol/cm2 *h; - number of pumps 0.15-1.5 * 106 1/cell; -pump rate 10-140 1/s

Basolateral intercellular space:

its osmolarity is higher by 3-7 mosmol then that of cytoplasm

3.5 Transporters for amino acids

2.0-4.3

Fig. 1. A schematic presentation of transepithelial Na+ transport routes. The rate of Na+ transport in the absence of nutrients (basal short circuit current) is taken as a unit. Relative rates of nutrient-dependent Na+ transport are evaluated as the ratio of maximal stimulating effect of listed nutrients on short circuit current through rat intestine to magnitude of basal short circuit current. For details, see Chapter 6.

Chapter 1. Transport processes in epithelial tissues€€€€€25

Fig. 2. Molecular models of two coupled co-transporters. On the left – mechanism of common mobile transporter for glucose and Na+. On the right – mechanism of common channel for glucose and Na+. For details, see text.

1.4. Absorption The term absorption designate all the phenomena providing transport of substances from an intestinal lumen into blood and lymph. It is agreed that absorption is the final stage of digestion based on complex biological processes of transport of substances from intestinal cavities (Fisher, Parsons, 1949, 1953; Ugolev, 1971). The human intestinal epithelium has a large resistance toward transfer of large and hydrophilic compounds (Lennernas, 2007). We believe that the term absorption is wrongly applied to xenobiotics and substances, the absorption rates of which are below some level (for example, ureas). Any substance, even at very low concentrations, will get from the intestinal lumen into blood sooner or later. More likely, it is not a true absorption, such as a specialized or active process, but the consequence of restricted mucosal permeability, although this issue is rather debatable. The absorption of nutrients is carried out mainly in the small intestine. Substances can be distinguished on the basis of their relative rates of absorption. Let us consider this issue in more details by using the example of sugars (Tab. 1).

26€€€€€Transport phenomena and membrane digestion in small intestinal mucosa Table 1. Relative initial rate of sugar absorption in the small intestine (Curie, 1926; after: Loewy, Siekevitz, 1969). Hexoses Galactose Glucose Fructose Mannose Pentoses Xylose Arabinose

Sugar

Relative initial rate of absorption, % 110 100 43 19 15 9

Note. Initial rate of glucose absorption is taken as 100%

We believe that all studied sugars can be classified into three groups on the basis of rates of absorption: well absorbed glucose and a galactose, moderately absorbed fructose and, poorly absorbed mannose, xylose, and arabinose. Sugars in the first group are absorbed through active or Na+-dependent transport (see below); sugars in the second group are absorbed through facilitated diffusion (see below); and sugars in the third group are absorbed through passive transport.

1.4.1. Passive transport Passive transport of substances through a monolayer of enterocytes proceeds without expenses of free energy and can happen along either transcellular or paracellular pathways (Fig. 1). Diffusion, osmosis, and filtration are among such types of transport. The driving force of diffusion of molecules (microparticles) of a solute is their concentration gradient. During osmosis, which is a form of diffusional transport, solvent (water) molecules move along their own concentration gradient. Filtration consists in the transport of a solution through a porous membrane under the action of hydrostatic pressure (Nozdrachev, 1991:385). Transcellular pathway means crossing passively (without an expense of free energy) through a brush border membranes of an enterocyte.

1.4.1.1. Passive transport of substances through a brush border membrane A few types of substance passive transport through membranes are distinguished: a) simple diffusion, direct passing through the hydrophobic part of a membrane; b) facilitated diffusion, transport by means of transporters, that is, special channels (pores) or carriers.

Chapter 1. Transport processes in epithelial tissues€€€€€27

All variety of transport mechanisms can be classified into two basic groups: 1) € each molecule is transported independently of others; effects of concentration saturation are absent. 2) € the transport of molecules is inhibited until the carrier is released from the transport of the previous molecule; effects of concentration saturation are present. It was experimentally shown that the transmembrane diffusional flux of uncharged molecules that are passively transported is directly proportional to the diffusion constant of the substance in the membrane D and to the substance distribution coefficient in a oil–water system, and it is inversely proportional to the membrane thickness. Studies following those of E. Overton have confirmed his data; however, it was found that the rule concerning solubility in lipids or oil is not valid for rather small molecules, such as water, methanol, formamide, and so forth. Nevertheless, urea, which is considered as a rather easily permeating substance, enters cells 100000 times more slowly than water does, and some ions pass through a cellular membrane 100000 times more slowly than urea does.

1.4.1.2. Facilitated diffusion, transport through transporters and by means of carriers In the elementary case, the transport of substances through pores with a rather large size does not depend on the concentration of the transported substance, and it is described by the equation for electrodiffusion. In this case, the effective factor of permeability P depends on the number of channels n, the unit of the membrane area, the radius of the channel r, and the diffusion constant of substances in water D: P = π€ r2 n D/l, where l is the length of the channel. If the channel contains charged groups, the concentration of ions in the channel will decrease or will increase. If the channel is narrow enough, ions can prevent each other to move. Facilitated diffusion, as well as simple diffusion, is carried out without expenses of free energy through a concentration gradient. At the same time, facilitated diffusion is a faster process which has a saturation threshold; it can be blocked by competitive inhibitors and hence is carried out with the participation of carriers or channels (Nozdrachev, 1991).

1.4.1.3. Transepithelial transport of water and solvent drag The phenomenon of solvent drag was discovered more than 35 years ago. The essence of this phenomenon can be described as follows (Parsons, 1975). The permeability

28€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

of intact frog’s skin to sucrose is very low. The skin permeability to sucrose (no penetration through the skin) significantly increases after the addition of urea in the external solution. An increase of transepithelial transport (from outside to inside) of other sugars and ions takes place because of the mechanism of solvent drag. Namely, urea diffuses from the external solution into the lateral intracellular space through the tight junctions. Local osmotic pressure within this space increases. Output of water from the neighboring cells increases the hydrostatic pressure in the intercellular space. As a result, urea follows the direction of the internal washing solution. Any molecule or ion that was included in the lateral intercellular space from the external solution by diffusion will be carried away by the flow along this space and will eventually be pushed into the internal solution. Hence, the action mechanism of solvent drag is determined by the generation of extra hydrostatic pressure in the lateral intercellular space, and the energy for its functioning is derived from the urea concentration gradient. This phenomenon has been supported by more evidences later (Evard et al., 1985; Godbillon et al., 1987; Lennernas, 1995). For example, the absorptions of an oxprenolole and a metoprolole are sharply increased in the presence of nutrients in the intestinal lumen (Evard et al., 1985). The isotonic absorption of water through the epithelium is caused by sodium transport through amiloride-sensitive channels in an apical membrane (Blaug et al., 2001). The driving force for the non-isotonic diffusional transport of water through the epithelium of intestine and nephrons is the gradient of osmotic pressures resulting from the transport of salts and organic compounds. The rate of water transport depends on the value of the osmotic pressure gradient and on the permeability of the epithelium for water also. Moreover it has been found that absorption of water is secondary in comparison with active sodium and nutrient (glucose) transport (Meinild et al., 1998; Wright, Loo, 2000; Loo et al., 2002). The stoichiometry of the transport of Nа +/glucose/Н2О is equal to 2:1:210. The mechanisms determining the rates and pathways of absorption of small compounds still remain unclear. For example, effective intestinal permeability for water and ureas are predicted to be due to their physical and chemical properties. In humans, the absorption of urea and creatinine (unlike other small molecules with a molecular weight of 60–4000 кDа) increases with solvent drug, but it does not change in rats (Fagerholm et al., 1999). It was shown that there is a significant effect of water absorption on mannitol absorption through pores in a cat small intestine (Bijlsma et al., 2002). Note that the promising data obtained on animals in vitro and in situ may not be correct (quantitatively) for humans, especially for drugs with a molecular weight above 200 кDа (Lennernas, 1995). However, it was found that the therapeutic effect of epirubicine can be increased with the use of low toxic excipients, increasing absorption because of the increase in bioavailability (Lo, 2003). So far, it still unclear whether the solvent drug transport mechanism happens through cells or through cell tight junctions (Fagerholm et al., 1999).

Chapter 1. Transport processes in epithelial tissues€€€€€29

Issues of permeability for water and changes in water flux directions in the intestine attract special interest owing to the development of oral rehydration therapy as a simple, cheap, and effective treatment for diarrhea-related dehydration. One diarrhea mechanism (such as in the case of cholera, which is a very dangerous form of profuse diarrhea) is enterotoxin interfering with enterocyte cAMP and G-proteins. The high affinity, slow wash-out, and external sites of lectin action support further development as antisecretory drugs for enterotoxin-mediated secretory diarrheas (Sonawane et al., 2007). It is agreed that mechanisms of bacterial diarrhea have the ability to convert total ion and water absorption to total secretion, both with thermostable and themolabile endotoxins upon morphologically intact mucosa. A secretory diarrhea arises as the result of such switching. However, water can still be absorbed by the SGLT1-transporter (sodium and glucose transporter) (see 1.4.2.). Preservation of the normal functioning of the mechanisms for coupled sodium and nutrients (glucose and amino acids) transport in widely used oral rehydration therapy assures success in such cases (Guandalini, 1988). It is suggested that the therapeutic efficacy of complex carbohydrate solutions determines their hypotonicity too (Thillainayagam et al., 1998).

1.4.1.4. An endocytosis and an exocytosis, a transcytosis. The endocytosis is a vesicular capture of a fluid, macromolecules, and small particles into a cell. There are three mechanisms of endocytosis (Fuller, Shields, 2006): 1. € pinocytosis: the term pinocytosis originates from the Greek words meaning to drink and cell and designates the phenomenon during which the cell seems to “drink”. Such mechanism refers to a clatrine-independent endocytosis also. 2. € phagocytosis: the term phagocytosis originates from the Greek words meaning “meal” and “cell”. 3. €receptor-mediated endocytosis or clatrin-dependent endocytosis: impairments of this mechanism result in the development of certain diseases. Many intestinal toxins, in particular cholera toxin, enter enterocytes by such a mechanism (Lencer, 2001; Lu et al., 2005). Pinocytosis is a cellular process that allows the transport of fluid from outside the cell through the membrane surrounding the cell into the cell. In pinocytosis, tiny cuppings called caveolae (little caves) in the surface of the cell close and are pinched to form pinosomes, little fluid-filled bubbles that are free within the cell cytoplasm. Owing to such process, a cell can absorb both large molecules and various ions, which are unable to get through the membrane itself. Pinocytosis is often observed in cells the function of which is related to absorption (epithelium of capillaries). Thus, bubbles (or vesicles) are formed on the cellular surface. They deliver substances into the cell and restore the plasmatic membrane. Such vesicles transfer water

30€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

and substances (small molecules and soluble proteins) from the fluid of the extracellular environment. The large number of vesicles allows them to deliver plenty of substances into the cell. Pinocytosis is extremely intensive process. In some cells, 100 % of the plasmatic membrane is used up and restored in only one hour. Pinocytic vesicles that are detached in cytoplasm are capable of fusing with each other, forming early endosomes. Moving deep into cells, such endosomes can fuse with neogenic lysosomes, forming late endosomes; the destruction of substances begins in its lumen. Hydrolases from Golgi complex are contained in them. Phagocytosis is the cellular process during which solid particles (up to 0.5 μm) are surrounded by the cell membrane to form an internal phagosome or “food vacuole”. The phagosome is usually delivered to the lysosome, an organelle involved in the breakdown of cellular components, which then fuses with the phagosome. The contents are subsequently degraded and either released extracellularly via exocytosis or released intracellularly to undergo further processing. The phagocytosis is carried out by a clatrin-independent actin mechanism. Polymerization of an actin, which happens after interaction of molecules with special superficial receptors of a cell, is necessary for this process. Phagocytosis is the basic mechanism of a host organism for protection from microorganisms. The phagocytosis of damaged or aged cells is necessary for tissue renewal and healing of wounds. The process of a phagocytosis plays an important role too in the reaction of an organism to an infection. Specialized leukocytes (phagocytes) swallow the bacteria after they have entered an organism, and then, they are destroyed by special enzymes. Aspects of membrane phenomena such as pinocytosis and phagocytosis show the dynamical nature of a membrane. Several kinds of membrane transformation take place: reorganization, transfer, fusion, replacement, and renewal. Receptor-mediated endocytosis uses specific superficial receptors for molecules transport. This mechanism has by the following properties. 1. € Specificity. Cells have superficial receptors providing selective binding of molecules from an extracellular solution. 2. € Ability to concentrate a ligand on the surface of a cell. Cells, selectively deleting a ligand from a washing solution, play the role of “water drainage” for these molecules. Finally, the ligand from a washing solution will be removed. 3. € Refractory period. If the specific receptor after binding of a ligand and its absorption on a membrane does not come back, the cell becomes refractory to the given ligand. That mechanism is of importance for the process of signal transmission and provides one way to cut off cellular signal. In the case of the GI tract, the endocytosis is surrounded by enterocytes from its extracellular fluid, including dissolved or suspended material. A portion of the brush border membrane is invaginated and pinched off, forming a membrane-bounded vesicle. By means of endocytosis vesicular mechanism, large molecular compounds,

Chapter 1. Transport processes in epithelial tissues€€€€€31

such as vitamin B12, ferritin, and hemoglobin, and small molecular compounds, such as Са ++, Fe++, and so forth, are absorbed (Loginov, Parfenov, 2000). The role of endocytosis is especially important in the early postnatal period. Antibodies contained in parent milk are absorbed in the child blood in a intact form (Loginov, Parfenov, 2000). In the adult humans, pinocytosis does not play an essential role for supplying an organism with nutrients. Cells carrying out exocytosis and releasing essential substances, for example, digestive enzymes or neurotransmitters, store these substances in vesicles located near the plasmatic membrane. When the appropriate signal is received, such vesicles fuse with the plasmatic membrane and release their contents. Sorting signals in vesicles selectively direct the substances to the apical or basolateral surface of the cell. The majority of the vesicles which do not have a specific sorting signal arrives at the apical surface through constitutive secretion , i.e., proteins secreted in the lumen lined by epithelial cells are transported to the surface by a constitutive pathway (Fuller, Shields, 2006). Transcytosis is a mechanism for transcellular transport in which a cell encloses extracellular material in an invagination of its membrane to form a vesicle and then moves the vesicle across to eject the material through the opposite cell membrane by the reverse process (Fuller, Shields, 2006). One of the most studied examples of transcytosis is the penetration of some parent antibodies through intestinal epithelium cells in the newborn. Parent antibodies enter the child organism with milk. The antibodies, bound with the receptors, are sorted by early endosomes of digestive tract cells, then pass through the epithelial cell by means of other vesicles, and fuse with the plasmatic membrane on a basolateral surface. Here, ligands are released from the receptors. Then, antibodies are collected in lymphatic vessels and arrive into the newborn blood. Upon transcytosis, neither the ligand nor the receptor are destroyed; the empty receptor comes back from the basolateral membrane to the apical surface of the cell to capture another ligand molecule.

1.4.1.5. Paracellular transport and persorption As indicated above, cell junctions are especially abundant in epithelial tissues. They provide contact between neighboring cells, between a cell and the extracellular matrix, or they built up the paracellular barrier of epithelia and control the paracellular transport. Basically, specific cellular interactions are provided by tight junctions, desmosomes, gap junctions, and plasmodesmosomes. Tight junctions form a barrier to paracellular diffusion of hydrophilic solutes. The phenomenon of adhesion of cells depends on desmosomes. Gap junctions allow free motion of ions and small molecules between adjoining cells. It is agreed that the motion of substances through tight junctions along intercellular spaces from one side of a cellular layer to another is impaired. But gonyautoxin 2/3

32€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

epimer penetrates the intestine through a paracellular pathway (Torres et al., 2007). Moreover, the paracellular route has a low contribution for compounds larger than an approximate molecular weight of 200 (Lennernas, 2007). Permeability of tight junctions depends on, or is determined by, absorption of various nutrients and their molecular structures and interactions with a cytoskeleton of cells (Ballard et al., 1995). Spermine (polyamine) can partly open the tight junctions of the epithelium (Sugita et al., 2007). Special interest to these problems has arisen after the appearance of a controversial hypothesis (Pappenheimer, 1990) stating that the main flux of absorpted glucose passes through tight junctions. However, such mechanism is possible, apparently, only with high permeability of tight junctions, very high glucose concentration in the lumen, and in the presence of a high-enough osmotic gradient for creation of a water flux (see 1.4.1.3). The ability of a mammal organism to absorb large-enough particles of starch and other substances of organic and inorganic origin has been demonstrated. This phenomenon is called persorption (Volkheimer, 1978). In the normal process of digestion, not only substances in solution are absorbed. Solid, undissolved food particles, in macrocorpuscular form, are regularly kneaded into the mucosa during their passage through the digestive tract. They pass between the epithelial cells into the subepithelial layer. From here, they are transmitted both by the lymph vessels and by the mesenteric veins into the circulation, where they remain for a considerable time. This process was discovered in 1844 (Volkheimer, 1993) and later confirmed by others but was generally considered impossible and therefore seen as an untenable theory because the passage of such large particles through the mucous membrane does not fit our conception of the physiological mechanism of absorption. Other particles of vegetable origin can also be persorbed. The phenomenon of persorption was also demonstrated in humans and animals with cellulose particles, pollen, spores, and fragments of plant hairs. After oral administration, yeast cells are found in the chyle, blood, urine, and cerebrospinal fluid (Volkheimer, 1993). The significance of persorption under conditions of increased permeability of the intestinal protective barrier has not been elucidated so far. It is not excluded that persorption takes place in pathogenesis of food allergies (Loginov, Parfenov, 2000). Unfortunately, Volkheimer’s effect has not been adequately investigated. However, the gastrointestinal uptake of micro- and nanoparticles has been the subject of recent efforts to develop effective carriers that enhance the oral uptake of drugs and vaccines (Hillyer, Albrecht, 2001).

1.4.2. Active, ion-dependent, or coupled transport processes In a quantitative sense, co-transport of Na+/glucose is the most powerful and specific mechanism of absorption in the small intestine (Fig. 1) that was suggested by successes of applying solutions for oral-rehydration therapy in case of diarrhea (Holtug

Chapter 1. Transport processes in epithelial tissues€€€€€33

et al.,1996). Transport processes coupled with sodium mediated€ by€ solute sodium symporters (SSS) (TC# 2.A.21) are extremely widespread in nature—subcellular organellas (nucleuses and mitochondrions), single cells (leukocytes, bacteria, erythrocytes, fibroblasts, etc.), nonepithelial tissues (brown adipose tissue, bone, a cartilage, brain, liver, cross-striated muscles, adrenal glands, etc.), and epithelial tissues (skin, GI tract, kidneys, etc.). From the theoretical and practical viewpoints, the major interest is in processes of Na+-dependent absorption of various nutrients in the small intestine. The resolution of the issue of coupled transport is directly related to finding out of the causes of metabolic diseases such as a malabsorption of sugars and amino acids, glucosuria, and so forth. Studies on dietotherapy, creation of ideal and synthetic diets, are impossible without the understanding of the mechanisms behind the active absorption of nutrients proceeding as well in a Na+-dependent manner. For a long time (up to 1991), only two basic models (or theories) of the coupled cotransporter —mobile carrier and common channel (Nozdrachev, 1991)—were discussed in university textbooks (Fig. 2). It was considered that there are two different categories of cotransporters capable of transporting monosaccharides and amino acids; however, they can compete for energy sources in a cell. To some extent, these models contradict each other. Within the framework of these models, facts such as the existence of non-integer numbers for stoichiometry transport coefficients of sodium and nutrients and the discrepancy of data about the binding sequence of sodium and nutrients with a cotransporter have not received any satisfactory explanation. It is common knowledge that, upon digestion of carbohydrates in mammal intestines, glucose, fructose and a galactose are formed and can be absorbed. Their absorption through the brush border and basolateral membrane of enterocytes is mediated both by Na+-dependent and by Na+-independent membrane proteins, respectively. Glucose and galactose transport through a brush border membrane is carried out by Na+/glucose (galactose) cotransporter (SGLT1) and passive transport of fructose-uniportrer (GLUT5). The passive output of all three sugars from cells through a basolateral membrane is takes place through two uniporters (GLUT2 and GLUT5). Mutations on SGLT1 cause significant insufficiency of glucose and galactose absorption (glucose–galactose malabsorption), and mutations on GLUT2 do not affect the absorption of these sugars. Studies on mice which are defective on GLUT1 and on patients with Fanconi-Bickel syndrome suggest that the absorption of glucose and galactose is apparently carried out through some alternative mechanisms. Any anomalies of fructose absorption are not been revealed so far (Wright et al., 2003). Recently, Kellett et al (Kellett, 2001; Kellett et al, 2008) have provided evidence that the passive component of glucose absorption exists, but it is in fact facilitated diffusion since it is mediated by the rapid, glucose-dependent activation and recruitment of the facilitative glucose transporter GLUT2 to the brush-border membrane. Apical GLUT2 (Kellett et al, 2005 ) is primed by a long-term diet containing high–glycemic index sugars, insertion is rapidly induced by simple dietary sugars, and the apical

34€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

GLUT2 component of absorption is several times greater than the active component at high glucose concentrations. Apical GLUT2 is tightly regulated by long- and shortterm supply of dietary sugars. Hence, apical GLUT2 can provide a major route of sugar absorption by which absorptive capacity is rapidly and precisely upregulated to match the dietary intake of sugars during assimilation of a meal. These experimental data were confirmed in several laboratories in particular (Gromova et al, 2006; Grefner et al, 2006).€ However, the interpretation of these results contradict the views of some researchers. So, Ferraris & Diamond (Ferraris, Diamond, 1997) maintain that the kinetics of glucose absorption can be explained solely in terms of SGLT1 and that a passive or paracellular component plays little, if any, part. Consequently, the hypothesis about the participation of transporter of GLUT2 in glucose transport across the apical membrane of enterocytes in principle no objection, although not in all laboratories, these data were confirmed. But assertion that under normal conditions of facilitated diffusion involving GLUT2 is the predominant mechanism for transport of glucose across the apical membrane, some researchers believe are not justified ( Gruzdkov et al, 2006). At best, these mechanisms (with the participation of GLUT2 and SGLT1) are comparable with large carbohydrate loads. Really mechanism of facilitated diffusion of glucose will be effective only at high concentrations of glucose in the gut of around 300 mM after the hydrolysis of maltose. But, if we take into account the fact of the high permeability of unstirred layer under normal conditions, such high glucose concentrations at the surface of the intestine can not be real (Gruzdkov, Gromova, 2004). These estimates coincide with those of other researchers (in particular,€Ferraris et al, 1990). Disaccharides, as well as monosaccharides (glucose and galactose), are absorbed apparently by an active Na+-dependent mechanism. The enzyme-transport ensemble model is suggested to explain the mechanism of hydrolase-dependent disaccharide transport. However, the connection between enzyme-transport ensemble and coupled cotransporter for glucose monosaccharide is unclear so far.

1.4.2.1. The coupled transport of water-soluble substances The ion-dependent transport of water-soluble substances in living organisms has a large range of applications. Among all ions, sodium is the most important as a cofactor of the coupled transport (Schultz, Curran, 1970). A sodium-dependent mechanisms transports many nutrients; a wide class of various substances enter living organisms from the environment (Schultz, Curran, 1970; Parsons, 1975), in particular glucose (Fisher, Parsons, 1949, 1953; Riklis, Quastel, 1958). Today, the study of Na+-coupled processes has two branches: first, the quantity of objects in which such a phenomenon is present grows, and second, the list of watersoluble substances capable to be transported through membranes by means of Na+-

Chapter 1. Transport processes in epithelial tissues€€€€€35

dependent transporters also increases. Data available today demonstrate the existence of an extensive network of transport processes in which substance accumulation in a cell depends on the presence of sodium in the cell environment. So, the presence of sodium is necessary for the accumulation of amino acids by many tissues. Amino acids may actively accumulate in various tissues. In contrast, only epithelial tissues are capable to actively accumulate glucose and other monosaccharides. Sodium-dependent accumulation of sugars is observed only in experiments on intestines, kidneys, and dog vascular plexus. Thus, it has been firmly established that absorption of a diversity of water-soluble organic substances in the small intestine depends on and is coupled with sodium absorption. The list of such substances includes D-hexoses, L-amino acids, di -and triglycerides, some vitamins, salts of bile acids, and a number of other substances. Hence, the Na+-coupled processes are features of any multicellular animal, from protozoans to the humans (Schultz, 1977; Schultz, 1981). The largest and most complete aggregate of Na+-dependent transport processes is present in various epithelial tissues. The modern view on indissoluble interlink between Na+-dependent nutrient transport (for example, glucose) and glucose-dependent sodium transport suggests that stimulating effects of glucose on active sodium transport should be widespread to a not lesser degree. We shall consider properties of epithelial tissues and epitheliocytes in more details.

1.5. Transport characteristic of epithelium and epitheliocytes In every living organism, epithelial tissues are composed of layers of cells which line the cavities and surfaces of structures throughout the body and cover significant surfaces. They form integument and line internal organs, carrying out functions of protection, absorption, secretion, and perception of irritation. The epithelium protects underlying cells from mechanical damages, harmful chemical substances and bacteria, and also from drying. Absorption of water and nutrients occurs through the cells of the intestinal epithelium. Other epithelial tissues are used for the excretion of various substances from an organism. Among the epithelial tissues are skin, tissues lining the GI tract, renal tubules, urinary and gall bladders, respiratory pathways, and also sensory (for example, olfactory) and glandular (secretion of milk, earwax, sweat, etc.) epithelia. In humans, epithelium is classified as a primary body tissue, the other ones being connective tissue, muscular tissue, and nerve tissue. The epithelial tissue presents a two-dimensional structure of tightly packed cells located on the supporting tissue, muscular or connective, consisting of collagen, laminin, heparan sulfate proteoglycan, and fibronectin (Lauris, Lebland, 1983). The thickness of epithelial layers does not exceed several cellular layers, and it is frequently equal to only one monolayer of cells (in organs such as intestines and renal tubules) (Fig. 1). On the external surface of the columnar epithelium of intestines, there is a

36€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

set of extensions, or microvilli. The microvilli are structures that increase the surface area of cells by approximately 600 (human), thus facilitating absorption and secretion. They largely increase the area of a cell surfaces, and hence, they provide a large surface for absorption. On each cell, there are a few thousand such villi, and 1 mm2 of intestinal surfaces accounts for up to 2x108 microvilli. Microvilli are covered with plasma membranes, which enclose cytoplasm and microfilaments. Although these are cellular extensions, there are little or no cellular organelles present in microvilli. Each microvillus has a dense bundle of cross-linked actin filaments, which serves as its structural core. 20 to 30 tightly bundled actin filaments are cross-linked by bundling the proteins fimbrin and villin to form the microvilli core. The actin filaments make the microvilli capable of contracting similarly to flexing of fingers, although the motion is limited. The surface of enterocytes from a lumen side is covered by glycocalix. As indicated above, cells at the border between apical and lateral membranes are connected with each other by the continuous structure named tight junction (Ussing, Windhager, 1964; Kottra, Fromter, 1983). Two other types of cell junctions occur in epithelial tissues: gap junctions and desmosomes; the latter are especially numerous in multilayered flat epithelia like skin. Electric resistance of epithelial tissues largely depends on organs and species. So, the resistance of a rabbit urinary bladder, considered as tight epithelia, is equal to 1000 Ohm сm2, and the resistance of a rat proximal tubule, considered as leaky epithelia, is equal to 5 Ohm сm2 (Fromter, Diamond, 1972; Ussing et al., 1974). The epithelium resistance is directly related with the resistance of its tight junctions (Fromter, Diamond, 1972) but without distinction in membrane properties of various epithelia types. At the morphological level, the epithelium of intestinal mucosa is different from other epithelia, such as that of urinary bladder and amphibian skin. It consists of several highly specialized types of cells which are tightly connected to each other in the region of a terminal bar, forming a layer with the thickness of one cell. The significant part of the lateral surface of the cells separated from the brush border is free and potentially limits large intercellular space (Fig. 1). The existence of this space can have enormous significance for the transport of solutes and waters. Epithelial tissues consist of polar cells which are characterized by distinct structural membrane components facing an organ lumen and blood vessels. These epithelial cells tightly adjoin to each other, forming layers separating various compartments. The zone of junction between epithelial cells is a highly specialized area which not only for keeps cells together but also provides a barrier to the penetration of proteins from a plasmatic membrane from one side of the cell to the other. Because epithelial cells transfer solutes and water through cellular layers, they are polarized in the sense that substances enter through one membrane, cross the cell, and are removed through another membrane. The zone of contact, or tight junction, is a barrier limiting the free diffusion of protein molecules along a plasmatic membrane. Such a criterion is very important to separate the membrane of each epithelial cell into two parts: luminal, or

Chapter 1. Transport processes in epithelial tissues€€€€€37

apical, membrane (facing the organ lumen) and basolateral membrane (facing blood vessels). These two parts of the membrane are characterized by their own set of proteins. In other words, plasmatic membranes of epithelial cells are highly specific. This specificity is intended to increase as much as possible the cellular surface accessible to transport and to provide extracellular compartments in which solutes may concentrate. The vector nature of transporting epithelia means that substances which have entered a cell through one membrane will leave the cell through another membrane. The epithelium sheet can transport solutes and water in two directions: absorption from the intestinal lumen to blood and secretion and opposite transport from blood into luminal compartments. The transported fluid can be iso-, hypo- or hypertonic in relation to the plasma. Ions can enter epithelial cells mainly through two pathways: ion channels and possibly carriers. The energy necessary for the input of ions is usually provided from a membrane electrochemical potential gradient of sodium generated by the work of a Na+-K+ATPase pump. Its crystal structure is now available (Ogawa et al., 2009). This pump is the basic tool for removing extra Na+ from and pumping K+ into cells; it is localized on a membrane facing the circulatory system. Owing to the work of such a pump, the intracellular sodium concentration decreases down to 10–20 mM, and the intracellular potassium concentration increases up to 90–120 mM. The energy released during the motion of sodium through an apical membrane following its electrochemical potential gradient can be used by other transport systems. Hence, the mentioned pump carries out two basic functions: it removes sodium from a cell and generates the electrochemical potential difference providing energy for solutes pumping in a cell. The motion of water through epithelial tissues is coupled with the transport of solutes. The water flux through the intestinal epithelium is described by the following equation: Jw = Lp x (ΔP-Δ), where Lp is the hydraulic permeability for water, ΔP is the difference of hydrostatic pressure, and Δ is the difference of osmotic pressure. For most epithelial tissues, ΔP is close to 0. Epithelia with high water permeability, i.e. capable of transferring a great quantity of water and solutes (for example, duodenum), are characterized by extended and highly folded lateral intercellular spaces. The area of the apical surface is usually increased because of the microvilli of the brush border. Membranes of all parts of enterocytes have high permeability for water (high Lp). Other epithelia (for example, colon) are characterized by a low water permeability (low Lp) of the apical membrane. Such cells are more oblate. They are covered by fewer and shorter microvilli and are capable of transporting of hypertonic solutions. A characteristic feature of epitheliocytes is the presence of highly specialized effective transcellular mechanisms for active transport of ions and some nutrients. There

38€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

are two clearly distinct kinds of transepithelial pathways by which ions or nutrients can cross the epithelium: cellular and paracellular (Parsons, 1972, 1978; Smyth, 1974) (Fig. 1). In the intestine epithelium, the cellular pathway implies movement through the basic (columnar) absorbing cells. The paracellular pathway, first, includes movement through the tight junctions cementing neighboring cells. Second, this pathway includes movement through regions where cells are desquamated, i.e. through defects of a cellular palisade. Transcellular transport of cations (for example, sodium) and active transport of nutrients are carried out by two different transport systems. In the first case, intracellular electrochemical activity of the transferred substrate is lower than in the surrounding environment. This is possible because of the work from the pumps directed outside and located in membranes, performing substrate excretion. Free energy is needed only for substrate excretion from a cell, but substrate absorption in epitheliocytes is passive. It is the classical system of Ussing (Ussing, 1971b, 1971c; Parsons, 1978). In the second case, the transferred substrate is concentrated in the cell because of pump directed inside. Cellular transport is finished by passive motion of the substrate down its concentration gradient because of the leakage in membranes that permits the substrate output. Such system forms the basis of the classical model of transport of monosaccharides and amino acids through epitheliocytes of intestinal mucosa and renal tubules (Parsons, 1978).

1.5.1. Transport of nutrients through a brush border Besides the above-mentioned system for sodium transport, which is sensitive to amiloride, in the brush border of an enterocyte, there is one more independent system for sodium transport, activated or stimulated by nutrients (for example, glucose) (Schultz, 1981; Wright et al., 2011). To better understand the properties of the latter system (Fig. 1) which ensures Na+-dependent transport of nutrients through the brush border of enterocytes, we shall consider its work in more detail in Chapters 5, 9, and 11.

1.5.2. Transport of other compounds through a brush border Transport of inorganic phosphate through the apical membrane of the small intestine via coupling with Na+ is a limiting stage of its absorption and responds to changes of physiological and pathophysiological factors (Murer et al., 2001). Inorganic phosphate is absorbed actively through a brush border membrane of the human body by means of a special Na+/phosphatic cotransporter (type IIb). For this transporter, the specific inhibitor is phosphophloretine (Peerce et al., 2003).

Chapter 1. Transport processes in epithelial tissues€€€€€39

Flavonoid glucosides are absorbed in the small intestine in a Na+-dependent way by means of the transporter SGLT1. Apparently, the lactase phlorizin-hydrolase takes part in this process (Day et al., 2003). Active Na+-dependent transport of nucleobases (hypoxanthine) through a brush border membrane also takes place (Theisinger et al., 2003). Furthermore, the Na+-dependent transporter for nucleosides is present in the enterocyte brush border membrane throughout€the€length of the small intestine in embryos and in adult individuals (Ngo et al., 2001). Sodium-dependent transporter of bile acids with a molecular weight of 48 kDa (SLC10A2 = ASBT) in a human ileum plays the main role, both under normal and pathologic conditions (Shneider, 2001; Hakannsson et al., 2002; Hulzebos et al., 2003); a specific inhibitor for it is the analogue of dimers of bile acids S 0960 (Schlattjan et al., 2003). The stoichiometry of transport sodium: bile acid is equal to 2:1 (Weinman, 1997). The cyclosporine A increases the reabsorption of bile acids in intestines and reduces the synthesis of cholate (Hulzebos et al., 2003). The participation of any vesicular transporters in transcellular transport of bile acids is considered as improbable (Agellon, Torchia, 2000). For absorption of glutamin in the villi and crypts of enterocytes, there is not only the Na+-independent system L, but also two Na+-dependent systems. These highly specific systems are not described anywhere so far (del Castillo et al., 2002). The transporters that carry out highly specific Na+- and Cl--dependent creatine absorption through the apical membrane are located along the villi of mammal and bird enterocytes (Speer, Ilundain, 2002). Some nucleosides (thymidine, guanosine) are capable to be transported against their own concentration gradients in the presence of a transmembrane sodium gradient (Theisinger et al., 2002). In human intestine, ribavirin (Glue, 1999) is absorbed by means of the Na+-dependent transporter for nucleosides N1. It was found that the system of co-transport Na+/L-carnitine operates in the apical membrane of enterocytes, and its properties are similar to those of the known transporter OCTN2 (Duran et al., 2002; Peral et al., 2002). Intestinal absorption of biotin occurs via a Na+-dependent carrier-mediated process that involves the sodium-dependent multivitamin transporter (SMVT; product of the Slc5a6 gene). The SMVT system is exclusively expressed at the apical membrane domain of the polarized intestinal epithelial cells. The role of the positively charged histidine (His) residues of the human SMVT (hSMVT) in transporting the negatively charged biotin was examined. The important role for His(115) and His(254) residues in hSMVT function, which is most probably mediated via an effect on level of hSMVT expression at the cell membrane, was shown (Ghosal, Said, 2010). Some substances can be absorbed because of other ions. Thus, in the human intestine, the neutral pH-dependent thiamine (vitamin) transporter, blocked by amiloride (Dudeja et al., 2001), is found out. For the transport of vitamin C in the small intestine, there are two systems: ascorbic acid is transported by a special system and dehydroascorbic acid is transported by a glucose cotransporter (Fujita et al., 2000). Antibiotic

40€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

D-cycloserine is transported through the apical membrane of human enterocytes by a Na+-independent, pH-dependent transport mechanism (Thwaites, Stevens, 1999). Metal ions are essential cofactors for a wealth of biological processes, including oxidative phosphorylation, gene regulation, and free-radical homeostasis. Failure to maintain appropriate levels of metal ions in humans is a feature of hereditary haemochromatosis, disorders of metal-ion deficiency, and certain neurodegenerative diseases. A metal-ion transporter in the rat, DCT1(divalent metal-ion transporter-1), which has an unusually broad substrate range that includes Fe2+, Zn2+, Mn2+, Co2+, Cd2+, Cu2+, Ni2+, and Pb2+, was identified. DCT1 mediates active transport that is proton-coupled and depends on the cell membrane potential. It is a 561-amino-acid protein with 12 putative membrane-spanning domains and is ubiquitously expressed, most notably in the proximal duodenum. DCT1 is upregulated by dietary iron deficiency and may represent a key mediator of intestinal iron absorption (Gunshin et al., 1997). Ca2+ is a low-affinity noncompetitive inhibitor, but not a transported substrate- of DMT1, explaining in part the effect of high dietary calcium on iron bioavailability (Shawki, Mackenzie, 2010 ). The human intestinal proton-coupled oligopeptide transporter hPEPT1 (see also Chapter 12) has been involved in the absorption of pharmacologically active compounds (Sala-Rabanal, Loo, Hirayama et al, 2006). It was indicated that the substrate selectivity of hPEPT1 is Gly-Sar > the neuropeptide N-acetyl-Asp-Glu, delta-ALA, bestatin > cefadroxil, cephalexin > ampicillin, amoxicillin. We have try to classify, at least partially, data that we found in the literature on the mechanisms and localization of transporters for various classes of substances in the intestine of mammals. The classification is presented in Tab. 2.

1.5.3. Transport processes in intestinal basolateral membrane As mentioned, above in order to get from intestinal lumen into the blood, nutrients must cross not only the apical membrane but also the basolateral membrane. Our knowledge about nutrient transport processes in intestinal basolateral membrane, unlike those relating to the apical membrane, is rather fragmentary. In addition to the above-mentioned Na+-K+-pump, there are various ion symporters and antiporters, ATP-dependent Ca2+ pump and transporters (See Table 2) for glucose, fructose, and galactose (GLUT2), for neutral amino acids- except proline (L), for phenylalanine, tyrosine, amd tryptophan (T), for short-chain polar amino acids (A), for alanine, cysteine, serine, and threonine (ASC), and for some vitamins, C€ and B12 (cobalamin). For example, glucose enters the enterocytes across the apical membrane (brush border membrane) because of SGLT1 and leaves the cell through the basolateral membrane through the facilitative glucose transporter GLUT2. The facilitative glucose

Chapter 1. Transport processes in epithelial tissues€€€€€41

Table 2. Specific mechanisms and pathways of transport of different classes of substances in the intestine Compound Glucose, galactose Fructose Glucose, fructose, galactose Sucrose Maltose Lactose Neutral and cationic amino acids, β-Ala Neutral amino acids Arginine, lysine, pyrrolysine, cystine Imino acids Neutral amino acids except proline Proline, glycine, alanine, GABA, β- Ala Phenylalanine, methionine Phenylalanine, tyrosine, triptophan Glutamic acid, aspartic acid Lisine, cysteine, basic amino acids Lysine, arginine, glutamine, histidine, methionine, leucine Neutral amino acids, cysteine Short-chain polar amino acids (alanine, glycine, proline, serine, cysteine, glutamine, asparagines, histidine, methionine) Alanine, cysteine, serine, threonine Glutamic acid, histidine, aspartic acid

Transport Na+ dependence mechanisms Monosaccharides SGLT1 + GLUT5 GLUT2 Disaccharides unknown + SGLT1 (in rats) + unknown + Proteinogenic amino acids В 0,+ +

Location Apical Apical Basolateral, apical Apical Apical Apical Apical

В0 b 0,+

+ -

Apical Apical

IMINO L

+ -

Apical Basolateral,

PAT (Imino acid)

-

Apical

РНЕ T

+ -

Apical Basolateral

X –A ,G Y+

+ +

Apical Apical

Y +L

+

Basolateral

L А

+

Apical Basolateral, apical

ASC

+

Basolateral

N

+-

Basolateral, apical

42€€€€€Transport phenomena and membrane digestion in small intestinal mucosa Compound Di- and tripeptides (intact) Di- and tripeptides

Water

Inorganic phosphate

Iron Flavonoid glucosides Hypoxanthine (nucleobase) Bile acids Nucleosides (thymidine, guanosine) Creatine

Transport Na+ dependence mechanisms Peptides PEPT1 Membrane digestion + (aminopeptidases) + transporters for amino acids Other compounds SGLT1, diffusion + aquaporins Phosphate + cotransporter (NaPi type IIb) DCT1 - (H+) SGLT1 + Special transporter + SLC10A2 - ASBT + Transporter N1 +

CT1, solvent drag, +Na+/Cl-/creatine transporter Iodide NIS ? + L- carnitine OCTN2 + Pharmaceutical [drug] substance RIBAVIRIN antiviral agent N1 + (prototype guanosine) Antibiotics (ampicillin, PEPT1 amoxicillin, cephalexin, cefadroxil), an antitumor agent (bestatin), inhibitors of the angiotensin converting enzyme (capropril, enapril), some prodrugs (valacyclovir, l-α-methyldopa) Fatty acids > 8 C (2- monoglycerides, Micelles phospholipids, cholesterol) (endocytosis?) < 8 C (fatty acids) Diffusion? -?

Location Basolateral, apical Apical

Apical, paracellular, basolateral? Apical

Apical Apical Apical Apical Apical Apical

Apical Apical Apical Apical

Apical, basolateral Paracellular

Chapter 1. Transport processes in epithelial tissues€€€€€43

Compound В1 (thiamine) Vitamin C (ascorbic acid)

Vitamin C (dehydroascorbic acid) Biotin B12 (cobalamin) Liposoluble vitamins (A, D, E, K)

Transport mechanisms Vitamins Thiamin transporters-1 and 2 (THTR-1 and 2) Vitamin C cotransporters, SVCT1 and SVCT2 SGLT1

Na+ dependence

Location

- (H+)

Apical

+

Apical

+

Basolateral Apical

SMVT Receptor-mediated endocytosis + facilitated diffusion Together with the lipolysis products

+ -

Apical Apical, basolateral

-

Apical

transporters belong to the Major Facilitator Superfamily (MFS) of transporter proteins. The crystal structures of three members of this family have been obtained. These are the Lactose Permease (Abramson et al., 2003), Glycerol-3-phosphate (Huang et al., 2003), and Oxalate transporters (Hirai et al., 2003). From these structures, homology structural and functional models for the GLUTs have been proposed. The recent advances in understanding the GLUT function has been reviewed by Carruthers et al. (Carruthers et al., 2009). GLUT1 is the first equilibrative glucose transporter (facilitated duffusion) to be identified, purified, and cloned. GLUT1 is a polytopic, membrane-spanning protein that is one of 13 members of the human equilibrative glucose transport protein family. Two fundamently different models have been suggested for protein-mediated sugar transport, the simple carrier and fixed-site transporter models (See 9.1). The authors conclude that some expeimental data (GLUT1 ligand binding) are compatible with the fixed-site transport mechanism, although simple carrier behavior is observed under special circumstances. There is evidence that, in this membrane transporters, some dipeptides are presented: for glycyl-L-proline f.e.- the uptake of glycyl-L-proline by the basolateralmembrane vesicles is stimulated by the presence of inwardly directed pH gradient, and this stimulation can be abolished by the proton ionophores (Dyer et al., 1990); for Gly-Sar - substrate affinity of the basolateral peptide transporter for Gly-Sar is apparently asymmetric, but pH-dependence and substrate specificity are symmetric for the two directions of transport (Irie et al., 2004); for L-alanyl-L-phenylalanine and L-phenylalanyl-L-alanine (Lister et al., 1995). Vascular perfusion of the photoaffinity label, [4-azido-D-phe]-L-ala, had no effect on the transepithelial transport of the non-

44€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

hydrolysable dipeptide - D-Phe-L-Gln from the lumen, its mucosal accumulation or wash-out into the vascular perfusate. These data are consistent with those of a basolateral transporter that is strongly asymmetric in its substrate binding and transport properties. This protein was novel with no obvious similarity to PepT1, the apical membrane transporter (Shepherd et al., 2002). In some cases, the intestinal basolateral peptide transporter can recognize and transport nonpeptidic compounds and play a definite role in the absorption of deltaaminolevulinic acid (Irie et al., 2001). At the basolateral side of the enterocyte, a saturable high-affinity transport system for polyamine was revealed too. Polyamine uptake from the circulation plays an important role in the maintenance of the intracellular polyamine content during extensive proliferation in intestinal mucosal cells (Milovic et al., 1998). The authors believe that polyamine uptake from the circulation plays an important role in the maintenance of the intracellular polyamine content during extensive proliferation in intestinal mucosal cells. As mentioned above, the Ca2+ extrusion occurs at the basolateral membrane. These extrusion processes include the operation of an ATP-dependent Ca2+ pump and a Na+/Ca2+ exchanger, as well as exocytosis as the terminal event in the proposed vesicular transport mechanism. Evidence for the presence of an ATP-dependent Ca2+ pump at the basolateral membrane is documented and illustrated with biochemical and immunological data from studies on the avian intestinal basolateral membrane (Wasserman et al., 1992).

1.5.4. Interaction between transport processes in apical and basolateral membranes Sodium absorption, a dominant transport mechanism in many epithelia, is explained by the Koefoed-Johnson and Ussing model. This model describes the absorption of sodium in terms of three processes only and postulates their restriction to one or the other face of the epithelial sheet: passive Na+€ entry to the cell membrane facing the lumen and passive K+ exit and€ active Na+ and K+ transport through the cell membrane facing the blood (Koefoed- Johnson, Ussing, 1958). The first indication of interaction or “cross-talk” (Diamond et al., 1982) between apical and basolateral membranes came in 1961. It was shown that inhibiting the basolateral pump reduces the ionic permeability of both the basolateral and the apical membranes (Macrobbie, Ussing, 1961). Stimulating the pump increases apical Na+€ permeability, whereas inhibiting the pump reduces this parameter (Diamond, 1982). The converse type of interaction (or cross-talk) is a regulation of the basolateral membrane by the apical one. There are only two examples. Blocking the apical Na+€ channel in frog or toad urinary bladders (tight epithelia) with amiloride decreases basolateral K+€ conductance (Davis, Finn, 1982). Addition of nutrients transported by€

Chapter 1. Transport processes in epithelial tissues€€€€€45

Na+-dependent mechanism, alanine or glucose, to the solution washing Necturus small intestinal mucosa rapidly increases the apical conductance (Gunter-Smith et al., 1982). In parallel, there is an additional flux of sodium which occurs in the mucosal solution in presence of the corresponding nutrient. Such additional sodium is also removed from the cell by the ouabain-sensitive mechanism of active Na+-К+-ATPase transport.€ The role of this mechanism is double. While removing from the cell the sodium entering together with a nutrient, the intracellular sodium concentration is maintained to a low level, creating the electrochemical driving force for the entry of nutrients. Furthermore, the suggestion has been made that the depolarization of the apical membrane with an increase of sodium input results in the depolarization of the basolateral membrane (because of the low resistance of tight junction) (Schultz, 1977). The latter stimulates Na+-К+-ATPase and increases the potential difference across this membrane, which is then propagated to the apical membrane, restoring the potential difference to its initial level. We can supplement this argumentation in the following way. Our estimate of the time needed for the total depolarization of the apical membrane after instant deenergization of Na+-К+-ATPase with the simultaneous addition of 10 mM glucose in the mucosal solution is approximately one minute. In real experiments, where Na+-К+ATPase is operating and the membrane potential is equal 40–50 mV, the shift of the membrane potential in response to the addition of 20 mM glucose is equal to only 3–4 mV and occurs during very small time intervals (Okada et al., 1977). According to our estimates, electrotonic changes of the membrane potential propagate along the cell surface more quickly than local changes of concentration in cytoplasm due to diffusion. Apparently, only the electrotonic potential changes are responsible for the minimization of the membrane potential. Schultz and collaborators succeeded in strengthening this assumption by more direct evidence (Schultz, 1981; Gunter-Smith et al., 1982). In his view (Schultz, 1977), the system accelerates its work to maximize the transepithelial movement of sodium and nutrients. However, such is not the case. If we imagine that the spontaneous transport of sodium into a cell is decreased for some reason, then, according to similar argumentations, it may be inferred that the hyperpolarization of both apical and basolateral membranes and the corresponding decrease of the rate of functioning Na+-К+-ATPase take place. Therefore, we believe that, in the case of the epitheliocytes of the small intestine, Na+-К+-ATPase has the properties of a natural molecular voltage clamp for the membrane potential. Electronic voltage clamps devices are widespread in biophysics. Finally, the presence in epithelial cells of the Na+-dependent transport of nutrients results in the following events: additional sodium absorption from the intestine lumen and absorption of a wide range of water-soluble organic substances with the energy of the pump which is responsible for the removal of sodium from the cells through the basolateral membrane and the change of potential difference on the epithelial sheet by few milliVolts. Owing to the presence of a molecular voltage clamp in the

46€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

epitheliocyte, the two systems can respond to external influence as a unified system. With the increase (reduction) of sodium concentration in the mucosal solution, the rate of transcellular sodium transport will accordingly increase (decrease). Therefore, the increased (decreased) sodium flux in the cell through the apical membrane will be compensated by an increased (decreased) sodium flux through the basolateral membrane. It is significant that, at the same time, the intracellular sodium concentration and the potential difference on apical and basolateral membranes will to be almost constant. What other signaling agent may be involved in these feedback circuits between apical and basolateral membranes? According to Diamond (Diamond, 1982), the leading contender is changes in intracellular Ca2+. But, to explain the observed feedbacks by Ca2+ mechanisms, one would also have to assume that raised cell€ Ca2+ increases basolateral K+ conductance but decreases apical Na+ conductance.

1.5.5. Sodium transport through the apical membrane and through the epitheliocyte One of the basic functional characteristics of epitheliocytes from various tissues is presence of a vector system of transcellular sodium transport consisting of two components, a passive component and an active one. In the modern view (Ussing conception) (Ussing, 1971b, 1971c; Levin, 1979; Macknight et al., 1980), various parts of the membrane of an epitheliocyte are not equivalent for sodium transport. The apical (external, mucosal, luminal) membrane has a high permeability for that ion, and the sodium pump (figure 1) is localized in the opposite basolateral (internal, serosal, peritubular) membrane. The basolateral membrane is characterized by a high permeability for a potassium. In histochemical and autoradiographic studies, it was found that the Na+-К+-ATPase is localized in the cells of most types of epithelia, mainly on a basolateral membrane (Dibone, Mills, 1978; Ernst, Mills, 1980). Now, the sodium pump and Nа+-К+ATPase are established to be the same. Sodium enters an epitheliocyte down the gradient of its electrochemical potential through the apical membrane and is removed from the cell by the pump through the basolateral membrane with the energy of ATP. Sodium that has entered a cell in such a way does not interact with other intracellular sodium, forming a so-called transport pool or compartment. The sizes of such compartments have been estimated by various authors. Their results differ dramatically. The average size is equal to 20 % of the total sodium in the cell (Macknight, Leaf, 1978). The output of sodium through the basolateral membrane occurs against the gradient of its electrochemical potential and is accompanied by an input of potassium in the cell. The operation of the sodium pump has a rheogenic character, because, within one cycle, more sodium ions are removed from the cell than potassium ions enter. In addition to the usual mechanism of passive sodium transport, the mechanism of coupled sodium transport and nutrients is also present in the apical membrane of

Chapter 1. Transport processes in epithelial tissues€€€€€47

epitheliocytes of some tissues. We shall consider in more details how the cellular mechanism of Na+-dependent absorption of nutrients in one of the best-understood object, the small intestine of mammals, is realized. This mechanism has the following features (Schultz, 1981). It takes place in the mucosa apical membrane which can couple the input of sodium and nutrients in a cell (Fig.1). In such a way, the movement of sodium down its electrochemical gradient provides energy for the movement of the nutrient (for example, glucose) against its concentration gradient. These conclusions have been made in studies on both intact preparations of small intestine and vesicles from an apical or brush border membrane (Kinne, Kinne-Saffran, 1978). € Data concerning ion transport through the apical brush border membrane are plentiful and sometimes contradictory. For example, data obtained on vesicles do not contradict as a whole the results obtained in electrophysiological studies. We shall consider the conclusions of the summarizing article (Tai, Decker, 1980), devoted to transport processes in a rat ileum brush border. It should be pointed out that all types of sodium transport phenomena occurred in various preparations under various conditions it is possible to describe assuming the existence of only three transport mechanisms (for ions) in the brush border membrane. Two of these mechanisms are electroneutral, the coupled transport from a serose to a mucose (secretion) and the equivalent exchange of mucosal chloride for serosal bicarbonate. The third mechanism, electrogenic active sodium transport in the direction from a mucose to a serose (Sellin et al., 1989), apparently, is similar to those found in tight epithelia (Metelsky, 1984а). Almost simultaneously, the suggestion has been made that only three mechanisms are needed to explain all the data on sodium transport through the intestinal epithelium: (i) active sodium absorption, which has not been related to the movement of other water-soluble substances, (ii) sodium absorption coupled with the absorption of a wide range of nonelectrolytes, and (iii) electroneutral absorption of NaCl, followed by two electroneutral processes, an exchange of chloride for bicarbonate and an exchange of sodium for a proton (Schultz, 1981). Evidently, conclusions of both works, in general, contradict each other, but a discussion about the reasons for these divergences is not our task. We shall only notice that the presence of electrogenic components in transcellular sodium transport is suggested in both works, and in (Schultz, 1981), it has been proposed that the electrogenic sodium transport through enterocyte is caused by two transcellular mechanisms, one not coupled with the transport of nonelectrolytes and another one coupled with the transport of nonelectrolytes. These hypotheses should be taken into account. Because the properties of not-coupled electrogenic sodium transport in enterocytes are not studied sufficiently, we shall consider the properties of the mechanism localized in tight epithelia. It has been established (Fuchs et al., 1977; Lindemann, 1980; Thompson et al., 1982) that passive sodium transport in epitheliocytes of tight epithelia is due to functions of

48€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

ion channels in their apical membranes, with basic characteristics apparently similar to those of sodium channels in exited membranes (Khodorov, 1975; Hille, 2001). One can summarize the data on the properties of sodium channels in the epithelial tissues obtained by various methods in the following way (Metelsky, 1984а). The number of sodium channels blocked by amiloride (ASSC, or ENaC), which has been estimated by various authors, varies from 50 to 200 per μm2. Those channels are localized on the surface of the apical (brush border) membrane. The life time of a channel, estimated by the binding of labeled amiloride in the presence of an inhibitor of protein synthesis (cycloheximide), is equal to 60 hours (Cuthbert, Shum, 1976). The channel is a pore in the apical membrane, and the carboxylic group (or groups) is localized at its entrance. Protoning of this group results in the reversible closing of the channel. There is the gate in the channel which may be closed in the presence of high sodium concentration. Such an effect is similar to a substrate inhibition in an enzymology and is unknown for sodium channels in exited membranes. Gate closing is prevented by p-chlormercuribenzoate and benzoyl-2-guanidine. The channel is open during 1 s and is closed during 6-10 s. A current of 0,3 pA flows through the channel which corresponds to a carrying capacity of the channel of 1–3 Nа+/μs. The permeability of a single channel is equal to 10-13 cm/s (Lindemann, 1976).

1.6. Transport of water This is a rather controversial area of knowledge, and it is difficult to conduct a thorough discussion. More than a century ago, it was reported that a number of epithelia are capable of absorbing fluid in the absence of an external, osmotic, driving force when viable, but that this ability is lost after procedures that destroy viability (Reid E.W., 1901 cited by Schultz, 2001). Years later, this pioneering€ finding was confirmed and extended€ by Curran and Solomon (Curran, Solomon, 1957), when it was shown that NaCl was absorbed “uphill” and water followed “passively” in isotonic proportion.€ Then, the following two points became obvious: the transport of water through the epithelium is described by more general laws and the transport of water, especially the quasi-isotonic transport across epithelial tissues, is important for any living organism so that is has its own mechanisms. Transport of water through the epithelium is described by the laws of physical chemistry.€ As a whole, the water flux through the epithelium is described by the following equation: Jw = Lp (ΔPh – ΔPo) where Lp is the permeability of the epithelium for water, ΔPh is the difference of hydrostatic pressure on both sides of the epithelium, and ΔPo is the difference of

Chapter 1. Transport processes in epithelial tissues€€€€€49

osmotic pressure. For the majority of epithelia, ΔPh is close to 0. But what then appeared is that there are specific mechanisms of transport of water in the intestine. For example, in the small intestine, water is absorbed even in the absence of any external driving forces. Let’s consider the proposed mechanisms for this quasi-isotonic water flows according to (Hill,2008). The Osmotic Coupling Theory It became clear that the best candidate for this local space would be the interspace, and this gave rise to the ‘‘standing gradient osmotic theory,’’ or SGOT (Diamond, Bossert, 1967). In this theory, interspace osmotic coupling can give rise to quasi-isotonic flow if the dimensions are right (Fig. 1). This dependence on geometry is due to the coupling space not being stirred; therefore, concentration gradients play a controlling part in fluid secretion. The modern form of this theory is as follows: If the osmotic permeability of the bounding membranes is high enough, SGOT does not need to be considered in details, and all transport will be quasi-isotonic. This is the current simplified version, precipitated by the discovery of aquaporins (AQPs), which have the potential to raise osmotic permeabilities to high values. The Cotransporter Model This is basically the osmotic coupling model of the previous paragraph but with a novel addition of ‘‘water pumps’’ at the membranes. One was always brought up to believe that water was never pumped (uphill) because it would not be worth it; the water permeability of the cell membrane would always be a massive leak pathway in parallel. However, in epithelia where water is moving down an osmotic gradient at a rate that is too slow, this argument does not apply. The theoretical and experimental basis of water cotransport has been laid out in some detail over the past several years (Loo et al, 2002; Loo et al., 1996; Zeuthen, MacAulay, 2002; Zeuthen, Stein, 1994). The situation has been summarized by the leading worker in this field (Zeuthen, 2002): ‘‘Cotransporters working as molecular water pumps could be important building blocks in epithelial models…and…would alleviate the problems inherent in the traditional models based on osmosis alone.’’ The Na-Recirculation Model Like the cotransporter model, this is an addition to osmotic coupling in which Na+ ions partially recirculate through the cell. In the cotransporter model, water is added to the osmotic flow; here, the problem of isotonicity is solved by clawing some of the salt back again as it leaves the epithelial interspace. The amount that is recycled is just enough to reduce what would be a hypertonic solution to an isotonic one.

50€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

The model was first developed qualitatively for fluid transport by the glands in frog skin (Ussing et al, 1996); but later, a quantitative assessment of the recirculation (expressed as a fraction of the Na+ ions reentering the cells) was made for toad small intestine (Nedergaard et al, 1999). It came to about 70%. The equations used, which are crucial to the argument, stem from theoretical work on membrane fluxes (Sten-Knudsen, Ussing, 1981) subsequently applied in a modified form to epithelia (Eskesen et al, 1985; Lim, Ussing, 1982). The recirculation number for toad intestine was later incorporated into a large computer model of the epithelium in which quasiisotonic flow was generated (Larsen et al, 2000, 2002). Certainly, this is a complex thread to follow. The Electro-Osmotic Theory Ions are pumped across the cell, generating a transepithelial potential. This draws the requisite counter-ion(s) through a selective paracellular route, setting up water flow by e-o in the tight junction. The extent of water to ion coupling then determines the tonicity of the emergent fluid. The theory can be divided in three parts: (1) the demonstration that volume flows respond to changing electrical polarization of the epithelium, (2) an electro-hydrodynamic model of e-o in the tight junction itself, and (3) a model of the epithelium with ion and water fluxes, showing how it can offer a better explanation of responses to changing protocols than the osmotic coupling theory. The Osmosensor-Feedback Theory This is a radical departure from the theories described above and is based upon two novel mechanisms. The first is the function of an osmosensor molecule in the membrane. The second is a mechanism for junctional fluid transfer, located in the junction but controlled by elements in the adjacent cell membrane. The rate of this is controlled by the osmosensor. The net result is that the emergent fluid from the epithelium is effectively osmo-clamped close to that of the source bath (Hill, Shachar-Hill, 2006). It is based upon salt pumping across the epithelium but with the osmosensor controlling the tonicity of the transported fluid by effectively mixing cellular and paracellular flows, which may be regarded individually as hyper-and hypotonic fluids. Cellular fluid flow is treated as osmotic and therefore hypertonic in origin, whereas paracellular flow is the forced convection of a solution through junctional channels which must discriminate against salt more than water and, thus, must be hypotonic. Crane’s discovery of cotransport of sodium and glucose across the intestinal mucosa led directly to the development of oral rehydration therapy. This treatment counter-balances the loss of water and electrolytes caused by cholera via a glucose containing salt solution that accelerates water and electrolyte absorption. Then, it has been found that such absorption of water is secondary in comparison with active sodium and glucose transport (Meinild et al., 1998; Wright, Loo, 2000). The stoichiometry

Chapter 1. Transport processes in epithelial tissues€€€€€51

of the transport of Nа +/glucose/Н2О is equal to 2:1:210; this may provide half of the daily amount of absorption in the small intestine (Meinild et al., 1998). In the proximal segments of the intestine, sodium and water absorption strongly depends on the presence of glucose. Evidence for active water transport by the Na+/glucose cotransporter has recently been obtained from molecular dynamics studies of the crystal structure of the bacterial homolog vSGLT. The simulations show that 80 water molecules are transported with each galactose sugar (Choe et al., 2010). Sodium which has entered cells together with glucose through the brush border membrane (transporter SGLT1) is pumped out into blood through the basolateral membrane by the 3Na+/2K+-pump. Glucose is transported into blood through the basolateral membrane because of the facilitated diffusion. As a result, water, glucose, and sodium pass through the epithelial sheet. The mechanism of coupling between sodium, glucose, and water is still insufficiently understood. Usually, it is agreed that local osmotic pressure in lateral intercellular space increases because of the transport of sodium (Fig. 1). Water in the intestinal lumen is aimed at equilibrating the gradient of the osmotic pressure arising between the lumen and intercellular space. A water flux through the epithelium is observed in the direction lumen-blood. However, a considerable amount of data about more direct coupling between sodium transport, glucose, and water has been accumulated recently: water is co-transported through a SGLT1 transporter together with sodium and glucose (Wright, Loo, 2000). In clinical studies, data on the influence of the absorption of nutrients on water movement through cell junctions are questioned (Thomson et al., 2001). All of the above models strongly favor the existence of quasi-isotonic transport of water, and now, none of them can be rejected. Indirect evidence in favor of the e-o hypothesis may have the opposite effect to the main one, “the demonstration that volume flows respond to changing electrical polarization of the epithelium” (Hill, 2008). Really, it is well known that, when you change the osmolarity of the solutions surrounding the epithelial tissue at least on 5 -10 mosmol, the SCC€ or potential difference is changed (Metelsky, 2007a).

1.7. Final remarks The mechanism through which epithelial tissues can transport a fluid in the absence of significant concentration gradients is the transport of water coupled with transport of solutes. 1) € It is agreed that the mechanisms providing the entrance of solutes through the apical membrane increase the osmolarity of cytoplasm by 2 mosmol (as compared to a washing solution);

52€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

2) € Owing to the work of the basolateral Na+-К+-ATPase pump, the osmolarity of intercellular spaces increases by 3–7 mosmol (as compared to cytoplasm) (Gebhardt, 1974; Stevens et al., 1982; Larsen, Mobjerg, 2006); 3) € The small gradient (~5.5 mosmol), which is practically impossible to measure, is enough to generate large water fluxes through highly permeable cellular membranes (Lp is very high). The lateral intercellular space of the epithelial tissues transporting a great quantity of solutes and water is rather extended and has a folded structure. The area of the apical surface is usually increased due to the microvilli. The entire surface of such cells has a high permeability for water (high Lp). The mechanism of water absorption, related to the presence of nutrients, is used for a long time, for example, upon a secretory diarrhea. The expressed secretory diarrhea caused by micro-organisms can be stopped partly by a peroral rehydration therapy. Such treatment has been developed on the basis of the physiology of transport mechanisms; in reality, some sugar and amino acids are actively absorbed in the small intestine by Na+-dependent secondary active transport mechanisms. Therefore, the intake of fluid containing high glucose concentration, amino acids, and sodium promotes the intestinal absorption of nonelectrolytes and electrolytes that result in the movement of water down its gradient from the intestinal cavity to a tissue. Such absorption of water counterbalances (in full or in part) the secretion of electrolytes, and water, with micro-organisms (for example, Vibrio cholerae), thereby maintains homeostasis and prevents dehydration which is life-threatening. Other epithelial tissues are characterized by a low permeability for water (low Lp of the apical membrane). The surface of such cells is smoother, their microvilli are shorter and are fewer. They are capable to transport hypertonic solutions. In some epithelial tissues, the permeability for water can be regulated. So, for example, the collective ducts of kidneys have very low permeability for water in the absence of antidiuretic hormone. An antidiuretic hormone increases the permeability for water, making collective ducts capable of reabsorbing water.

Chapter 2. Investigative techniques of sodium transport in the epitheliocyte€€€€€53

Chapter 2. Investigative techniques of sodium transport in the epitheliocyte Transcellular sodium transport (especially its initial stage, the crossing of the apical membrane) is basic transport process of great importance, peculiar to any epitheliocyte. Before everyone decided to study sodium transport or Na+-dependent transport of substances through the apical membrane, the question aroused which technique should be used. Therefore, we shall consider briefly the basic investigative techniques of sodium transport in epitheliocytes. It should be pointed out that the number of such techniques is large and constantly increases (Metelsky, 1984b, 2007a). The investigative techniques of transport of salts and water on the level of a single membrane have been developed too. They essentially facilitate interpretation of the obtained results. Among these techniques are ultrastructural studies, intracellular microelectrodes, x-ray microanalysis, and study of transport in the isolated membranes. But some of them are time consuming (x-ray microanalysis and freeze-fracture microscopy), others (vesicles) are biochemical rather than physiological, and still others (microelectrodes) are accessible and convenient enough, but their application was strongly criticized (Ehrenfeld et al., 1976; Palmer et al., 1978). At the same time, there are physiological (biophysical) techniques which, under certain conditions, allow to study ion transport on the level of the apical membrane.

2.1. Isotope studies Under the conditions of a stage limiting process of sodium transport through a cell, where the passage through a channel apparently takes place in tight epithelia (Gurran, Gill, 1962; Lewis et al., 1977; Macknight et al., 1980), classical investigative techniques of active transport and sodium channels are applicable. Assay of unidirectional fluxes of sodium by means of isotopes is best suited for such studies. The first who has applied isotopes to study active transport was Ussing (1950). Later, the method, with various updated, was widely used both for independent studies and

54€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

to check indirect methods (Schultz et al., 1967) (see below). Many important results on the influence of various substances and their pharmaceutical compositions on the permeability of the apical membrane for sodium have been obtained with this technique. However, it is not the most simple and convenient technique. We have mentioned it only because of its methodological value, in theoretical equations describing transport phenomena in membranes, especially unidirectional fluxes (Ussing, 1971a; Wolf, Essig, 1977) appear.

2.2. Methods of molecular biology Although discussion on the opportunities of molecular-biology techniques are a little away from the main theme of our book, we should at least briefly describe the achievements obtained in the study of transport mechanisms. Molecular-biological approaches to studying transport processes across the intestine have been developed in the past 22 years (Parent, Wright, 1993; Broer, 2008a, 200b; Kleta et al, 2004; Page & DiCera, 2006). Many of the channels and transporters discussed in the book, such as the amiloride-sensitive Na+ channel (ENaC), aquaporins (AQPs), the facilitative glucose transporters (GLUTs), the Na+/glucose (SGLTs), Na+/phosphate (Na+/Phosphate), and H+/dipeptide (PEPT) cotransporters, have been cloned and sequenced. Over-expression in heterologous expressions such as Xenopus laevis oocytes and cultured cells has enabled their kinetic properties, such as substrate specificity, affinities, maximal transport rates, and temperature dependences, to be studied with high resolution. Members of the family of solute sodium symporters (Wright et al, 2004) are important in human physiology and disease where mutations in glucose and iodide symporters (SGLT1 and NIS) result in the congenital- metabolic disorders glucosegalactose malabsorption and iodide transport defect (Wright et al, 2007; Reed-Tsur et al, 2008). SGLT1 is the rationale for oral rehydration therapy, and SGLTs are currently being targeted in drug trials for type II diabetes. Of particular interest are studies that take advantage of the complex modern methods of molecular biology that provide very important results. Thus, in recent years, many methods for investigating the crystal structure of sugar-binding proteins, and especially Na+-binding proteins (Page & DiCera, 2006), appeared. Therefore, by using the crystal structures of molecules related to the ENaCs (chicken ASIC-1) and CFTR (bacterial multidrug ABC transporter SAV1866), a simple representation of the channels with fused fluorescent proteins was created with Visual Molecular Dynamics (Humphrey et al 1996). In our view, an example of such a study is the article of (Faham et al, 2008). Let us consider this article in more detail. To gain structural insight into the mechanistic details, the structure of vSGLT in the presence of Na+ and galactose was solved. As predicted by experimental and in

Chapter 2. Investigative techniques of sodium transport in the epitheliocyte€€€€€55

silico studies, vSGLT has 14 membrane-spanning helices with extracellular amino and carboxy termini (Turk, Wright, 1997; Turk et al, 2000). Optimized crystals of SGLT from V. parahaemolyticus (Faham et al, 2008) displayed anisotropic diffraction, with Bragg spacing of ~3.0 Å. Of the 548 residues of vSGLT, 512 were built; the remaining residues were in disordered loop regions. The structure is composed of a central group of seven helices (TM2, TM3, TM4, TM7, TM8, TM9, and TM11) that contribute side-chain interactions for ligand selectivity, along with seven supporting helices that stabilize these central helices.€ A striking feature is the two discontinuous TM helices, TM2 and the symmetrically related TM7, in the center of the protomer. In TM2, there is a break in the hydrogen-bonding pattern around residues I65, S66, and A67, dividing it into roughly equivalent intracellular (TM2I) and extracellular (TM2E) components loop regions. Data suggest that the inward-facing conformation of vSGLT hosts a large cavity exposed to the cytoplasm that requires simple displacement of an intracellular gating residue (Y263) for release of galactose. Modeling reveals an external pathway to the substrate-binding site formed by TM2E, TM3, TM7E, TM11, and helix EL8b in the extracellular loop. External Na+ binds first (Veenstra et al, 2004), and it was postulated that this facilitates molecular rearrangements in TM2 to form the substrate-binding site. A possible link between the Na+ and substrate-binding sites is residue N64, which is in hydrogen-bonding distance to the C2-OH of galactose. Galactose binding will induce the formation of the extracellular gate (Y87, F424, and M73), closing the cavity through bends in TM3 and TM11. These structural rearrangements are facilitated by conserved glycine and proline residues (TM3 G99 and P104; TM11 P436 and G437). Consistent with this model, the corresponding helices of LeuT have conserved glycine residues in the same regions. Authors conclude that to expand on this transport mechanism, further structural and functional analyses are required. It is of up most importance (see Chapter 9) that the authors (Faham et al, 2008) were able to identify a plausible Na+-binding site on the basis of a comparison with the LeuT structure, conservation of sequence amongst solute sodium symporters proteins, and a mutational analysis. It is surprising that Na+ coordination in the LeuTAa (from Aquifex aeolicus) involves no water in the rigid coordination shell. In this transporter, two Na+ are transferred with Leu with antiport of Cl– (Page & DiCera, 2006). Structural alignment with LeuT revealed a possible Na+-binding site at the intersection of TM2 and TM9, ~10 Å away from the substrate binding site (Faham et al, 2008). Yet, at the moment, it is hard to imagine how such research can make a significant contribution to the study of dynamics of change in the states of coupling mechanisms for intestinal transport processes which occur in a very short time, on the millisecond scale. However, the study by Li (Li, Tajkhorshid, 2009), which attempted to compensate for this disadvantage by using repeated molecular dynamics simulations, should be noted. Further analysis of the trajectories and close structural examination, in particular,

56€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

comparison of the Na+-binding sites of vSGLT and LeuT, strongly indicates that the crystal structure of vSGLT actually represents an ion-releasing state of the transporter. The observed dynamics of the Na+ ion, in contrast to the substrate, also suggests that the cytoplasmic release of the Na+ ion precedes that of the substrate, thus shedding light on a key step in the transport cycle of this secondary transporter. An interesting approach is used in the Lab of Prof. E.M. Wright. Rabbit or human SGLT1 (hSGLT1) was expressed in Xenopus laevis oocytes, and experiments were performed on oocytes 5 to 14 days after injection of complementary RNA (cRNA). Membrane currents were measured by using a two-electrode voltage clamp. The system chosen was the cloned SGLT1 expressed in Xenopus oocytes, and the advantages are as follows: (i) the cloned SGLT1 protein is expressed at high levels in the plasma membrane of oocytes, greater than 1011 copies per cell; (ii) the coupled transport of Na+ and glucose and the number of transporters expressed in the plasma membrane can be monitored by electrophysiological methods; (iii) Na+/glucose cotransport may be rapidly blocked by phlorizin; and (iv) the rate of Na+/glucose cotransport can be controlled by membrane voltage€ (Birnir et al, 1991). The kinetics SGLT1 do not depend on the expression system used.€ Human SGLT1 has been expressed in mammalian HEK cells and Xenopus laevis oocytes, and the kinetic properties have been found to be the same in both systems (Hummel et al., 2011). By using this approach, the authors managed to obtain many important results. For example, they were the first to discover the new fundamental mechanism of isotonic coupled water transport by cotransporters for ions and sugars (Loo et al, 1996; Meinild et al, 1998). It is possible that such mechanisms of isotonic water transport are very widespread in nature.

2.3. Optical studies With the help of optical methods, the conformations of the Na+/glucose cotransporter (SGLT1) during sugar transport by using charge and fluorescence measurements on the human SGLT1 mutant G507C expressed in Xenopus oocytes can be studied (Loo et al., 2006). Changes in charge and fluorescence (Cys507 of hSGLT1 was labeled by tetramethylrhodamine-6-maleimide) in response to rapid jumps in membrane potential in the presence and absence of sugar or the competitive inhibitor phlorizin were recorded. Authors succeeded to isolate an electroneutral conformational change that has not been previously described. This rate-limiting step at maximal inward Na+/ sugar cotransport (saturating voltage and external Na+ and sugar concentrations) is the slow release of Na+ from the internal surface of SGLT1. The high affinity blocker phlorizin locks the cotransporter in an inactive conformation. As seen from the example mentioned above, such studies are often used in conjunction with the methods of molecular biology and electrophysiological techniques

Chapter 2. Investigative techniques of sodium transport in the epitheliocyte€€€€€57

2.2. Electrophysiological studies 2.2.1. Short circuit current method and the rate limiting step for trans� epithelial transport of Na2+ The results obtained in isotope studies can be interpreted rather easily. However, this method requires special conditions and equipment and consequently has limited applications. More often, active sodium transport is measured by the short circuit current (SCC) method (Fig. 3), developed by Ussing and Zerahn and widely used until now (Ussing, Zerahn, 1951; Costa et al., 2000; Taylor et al., 2001; Kuge et al., 2001; Hardcastle et al., 2001; Patacchini et al., 2001; Zareie et al., 2001). The SSC method is a kind of mode of voltage-clamp method when the holding potential is 0. This SCC method to measure active sodium transport has been found to yield results adequate to data obtained in isotope studies (Lewis, Diamond, 1976; Mandel, Curran, 1973). After discovering the fact that nutrients, which can be absorbed from the intestine lumen by active Na+-dependent transport, are also capable to stimulate active sodium transport in a mucosal solution (Schultz, Zalusky, 1964b), some authors have offered

SCC PD

Na+

Na+

М

S

Frog skin Fig. 3. Method of short circuit current for the frog skin. To reduce the potential difference on the preparation down to 0 mV, an external electric current is passed through it; the short circuit current is caused by ion active transport.

58€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

to use a SCC increase in response to addition of nutrients as a measure of active transport of such substances (Kohn et al., 1968; Syme, Levin, 1976; Smith et al., 1981). There were experimental and theoretical bases for this. There is a resemblance between the properties of active glucose transport (for example, determined in biochemical studies) and effects of glucose on active sodium transport (recorded by the SSC method). On the one hand, it has been found that active glucose transport and induced effects of glucose on the SCC depend on the glucose concentration with saturation. The kinetic parameters are in close agreement, and they are inhibited by identical concentration of phlorizin. On the other hand, this method was supported by the dominating concept about indissoluble coupling between active fluxes of sodium and glucose through the apical membrane, formulated as a hypothesis on common carrier (Crane, 1962, 1965). It is believed that studying SCC responses is a fast and specific method to determine the characteristic of highly specific transport systems for amino acids in the enterocytes of various mammals (Smith et al., 1981). So, the method of measurement of Na+-dependent absorption has been proven theoretically and has obtained numerous experimental evidences (Kohn et al., 1968; Syme, Levin, 1976; Smith et al., 1981; Hollander, Dadufaza, 1983; Green et al., 2000; Alexander, Carey, 2001; Kroesen et al., 2002). Studying the active transport of nutrients by measuring the SCC response amplitude upon addition of these substances in a mucosal solution is now a widely used method (Kohn et al., 1968; Syme, Levin, 1976; Smith et al., 1981; Hollander, Dadufaza, 1983; Metelsky, 1984b, 2004a; Metelsky, Dmitrieva, 1987; Polyakov, Danilevskaya, 1989; Green et al., 2000; Alexander, Carey, 2001; Kroesen et al., 2002). Now it is commonly accepted that the measurements of Na+-dependent nutrient absorption magnitude do not dependent on the techniques used (electrophysiological or biochemical assays). Although the measurement of SCC is an indirect method, it used rather widely because of its simplicity and availability. In such measurements, a preparation of epithelium is mounted in a Ussing chamber where it serves as a diaphragm separating two compartments in which electrodes are placed. It should be pointed out that the SCC is measured by the voltage clamp technique. The main point of that technique is that the potential difference in a preparation is held at a predetermined level irrespectively of changes of its electromotive forces (EMF) and resistance thanks to a special electronic. In this case, the potential difference can be considered as an independent variable. The current necessary to maintain the potential difference in a preparation, equal to 0, is known as the SCC. The value of the SCC within an electron charge is equal to the flux of actively transported sodium which is calculated as the difference of two unidirectional fluxes of this cation. Owing to the use of the voltage clamp technique, it was possible to demonstrate the presence of a regulatory center near the sodium channel and to introduce the concept of permeability for objects as complicated (in the morphological relation) as frog skin (Fuchs et al., 1977) and a colon (Thompson et al., 1982; Bize, Horisberger, 2007).

Chapter 2. Investigative techniques of sodium transport in the epitheliocyte€€€€€59

Results of the pioneering works of Ussing, Zerahn (Ussing, Zerahn, 1951) and Schultz, Zalusky (Schultz, Zalusky, 1964b) implied that the rate limiting step for transepithelial transport of Na+ is Na+€ entry into cells across the cell apical membrane. Further studies showed that the assumption of the localization of the limiting stage of Na+ transport at the apical membrane is valid for frog skin and intestinal epithelium. On a frog skin (Ussing, 1971b), the rate by which chloride is capable to follow Na+ can determine the rate of the active Na+ transport (certainly, under the condition when a limiting stage is the crossing by Na+ of an apical membrane). Noise in biological systems can be treated as a process reflecting fundamental properties of the object (Lindemann, 1980; Chen, 1981; Awayda et al., 2000). Usually, the characteristics of noise of an electric current passing through an object are studied by the voltage clamp technique. In one of these studies (Van Driessche, Lindemann, 1976), the authors reach the following conclusion after analyzing their data: the ampÂ� litude of fluctuations is proportional to the Na+ concentration in the external solution washing frog skin; passive electrodiffusion of Na+ through pores which are open and closed randomly takes place. In studies of the noise on the potential difference on frog skin, fluctuations of the potential difference at low Na+ concentration in the external solution have been found to reflect the noise of absorption of this cation on the skin’s external surface (See 2.2.3). Artificial compounds which result in an increase in permeability of the apical membrane when they are added into a solution used to wash the external surface of frog skin have also been studied (Zeiske, Lindemann, 1974). The presence of such compounds points to the fact that the sodium pump in these tissues does not function at the maximal rate, and the rate of transcellular Na+ transport is limited by its passage through the apical membrane (Metelsky, 2007a) (See 2.5.2). Owing to the use of the voltage clamp technique, it was possible to demonstrate the presence of a regulatory center near the Na+ channel and to introduce the concept of permeability for objects as complicated (in the morphological relation) as frog skin (Fuchs et al., 1977) and the colon (Thompson et al., 1982; Bize, Horisberger, 2007) (See 2.2.1). Active Na+ transport and active glucose transport at temperatures below 16°С are dissociated (Metelsky, 1987). Because glucose at low temperatures does not permeate enterocytes, it stimulates Na+-transport from the external surface of an apical membrane. Therefore, one is inclined to think about the superficial mechanism of an induction of Na+ transport by glucose when the phenomenon of an induction itself is caused by the binding of glucose with some nearby receptor of the sodium transporter. The presence of a stimulating glucose effect upon its binding with the external surface of an apical membrane shows that Na+ transport through a brush border in the absence of glucose proceeds at a rate slower than the maximal rate and that a stage limiting transcellular active Na+ transport is the stage of Na+ crossing a brush border. In actuality, if we assume that the limiting stage is the crossing of a basolateral membrane, it is difficult to imagine a mechanism according to which the acceleration

60€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

of a fast stage of crossing an apical membrane could result in acceleration of active transcellular Na+ transport (See 9.4.1). The conclusion that crossing an unstirred layer and an apical membrane is a limiting stage of some processes is consistent with the conclusion obtained earlier about activation of special transport systems in a brush-border membrane for Na+ as glucose molecules approach them (See 9.4.1). The observed (Metelsky, 1987) values of activation energy are rather high, but it has long been known that, for transport processes through membranes, the value of activation energy is 80 kcal/mol (Kotyk, Janacek, 1977). When proceeding from high (in comparison with diffusion) values of activation energy (21.95 and 49.74 kcal/mol), we consider that the energy barrier to Na+ and glucose transport is localized at a level of passage of a brush-border membrane, and it is a limiting stage of processes of coupled transport. Renal and small intestinal (re-)absorption contributes to the overall phosphate(Pi)homeostasis. In both epithelia, apical sodium (Na+)/Pi-cotransport across the luminal (brush border) membrane is rate limiting and the target for physiological/pathophysiological alterations (Murer et al., 2001).

2.2.2. Transient characteristics measurement technique There are techniques which allow to isolate part of the process caused by sodium transport through channels in the apical membrane (Van Driescche, Borghyreef, 1975; Tarrin et al., 1979). In one of such techniques (Van Driessche, Borghyreef, 1975), an electric current with an amplitude of about 10 μА is passed through a preparation, and the dynamics of the potential difference changes on the epithelium is recorded. It turns out that the time-dependence of the potential difference after a current passes through a preparation is described by the sum of two exponents with time constants 3 and 100 μs. Replacing Сl- in a solution that washes the external surface of frog skin with SO42- or gluconate results in an increase of the contribution of the slow exponent, and its time constant increases up to 20 minutes. The decrease in sodium concentration in a solution by washing the external side of the preparation causes the reduction of the contribution of the slow exponent and the decrease of its time constant. In the presence of amiloride, the slow component disappears. On the basis of the analysis of experimental data, authors conclude that the mechanism responsible for the low-frequency component of the transient process is sensitive to the sodium content in the external solution; the mechanism takes place on the external side of the preparation surface. These data also show that the crossing by a sodium cation of the apical membrane is a process that limits transcellular sodium transport in frog skin. Thus, this simple technique allows to study sodium transport through the apical membrane. However, it should be pointed out that despite obvious advantages of such an approach, the interpretation of the studied effects is not simple.

Chapter 2. Investigative techniques of sodium transport in the epitheliocyte€€€€€61

2.2.3. Analysis of noise The following approach is devoid to some extent of such disadvantages, and its use in biology increases constantly. Noise in biological systems can be treated as a process reflecting fundamental properties of the object (Lindemann, 1980; Chen, 1981) – a fluctuation analysis (noise analysis) (Awayda et al., 2000). Usually, the characteristics of noise of an electric current passing through an object are studied by the voltage clamp technique. Then, the mathematical model for the basic properties of the channels (distribution in length, time of their operating in the open and closed states, etc) is elaborated. Parameters of the channels vary in such a way that the model will describe experimental data as well as possible. This approach has been applied to study the properties of sodium channels in epithelial tissues (Van Driessche, Lindemann, 1976; Li et al., 1979). In one of these studies (Van Driessche, Lindemann, 1976), the authors reach the following conclusion after analyzing their data: the amplitude of fluctuations is proportional to the sodium concentration in the external solution of frog skin; passive electrodiffusion of sodium through pores which are open and closed randomly takes place. In studies of the noise on the potential difference on frog skin, fluctuations of the potential difference at low sodium concentration in the external solution have been found to reflect the noise of absorption of this cation on the skin’s external surface. Such a technique is rather complex; however, this approach allows to get unique information on the functioning of a single channel, such as life-time in open and closed conditions, channel length distribution, etc.

2.2.4. Patch-clamp technique This technique, which was first applied to study exited membranes, started with the observation that, after pressing a glass microelectrode polished with fire to a clean cell surface, there is such a strong adhesion between the cell and the microelectrode that when the cell is removed, a microsite with size similar to the aperture of the microelectrode is pulled out from the cell. Because the area in question is equal to ~1 μm2, it is possible that only one channel is built at the tip of the microelectrode (Hamill et al., 1981). Now, this technique is a unique and direct method, giving information about single ion channels in a biomembrane. Thanks to this technique, basic results on noise studies have been confirmed. Indeed, channel conductivity takes only discrete values (Sakmann, Neher, 1983). When the patch-clamp technique is applied to epithelial tissues, it is possible to show that the functioning of ion channels in epitheliocytes does not differ much from the functioning of ion channels in exited membranes (Seip et al., 2001; Blaug et al., 2001). To use this technique, it is necessary to have electronic equipment with an electric noise lower than 1 nA in a range of several kHz (Stevens,

62€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

1984). Patch clamp techniques, such as excised inside-out patches and whole cell recordings, allow one to manipulate the composition of the solutions on both sides of a membrane. For example, by using the giant patch, it was possible to alter the composition of cytoplasmic compartment and to study the Na+/glucose cotransporter working in the reverse direction. This allows the authors to determine the internal kinetics, such as the affinity of the transporter for substrates Na+ and glucose in the cytoplasm. (Eskandari et al., 2005; Hummel et al., 2011).

2.3. Non polarized preparation This interesting approach, which allows to determine on which membrane, apical or basolateral, the studied compound acts, was used by Janacek (Janacek, Rybova, 1970). These researchers used a “non polarized” preparation of an epithelial tissue in their experiments, i.e. the one side of the preparation is in contact with a water phase and the other side with a hydrophobic phase. The measured parameter in that technique is the intracellular content of sodium ions. In such a modeling system, one can study the influence of various compounds on sodium transport only through one border, apical or basolateral, which is chosen in advance, because the transport of ions across a border washed with a hydrophobic phase is complicated. With this technique, it has been found that the amilorides influence exclusively the apical membrane, and cardiac glycosides, in particular, ouabain, influence only the basolateral membrane. Hormones, drugs, and anesthetics can change, apparently, the properties of both membranes (Janacek, 1975). Seemingly, this approach is not used anymore.

2.4. Fractionation techniques Epithelial tissues are in the morphological and functional aspects unique objects, the study of which requires the development of non-standard techniques (measurement of the SCC, transient characteristics technique, non-polarized preparation). The techniques allowing to study sodium transport through epithelial are described too. The case under study is the preparation of suspensions of isolated epithelial cells and vesicles of plasmatic membranes, the analysis of which is possible with classical techniques (Ugolev et al., 1969; Korn, 1975; Lamers, 1975; Sylber et al., 1975; Gall, Chapman, 1976; Hopfer, 1977; Murer, Kinne, 1980; Sachs et al., 1980; Lodish et al., 2000). These approaches should be considered as rather interesting, because they allow to simplify considerably the object structure and to study the basic functional and structural unit of the epithelium (cell or only a fragment of its membrane).

Chapter 2. Investigative techniques of sodium transport in the epitheliocyte€€€€€63

2.5. Pharmacological analysis 2.5.1. Inhibitors In any biological studies, detection of compounds that are capable to selectively suppress certain functions of a system in small concentration is an extremely productive approach to for the analysis of such functions. Therefore, the best compound to study sodium transport through the apical membrane of epithelial tissues is the weak basis amiloride. In numerous studies, it has been found that this compound competitively blocks the process of sodium transport from an external solution into the transport compartment of an epithelial cell (Benos et al., 1976, 1979). Thus, on the basis of these data, it may be inferred that amiloride only plugs the channel entry and doesn’t induce any chemical changes (Lindemann, 1980). The inhibitor of sodium transport through epithelial tissues -2,4,6-triaminopyrimidine, the structure of which is similar to that of classical inhibitors such as amiloride and triamterene, was investigated. It has been found that the cationic form of this compound competitively blocks sodium transport (ENaC) through frog skin, toad urinary bladder (Moreno, 1975; Zeiske, 1975), horse small intestine (Cehak et al, 2009), and ovine fetal small intestine (Keller-Wood et al, 2009) and in rat small intestine after total proctocolectomies (Fukushima et al, 2005) and cultured monolayers of dog jejunum (Weng et al, 2005). The inhibition constant is equal to 0.5 mM, i.e., 2,4,6-triaminopyrimidine is less effective than amiloride by a factor of 100. However, it was found out that although this compound blocks sodium transport through a cell tight junction (Simons, Naftalin, 1976), the rate of transcellular sodium transport remains constant. Thus, 2,4,6-triaminopyrimidine gives, apparently, the unique opportunity to increase the relative contribution of a transcellular pathway in comparison with a paracellular pathway; this may be of importance in studies of sodium transport in leaky epithelia (Balaban et al., 1979; Fanestil, Vaughn, 1979; Metelsky, 2007a). All the listed compounds block sodium transport in epithelial tissues reversibly; in reality, active transport increases up to the initial value when these compounds are removed from the solution. Except for the listed compounds, Ba2+, Mn2+, and some derivatives of benzodiazepine have an inhibiting action on sodium transport through the apical membrane (Hajjar, 1975; Rubery-Schweer, Karger, 1975;€ Ramsay et al., 1976). Now, agents that are capable of selectively modifying certain functional groups of proteins when participating in sodium transport are used more actively and more widely. Therefore, it has been found that if the carboxylic groups localized on the external surface of frog skin are modified with water-soluble carbodiimide (CMCD), the SCC through the preparation decreases down to 0 (Zeiske, Lindemann, 1975). This led authors to draw the essential conclusion that sodium at a channel entry interacts with carboxylic groups. This conclusion has been confirmed in studies of the pH dependence of SCC through frog skin as well. It has been established that with lowering

64€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

the рН of the incubation solution, SCC decreases, and the point of inflection is at рН 4.1. That pH corresponds to рК of carboxylic group.

2.5.2. Stimulants Artificial compounds which result in an increase in permeability of the apical membrane when they are added into a solution used to wash the external surface of frog skin have also been studied (Zeiske, Lindemann, 1974). The presence of such compounds points to the fact that the sodium pump in these tissues does not function at the maximal rate, and the rate of transcellular sodium transport is limited by its passage through the apical membrane (Metelsky, 2007a). The fact that these compounds increase the permeability of sodium channels results from the presence of a regulatory center in the immediate vicinity of a channel entry. The existence of such a center results in a decrease in the channel permeability with an increase of sodium concentration in the solution washing the apical membrane (Zeiske, Lindemann, 1974; Bize, Horisberger, 2007). Therefore, the compounds blocking sodium binding with that center lead to an increase in channel permeability. The higher the sodium concentration in the external solution, the largest the effect. Among such effective compounds are benzoyl-2-imidazole2-guanidine and p-chlormercuribenzoate (PCMB) (Lindemann, 1976; Spooner, Edelman, 1976). Addition of the latter compound in a concentration of 10 mM to an external solution results in the vanishing of the dependence of sodium channel permeability on the sodium content. Apparently, the structure of the regulatory center includes a sulfhydric group blocked both in the presence of Со2+ and pchlormercuribenzoate, a reagent on sulfhydric groups of proteins; in this case, the SCC through frog skin increases too (Hillyard, Gonick, 1976). We emphasize that sodium channels controlled by external sodium concentration are characteristic, apparently, of epithelial cells only, because known ion channels in exited membranes are controlled by a voltage. Uranyle ions in a concentration of 10 mM increase the SCC through frog skin by a factor of two. The apparent affinity constant of its binding with skin is equal to 1 mM. Authors believe that uranyle interacting with phosphatic groups of proteins in the vicinity of a channel changes the quaternary structure, thus inactivating the regulatory center (Zeiske, 1978). Apparently, copper ions act similarly on frog skin. (Ferreira et al., 1979). It should be pointed out that uranyle ions and mercury affect a turtle urinary bladder in the opposite manner; this can be followed by affecting the paracellular pathway or by the absence of the regulatory center near bladder channels. In this case, it is interesting to obtain data about the influence of p-chlormercuribenzoate and benzoyl -2-imidazole-2-guanidine on sodium transport in a urinary bladder.

Chapter 2. Investigative techniques of sodium transport in the epitheliocyte€€€€€65

2.6. Limitations of the used realizations of the short circuit current technique Hence, the most suitable method for the measurement of active sodium transport is the SCC technique. However, we are still lacking a complete description of its updates which would be suitable for studying the small intestinal epithelium of small laboratory animals. After analysis of publications devoted to that technique, some basic points remain€obscure. SCC in a small intestine of mammals, unlike in the case of frog skin, is caused mainly by active transport of the following ions: sodium (absorption), chloride (absorption), and bicarbonate (secretion). How should a technique be modified so that the SCC will measure mainly sodium transport? And would such modifications affect the preparation viability? Two types of preparations are used in the SCC technique. These are segments of intestine in the form of sacs with a length of 5–10 cm or segments of intestine opened along the mesenteric border and mounted in a Ussing chamber. No study discuss the advantages or disadvantages of one type of a preparation over the other, even though it is obvious that they are by far not equivalent. Furthermore, the resistance of subepithelial tissues distorts the obtained results. To reduce this effect, the serosa and part of the muscular layers are sometimes removed from a preparation. This method has obvious disadvantages. In most cases, the perfusion of a preparation is used on the closed cycle. It is apparent that with this perfusion mode, metabolism products which may be toxic may be concentrated in the system; in an organism these products would be removed with blood. Only in one realization of the SCC technique, solutions under the influence of gravity perfuse the chamber with rate of 1 mL/min. The solution which has left the chamber repeatedly is not used. Hence, in this case, the perfusion is carried out on the open cycle. In all realizations of the SCC technique, except for the one mentioned above, the perfusion rate is not indicated at all, and this is a neglect. There are unstirred layers of water near a preparation surface (Pidot, Diamond, 1964; Thompson, 1979; Metelsky, 2007a, 2007b, 2007c), which strongly distort the results. Besides, it is unclear whether the perfusion rates are sufficient for the adequate registration of the dynamics of the SCC response. It is apparent that the SCC response to a nutrient will be the slowest if a nutrient is added to a mucosal solution without any stirring. With stirring, the response rate will increase; it remains obscure whether the maximal response amplitude is achieved with any perfusion rate, and if so, which rate it will be. Thus, we reach the conclusion that the most suitable technique for studying sodium transport and effects of nutrients on it is the SCC technique. This technique has the following advantages: continuous record, simplicity in utilization, availability of the equipment, simplicity in obtaining preparations (in contrast to, for example, preparation of vesicles), and potentially good time discrimination of the dynamics

66€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

of the SCC response to nutrient addition. However, all the described updated of the SCC technique have some disadvantages which impair the study of coupled sodium and nutrients transport in adequate conditions.

2.7. Final remarks Among all the available investigative techniques for processes of coupled sodium and nonelectrolytes transport, the SCC technique is the most effective, and indeed, it has been widely used so far (Green et al., 2000; Diaz et al., 2000; Alexander, Carey, 2001; Kroesen et al., 2002; Metelsky, 2007a). It allows to characterize not only sodium transport and the effects of nutrients, but also Na+-dependent transport of nutrients. This is extremely important. However, the SCC technique, developed to investigate the indicated issues, is insufficiently elaborated both in methodological and theoretical aspects. This technique, perhaps, will become extremely informative and rapid, but so far it has been used only to get information such as “upon addition of a nutrient, the SCC has increased by such a value”, which is be then correlated with the nutrients transport intensity. It is appropriate first to carry out careful theoretical analysis of SCC measurement principles through a small intestinal wall with the voltage clamp technique. Because such a study is not published so far, there is no reason to consider the existing updates of the SCC technique as a completely adequate tool. On the basis of the theoretical analyses, it will be necessary to develop adequate updates of the SCC technique, suitable for measurements of sodium transport through the intestinal wall of small laboratory animals. Widening the scope of the SCC technique is also a vital issue. Therefore, it would be optimal to determine the kinetic parameters describing the interaction of a nutrient with sodium transport in a preparation upon addition of a single substance into the washing solution. The fact that one could determine the thickness of an unstirred layer of a fluid near a preparation from the single SCC response to an nutrient is important too. To do this, a mathematical model describing the SCC response dynamics needs to be developed.

Chapter 3. Some aspects of an adequate short circuit current techinque€€€€€67

Chapter 3. Some aspects of an adequate short circuit current techinque As it was mentioned earlier, to reduce the spontaneous potential difference on epithelial tissues down to 0, an external electric current must pass through the tissue in the corresponding direction. Usually, for this purpose, the voltage clamp technique is used. For better visualization of the essence of the SCC technique, we shall consider theoretical bases of the voltage clamp technique on an epithelial tissue. To reduce the potential difference on an epithelial tissue down to 0 for a long time (a few hours), significant currents (up to 120 μА/cm2) must pass through the tissue. At the same time, an electric charge passes through the setup electrodes; this electric charge is two orders of magnitude larger than the charge that passes through exited membranes. In studies of exited membranes, three electrodes are used: one electrode for the potential measurement, a second electrode for the current, and a third electrode both for the potential measurement for the current. Upon voltage clamping on epithelia, this modification of the technique is unacceptable. In the case of epithelial tissues, the charge passing through current electrodes is so large charges that the electrodes may become polarized. As this takes place, it is impossible to use of only one electrode both for the potential measurement and for the passing of the current. Therefore, to measure the SCC on epithelial tissues, four electrodes must be used, two separate electrodes serve only for the potential measurement and two separate electrodes serve only for the passing of the current. Functioning, accuracy, and stability of three electrode voltage clamps are well analyzed (Maksimov et al., 1975; Osipchuk, Timin, 1984), whereas not enough is known on the functioning of four electrode voltage clamps to measure the SCC. A detailed description of the adequate SCC technique will be presented in the next book provisionally entitled ”The short circuit current method”. At this point, the adequate SCC technique by the example of choice of experimental chamber construction, of its rate of perfusion and analysis of influence of subepithelial tissues on obtained results is considered only.

68€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

3.1. The chamber construction Each half chamber (Fig. 4) represents an indivisible construction made of Plexiglas (Metelsky, 1984b, 1987). The half chamber consists of a vertical load-bearing skeleton that can slide along guide rails; the basic part of the half chamber includes a water jacket and an experimental compartment€ attached to it. On the right of the half chamber, a latex ring is pasted. The physiological solution enters the silicone tube, which is in the water jacket, through an input fitting. For the best heat exchange, the tubes makes a few coils in the jacket. The physiological solution passing through the experimental compartment and then through the output fitting and a short polyethylene tube freely flows out in special capacity. Outputs of chamber channels for current electrodes for the best spatial voltage clamp are located concentrically with round half chambers. Outputs of the chamber channels for measuring electrodes are located in the immediate proximity of the preparation (~0.5 mm). In some experiments, a special rare nylon mesh was applied to restrict the displacement of the preparation in the chamber; however, because the record quality of the SCC did not improve, the mesh was not used (Metelsky, 1987). The volume of the left half chamber amounts to 30 (rat) or 100 (turtle) µL.

3.2. Rate of perfusion The influence of the perfusion rate and, accordingly, the thickness of unstissed layer of a fluid (see Chapter 7) on a SCC response to the addition of nutrients (and also on the coupled transport, during absorption), is insufficiently analyzed in the literature; however, it is common knowledge that processes of glucose transport in the intestine depend on the stirring intensity (Pidot, Diamond, 1964; Thomson, 1979; Thomson, 1983; Thomson, Dietshy, 1980; Winne et al.,€ 1979; Metelsky, 1987; Barry, Diamond, 1984). It is possible that SCC responses to glucose depends on stirring very strongly and then small uncontrollable changes in conditions of the solution flow near a preparation would result in changes of the SCC response. We must choose such perfusion rate so that the SCC response amplitude on glucose will be constant. According to the data obtained on rat small intestine (Metelsky, 1984b, 1987; Metelsky, Dmitrieva, 1987), the SCC response amplitude on 10 mM of glucose increases linearly with an increase of perfusion rate from 0 up to 1.5 ml/min, and the response value practically does not depend on the perfusion rate for higher rates (3–4 ml/min). A rate of 3–4 ml/min was considered as optimal (Metelsky, 1984b, 1987; Metelsky, Dmitrieva, 1987; Danilevskaya 1987, 1989; Danilevskaya, Polyakov, 1988). Apparently, the absolute perfusion rate is not important, but the important parameter is how quickly the solution is replaced in a half chamber – the ratio of experimental chamber volume to perfusion rate (Fig. 5).

Chapter 3. Some aspects of an adequate short circuit current techinque€€€€€69

Oscillograph

VCC Pulse stimulation

CA

CA

Recorder

VA

- Agar/AgClelectrodes - Solutions

Pump

Specimen Fig. 4. Set-up for short circuit current measurements. The preparation is mounted as a diaphragm in the Ussing chamber. Through both halves of the chamber, physiological solutions are pumped independently. VA – voltage amplifier for potential-difference control of the preparation; CA – current amplifier for short-current measurement; VCC – voltage clamp cascade. The measured short circuit current is recorded by an automatic recorder and controlled by an oscillograph.

70€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

Kt, mM (__ )

l, µm (_ _ )

500 25

400

20

300

15 200

RAT

10

100

5

TURTLE

0.020.030.05

0.1 0.20.30.5

1.0 2 3 5

10

tch, s

Fig. 5. Transport constant (Kt, solid line) and unstirred layer thickness (l, dotted line) near the surface of the small intestine of the rat (black circles) and turtle (open circles) in relation to the constant time of solution replacement in the experimental chamber (tch, s). For details, see text.

These results agree well with the data of Barry, Diamond, 1984, Fig. 2; Westergaard, Dietshy, 1974. Although different methods to measure the layer thickness were used (using the mannitol and the glucose) and the different measuring methods (SCC technique and potential difference recording) the obtained results were amazingly close to 220 µm. Kt for glucose in rat intestine, in Metelsky, 1987, decreased with chamber perfusion rate (0.6-36 ml/min) from ~18 mM down to 7-8 mM and in turtle (0.5-8.0 ml/min) from ∞ down to 2.2-2.3 mM. The above data are in agreement with results in rabbit ileum: when increasing rate of stirring from 0 to 600 rpm (Thomson, Dietshy, 1980), apparent Km for glucose decreases from 17.7 down to 1.5 mM.

Chapter 3. Some aspects of an adequate short circuit current techinque€€€€€71

3.3. Effect of subepithelial tissues on the results obtained For any voltage clamp, the point of elimination of the influence of the specimen series resistance, which in our case, consists of the solution resistance and mainly the subepithelial tissues resistance, is of first importance. On epithelial tissues, this problem is solved by two ways: first, by working out the clamps which automatically take into account the series resistance, and second, by removing a part of subepithelial tissues in a mechanical way. Two types of voltage clamps of similar type were described; they were constructed for tight epithelia and have not been applied so far in any studies. The principle of work of a voltage clamp with a discrete feedback (Brenneke, Lindemann, 1974) consists in a fast switching of two cycles. During the first cycle, the current does not flow through the preparation, which makes it possible to measure exactly the potential difference. In the following cycle, the current proportional to the difference between the measured and the holding potentials flows through the preparation. “Storage” of the potential fixed on a preparation takes place on the electric capacity of its membranes. Hence, such a clamp acts as if it does not notice the series resistance because it is not connected with the electric capacity. The functioning of the second voltage clamp (Gebhardt, 1974) is also based on the switching of two cycles. During the first cycle, the alternating high-frequency current (100 kHz) passed through the preparation. Only the series resistance is measured, and the membrane resistance is shunted through its capacity. In the following cycle, the voltage clamp works in the usual mode, but the flowing current automatically increases by taking into account the voltage drop on the series resistance. Hence, such a voltage clamp measures and compensates the series resistance in each cycle. In voltage clamp circuits with 3 electrodes, the compensation of the series resistance is carried out manually because of the increase of the positive feedback depth until the moment after which the circuit is excited (Osipchuk, Timin, 1984). Any of these ways of compensation the influence of the series resistance is not suited for us. In the case of a small intestine, we have to deal with a two-membrane system (in the case of exited membranes, the potential is clamped on one membrane only). Moreover, a system of two consecutive membranes is shunted by a small resistance. The theory of compensation of the series resistance for such objects is not developed yet. Apparently, the application of the listed techniques is not appropriate so far. To resolve this problem, other approaches are necessary.

3.4. Updating the SCC techinque for clinic study The SCC technique is well suited for experiments and, certainly, sooner or later, it should be used for clinic studies, where gut biopsies for morphological and, some-

72€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

times, for biochemical assays are regularly tested (for improvement of diagnostics). It is significant that during endoscopy, forceps for biopsy do not enter the internal environment of the organism (peritoneum or blood) but only tear off a piece of mucosa from the intestine surface. There is a wide range of diseases related with malabsorption, and therefore, the needs for clinical approaches allowing to determine Na+-dependent transport of nutrients, and hence, the basic mechanisms of intestinal absorption are huge (Loginov, Parfenov, 2000). There is a paradoxical picture; in clinical studies, a malabsorption is often diagnosed by indirect signs, but such diagnosis is rarely supported by modern methods. For example, in gastroenterology, loading tests (Loginov, Parfenov, 2000) with nutrients are applied only occasionally. When adapting the SCC technique for clinical research, two problems had to be overcome. First, in studies of animal GI tract, the researcher, as a rule, can determine the optimum size of resected tissue; but under clinical conditions, the size of a biopsy specimen is rigidly determined by the forceps features and not so much by the researcher (1.5-2.0 mm). It is pretty difficult to work with such a piece of tissue. Secondly, unlike in the case of resected preparations of animal intestine, where the researcher deals with the total thickness of the intestine wall, in the case of a human biopsy specimen, he works with a fragment of mucosa only, torn off from vessels and muscular layers. The SCC technique for studying biopsies of the GI tract under clinical investigations was applied for the first time in 1995 (Schulzke et al., 1995). The main point of this technique is the following (Fig. 10). The spread biopsy specimen of an intestinal mucosa cut during gastroenteroscopy is placed and then glued on a flat rubber ring which is then mounted in the miniature Ussing chamber. It is apparent that due to the small sizes of biopsies, there is no problem with the spatial voltage clamp.

3.5. Final remarks So, an adaptation of the SCC technique is developed for the adequate studying of sodium transport in the small intestine of various animals and human. The system is supplied by a reliable device for the stabilization of a preparation temperature. It is found that the value of the SCC responses upon addition of nutrients does not depend on holding the potential in a range ± 1 mV (Metelsky, 1987). Most studies demonstrate that short circuit current = active ion transport (Hirota, McKay, 2006; Metelsky, 2007a). The standard procedure of the maximal removal of ions from washing solutions (chloride and bicarbonate) is applied for a better determination of sodium transport among of electrogenic components of the transport of others ions (Cl- and HCO3-). As this takes place, it has long been known that the SCC becomes equal to a flux of actively transported sodium (Gonzalez et al., 1967; Quay, Armstrong, 1967; Sellin, Field, 1981). So, for perfusion of chambers, it is appropriate to use the peristaltic pump, because a greater precision of measurements and stability of perfusion is achieved; this is of

Chapter 3. Some aspects of an adequate short circuit current techinque€€€€€73

View from above

Intestinal biopsy material Just after biopsy

Spread

+

= Rubber ring Bottom view

Fig. 6. Preparation of a biopsy specimen before its mounting into modified miniaturized Ussing chamber. Small pieces of jejunum were glued on elastic rings.

importance for a better reproducibility of the results. It is of first importance that in this case, one can ensure the isoosmotic addition of the studied nutrients (see Chapter 4). The effects of addition of any substance result from its direct effect on the studied process and its indirect effect on the solution osmolarity. A non-isoosmotic addition results in changes of water fluxes through membranes and inevitable yields artifact of data. But in an overwhelming majority of studies, the addition of substances is made non-isoosmotically. Unexpectedly, it turns out that a number of the important issues are not analyzed or are analyzed insufficiently in the literature. This is the case with the choice of optimum rate of perfusion, for example, in studies realized with the SCC technique. So far, the majority of researchers does not provide any information on important characteristics of the technique, such as the rate of perfusion (Grubb, 1999; Yang et al., 1999; Winckler et al., 1999; Kroesen et al, 2002). In the studies where that parameter is given, the choice of perfusion rate is not grounded (Clarkson, Toole, 1964; White, Armstrong, 1971). This is rather surprising, because it is common knowledge that the perfusion rate influ-

74€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

ences both the oxygen supply to tissues and the thickness of unstirred layers, which play an important role in transport processes (Winne et al., 1979; Thomson, Dietshy, 1980; Thomson, 1983; Metelsky, 1987). It turns out that to obtain adequate results, the preparation should be perfused with rates no less than 3–4 mL/min. Maybe, the perfusion rate itself is not important, but the ratio of the volume of the mucosal halfchamber to the perfusion rate (tch) (Metelsky, 1987), describing the speed of solution replacement, is important. In some studies (Metelsky, 1987), this value is equal to 0.56 s. This perfusion rate (Metelsky, 1987) agrees closely with the maximal rate of stirring achieved in studies of active glucose transport (Thomson, Dietshy, 1980). In all known realizations of the SCC technique, the perfusion rate is much lower than in White’s study (White, Armstrong, 1971). Only in Clarkson’s study (Clarkson, Toole, 1964) on the creation of solution circulation with a peristaltic pump the rate was very high (5 mL/ min). However, this preparation used an everted sac with a length of 8 cm. Our assessment of the volume of such a sac gives ~1 mL. Hence, the time constant of the solution change (tch) in such a sac is 1 mL/5 ml/min = 12 s. A time constant of 12 s corresponds to a segment of the curve (Fig. 5) where the SCC response upon addition of glucose strongly depends on the perfusion rate. In such a range of rates, any improvement or deterioration of the conditions of the solution flow near the preparation will result in an increase or decrease of the SCC value. On the contrary, in (Metelsky, 1987), owing to the choice of the perfusion rate near a plateau (Fig. 5), any change of conditions of the water flow near the preparation (for example, its deflection in any direction) will not result in an error in the measurement of the SCC. Hence, the maximal perfusion rate in (Metelsky, 1987) is achieved by taking into account the significance of the time constant of the solution replacement in the chamber The conclusion of the theoretical analysis (Metelsky, 2007a) that, if the resistance of subepithelial tissues is close to 0, the SCC does not depend on the nature of the resistance shunting the epitheliocyte, turns out to be of first importance. Now, when there is an opportunity to use the low resistance of rat vascular system, that conclusion leads us to far-reaching consequences. We shall note in the beginning that, as the nature of the resistance shunting an enterocyte sheet is not stipulated, it can be anything. Shunting of an epitheliocyte may be due to cell junctions, a cell desquamation, or edge damage. If, as shunting resistance, the resistance of cell tight junctions is understood, it may be inferred that the SCC does not depend on the resistance of cell junctions. This is of prime importance for long experiments, where the state of cell junctions is certainly disturbed. Close values of the SCC on tight (shunting paracellular resistance is high) and leaky (shunting resistance is low) epithelia indirectly suggest such a paradoxical conclusion from the theory. Thus, on frog skin, the SCC is usually equal to 300 μA/ cm2, and on rat small intestine (in the presence of glucose), it is 200 μА/cm2. Agreement is rather good when taking into account that the specific resistances of these preparations are distinguished by two orders of magnitude.

Chapter 3. Some aspects of an adequate short circuit current techinque€€€€€75

In some parts of the intestinal epithelium, cells can be desquamated, and on a preparation different from (Metelsky, Ugolev, 1983), a spatial voltage clamp can be lacking near such areas. An edge damage in a preparation is a real phenomenon, not only on epithelium of a small intestine (Crane et al.,1965; Stockmann et al., 1999; Larsen et al., 2001) but even on tight epithelia (Dobson, Kidder, 1968; Walser, 1970). On the preparation used in (Metelsky, Ugolev, 1983), the SCC should not be changed if there is an edge damage. We emphasize that that phenomenon was already observed, but no one attaches any importance to this matter. So, in (Dobson, Kidder, 1968), the edge damage on a frog skin preparation was controlled (by means of light microscopy). Without discussion, authors note that the SCC value does not depend on the degree of edge damage! This is easy to understand on the basis of the theoretical analysis. Perspectives of application of the SCC technique in clinical research (see Chapter 15) are of particular interest.

76€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

Chapter 4. Osmotic phenomena and water fluxes€€€€€77

Chapter 4. Osmotic phenomena and water fluxes 4.1. Effects of osmotic pressure gradient through two types of epithelia Epithelial tissues can be subdivided into tight and leaky epithelia. Tight epithelia are characterized by electric resistance above 100 Ohm*cm2, and leaky one have a resistance equal or below 100 Ohm*cm2. In vivo epithelia, both tight and leaky, separate milieus, as a rule, having various osmotic and, to a lesser degree, hydrostatic pressures. Therefore, the osmotic phenomena should play an important role in function processes of all epithelial tissues in general and intestinal epithelium in particular. Let us suppose that a biomembrane separates (at the beginning) two solutions with different osmotic pressures: the osmotic pressure on the left side of the membrane is lower than that on the right. It means that the concentration of water on the left side of the membrane is larger than that on the right. Therefore, water will start to flow through a biomembrane from the left compartment to the right one. This will happen until the difference of osmotic pressures on both sides of the biomembrane becomes 0.

4.1.1. Influence of the gradient of osmotic pressure upon electric characteristics The effects of a gradient of osmotic pressure on two types of epithelia (tight and leaky) for a long time were developed independently; each type of epithelium had its own concepts and hypotheses. For example, for leaky epithelia, for a long time, the influence of a gradient of osmotic pressure on electric parameters of the tissue was discussed in the context of so-called streaming, or electrokinetic potential.

78€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

4.1.1.1. Streaming potential The streaming potential was found for the first time in 1861 by G. Quincke, and these data have been confirmed in the following 100 years by studies of rabbit urinary bladder (Diamond, 1962; Dietschy, 1964). Causes of osmotic potential are as follows (Pidot, Diamond, 1964). The membrane matrix carries an excessive negative charge; therefore, the channels through the membrane contain more mobile cations than anions. Upon creating an osmotic gradient, the water concentrations in both compartments become unequal, and water starts to flow through the preparation from the solution with the smaller osmotic pressure to the solution with the greater osmotic pressure. The water flux drags away fluid from the channels with an excess of cations. Thereby, the preparation side receiving this flow acquires a charge opposite to that of the membrane matrix. Notably, sucrose, raffinose, and mannitol produce the same results. Moreover, the curves expressing the dependence of water flux through a wall and the induced potential difference on substance concentration are parallel. A similar phenomenon was revealed in rat small intestine. It has been found (Smyth, Wright, 1964) that in the presence of a mucosal solution of 28 mM glucose, the potential difference on the intestinal wall is 14 mV. Upon addition of portions of mannitol up to a final concentration of 48 mM in a mucosal solution, the potential difference on the preparation gradually and linearly decreases with a coefficient of 1 mV/18 mM of mannitol. Authors reach the conclusion that recorded changes are caused by a streaming potential through negatively charged pores. The streaming potential is found out in an absence of hexoses. At a later time, it has been found (Smyth, Wright, 1966) that the addition of mannitol from the mucose side results in reducing both the spontaneous potential difference on the small intestinal wall with a coefficient of 2 mV on 1 mosmol/L and the fluid transport with a coefficient of 0.015 μL/mosmol hour. The value of the potential difference change does not depend on the presence of 28 mM glucose on both sides. In the absence of glucose upon addition of mannitol the potential difference changes its sign on opposite. In the presence of glucose, the changes in the potential difference caused by addition of mannitol (118 mM) do not vary over time. The same is observed for the responses of the potential difference to multiple additions of 28 mM mannitol in the mucosal solution not containing glucose, up to the 5th addition. However, in response to the 5th and 6th additions of mannitol, the potential difference on the intestinal wall after the initial decrease comes back almost to the initial value. Authors reached the conclusion that the movement of a fluid induced by glucose is not carried out through the pores responsible for the occurrence of the streaming potential. The streaming potentials give information about pore structure like electroosmosis does, and these may be used to distinguish between aqueous€ channels and ion carriers (Barry, Diamond, 1984). At a later time, one more mechanism for the potential difference on an intestinal epithelium decrease upon increasing osmolarity in a mucosal solution (in comparison

Chapter 4. Osmotic phenomena and water fluxes€€€€€79

with a serosal solution) has been offered. According to this mechanism, the occurrence of the resulting water flux down to its concentration gradient from a serosal solution to a mucosal solution is essential too. The water flux variously influences the salt concentration in two unstirred layers –€near the mucosa and in the subserosal layer including subepithelial tissues and unstirred layer near serosa. The dilution of salts occurs in the former unstirred layer, and in the latter one, salts are concentrated. The total effect of the water flux will be observed as an increase in salt concentration in the subserosal unstirred layer. As this takes place, there is an additional diffusion of salts through the epithelium in the direction from the serosal solution to the mucosal solution. Owing to the presence of some cationic selectivity in the small intestine epithelium, an additional potential difference is generated on the epithelium (plus in a mucosal solution) (Wedner, Diamond, 1969; Wight et al., 1972).

4.1.1.2. Opening of tight cell junctions Other mechanisms have been offered as an explanation for osmotic effects in tight epithelia. The significant reversible decrease in the potential difference on frog skin was observed for the first time in 1956 upon increasing the external solution hypertonity. Later, it has been assumed that the hypertonic solution causes the opening of a tight junction (Ussing, Andersen, 1956). At a later time, this assumption has been confirmed. Now, the opening of tight junctions between frog skin epitheliocytes under the action of a hyperosmotic solution is considered as a firmly established fact (Ussing, Windhager, 1964; Erlij, Martinez-Palomo, 1972; Ussing et al., 1974). Correlation between morphological changes and changes in the conductivity of a tissue has been found upon generation of a gradient of osmotic pressure through a leaky epithelium (Loeschke et al., 1970; Ussing et al., 1974; Bobrycki et al., 1981). Then, it was found that when increasing the osmolarity of the solutions washing the isolated frog skin by 400 mosmol (by addition of urea), there is a compression of cells and tight junction opening. The potential difference on the preparation and its resistance quickly and reversibly decrease, from 85 down to 4 mV and from 1800 down to 100 Ohm*cm2, respectively (Erlij, Martinez-Palomo, 1972). Hence, in these experiments, the opportunity of reversible transformation of a tight epithelium to leaky one (at least, from the viewpoint of electric resistance) is shown. These facts are evidence that the difference between tight and leaky epithelia is smaller than it seems to us and suggest that a change of osmotic pressure can change cell junctions in leaky epithelia (like intestinal one) too. In reality, it has been found in the case of rat jejunum that, upon addition of 170 mM mannitol in a mucosal solution, the same decrease in potential difference on the preparation is observed, as well as upon addition of a 170 mM mannitol in both solutions –€serosal and mucosal. Upon simultaneous addition of a 170 mM mannitol

80€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

in both solutions, the water concentration remains the same, and consequently, the resulting water flux through the wall should be absent. Hence, the decrease in the potential difference is not caused by the development of a streaming potential. That fact that a change of the potential difference in response to the addition of mannitol is directly proportional to the initial potential difference on the preparation supports this assumption (Garcia-Diez, Corcia, 1977). This fact suggests that a change of potential difference is not caused by the development of a diffusion potential (Garcia-Diez, Corcia, 1977), which should be proportional to the ratio of sodium concentrations in unstirred layers. In reality, the changes of this potential are proportional to the logarithm of the ratio of sodium concentrations. The elementary explanation for the obtained results is that under the action of an extra mucosal mannitol, the electric resistance of a shunting paracellular pathway decreases. The resistance of that pathway consists of the resistance of the tight junction and of the resistance of the lateral intercellular space (Ussing et al., 1974). With an increase of the solution osmolarity, there is a decrease of the resistance of the lateral intercellular space in a leaky epithelium because of its expansion. As a result, the resistance shunting the potential difference on the intestinal epithelium sheet decreases. This is manifested as a reduction of the initial potential difference. The authors think that the assumption about the decrease in the value of shunting resistance in a paracellular shunting pathway without changing its EMF is enough to explain the obtained results (Armstrong et al., 1975; Garcia-Diez, Corcia, 1977).

4.2. Osmotic effects in small intestine Indications on regular study of the influence of the osmotic phenomena on the SCC in leaky epithelia are not revealed. However, one can compare the properties of the reaction mechanism of small intestine transmural potential differences upon addition of mannitol with the properties of that for the SCC. The addition of mannitol in a mucosal solution results in the reduction of the SCC through a small intestinal wall (Metelsky, 1987) that corresponds to a decrease in transmural potential difference (Barry et al., 1964; Smyth, Wright, 1964; Garcia-Diez, Corcia, 1977). Under the action of the mannitol added in a serosal solution, the SCC increases, and this corresponds to an increase in the transmural potential difference (Garcia-Diez, Corcia, 1977; Naftalin, Tripathi, 1982). Moreover, there is a significant quantitative asymmetry between the effects of equal concentrations of mannitol in serosal and mucosal solutions (Metelsky, 2007a): the mannitol from a mucosal solution causes much greater effects than that from a serosal solution. This is in agreement with (Garcia-Diez, Corcia, 1977; Decker et al., 1981).

Chapter 4. Osmotic phenomena and water fluxes€€€€€81

When the probe solute (e.g., mannitol) is added to mucosal or serosal solutions, except for the quantitative differences noted above, there are some qualitative differences because the two unstirred layers are actually asymmetric (thicker on serosal side of intestine than on the other one). If the probe solute (e.g., mannitol) producing the osmotic flow is added to the serosal solution, the following result can be obtained: the flow increases with time (Barry, Diamond, 1984). When the osmotic probe (mannitol) is added to the intestinal mucosal side, the results are qualitatively very different: there is a transient overshoot in volume-flow component because, now, the diffusional delay time in the unstirred layer is shorter than the time needed for sweeping- away effects to become significant in the thick unstirred layer (serosal) (Barry, Diamond, 1984). These data are in agreement with results (Metelsky, 2007) where it was shown that a transient overshoot of osmotic response of SCC on mannitol really takes place when its concentration is higher than 30-40 mM. At mannitol concentration 20 mM or less, a transient overshoot in SCC response is not observed (Metelsky, 1987a). € Upon addition of small concentrations of mannitol (10-20 mM) in a mucosal solution, both the SCC and electric potential differences decrease to a new level, which does not change for a long time (Dietschy, 1964; Smyth, Wright, 1964, 1966; Metelsky, 1987). Addition of mannitol in a concentration equal to or higher than 100 mM in a mucosal solution results in the fast decrease of the potential difference or the SCC down to 0; then, these parameters change their signs and begin to increase in the opposite direction. When they reached an optimum value, they begin to increase slowly (Smyth, Wright, 1966; Metelsky, 1987). An important characteristic of osmotic responses of transmural potential differences is their independence of the nature of the substance causing such response (mannitol, sorbite, xylose, sucrose, fructose etc) (Levin, 1966; Metelsky, 1987). In a wide range of concentrations of added substances, the dependence of the osmotic effect is linear, and the factor of proportionality is 18-62 μV/mosmol (Diamond, 1962; Dietschy, 1964; Smyth, Wright, 1964, 1966) or 0.13-16 μA/mosmol (Metelsky, 1987, 2007a). Hence, from the qualitative and quantitative similarity of the effects of osmotically active substances on small intestinal transmural potential difference and on the SCC, it may be inferred that the cellular-tissue mechanisms responsible for these effects in both cases are very similar or even identical. A close link exists between osmotic SCC responses and SCC responses to glucose, which is confirmed both by the correlation between values of the osmotic response and the response to glucose and by the increase in the SCC response to glucose after a short-term unilateral incubation of the preparation with a hypotonic solution (Metelsky, 1987). Apparently, it is not possible so far to offer any adequate explanation of these phenomena. Perhaps, additional experiments are needed to explain both the existence of the earlier-mentioned correlation and the effect of a hypotonic shock. Perhaps, it can be inferred that the important physiological mechanism may be implied in the effect of a hypotonic shock (human and animals often drink water).

82€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

4.3. influence of water fluxes on absorption What is the mechanism of influence of osmolarity changes of washing solutions on the SCC? It is common knowledge that in response to an increase in osmolarity of a mucosal solution on 100 mosmol, the water flux entering through the mucosal membrane, equal (Newey, Smyth, 1962; Weldner, 1975) to 26 μL/hour*cm2, changes its sign and increases up to a level that is lower than the initial one. It should be pointed out that the water flux outflowing through the serosal border remains constant upon such sharp changes of water transport through the mucosal border (see Fig. 7 insert above, a dotted line) (Naftalin, Tripathi, 1983b). These results have generated some doubts on the basic argument (Garcia-Diez, Corcia, 1977) against the participation of transmural potential differences in the formation of the osmotic response. It has been found that the addition of mannitol in a mucosal solution causes the same change of the potential difference, as well as simultaneous addition of mannitol into both solutions. Water fluxes through mucosal and serosal borders are independent (Naftalin, Tripathi, 1983b). Because the mannitol from the serosal side changes the transmural potential difference much less effectively (compared with that from mucosal one), the water transport through the mucosal border in response to an increase in the mucosal solution osmolarity should change a little with a simultaneous increase in the serosal solution osmolarity. Unfortunately, such experiment (Garcia-Diez, Corcia, 1977) was not realized. However, this assumption is supported by experiments on SCC measurement. According to (Garcia-Diez, Corcia, 1977), the SCC should not change with simultaneous addition of mannitol in mucosal and serosal solutions. On the contrary, according to the stated assumption, the simultaneous increase in osmolarity of both solutions should cause changes of the SCC, and the sign of these changes should be same as upon addition of mannitol only in the mucosal solution. Results of experiments with addition of a 100 mM mannitol (Fig. 7) (Metelsky, 1987) support this assumption (Garcia-Diez, Corcia, 1977). Besides, the explanation (Garcia-Diez, Corcia, 1977) that osmotic effects on transmural potential differences are due to the change of the resistance of shunting cellular conductivity only, with constant EMF of a shunting pathway, has one more serious disadvantage. This explanation assumes the independence of the shunting pathway EMF and its resistance. However, it is difficult to imagine a reduction of this resistance (i.e. weakening of the link between cells) that would not be accompanied by a change of EMF of the shunting pathway localized in this area. Therefore, we believe that the streaming potential participates in the formation of the SCC response to change the osmolarity of a mucosal solution. This, certainly, does not exclude that this process plays a role and changes the resistance of the shunting paracellular pathway. The important argument in favor of the participation of the streaming potential in the formation of the osmotic SCC response is its similarity with the dynamics of

Chapter 4. Osmotic phenomena and water fluxes€€€€€83

Short circuit current

Mannitol 100 mM

15

Water flow, µl/cm2 * h

0

Time, min

15 30 0

0

5

10

10 µA/cm2

200 s Fig. 7. Changes in short circuit current response across rat small intestine on mucosal solution osmolality elevated by 100 mosmol (mannitol). Top – changes in water absorption across mucosal border of rat small intestine under mucosal solution osmolality elevated by 100 mosmol. Dotted line – water flow across serosal border. Adapted after Naftalin & Tripathi (1983: fig. 3).

water flux through a mucosal border and with the change of the SCC in response to an increase in a mucosal solution osmolarity (Fig. 7) (Metelsky, 1987; Metelsky, 2007a). In reality, before addition of mannitol, there is a streaming potential (or EMF) of the paracellular pathway (possibly equal to zero) caused by the flow of a fluid through tight junctions having cationic selectivity (Schultz et al., 1974). After an increase in a mucosal solution osmolarity, the flow of the fluid through tight junctions temporarily changes its direction, resulting in a change of the spontane-

84€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

ous potential difference on the preparation and, hence, a reduction and temporary change of the SCC sign. These conclusions are supported by the study of the link between the value of the osmotic SCC response and the preparation resistance (Fig. 8). Unexpectedly, it turns out that this relationship has a biphase character (Metelsky, 1987). When increasing the preparation resistance (from 0 up to 22 Ohm*cm2), the SCC response amplitude increases, and with a further increase of the resistance (above 23 Ohm*cm2) – (Metelsky, 1987), the SCC response amplitude decreases. This dependence is explained in the following way. The resistance of a shunting pathway consists in two resistances connected in series (Ussing et al., 1974), namely, the resistance of the tight junction and the resistance of the intercellular space, which can be changed during experiment. The low resistance of a preparation (ascending portion of the curve) means that the resistances of the tight junction and intercellular space are low, separately. Hence, although the paracellular water flux can be very large, it cannot develop a considerable streaming potential, because its selectivity, in view of the large size of intercellular spaces, is low. Upon

A, µA/cm2 30

20

10

0

10

20

30

40

R,Ohm*cm2 50

Fig. 8. Relation between short circuit current osmotic response on 10 mM mucosal mannitol and electric resistance of specimen. The peak on the curve corresponds to 22–23 Ohm*cm2.

Chapter 4. Osmotic phenomena and water fluxes€€€€€85

low resistance (ascending portion of the curve), water fluxes (the driving force of streaming potential) through a preparation are great. Therefore, when increasing of a preparation resistance (possibly through narrowing the tight junction), its cationic selectivity and, hence, streaming potential increase in parallel. This results in an increase of the SCC response. When increasing a preparation resistance above 23 Ohm*cm2 (descending portion of the curve), its permeability for water decreases. Although the selectivity of tight junctions is high, the streaming potential decreases, because its development is restricted by water fluxes. At a given resistance of the preparation (22.5 Ohm*cm2), the paracellular pathway has both optimal permeability for water and high-enough cationic selectivity; therefore, it is capable of developing the maximal streaming potential. When decreasing only one of these factors, the streaming potential and, hence, SCC responses upon addition of mannitol in a mucosal solution decrease. Because water fluxes perpendicular to membrane surfaces can strongly affect the results, the influence of these fluxes on glucose-dependent sodium transport has been estimated. The rate of water absorption depends almost linearly on the solution osmotic pressure. One is inclined to think that the water flux directed from the mucosal solution to the serosal solution will increase the SCC response to glucose a little, sweeping effect (Barry, Diamond, 1984), and the water flux directed in the opposite direction, sweeping- away effect (Barry, Diamond, 1984), will reduce the value of the response. The results obtained in studies (Metelsky, 1987, 2005d) on the influence of the simulation of intestinal water secretion and absorption on coupled transport (Tab. 3) agree closely with the results on modeling the influence of water fluxes on absorption (Gusev et al., 1983). The revealed effect of a water flux directed to an intestinal cavity on nutrient absorption, apparently, models the clinical case of secretory diarrhea. As this takes place, sharp inhibition of absorption can promote the passing of not-absorbed nutrients into the colon where they become a prey for microorganisms (in particular, E. сoli), which can then reproduce. It is common knowledge that toxin E. coli causes diarrhea development.

Table 3. The effect of osmotic pressure gradient across the rat small intestine on the SCC response on addition of 10 mM glucose (Metelsky, 2005d). Osmotic pressure gradient, mM 0 - 50 (serosa) + 100 (mucosa) 1

p< 0.02 versus 0 mM

Direction of total water flow across mucosa Absent From mucosal to serosal side From serosal to mucosal side

SCC response on addition of 10 mM glucose, µA/cm2 20.15 ± 1.81 (6) 22.3 ± 4.9 (3) 13.31 ± 1.45 (3)

86€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

4.4. Final remarks One can see that the osmotic phenomena are related with the process of water transport. Special conditions or solutions are not required for their observation. In fact, the osmotic component is present in any electrophysiological response to any substance in high concentration. The osmotic response does not depend on the nature of the added substance. Moreover, it seems to not be important that the added substance may penetrate to some extent through the brush border membrane. Therefore, SCC responses to 20 mM of mannitol which poorly penetrates through a membrane agree closely with those of 20 mM of fructose, for which a special system of passive transport exists. To reduce the osmotic component of the response, the tested substance should be added so that the solution osmolarity, especially in the case of a mucosal solution, does not change, i.e. in an isoosmotical way. Osmotic responses are somehow related with the SCC response to glucose, i.e., on the nutrient stimulating sodium transport. The correlation between osmotic and glucose SCC responses (Кcorr = 0.818) and the influence of unilateral short-term incubation of a preparation with a hypotonic solution on a SCC response to glucose are arguments in favor of it too. With the dynamics of the SCC response to mannitol (Diamond, 1966), one can determine the thickness of the unstirred layer of a fluid near a preparation. The mechanisms participating in the formation of osmotic responses are localized on cellular – tissue levels. The basic part of such mechanisms is situated in the socalled paracellular pathway formed by a tight junction and a lateral intercellular space. For the development of the osmotic response, it is important that two contradictory conditions are simultaneously satisfied: the permeability of the paracellular pathway for water should be high enough, and the tight junction should be at the same time narrow enough to distinguish cations and anions. Then, sodium ions move through the cell tight junctions (due to solvent drag), generating an electric current through the highest-resistance part of that pathway; this results into reducing the transmural potential difference. The importance of this two factors to the development of the osmotic response is suggested by the non-monotonic dependence of the response value on the preparation resistance. The optimum of that dependence is a resistance of 22.5 Ohm*cm2. The ascending portion of this curve (Fig. 8) is caused, apparently, by an increase in the tight junction selectivity, and the descending portion is caused by a reduction of the permeability of the paracellular pathway for water. Therefore, two main states of the intestinal wall were discovered; the first one is characterized by a low selectivity of the paracellular pathway and a high permeability for water, and the second one, on the contrary, is characterized by a high selectivity and a low permeability for water. This, at least partially, explains the differences in data concerning the cellular and tissue mechanisms of development of the osmotic response. In studies of preparations with a low resistance (ascending portion of a curve), researchers will reach the conclusion that the role of the streaming potential is determining, and in

Chapter 4. Osmotic phenomena and water fluxes€€€€€87

studies of high-resistance preparations (descending portion of a curve), one reaches the conclusion that the role of the resistance of intercellular space is the main component. The sensitivity of an intestinal epithelial sheet to changes in osmolarity of a mucosal solution is surprisingly high; the addition of 5-10 mM of mannitol only is enough for a reliable registration of SCC and potential difference responses. If the total osmolarity of a mucosal solution is about 300 mosmol, the epithelium can feel its insignificant changes (5-10 mM)/300 mosmol, i.e., 1.7-3.3 %. This sensitivity is not achieved in any measuring device created by the human. Sometimes, to explain certain data, the presence in intestine of osmoreceptors is postulated. For example, it is agreed that the an increase in water absorption and electrolytes in response to food loading is caused by neuro-humoral mechanisms, promoted by osmosensing and osmosignaling of upper the parts of the small intestine (Bastidas et al., 1992; Lim et al., 2007). From our viewpoint, all intestinal mucosa can be considered as a huge system of extremely sensitive osmoreceptors.

88€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

Chapter 5. Transport of monosaccharides and the contribution to its study made ...€€€€€89

Chapter 5. Transport of monosaccharides and the contribution to its study made by electrophysiological techniques 5.1. Transport of sugars through a brush border and a basolateral membrane Experiments performed on an intact tissue (Fisher, Parsons, 1953; McDougal et al., 1960) and confirmed in autoradiographic studies (Kinter, Wilson, 1965) and also experiment carried out on isolated vesicles (Murer, Kinne, 1980) have shown that the energydependent stage of sugar transport localized on the brush border membrane. Sugars first concentrated in an enterocyte in an active manner and then leave subepithelial tissues into vascular system down a concentration gradient. Movement of sugars in cells means the existence of a secondary active transport mechanism coupled with sodium. D-isomers of glucose and galactoses, and also a number of derivatives of Dglucose, 3-О-methyl glucose, 6-deoxy-D-glucose, 3-deoxy -D-glucose, 1-deoxy-Dglucose, 5-thio-D-glucose, and α- and β-methyl glucosides are transported against a concentration gradient. They can be transported by means of a Na+-glucose cotransporter in the small intestine and some other substances, for example, thioglycoside (Mizuma et al., 2000). In the apical membrane of a chicken small intestine, there is an electrogenic Na+dependent transport mechanism for mannose, distinct from glucose transporter SGLT1 (Cano et al., 2001). At the same time, some sugars such as mannitol, D-arabinose, Lramnose, L-mannose, and L-fructose are not transported actively. The existence of a separate passive transcellular system for fructose has been confirmed by experiments with an isolated brush border membrane of rat enterocytes and in in-vivo experiments (Sigrist-Nelson, Hopfer, 1974; Davidson, Leese, 1977). On the contrary, it is agreed that the flux of nutrients from a cell through a basolateral membrane is directed down its gradient from the compartment where its concentration is high to the compartment with low concentration; consequently, metabolic energy is not required at this stage (Brot-Laroche, Alvarado, 1983). The

90€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

exit of a nutrient from cells by the mechanism of simple diffusion is represented to be extremely improbable. Undoubtedly, experiments on basolateral membrane vesicles showed the participation of such transporters for sugar exit, the properties of which agree closely with those in other types of cells. The system of sugar transport, localized at the brush border, determines the number of the common transport characteristics of intact tissues. It turns out that absorption of sugar by an intact tissue depends on the sugar concentration (Michaelis –€Menten –€Henry kinetics) and is competitively blocked by the plant glycoside phlorizin (BrotLaroche, Alvarado, 1983). Transport of most nutrients into enterocytes is an active process; therefore, the presence of oxygen and free metabolic energy are necessary for its functioning. Under anaerobic conditions, the transport of nutrients against a concentration gradient is inhibited, and its efficiency brings it nearer to diffusion (Ugolev et al., 1970; Lerner, Burril, 1971; Kushak, 1983). Inhibition of nutrients transport is observed also after addition of tissue respiration inhibitors in the incubation medium, such as 2, 4-dinitrophenol, sodium azide, and so forth. Sodium fluoride (Kushak, Ugolev, 1966; Ugolev, Kushak, 1966), cyanide (Agar et al., 1953; Reiser, Christiansen, 1965) and dinitrophenol are inhibitor processes of amino acid and sugar absorption by enterocytes and do not affect their exit in the incubation solution (Agar et al., 1953; Reiser, Christiansen, 1965). From what source is the energy for sodium active transport derived? It has been proposed that the energy necessary for the generation of a concentration gradient of sugar through an apical membrane is taken from the sodium flux through that membrane down the gradient of its chemical potential (Schultz, Curran, 1970). According to (Crane et al., 1965), there are two centers on a carrier capable to move across a membrane, binding glucose and sodium. Results of kinetic experiments have shown that sodium changes the transport constant Кt and does not influence the maximum rate of sugar transport. The carrier has a higher affinity to glucose when the sodium center is occupied. Active transport of sugar occurs owing to the asymmetry of the carrier affinity to sugar on two membrane surfaces (Crane et al., 1965; Lyon, Crane, 1966; Curran et al., 1967). Upon exposition into an extracellular medium where sodium concentration is high, the corresponding binding center of the carrier is occupied by sodium; therefore, the carrier has a high affinity to sugar. When transferred to the internal surface of the membrane near which the sodium concentration is low, sodium dissociates from the carrier, its affinity to sugar decreases, and consequently, sugar dissociates from the carrier into the intracellular fluid (Crane et al., 1965; Kimmich, 1981). This mechanism will work until there is a sodium gradient concentration through the membrane. In contrast to the above data obtained on hamster intestine, in studies of rabbit ileum and chicken isolated enterocytes (Goldner et al., 1969; Kimmich, Randles, 1975), it turns out that sodium does changes the transport constant Кт but the maximal rate

Chapter 5. Transport of monosaccharides and the contribution to its study made ...€€€€€91

of sugar transport. However, the hypothesis of sodium gradient has been adapted for that case. It has been proposed that sodium first binds with a carrier, and the second substrate (sugar) binds with the carrier; this ternary complex, capable to move through the membrane, is formed as a result. It is vital to note that any binary complexes cannot move through the membrane. Therefore, the driving force for glucose transport is the gradient of sodium chemical potential. However, data showing insufficient energy stored in the sodium chemical gradient for active transport of nutrients gradually began to accumulate (Jacquez, Schafer, 1969; Kimmich, 1970; Potashner, Johnstone, 1971). In this respect, an additional mechanism of energization of that process has been proposed (Kimmich, 1970). As a result of long searches, it has been found that the potential difference on the apical membrane is responsible for this additional mechanism of energization (Gibb, Eddy, 1972; Murer, Hopfer, 1974; Reid et al., 1974; Hopfer et al., 1975; Carter-Su, Kimmich, 1980). Estimations have shown that for the creation of a 300-fold concentration gradient of sugar (this exceeds the values observed in experiment) between intra- and extracellular media, the electrochemical potential of sodium existing on the apical membrane is enough (Kimmich, 1981, 1983). Knotty problem, there was a point on the stoichiometry of the coupled transport. According to the first studies in this field (Goldner et al., 1969; Turner, Mогan, 1982), the stoichiometry of sodium and glucose transport is 1:1. However, in these results, the systematic error, consisting in underestimating the influence of the membrane potential, was found out later, and these data have been revised (Kimmich, 1981; Moran et al., 1982). Some data show that sodium and glucose are transported through the brush border with a stoichiometry of 2:1 (Kimmich, 1981; Kimmich, 1983). The situation became even more confusing when evidence in favor of the existence of a fractional stoichiometry of the transport began to appear. In one of the studies, carried out at 22°C on vesicles of a brush border of rabbit small intestine, the stoichiometry of sodium and glucose transport is 3.2:1.0 (Wright et al., 1983). Earlier data about a stoichiometry of 1.4:1 and 4.6:1 were published (Paterson et al., 1980; Sepulveda, Burton, 1982). This variety in stoichiometry for the same glucose transporters and especially the fractional coefficients, which are difficult to interpret from the viewpoint of modern theoretical concepts, point out that we are still far from understanding the mechanism of Na+-dependent nutrient transport. Essential contribution to the understanding and the resolution of the mentioned issues has been brought by the results obtained by the SCC technique.

5.2. Data on sugar transport obtained by the SCC techinque The measurement of Na+-dependent absorption of nutrients with SCC responses is an indirect method; but this is the unique technique, allowing to record and to measure

92€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

absorption of nutrients online. This technique, unlike any other, gives the possibility to study the very first stages of the approach of nutrients to the surface of mucosa and its interaction with the corresponding transporters. The ability to record online the dynamics of the beginning of nutrient absorption gives a unique opportunity to supplement data on biochemistry with the results obtained by the SCC technique. As we shall see later, important information on mechanisms and conditions of absorption is contained in the dynamics of the development of the SCC response to nutrients. One of the basic lines of the book is a comparison of the data obtained by any other methods with the results obtained by the SCC technique.

5.2.1. SCC responses to glucose Upon addition of 10 mM glucose to a mucosal solution, the SCC starts to increase quickly from a basal level (Fig. 9), then the speed in the SCC development gradually decreases down to 0. After that, the SCC flattens out at a new, higher level. At the new level, the SCC can keep for a long time in the presence of glucose, at least 40–60 minutes (Metelsky, 1987, 2007a). In response to glucose removal from the solution, the SCC starts to decrease, at first fast, then more slowly, eventually achieving the basal level of the SCC or close to it. Initial rates of development and wash-out of glucose effect considerably vary from preparation to preparation. On the contrary, relative rates of glucose effect development (α) and wash-out (β) (see the Chapter 6), equal to the corresponding absolute rates divided by the response value (A), vary much less from preparation to preparation. From this viewpoint, one can say that SCC responses to 10 mM of glucose have very different absolute values but are similar in forms. By comparing rates (both absolute and relative) of development and wash-out, it was unexpectedly found out that the rate of development of glucose effect is always much higher (sometimes ten-fold) than the rate of wash-out of the substance; on the average, at 26°С, the ratio α/β is equal to 4.5. The second SCC response to addition of glucose can be recorded at any time after the first one (if, certainly, the preparation has not deteriorated during this time). Moreover, if the addition of the new portion of glucose takes place during the wash-out of the first SCC response, the SCC will start to increase again from the level where it was found at the time of the second addition up to the level of the first response. The dynamics of wash-out of the second SCC response to glucose coincides with that of the first one. Hence, there is no lag period (or the period of “silence”) of SCC responses (unlike the action potential on exited membranes). The known highly specific competitive inhibitor of glucose active transport, phlorizin, when added with a concentration of 0.1 mM in a mucosal solution, causes the fast reduction of the stimulated SCC, almost down to the basal level. The surprising thing is that the inhibiting effect of phlorizin develops very quickly. For example, the relative rate of development of inhibiting effect (a) (the ratio of absolute rate of

Chapter 5. Transport of monosaccharides and the contribution to its study made ...€€€€€93

+ Glucose

β

A

2 µA/cm2

5 min

α - Glucose Fig. 9. Short circuit current response across rat small intestine on 10 mM glucose addition – solid line. SCC response parameters: response magnitude A = 80 mA/cm2; relative initial rates of effect development (α) and wash-out (β) equal to 3.9*10-2 and 0.81*10-2 1/s, respectively. Dotted line – theoretically expected response dynamics calculated from the following: Kt = 8.4 mM; Amax = 147.2 mA/cm2 (calculated from the above parameters A, α, and β).

development of the effect to the value of its maximal inhibiting effect) is equal to 3.4 x 10-2 s-1 and in some cases can be even more than that of glucose stimulating effect in spite of the fact that its concentration is less than that of glucose by two orders of magnitude (see Chapter 6). Hence, SCC responses upon addition of glucose (or other substance) can be characterized by the following parameters: the SCC value response (A) in μА/cm2, i.e., the difference between two stationary levels of the SCC with and without glucose and the initial rate of increase in the SCC (Vd). Because spontaneous changes of the basal SCC are characterized by low speeds of SCC changes and the rate of development of

94€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

the SCC response is higher by two orders, the accuracy of measurement of the initial rate is higher than the values of the response of A. The third parameter (a) characterizes the degree of similarity of the responses. It is calculated as the ratio of the initial rate of development of effect (Vd) to the value of the response A (s -1); a is, in essence, a measure of the relative speed of increase of the SCC in response to the substance addition. This parameter, apparently, is also measured with a greater accuracy than the value of responses A, and it is more a stable characteristic of the response than A and Vd separately. Besides, the preparation is characterized by the value of the initial rate of the “wash-out” of the effect Vw and by the value of the relative initial rate of the wash-out of effect β= Vw/A. For these two parameters, the above remarks for the corresponding parameters describing the development of the effect are applicable. In a series of 34 experiments (Metelsky, 1987, 2007a) (carried out at a temperature of 26 o C), in response to the addition of 10 mM glucose, A varies within 0.65-20.8 μА (the ratio of the maximum to the minimum is 32), Vd varies within 0.1-1.4 μА/4 s (the ratio of the maximum to the minimum is 14), a varies from 1.1 x 10-2 up to 6.4 x 10-2 s-1 (the ratio of the maximum to the minimum is 5.8), Vw varies from 0.014 up to 0.52 μА/4 s (the ratio of the maximum to the minimum is 37.1), and β varies from 0.22 x 10-2 up to 2.1*10-2 s-1 (the ratio of the maximum to the minimum is 9.5). The mentioned parameters are inherent recently prepared preparations and for preparations stored for a short time. During storage of preparations in a solution at a temperature of 1-3°С, the spontaneous potential difference and SCC responses upon addition of glucose gradually decrease: one can obtain the SCC responses to glucose on the preparations stored for no more than 70-75 min. It is not possible to record the stimulating glucose effect for preparation stored over 90-120 minutes (Metelsky, 1987).

5.2.2. The one-sideness of the glucose response Just as the osmotic effects considered above, the glucose effect is asymmetrical. The addition of 10 mM glucose in a serosal solution almost does not affect the SCC. The minute inhibiting effect developing in this case is caused, perhaps, by osmotic effect. Similarly, upon addition of 10 mM glucose in a serosal solution in the presence of 10 mM glucose in a mucosal solution, the SCC response does not change (Metelsky, 1987). Under conditions of bilateral oxygenation of the preparation, the values of the SCC response to simultaneous addition of 10 mM glucose in mucosal and serosal solutions and upon addition of glucose only in the mucosal solution were compared. It turns out that the amplitude of the SCC responses on glucose in these two cases correlated as 1:0.99 (n = 4), respectively. In one experiment, the stimulating effects of mucosal glucose were recorded continuously for a long time when there was constantly 30 mM of glucose in a serosal solution. It turns out that the long presence of high glucose concentration in a serosal solution does not affect the stimulating effect of mucosal glucose (Metelsky, 1987).

Chapter 5. Transport of monosaccharides and the contribution to its study made ...€€€€€95

5.2.3. Time-dependence of SCC responses to glucose in experiment 5.2.3.1. Increase in SCC responses to glucose on the background of fast initial decline of basal SCC. The basal SCC during the first 20-30 minutes after chambering of a preparation decreases quickly down to a certain stationary value (Metelsky, 1981, 1983). Upon addition of 10 mM of glucose in a mucosal solution at once after the beginning of an experiment on the background of the fast decrease of a basal SCC, an increase in the SCC can be seen, which after going through a maximum decreases, approximately following the dynamics of decrease of the basal SCC. After removal of glucose from the mucosal solution, the SCC starts to decrease with a speed greater than the speed of decrease in a basal SCC, reaching the same value as that which would be reached for a basal SCC without any addition. The second response to the glucose, obtained on the background of fast change of the basal SCC, is always more than the first one. The second response, as the first one, reaches a point where the response after wash-out decreases to a certain level, characteristic of the basal SCC. The beginning and the end of the response are identified precisely. The beginning of the response development could be inferred by a sharp deviation of the basal line of the SCC from its initial direction. The moment when the glucose effect is completely washed-out can be found at the point where the line of the basal SCC (recorded during the response to glucose) is crossed by the real line (recorded at the wash-out of the effect of glucose). This moment can also be found at the time corresponding to the last moment of omitting glucose from the mucosal solution, because development time and the time of effect wash-out are approximately proportional. On average, for 17-25 minutes, SCC value responses to 10 mM glucose increases by a factor of ~2.5 (Metelsky, 1984b).

5.2.3.2. Increase in SCC responses to glucose on the background of quasistationary reduction of the basal SCC Besides fast or initial build-up of the SCC responses occurring during the phase of fast decrease of the SCC described above, one more type of behavior of stimulating the effect of glucose exists, which is characteristic of the phase of the quasi-stationary SCC. Consecutive addition and removal of 10 mM glucose in and from a mucosal solution after the basal SCC has stopped to decrease quickly results in the development of consecutive SCC responses (Metelsky, 1987). If the preparation does not die in the chamber within 2 hours, each subsequent SCC response, as a rule, is a little higher than previous one. The next response distinctions (unlike the next SCC responses registered on the background of the fast reduction of the basal SCC) are not so significant, the difference being only 10-20 %. Eventually, distinctions between

96€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

neighbor responses gradually decrease; therefore, the responses during some time (2-3 responses) do not change and then start to decrease. In contrast to effective build-up of responses on the background of the fast decrease of the basal SCC (by a factor of 2.5 for 20-25 minutes), build-up responses in the quasi-stationary phase of the basal SCC are insignificant, about a factor of 1.5 for 90-100 minutes. Despite the increase in absolute value of the responses, the relative initial rate of development of SCC responses (a) can even decrease with time. If the lifetime of a preparation in the chamber, estimated by its ability to respond to the addition of glucose, is less than 4 hours, the value of the consecutive responses starts to fall practically at once after the SCC reaches the quasi-stationary level. The duration of the periods of time when an increase and decrease in consecutive responses are observed as well as the relative values of the change of two consecutive responses from preparation to preparation strongly varies. As shown earlier, there is a correlation between osmotic responses and SCC responses to glucose (Metelsky, 1987, 2007c). A question arises from the connection with the effect of build-up of SCC responses to glucose: is the correlation between osmotic and glucose responses kept when SCC responses to glucose spontaneously increase. Seven consecutive pairs of SCC responses to 20 mM of mannitol and 10 mM of glucose (Metelsky, 1987) were compared. It turns out that during 140 minutes of observation, in parallel with a double increase in glucose responses, osmotic responses increase in the same proportion. The qualitative behavior of responses is approximately the same and includes the following phases: initial strong build-up of responses during the fast decrease in the basal SCC (~30 minutes), then less obviously expressed and longer build-up (1-3 hours), and phase of reduction of the SCC response amplitude. At a later time, under the SCC response to glucose, the responses measured in the phase of quasi-stationary basal current phase are considered.

5.2.4. Dependence of stimulating glucose effect on its concentration The dependence of SCC responses to glucose concentration (Metelsky, 1987) has been studied in a wide range of concentrations (from 1 up to 40 mM, Tab. 4). Processing of this dependence with the procedure of double reciprocal coordinates (Fig. 10) gives values of the transport constant Кt and the maximal effect Аmax equal to 4.4 mM and 67.6 μА/cm2, respectively (Fig. 10, 11). It turns out that relative rates of development (a) and wash-out (β) of the effect also depend on glucose concentration but are changed in a much smaller degree. Hence, similarity between the SCC responses exists only at a given concentration, and with a change of glucose concentration, the similarity between responses is lost. Relative rates of development and wash-out of an effect depending on glucose concentration are changed in the opposite manner: with increasing glucose concentration from 1 up

Chapter 5. Transport of monosaccharides and the contribution to its study made ...€€€€€97

Table 4. Parameters of short circuit current response on glucose, at different glucose concentrations (t° = 26°С) (Metelsky, 1987) Glucose, mM 1 2 5 10 20 40

SCC response amplitude, A, µA/cm2 13.9±1.8 (5) 22.4±3.5 (13) 35.0±11.7 (2) 45.5±5.5 (21) 56.5±13.9 (6) 65.0±8.3 (6)

Relative initial rate of SCC response development, α*102 s-1 1.19±0.05 (4) 1.63±0.20 (13) 2.15±1.44 (2) 2.60±0.28 (21) 2.63±0.37 (6) 2.59±0.52 (6)

Relative initial rate of SCC response wash-out, β*102 s-1 0.95±0.06 (4) 0.89±0.10 (12) 0.75±0.41 (2) 0.58±0.07 (15) 0.39±0.04 (6) 0.28±0.05 (6)

to 20 mM, a monotonously increases from 1.19*10-2 up to 2.63*10-2 s-1, and β monotonously decreases from 0.95*10-2 down to 0. З9*10-2 s-1 (Tab. 4). The hyperbolic relation between the value of the SCC response to glucose (A) and glucose concentration has been analyzed for turtles. With an increase in glucose concentration from 1 up to 10 mM, the value of the effect gradually increases from 14.0 up to 26.0 μА/cm2. Processing of this dependence graphically by the procedure of double reciprocal coordinates gives values for the transport constant and the maximal effect equal to 1.2 mM and 28.6 μА/cm2, respectively.

1/A

1/Amax

1/Kt

1/C

Fig. 10. Determination of kinetic parameters for enzymes and transporters by the method of double reciprocal coordinates.

98€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

1/А μA/cm2 0.08

0.04

0.02

0 0.1 0.25

0.5

1.0 1/С mM

Fig. 11. Concentration dependence of short circuit current response on glucose plotted in double reciprocal coordinates (26oC). Kt = 4.4 mM; Amax = 67.6 mA/cm2.

5.2.5. Dependence of the parameters of the SCC response to glucose from rate of perfusion Above, we mentioned that the parameters of the SCC response in rat small intestine on 10 mM glucose strongly depend on the perfusion rate of preparation. It is appropriate to compare such dependences for two various species of animals, rats and turtles. With an increase in perfusion rate, the SCC value response in rat small intestine on 10 mM of glucose gradually increases (Tab. 5). Relative initial rates of development and wash-out of the effect similarly changes too, and the effects of the perfusion rate prove to be rather significant. Therefore, the increase in rate of perfusion from 0.6 up to 36 mL/min results in that a grows from 0.88*10-2 up to 6.0*10-2 s-1, and β grows from 0.34*10-2 up to 1.28*10-2 s-1. All parameters increase with increasing perfusion

Chapter 5. Transport of monosaccharides and the contribution to its study made ...€€€€€99

Table 5. Effect of specimen perfusion rate on short circuit current response across rat small intestine on addition of 10 mM glucose (t= 26°С) (Metelsky, 1987). Specimen perfusion rate, ml/min 0.6 (3) 1.2 (3) 2.4 (3) 4.6 (4) 9.2 (4) 18.0 (6) 36.0 (8)

SCC response amplitude, A, µA/cm2 5.0±1.0 5.5±1.5 6.0±1.2 6.4±1.6 7.0±1.3 7.5±1.5 8.2±1.1

Relative initial rate of SCC response development, α*102 s-1 0.88±0.07 1.45±0.23 3.01±0.73 3.22±0.33 4.20±0.59 5.24±0.80 6.0±0.93

Relative initial rate of SCC response washout, β*102 s-1 0.34±0.02 0.47±0.07 0.70±0.07 0.82±0.08 0.85 ± 0.13 1.10±0.24 1.28±0.26

rate from 0.6 up to 2.4 mL/min more sharply. With a further increase in perfusion rate, these parameters changed less. It is important to note that with increasing the perfusion rate, the values a and β do not increase proportionally. Therefore, with a rate of perfusion of 0.6 mL/min, the ratio a/β is equal to 2.6, and with a rate of 36 mL/ min, this ratio is equal to 4.6. The stimulating glucose effect on the SCC through the small intestine of a turtle depends a little less on the perfusion rate (Metelsky, 1987). When increasing the perfusion rate from 0.5 up to 2 mL/min, the value of the response increases from 6.0 up to 9.0 μА/cm2 (Tab. 6). With a further increase in perfusion rate up to 8 mL/min, this parameter changes slightly. On the contrary, the relative initial rate of the response development (a) increases in the same range of the rates of perfusion, by more than a factor of 10, from 1.07*10-2 up to 12.95*10-2 s-1. The increase in the relative initial rate of wash-out of the effect (β) is less apparent than for a, but nevertheless, it is considerable, from 1.12*10-2 up to 6.32*10-2 s-1. Table 6. Effect of specimen perfusion rate on response parameters of short circuit current across turtle small intestine on 1 mM glucose addition (only two measurements for every rate) (Metelsky, 1987). Specimen perfusion rate, ml/min 0.5 1.0 2.0 4.0 8.0

SCC response amplitude, A, µA/cm2 6.0 8.0 9.0 9.3 9.9

Relative initial rate of SCC Relative initial rate of response development, SCC response washα*102 s-1 out, β*102 s-1 1.07 1.12 2.34 1.65 4.98 3.22 8.31 3.83 12.95 6.32

100€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

It is important to keep in mind that, in the case of a turtle small intestine, when increasing the rate of perfusion, the increases in initial relative speeds of development and wash-out of effect are disproportionate. With a rate of perfusion of 0.5 mL/min, the ratio of the relative rates of development and wash-out of the effect (a/ β) is close to 1, and with the maximal rate of perfusion, 8 mL/min, that ratio is equal to 2.04.

5.2.6. Influence of some physiological factors on stimulating effect of glucose 5.2.6.1. A proximo-distal gradient of stimulating effect of glucose How far does the affinity of properties between SCC responses to glucose and active transport of this sugar extend? It is common knowledge that active glucose transport in various parts of the GI tract occurs with various intensities. The stimulating glucose effect is well manifested in proximal and medial parts of the small intestine and considerably (Metelsky, 1987) decreased in its distal part. It is important to note that in proximal segments of the colon, the stimulating glucose effect on the SCC is completely absent. If a rat small intestine is separated into three equal parts (in length), and the SCC response upon addition of 10 mM glucose in the mucosal solution in the medial part is 100 %, the relative values of the stimulating glucose effect from the duodenum to the beginning of the colon will be, accordingly, equal to 57:100:31:0 (Metelsky, 1987).

5.2.6.2. Influence of thermal stress on transport of sugars and on stimulating effect of glucose Under stress condition, the active transport of glucose and the influence of glucose on the SCC were determined simultaneously. It was possible to compare for the first time transport and stimulating glucose effect not only under normal conditions (which is well-known) but also under stress. In studies of the link between glucose and fructose transport in rat small intestine and SCC responses to glucose, the following results (Metelsky, 1987) (Tab. 7) have been obtained. Accumulation of glucose is maximal in the tissue of the control rat intestine,14.73 mM. In animals that were subjected to thermal stress, accumulation of glucose was lower (in the first rat, by a factor of 1.6 and in the second one, by a factor of 2.0). The fructose content in the tissue of rats is much lower in animals that were subjected to stress than in the control animal. Stimulating glucose effect is maximal for the control rat (40 μА/cm2), and for the first and second experimental rats, the values of this effect are equal to 22.0 and 17.0 μА/cm2, respectively. It is important to note that in the first and second experimental

Chapter 5. Transport of monosaccharides and the contribution to its study made ...€€€€€101

Table 7. Effect of heat stress on glucose and fructose accumulation in rat small intestine tissue and in serosal solution, as well as on stimulation of short circuit current response by 10 mM glucose addition (Metelsky, 1987).

State Stress Control

Number Concentration in of animals tissue, mM (amount of Glucose Fructose everted sacks) 1 (3) 9.22± 0.25 4.72± 0.22 2 (3) 7.31± 0.87 3.22± 0.84 1 (3) 14.73± 0. 9 6.28± 1.20

Ratio of SCC response tissue glucose on addition of 10 mM glucose, accumulation to SCC response on glucose µA/cm2 22.0 0.42 17.0 0.43 40.0 0.36

rats, the values of the stimulating glucose effect are lower than those for the control animal by a factor of 1.8 and 2.35, respectively. More importantly, SCC responses to glucose in these experiments changed by more than a factor of two and, despite of this change, one can estimate the accumulation of glucose in the tissue, taking into account that the conversion factor between μА/cm2 and mM is equal (at least, for these experiments) to 0.36–0.43 (Metelsky, 1987). So, the observed effects of glucose on the SCC correctly reflect the physiology of the mechanism of active glucose transport both under normal and stress conditions.

5.3. Final remarks The stimulating action of glucose on the SCC through the small intestine wall of rats, rabbits, turtles, frogs, and other animals can be repeated. It is a reliable and established effect. Because in the modern view, the link between glucose-dependent sodium transport and active glucose transport is indistinguishable, from the obtained data follows that in the intestine of these animals, the active transport of glucose should be observed. In reality, in intestines of all four species, glucose is absorbed in the active manner (Schultz, Curran, 1970; Schultz, 1977; Parsons, 1978; Kushak, 1983). As the studied animals occupy various positions on the scale of ranking, one is inclined to think that the well-known concept is once again confirmed: the Na+-dependent glucose transport is an extremely widespread process in fauna. How strong is the relation between glucose-dependent sodium transport and active transport of glucose? Does such coupling take place in all parts of the gut? In reality, there are no bases to believe that various parts of the GI tract are equivalent in functional and morphological relations (Fisher, Parsons, 1949; Wilson, Wiseman, 1954; Parsons, Prichard, 1956; Smyth, Taylor, 1957; Annegers, 1964; Schultz et al., 1974; Alvarez, 1979). Let’s consider in more detail the findings of in vitro studies of proximo-distal gradient both of glucose transport and of electrophysiological responses to glucose

102€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

(transmural potential differences and SCC). Such a study has been carried out for the first time in 1953 on an preparation of a resected small intestine (Fisher, Parsons, 1953). It was found that Kt for proximal and distal parts are identical and equal to 8.3 mM, but the rate of glucose absorption decreases in the direction toward ileocolonic sphincter. In that study, a small intestine was divided into two parts, and consequently, the segmental resolution of the procedure was low. In this other study (Barry et al., 1961), an intestine was divided into five parts, and more appreciable regional differences in transport of glucose were found. If the maximal rate of glucose absorption in the third segment is taken as 100 %, the values of proximal-distal gradient will be 69, 87, 100, 54, and 13. Thus, in these studies, it has been found that the maximum of glucose transport activity in an intestine is on the medial segment. In the GI tract of mammals, a potential difference, the value of which considerably varies in various segments, has been found (from 0.5 up to 2.5 mV, plus on a serosal surface); such potential considerably increases in the presence of glucose in a mucosal solution, up to 4-12 mV (Barry et al., 1961; Clarkson et al., 1961a, 1961b; Schachter, Britten, 1966; Metelsky, 1987). It is significant that the maximum of the sugar-dependent potential difference falls on the medial part of a small intestine (Barry et al., 1961; Clarkson et al., 1961b). The distribution of the sugar-dependent potential difference along a small intestine has been studied in more details (Barry et al., 1964). For this purpose, the entire small intestine was divided into five parts, and the sugar-dependent potential differences on the wall of the everted sacs made of the 1st, 3rd, and 5th segments of an small intestine were measured. Values of the responses to sugar were equal to 4.8, 10.5, and 4.3 mV, respectively. If one takes the value of the response in the medial segment as 100 %, the distribution of sugar-dependent potential-generating activity is 46, 100, and 41, respectively. By comparing that proximal-distal gradient with the distribution of glucose transport in the same segments (69, 100, and 13) (Barry et al., 1961), one can see a qualitative similarity between these two distributions. The medial part of an intestine has the maximal capacity both for active transport and for generation of a sugar-dependent potential difference. However, in proximal segments, the relative values of the activity of glucose transport and the sugar-dependent potential difference are in relatively good agreement (69 and 46), but in the distal parts, these values differ from each other by more than a factor of three (13 and 41). The results (Metelsky, 1987) are in good agreement with the distribution of both activities. In reality, in relative units, the SCC value response to 10 mM of glucose in proximal, medial, and distal parts of rat small intestine (corresponding to the 1st, 3rd, and 5th segments of (Barry et al., 1961, 1964)) proves to be equal to 57:100:31. Thus, the maximum of sugar-dependent SCC is on the medial small intestine, and the minimum is on the distal segment. The proximal segment in this respect occupies an intermediate position, and in the beginning of the colon, there are no glucose-induced effects on the SCC. One can see that the obtained distribution (Metelsky, 1987) – 57:100:31 corresponds closely to the distribution of glucose-transport activity (69:100:13) and to the distribu-

Chapter 5. Transport of monosaccharides and the contribution to its study made ...€€€€€103

tion of sugar-dependent potential (46:100:41). The insufficient agreement between the distribution of the potential difference (46:100:41) (Barry et al., 1964), the distribution of glucose-transport activity (69:100:13) (Barry et al., 1961), and the data (Metelsky, 1987) (57:100:31) once again shows that the potential difference has no physiological analogue (such analogue in the case of the SCC is ion transport). Moreover, the location of the study of SCC responses to glucose (Metelsky, 1987) is extended prior to the beginning of the colon where, it has long been known that active glucose transport is absent (Cooperstein, Hogben, 1959; Levin, 1966; Phillips, 1984). According to these data (Metelsky, 1987), it was found that in a colon, the capacity of its specimens to respond to glucose is lost. From the similarity of the distributions in rat small intestine of active glucose transport and stimulating glucose effect on active sodium transport (69:100:13 and 57:100:31, respectively), it may be inferred that coupling between active glucose transport and glucose-dependent sodium transport takes place not only in single segments (that is known) but throughout the length of the GI tract. Is coupling between the two studied transport processes maintained under a pathological state? It is common knowledge that 16 hours after thermal stress, glucose transport rate in the small intestine is strongly inhibited. However, it remains unknown, whether the coupling between the two discussed transport processes under such conditions is maintained. Under such stress, the accumulation of glucose in a serosal solution and, especially, in an intestinal tissue sharply decreases, and apparently, the damage of the transport mechanism does not occur at the level of bioenergetics but occurs at the level of the brush border membrane (Metelsky, 1987). In reality, both in a serosa solution and in a tissue, there is a parallel sharp decrease in absorption of other sugars such as fructose which, it has long been known, is passively transported through the brush border membrane, through the mechanism of facilitated diffusion (Schultz, Strecker, 1970; Kushak, 1983). As indicated above, the mechanism of coupling between sodium and glucose transport is localized in the brush border membrane; therefore, it is especially important to elucidate what the SCC responses to glucose will be in such a case. The accumulation of glucose in the intestinal tissue the control rat was equal to 14.73 mM, and it was equal to 9.22 and 7.31 mM in the tissues of the first and second experimental rats, respectively. The stimulating effect of 10 mM glucose in the control rat is equal to 40.0 μА/cm2, and it is 22.0 and 17.0 μА/cm2 in the first and second experimental rats, respectively. The ratio of the accumulation of glucose in tissues and the stimulating glucose effect for the control, first, and second experimental rats are equal to 0.36, 0.42, and 0.43 mM/μA/cm2, respectively (Metelsky, 1987). Hence, the coupling between transport processes is maintained upon damage of the brush border membrane (thermal stress). The effects of glucose observed in an intestine are essentially asymmetrical (Levin, 1966; Metelsky, 1981, 1987). This corresponds to the idea that serosal-mucosal glucose transport does not depend on sodium (Ugolev, Roshchina, 1982) and that the mechanism of increasing sodium transport in response to the addition of glucose is localized

104€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

in the brush border membrane (Kushak, 1983; Parsons, 1978). It is important to note that the barrier function of the intestinal wall (Metelsky, 1987) is maintained for a long time. If such function were disturbed, it would be possible to see an increase of the basal SCC or a decrease of the SCC response to mucosal glucose upon long incubation with glucose in a serosal solution because of the penetration of serosal glucose into the binding site of the coupled transporter. Thus, SCC responses to glucose reflect the physiological reality and provide information on the mechanism of coupling of sodium and glucose transport.

Chapter 6. The single response method€€€€€105

Chapter 6. The single response method SCC responses upon addition of glucose were already measured almost 50 years; however, the information obtained from those experiments, in most cases, is obviously insufficient. As a matter of fact, the only measured parameter is the value of changes of the SCC in response to addition of a nutrient (Grabb, 1999; Ferraris, Carey, 2000; Kroesen et al., 2002; Kozar et al., 2002; Ducroc et al., 2005; Troeger et al., 2007). At the same time, it seems that for SCC responses to glucose, not only the response value contains information. The attempt to estimate the information contained in the single SCC response to a nutrient has been undertaken. To understand better the parameters on which the form of the response depends, a mathematical model of the observed phenomena has been developed (Metelsky, 1987b, 2004a, 2007a, 2007c). Its basic assumptions consist in the following. In a brush border membrane, the transporters of sodium that are capable to be activated as a result of adsorption of a glucose molecule on their gate mechanism (like the opening of sodium channels by acetylcholine in neuromuscular transmission) are localized. The rate of sodium transport by the transporter is high enough and does not limit the rate of development of glucose effect. On the contrary, the diffusion rate of glucose from the bulk through an unstirred layer is small and limits the development of stimulating effect. Under such assumptions, one can describe the dynamics of development of SCC responses.

6.1. The principles of the method Until 1987, the main and only parameter measured by using SCC technique was the magnitude of the response (A) in μА/cm2 on nutrient addition. Then, four new parameters describing the SCC response to glucose have been introduced (Metelsky, 1987b, 2004a, 2007a, 2007c ): initial rates of development Vd and of wash-out Vw of the effect measured as a deviation of a recorder pen in the first 4-12 s after addition or removal of nutrient to a solution in μА/cm2/s and relative initial rates of development (a) and wash-out (β) of the effect determined as Vd/A and Vw/A correspondingly. Therefore, the single SCC response to the single isotonic addition of a nutrient provides the fol-

106€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

lowing information: the relative initial rate of the effect development (a, s-1) and the relative initial rate of decline upon removal of nutrient (effect wash-out) from a solution (β, s -1). These parameters contain different information; in practice, only the latter two parameters are used. With these two parameters from the single SCC response to the addition of glucose, it is possible to calculate the thickness of the unstirred layer of a fluid in the vicinity of a preparation (δ), the transport constant for glucose (Kt), and the value of its maximal effect (Аmax). For brevity, the above modification of the classic SCC technique was called the single response method (Metelsky, 1987). In order to better understand the benefits of the above parameters, we shall consider the simplified model of the phenomenon with the assumption that the surface of the preparation is flat (Metelsky, Dmitrieva, 1987; Metelsky, 2004a, 2007a). It has been proposed that the primary factor determining the dynamics of the development of the SCC response to glucose, is the rate of the glucose transfer to the surface of a brush border membrane; the form of the response has been calculated on the basis of this factor. The final expressions (Metelsky, Dmitrieva, 1987; Metelsky, 2004a; 2007a) are obtained after resolution of the diffusion equation for appropriate boundary conditions: Kt = Cо / (√¯ (a/β)-1),€€€ Аmax = A (Cо+Kt) / Cо, δ =π/2 √D / √√ (aβ),€€ where Co is the bulk concentration of the added substance, Кt is the transport constant of the nutrient, Аmax is the maximal value of the response, δ is the thickness of the unstirred layer, and D is the diffusion constant of the nutrient.

6.2. Dependence of kinetic parameters of coupled sodium and glucose transport on the rate of perfusion From the concentration dependence of the stimulation effect of the SCC by glucose, it has been found that Kt it is equal to 4.3 mM for glucose (Metelsky, 1987). In biochemical studies, it was found that glucose transport is characterized by transport constants from 2.5 to 9.0 mM (Fisher, Parsons, 1953; Barry et al., 1964; Thomson, Dietshy, 1980). For a rabbit small intestine, it is found that half the maximal stimulation of the SCC response is observed with concentration of glucose equal to 4.0 mM (Schultz, Zalusky, 1964b) or 6.9 mM (Larsen et al., 2001). However, calculation of Kt from the SCC response to 10 mM of glucose gives a higher value, 9.0 ± 1.4 mM (Tab. 8). One of the possible reasons for these different results could be an ineffective change of the solution in the chamber; indeed, it is common knowledge that the thickness of the unstirred layer strongly affects the measurement of kinetic parameters in a small intestine.

Chapter 6. The single response method€€€€€107

Table 8. Response parameters of short circuit current across rat small intestine on glucose, kinetic parameters of glucose effect and unstirred layer thickness at different glucose concentrations (t° = 26°С) (Metelsky, 1987). Glucose concentration, mM

SCC response amplitude, A, µA/cm2

1 2 5 10 20 40

13.9±1.8 (5) 22.4±3.5 (13) 35.0±11.7 (2) 45.5±5.5 (21) 56.5±13.9 (6) 65.0±8.3 (6)

Relative initial rate of SCC response development, α*102 s-1 1.19±0.05 (4) 1.б3±0.20(13) 2.15±1.44 (2) 2.60±0.28 (21) 2.63±0.37 (6) 2.59±0.52 (6)

Relative Maximal Unstirred initial rate of Transport stimulatlayer SCC response constant, ing effect, thickness Kt, mM wash-out, Amax, δ, µm β*102 s-1 µA/cm2 0.95±0.06 (4) 8.6±3.1 132.8± 46.3 340±7 0.89±0.10 (12) 5.7±1.8 85.8±24.2 320±13 0.75±0.41 (2) 7.2±9.1 92.4±101.2 312 0.58±0.07 (15) 9.0±1.4 86.2± l2.2 316±13 0.39±0.04 (6) 12.5±1.8 91.9±23.2 349±15 0.28±0.05 (6) 19.6±3.9 96.8±13.9 380±26

As one would expect (Thomson, Dietschy, 1980; Barry, Diamond, 1984), Kt in rats decreases with an increase in efficiency of the solution change, until the curves flatten out with a time constant of solution change of 0.2 s, reaching 8.2 ± 1.5 mM (Tab. 9, Fig. 5). With further increase in perfusion rate, Kt almost does not change. In studies of such dependence in a turtle, the following picture was observed (Tab. 10, Fig. 5). With a decrease of the time constant of a solution change, the magnitude of Kt gradually decreases, and the curves flattens with a time constant equal to 1.5 s, reaching 2.2 mM. At the same time, the value of Kt on a plateau and the Table 9. Effect of specimen perfusion rate on response parameters of short circuit current across rat small intestine on addition of 10 mM glucose (t= 26°С) (Metelsky, 1987). Relative Relative Maximal Time SCC initial rate initial rate Specimen stimulating constant of Transport response, of SCC perfusion of SCC effect of solutions constant, A, response rate, ml/ replacement in response glucose, Kt, mM µA/cm2 development, wash-out, min Amax, experimental α*102 s-1 β*102 s-1 µA/cm2 chamber, tch, s 0.6 (3) 3.0 5.0±1.0 0.88±0.07 0.34±0.02 16.4±2.1 13.2±2.9 1.2 (3) 1.53 5.5±1.5 1.45±0.23 0.47±0.07 13.2±3.3 12.8±3.9 2.4 (3) 0.76 6.0±1.2 3.01±0.73 0.70±0.07 9.4±2.4 11.6±2.7 4.6 (4) 0.40 6.4±1.6 3.22±0.33 0.82±0.08 10.2±1.5 12.9±3.4 9.2 (4) 0.20 7.0±1.3 4.20±0.59 0.85 ± 0.13 8.2±1.5 12.7±2.6 18.0 (6) 0.10 7.5±1.5 5.24±0.80 1.10±0.24 8.5±2.1 13.8±3.2 36.0 (8) 0.05 8.2±1.1 6.0±0.93 1.28±0.26 8.6±2.0 15.2±2.6

108€€€€€Transport phenomena and membrane digestion in small intestinal mucosa Table 10. Effect of specimen perfusion rate on response parameters of short circuit current across turtle small intestine on 1 mM glucose addition (two measurements at every rate) (Metelsky, 1987) Relative Relative Maximal Time constant SCC initial rate initial rate Specimen of solutions stimulating Transport of SCC perfusion replacement in response, of SCC effect of constant, A, response rate, ml/ experimental response glucose, Kt, mM µA/cm2 development, wash-out, min Amax, chamber, α*102 s-1 β*102 s-1 µA/cm2 tch, s 0.5 12 6.0 1.07 1.12 ∞ ∞ 1.0 6 8.0 2.34 1.65 5.2 49.6 2.0 3 9.0 4.98 3.22 4.1 45.9 4.0 1.5 9.3 8.31 3.83 2.2 30.0 8.0 0.75 9.9 12.35 6.32 2.3 32.8

value of Kt determined by a traditional procedure (double reciprocal coordinates or Lineweaver-Burk plot) are equal to 2.2 and 1.3 mM, respectively, and differ only by 0.9 mM! According to Barry, Diamond, 1984, effects of unstirred layers on active transport include overestimating the Kt determined by Lineweaver-Burk plot by factors that are frequently very large. In Metelsky, 1987a, Kt is determined by the single response method. It is unclear whether such overestimate of the Kt values occurs when using this method.€

6.3. Concentration dependence of the constant of binding of a nutrient with the transporter The second possible reason for the divergence of results could be an unsuccessfully chosen glucose concentration equal to 10 mM. In studies of rat intestines, it was shown that with an increase of glucose concentration from 1 up to 40 mM the value of Kt, determined by the single response method, tends to increase from 8.6 ±Â€3.1 up to 19.6 ±Â€3.9 mM. In other words, the value of Kt determined with this technique increases with an increase of glucose concentration in a mucosal solution. It seems that with a small concentration, the true value of Kt is measured, and for measurements with higher concentrations, some mechanism leading to impairment of Kt is triggered off. To be convinced of this fact, the same experiments (Metelsky, 1987) have been carried out at temperatures which are different from 26°С (temperature dependences will be discussed in more detail below, Chapter 9). In all 4 series of experiments, the value of Kt, determined with the single response method, increases with an increase of glucose concentration.

Chapter 6. The single response method€€€€€109

Thus, it may be inferred that the technique allows us to measure the true Kt only at low glucose concentration. However, the use of very low glucose concentration is risky. Already with concentration of added glucose of the order of 1 mM, the diffusion rate to the surface is equal to the rate of glucose transport through the epithelium. The divergence of results on the measurement of Kt by the single response method with low and high glucose concentration is not caused by a lower superficial glucose concentration due to the greater contribution of transport through the epithelium with low rather than high concentration, because such distinction is maintained under conditions where the transport is lowered to a minimum. Also, the divergence is not caused by water fluxes perpendicular to surfaces: (1) such water fluxes should be essentially nonlinear already upon addition of 10 mM glucose, which is unlikely; (2) during measurement of the initial rate, 4–12 s, fluxes are not likely to change significantly. It is not inconceivable that the mentioned divergences are related with the imperfection of the mathematical model used for the phenomenon because during theoretical analysis, a number of factors have not been taken into account. The main factor is that the villus surface is not a plane. Possible water fluxes, the architecture of which can be extremely complex, are not considered; it is not clearly, what role the movement of villi plays in the studied phenomenon. Proceeding from that, it is necessary to draw the conclusion that for the degree of simplification of the phenomenon which exists in above analysis (Metelsky, 1987, 2004a), the results calculated by a single response method are in agreement with those (values of Kt) obtained from concentration dependence. To better understand the degree of divergence and its reason, there are two approaches: first, the development of the mathematical model, and second, the physiological modeling, allowing to take into account some factors more carefully. Hence, the elimination of only one factor from several factors—villus (turtle)—results in that the values of Kt determined from concentration dependences and from the SCC response to glucose are practically the same. It is useful to analyze SCC responses to other substances with a Kt different from that of glucose. For this purpose, the SCC responses to nutrients (monosaccharides, amino acid, and disaccharide) and the inhibiting effect of a phlorizin (Metelsky, Dmitrieva, 1987) (Tab. 11) are analyzed in more detail. SCC responses to 10 mM of glucose or galactose are approximately identical. The calculation of Kt from the SCC response to galactose gives a value that is higher than that for glucose by a factor of ≈3.8. The ratio of Kt for galactose and glucose, determined from concentration dependences, is equal to 2.5 (Schultz, Zalusky, 1964b). The single response method can be applied to the analysis of the inhibiting effect of substances too (Metelsky, 1987). The initial rate of development of the effect of a phlorizin in this case (relative development of glucose effect) is directed to the opposite side. From the comparison of relative initial development rates of effects of phlorizin and glucose, a value of Ki for a phlorizin equal to 20 μM is obtained. This

110€€€€€Transport phenomena and membrane digestion in small intestinal mucosa Table 11. Kinetic constants and unstirred layer thickness near mucosal surface determined by five substances (Metelsky, Dmitrieva, 1987). Relative Relative Transport Maximal Unstirred initial rate initial rate SCC (Kt) or effect of layer of SCC of SCC response, Substance inhibition substance, thickness, response response A, (Ki) constant, Amax, δ, µm µA/cm2 development, wash-out, 2 mM µA/cm α*102 s-1 β*102 s-1 Glucose 40 5.1 0.83 7.0 65 250 Maltose 42 2.2 0.81 5.0 84 301 Galactose 39 1.8 0.96 27.0 144 271 Glycine 53 1.9 0.66 57 129 297 Phlorizin -30 -3.5 -1.6 0.02 -45 250

value coincides with that of the inhibition constant for a phlorizin, equal 20 to μM and determined by the usual techniques (Brot-Laroche, Alvarado, 1983). According to autoradiography data, Ki for phlorizin for rat and hamster intestine (Stirling, 1967) are equal to 3 and 13 μM, respectively. So, in studies of five substances it has been found that though the Kt for them are distinguished more than by three orders, it is possible by the single response method to determine values of Kt or Ki consistent with other data (Metelsky, 1987, 2007a).

6.4. Time dependence of SCC responses on glucose Advantages of the single response method are demonstrated on consecutive cycles of addition and removal of glucose (Metelsky, 1987, 2007a). For each response, kinetic constants and layer thicknesses were calculated, and they were relatively stable during all experiments. By the single response method, the effect of SCC changes responses to glucose with time is analyzed. As it can be seen from Tab. 12, although the effect of “build-up” of responses on the background of fast initial SCC decrease is rather large (only for 19 minutes a SCC responses amplitude on 10 mM of glucose grows by a factor of 2.3), Kt tends to decrease slightly, from 14.7 ± 3.8 mM in the beginning down to 11.0 ± 1.3 mM at the end of measurement. The maximal value of the effect, Аmax, increases almost by a factor of two, and the thickness of the unstirred layer tends to increase (Chapter 7, Tab. 17). Probably, this suggests that for glucose diffusing to the surface of an intestine from the bulk, the enterocytes laying closer to the base of the villus become more accessible. Similarly, the effects of an increase of SCC value response to glucose were observed on the background of quasi-stationary or basal SCC (Tab. 13). Although the effect of

1 2 3

9.8±5.1 16.5±8.8 22.5±10.0

Number SCC of response, A, response µA/cm2

Initial rate of SCC response development, Vd, µA/cm2€s 0.45±0.21 0.69±0.39 1.0±0.46 0.144±0.041 0.20±0.08 0.25±0.09

Initial rate of SCC response wash-out, Vw, µA/cm2€s

Relative Relative initial initial rate of Transport rate of SCC SCC response constant, response wash-out, Kt, mM development, 2 -1 2 -1 β*10 s α*10 s 5.19±0.46 1.83±0.35 14.7±3.8 4.03±0.27 1.36±0.23 13.9±3.0 4.41±0.21 1.21±0.12 11.0±1.3 Maximal stimulating effect, Amax, µA/cm2 24.2±13.1 39.4±21.6 47. 3±21.2

Number of response

1 6 13 16 18

Time since beginning of experiment, min

30 64 126 158 189

SCC response on addition of 10 mM glucose, A, µA/cm2 14.0 18.0 28.0 42.0 36.0

Relative initial rate of SCC response development, α*102 s-1 5.4 8.3 8.0 5.0 5.6

Relative initial rate of SCC response wash-out, β*102 s-1 1.1 1.1 0.89 0.71 0.69 41 33 37.5 53 51

Time constant of response development, τ, s

8.3 5.7 5.0 6.1 5.4

Transport constant, Kt, mM

25.6 28.3 42.0 67.6 55.4

Maximal stimulating effect, Amax, µA/cm2

Table 13. Time dependence of parameters of short circuit current response across rat small intestine on addition of 10 mM glucose (Metelsky, 1987).

3.3 (3) 10.0 (3) 19.0 (3)

Time since beginning of experiment, min

Table 12. Changes in parameters of short circuit current response across rat small intestine on addition of 10 mM glucose against the background of a fast initial short circuit current decline during the first 19 min (Metelsky, 1987).

Chapter 6. The single response method€€€€€111

112€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

“build-up” of the response in this case is significant too, the thickness of the unstirred layer (Chapter 7, Tab. 18) and Аmax also increase; this shows that more deeply localized cells participate in the development of the SCC response with time.

6.5. Final remarks The essence of the single response method is the following. From the single SCC response to addition of a nutrient, we can obtain the following information: the value of the response (A) in μА/cm2, the relative initial rate of the effect development (a), measured as the ratio of initial rate of the effect development to the value of the response A in s-1 , and the relative initial rate of the effect wash-out upon removal of the nutrient from the solution (β), measured as the ratio of initial rate of the effect wash-out to the value of a response in s-1. After determining all three values from the single SCC response to addition of a nutrient, one can calculate the kinetic parameters describing the Na+-dependent transport of substances (Кт, Аmax) and the thickness of unstirred layer of a fluid at the surface of a preparation (δ). This circumstance qualitatively changes the treatment of the results obtained by the method of single SCC response and allows to discuss the obtained results in terms of molecular mechanisms (Fig. 12). In reality, the increase in the SCC response to a nutrient can be caused both by a decrease in Kt and by an increase in Аmax. The single response method allows to distinguish such possibilities (see Chapter 15). The results obtained by that technique are in agreement with other data and offer a clearer view of some aspects of Na+-dependent transport of substances.€

Chapter 6. The single response method€€€€€113

Scientific information that one can draw out from a single experiment (with single specimen) Classical SCC method Measured parameters

Calculated parameters

SCC response magnitude

No

Method of single response of SCC SCC response magnitude Relative initial rate of SCC response development (α) Relative initial rate of SCC response wash-out (β) Kt and Amax: for 5-10 specimens Unstirred layer thickness (δ): for 5-10 specimens Absorption profiles for nutrients

Additional information

No

Sequence of Kt for nutrients for each preparation

Fig. 12. Comparison of informative possibilities of the classical short circuit current method and the method of single response of short circuit current.

114€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

Chapter 7. Unstirred layers of the fluid at the mucosa surface€€€€€115

Chapter 7. Unstirred layers of the fluid at the mucosa surface 7.1. A phenomenon of unstirred layer The nutrients that have formed as a result of cavital digestion and also other substances which have appeared in intestinal cavities (for example, medicines) during the transfer from the bulk to the surface of the intestine where the membrane digestion and absorption take place should overcome a diffusion barrier. The concept of unstirred layers (House, 1974; Smith et al., 1983; Barry, Diamond, 1984) distinguishes two parts in the diffusion barrier: a step that can be somehow controlled (e.g. by vigorously stirring the solution) – substrate supply from the bulk to the unstirred layer boundary – and a step that cannot be influenced by the researcher, determined through simple diffusion through the unstirred layer to membrane (Fig. 13). The existence of unstirred layers at the biomembranes is a general but poorly studied phenomenon, which may lead to ordering adjacent water layer, resembling the formation of hydration shell about dissolved molecules and ions. During the most vigorous stirring of a solution at any, even ideally smooth, surface, there are immobile layers of water. Such layers of course are found out of and near the mucosa surface of the gastrointestinal tract. Until recently, the concept of unstirred layers has had only theoretical interest, and it was not used in clinical studies, in particular in gastroenterology. Now, clinicians have learned to successfully reduce the rate of absorption of some nutrients by giving pectin and guar gum to patients (Gerencser et al., Guild, 1984; Cerda et al., 1987), increasing the thickness of unstirred layers (Thomson et al., 2001). What mechanisms underlie the action of pectin and guar gum and why gastroenterologists are now interested in unstirred layers?

116€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

Water flow velosity

Transporters

Unstirred layer thickness

Nutrient molecules

Enterocyte membrane

Fig. 13. Unstirred liquid layer near smooth specimen surface. The arrow length reflects the velocity of solution movement along the surface.

7.2. Measurement of the thickness of unstirred layer Let us consider this issue in more detail according to (Metelsky, 1987, 2007a, 2007b, 2007c). Experiments were performed on sections of medial intestine (spread along the contramesenterial line) from Wistar male rats which had not been fed for 16–18 h but had with water ad libitum. (1) In the mannitol assay (Diamond, 1966), the half-response time (t1/2) is determined, and the layer thickness l is calculated as € √t1/2 √D δmn = -------------------; 0,616 where δ is the thickness of the unstirred layer, and D is the diffusion constant for mannitol . (2) The assay (Metelsky, Dmitrieva, 1987; Metelsky, 2004a, 2007a) was based on SCC reflecting active sodium transport (see Chapters 3, 6). Briefly, the single SCC response to isotonic addition of a nutrient yields the following information: magnitude of the response (A) in μА/cm2, the relative initial rate of the effect development (a, s-1), and

Chapter 7. Unstirred layers of the fluid at the mucosa surface€€€€€117

the relative initial rate of decline upon removal of nutrient (effect wash-out ) from a solution (β, s -1). Then, the layer thickness δ is calculated as δ = (π/2) √D/√√a β, where D is the diffusion constant of the added substance, taken from reference books. There is a correlation between the thickness of unstirred layers, determined in two ways (half-time of development of the osmotic response (response to an increase in osmolarity of a mucosal solution on 20 mM) to mannitol (range of changes of the layer thickness from 185 up to 450 μm)) and the relative rates of development and wash-out of a SCC response upon addition of 10 mM glucose (Кcorr = 0.929, n = 19). The linear regression is described by the expression δgl = 0.997 lmn (Metelsky, 2004a). The half-time of the osmotic response, t1/2, to 10, 20, and 50 mM mannitol, proved to be the same, 38.0, 35.8, and 35.8 s, respectively, for unstirred layer thicknesses of 223, 217, and 217 μm, respectively; the magnitude of the osmotic response increased linearly with mannitol concentration. In contrast, the values obtained with the nutrient assay were somewhat higher than the values mentioned above, even at lowest concentration tested and, strikingly, raised with the concentration; with 10 mM of either substance, the increment was roughly 30 μm with every concentration doubling. The thickness of the unstirred layer of a fluid, determined for amino acid glycine with increasing its concentration from 5 up to 40 mM, increases from 247 ± 12 up to 319 ± 14 μm (p <0.001, (Tab. 14). Hence, the layer thickness determined on glycine with its concentration, unlike mannitol, increases. The thickness of unstirred layer, determined for glucose, with a 20-fold increase in concentration from 2 up to 40 mM tends to increase (Tab. 14) by

Table 14. Unstirred layer thickness determined by glycine and glucose in relation to their concentration (Dgly = 4х10-6 cm2/s, Dglu = 5х10-6 cm2/s) (Metelsky, 2007d). Nutrient concentration, mM 2 5.0 7.5 10.0 15.0 20.0 40.0

Unstirred layer thickness determined by glycine, δ, µm 247 ± 12 g (8) 224 ± 8 b (3) 254 ± 11 f (21) 240 ± 10 b (11) 290 ± 12 a (21) 319 ± 14 (17)

Unstirred layer thickness determined by glucose, δ, µm 320 ± 13 (13)

316 ± 13 c,e (21) 349 ± 15 d (6) 380 ± 26 c (6)

p < 0.02 versus 5 mM; b p < 0.001 versus 5 mM; c p < 0.05 versus the same glycine concentration; d p < 0.02 versus the same glycine concentration; e p < 0.05 versus 40 mM glycine; f p < 0.01 versus 40 mM glucose; g p < 0.001 versus 40 mM glycine a

118€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

19€%, and the value of δ determined for glycine (5-40 mM) increases by 22.5 %. However, in the case of mannitol, the layer thickness does not change when increasing its concentration from 10 up to 50 mM, and in the cases of glucose and glycine, the layer thickness increases from 316 ± 13 up to 380 ± 26 μm and from 254 ± 11 up to 319 ± 14 μm, respectively, with an increase of their concentration from 10 mM up to 40 mM, respectively (Tab. 14). Notably, the thickness of the unstirred layer determined with an amino acid was always ~60 μm smaller than that with the same concentration of glucose. Between the two techniques of determination of thickness of the unstirred layer, there are basic differences. Upon addition of mannitol in a mucosal solution, the potential difference on the intestinal wall and the SCC across the wall decrease, whereas they increase upon addition of nutrients. It is even more important that mannitol (impermeant sugar) is practically not consumed at the intestinal surface, whereas the nutrients are actively transported into enterocytes so that their concentration at the surface is lower than their concentration in the bulk. It is important to note that in the latter assay, it is not necessarily to use a high concentration of the tested substance. Any nutrient concentration can be used for the measurement of the layer thickness, including concentrations smaller then 10 mM. The surprising thing is that both assays of layer thickness yield similar results. This can be seen by the slope of the line of linear regression, 0.997 at high coefficient of correlation. Upon addition of a nutrient, the electric parameters of the mucosa are changed because of the activation of the additional mechanism of sodium transport through the brush border membrane. For this, it is not mandatory that the coupled transporters for sodium and the nutrient are uniformly distributed along all villus surface. The thickness of the layer must be comparable with the villus height (Metelsky, 2007c) which, as it has been long known, is equal 300-500 μm. It is important to note that because of the values of δ determined with mannitol and glucose are the same, transporters for glucose might also function on any villus height. Hence, the evidence to the hypothesis is obtained that the thickness of an unstirred layer in the intestine may reflect the morphology of its surface, for example, the villus height (Gusev et al., 1983; Metelsky, 1987). This might be different in the case of glycine; the value of δ with any concentration is smaller by ~60 μm than the value obtained with glucose. The dependence of the thickness on the nutrient concentration may have two explanations. One is a nonuniform distribution of specific transporters along the villar surface; indeed, for some nutrients, the transport ensembles have been reported to be present only on the upper third of the villi (Cremasceid et al., 1972; Gervey et al., 1976; James, Smith, 1981; Smith et al., 1983; Sangild et al., 1995). The other explanation is that, even upon a uniform distribution of transporters, the surface concentration will decrease along to the villar base because of nutrient absorption. If this is true, the layer thickness determined in this way should be the

Chapter 7. Unstirred layers of the fluid at the mucosa surface€€€€€119

smallest for the highest nutrient absorption rate. In other words, there should be a negative correlation between the layer thickness€ and the maximal SCC response (A). However, the data available (Tabs 38, 40) demonstrate a weak positive correlation (r ~ 0.35 and 0.55 in old rats and young rats, respectively) between these values obtained with glucose, maltose, sucrose, alanine, leucine, and Gly-Ala. This contradicts the latter interpretation but is consistent with the nonuniform distribution of transporters; moreover, this assay may perhaps be adapted as a tool for examining the topography of particular transporters on the villar surface (Metelsky, 2007a, 2007b, 2007c).

7.3. Dependence of the thickness of an unstirred layer on the rate of perfusion€€ With the rate of solution change in the chamber (Tab. 15, Fig. 5) of 0.6 mL/min, the layer thickness in rats is equal to 474 μm (Metelsky, 1987). Upon increasing the perfusion rate, the layer thickness gradually decreases, and with a rate of 36 mL/min, the layer thickness is 210 μm. These results agree well with the data of Barry, Diamond, 1984, Fig. 2; Westergaard, Dietschy, 1974.€ In these studies, the minimum thicknesses of the unstirred layer near a rabbit jejunum (5 min preincubation) proved to be amazingly close to ~220 µm. An interesting phenomenon was found when examining the influence of the thickness of unstirred layer near the intestinal mucosa on the Kt for glucose (Thomson, Dietschy, 1980; Barry, Diamond, 1984). With a decrease in resistance of the unstirred layer near the rabbit ileum (which is linearly related to the unstirred layer thickness), Kt linearly decreases from 17.7 down to 1.5 mM. In Metelsky, 1987, Metelsky, 2007a, the mentioned phenomenon was also detected. When reducing the thickness of the unstirred layer in the vicinity of the surface of the small intestine of rats and turtles, Kt for glucose decreases linearly from 17 down to 8.3 mM (r = 0.95, n= 7) and from 8.5 down to 2.2 mM (r = 0.974, n= 5), respectively. It has long been known that the Table 15. Effect of specimen perfusion rate on unstirred layer thickness at rat small intestine surface (Metelsky, 1987) Specimen perfusion rate, ml/min 0.6 (3) 1.2 (3) 2.4 (3) 4.6 (4) 9.2 (4) 18.0 (6) 36.0 (8)

Unstirred layer thickness, δ, µm 474±12 386±121 291±19 275±10 255±13 226±15 210±13

120€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

small intestine of a turtle differs from a rat intestine by a few anatomic features. For example, in a turtle intestine, villi are absent, and instead, relatively flat crests are present (Gilles-Ballien, 1983). Therefore, one can expect that for a turtle intestine, the thickness of the unstirred layer will be smaller. This is indeed the case (Metelsky, 1987). With any perfusion rate, the thickness of the unstirred layer in a turtle is smaller than that in a rat by at least by 110-140 microns (Metelsky, 1987) (Tab. 16, Fig. 5). It should be pointed out that the length of the curve portion with a fast decrease in layer thickness in a turtle is larger than in a rat with rate of 2.0 mL/min. However, in turtles, the curve flattens more slowly than that in rats upon further increase of the rate of solution change in the chamber.

7.4. Does the thickness of the unstirred layer change during experiment? Before chambering a resected preparation of a rat intestine, one can observe a mucous application with a binocular magnifier. However, if a preparation stays in the chamber more than 20 min, such applications disappear or are washed out. Does this affect the results obtained? It is thought that in the presence of mucous applications, the thickness of the unstirred layer is large, and with a gradual removal of mucous (as a result of perfusion), the layer thickness decreases. However, it turns out that 19 minutes after the beginning of the experiment (Tab. 17), the thickness of the unstirred layer practically does not changed and even (Metelsky, 1987) tends to increase. This is surprising. The mucus is not removed before chambering of an intestine preparation, and after a long experiment, all mucus is absent. Hence, mucus is washed away during the first minutes, and this could result in the reduction of the unstirred layer thickness. However, this effect is absent. During all experiments (Tab. 18), the thickness of the unstirred layer determined with glucose stable.

Table 16. Effect of specimen perfusion rate on unstirred layer thickness at a turtle small intestine surface (two measurements at each rate) (Metelsky, 1987) Specimen perfusion rate, ml/min 0.5 1.0 2.0 4.0 8.0

Time constant of solutions replacement in experimental chamber, tch, s 12 6 3 1.5 0.75

Unstirred layer thickness, δ, µm 335 250 175 147 116

Chapter 7. Unstirred layers of the fluid at the mucosa surface€€€€€121

Table 17. Changes in unstirred layer thickness at a rat small intestine surface against the background of fast initial short circuit current decline (Metelsky, 1987) Time since beginning of experiment, min 3.3 (3) 10.0 (3) 19.0 (3)

Number of response 1 2 3

Unstirred layer thickness, δ, µm 200 ± 11 229 ± 10 231 ± 6

Table 18. Time dependence of unstirred layer thickness near small intestine surface (Metelsky, 1987) Time since beginning of experiment , min 30 64 126 158 189

Number of response 1 6 13 16 18

Unstirred layer thickness, δ, µm 224 201 215 256 250

7.5. Dependence on temperature of the unstirred layer thickness The influence of the temperature on the activity of digestive enzymes has been studied since the end of the 19th century. It has been found that at temperatures close to 0 o С, warm-blooded animal enzymes, unlike fish enzymes, lose their activity (Ugolev, Kuzmina, 1993). Under the assumption that the thickness of an unstirred layer is not affected by temperature changes, the dependence 1 /τ on temperature reflects the dependence of the diffusion constant of glucose D = 4δ2 /π2 τ (see 6.1). This dependence is characterized by a direct line with an activation energy of 5.6 kcal/mol which corresponds to Q10 = 1.34. As it has been found (Metelsky, 1987), the time constant of the glucose effect development decreases with temperature. Such result should be expected, when taking into account the physical sense of the parameter τ = 4δ2 /π2 D (δ is the thickness of the unstirred layer); the diffusion constant (D) increases with an increase of temperature. Because Q10 = 1.2 for glucose diffusion in water, one would expect that its activation energy will be insignificant. Under the assumption that the thickness of the unstirred layer does not depend on temperature, the Arrhenius plot for ln(1/τ) reflects the temperature dependence (1/Т) of the diffusion constant. The activation energy for the = diffusion constant is equal to 5.6 kcal/mol, which corresponds to Q10 = 1.34; i.e., the value is actuality close to the activation energy for diffusion processes.

122€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

The activation energy of enzymes, it has long been known, is one of their major characteristics. The value of the activation energy of enzyme shows which minimal energy (counting upon 1 mole) particles must have to react (Ugolev, Kuzmina, 1993). However, the thickness of the unstirred layer, perhaps, should increase when the temperature decreases because of an increase in interactions between water molecules. If we now take into account that both the increase in layer thickness and the reduction of the diffusion constant result in an increase of τ, it is apparent that the Q10 for glucose diffusion measured by (Metelsky, 1987) agrees closely with the theoretical value of 1.2. This conclusion once again demonstrates the correctness of the procedure for determining the thickness of the unstirred layer by single SCC response.

7.6. Final remarks The thickness of the unstirred layer can be determined both with the response of electric parameters (in particular the SCC) upon addition of impermeant sugar (mannitol) and with the nutrients transported in the Na+-dependent manner. The values of the layer thickness determined with mannitol and with glucose or glycine are close. The nonspecific effect of mannitol is reproducible with any other impermeant or poorly permeating sugar, for example, fructose. Nevertheless, the layer thickness depends on the nature of the nutrient. The main point of the mannitol assay is as follows. The thickness of the unstirred layer can perhaps be measured by using any diffusion-limited reaction. For instance, in experiments, it is routinely determined from the dynamics of the electrophysiological response of the intestinal wall or from the difference in osmotic pressure on the two sides of a specimen upon supplementing the mucosal solution with a nonpermeating substance (osmotic response due to emergence of streaming potential) or a substance absorbed in a Na+-dependent way (electrogenic Na+ transport across the enterocyte apical membrane). The osmotic response (see Chapter 4) is explained as follows (Pidot, Dimond, 1964). The membrane matrix carries an excessive negative charge; therefore, the transmembrane channels contain more cations than anions. Upon creating an osmotic gradient, the concentration of water in the two compartments becomes unequal, and water starts to flow through the specimen, carrying with it the cation-rich intrachannel liquid. Thereby the side receiving this flux acquires a charge opposite to that of the matrix. Notably, sucrose, raffinose, and mannitol produce the same result. Moreover, the concentration dependences for the transmural water flow and the induced potential are parallel. A similar phenomenon was reported for the rat small intestine (Smith, Wright, 1964; Metelsky, 1987, 2007a, 2007b, 2007c). The same phenomenon can be described in several other terms (Barry, Diamond, 1984). In most of the listed epithelia (gallbladder, small intestine, renal proximal

Chapter 7. Unstirred layers of the fluid at the mucosa surface€€€€€123

tubule, and choroid plexus), the sign of diffusion potentials implies that the cation permeability exceeds the anion permeability. The€ sign of the€ apparent streaming potentials is that the side toward which water is flowing becomes electrically positive, as one expects intuitively for a pseudo streaming potential (because it is actually a diffusion potential) and may also generally expect for a true streaming potential (Barry, Diamond, 1984). Another mechanism was proposed (see Chapter 4) for the attenuation of the epithelial potential upon increasing the osmolarity of the mucosal solution (relative to the serosal solution). It essentially implies a resulting water flux along the gradient, which differentially affects the salt concentration in the two unstirred sections of the epithelial stratum, perimucosal and subserosal (including the subepithelial tissue); because the subserosal layer is thinner, the overall effect of the water flow is a salt accumulation therein. This entails additional diffusion of salt through the epithelium toward the mucosal solution. Owing to cation selectivity inherent to the intestinal epithelium, an additional potential is generated (plus in the mucosal solution). In mannitol assay, the concentration of the added substance should be more than 10 mM, otherwise a response can be not observed. On the contrary, for measurements of the layer thickness with a nutrient or with its transport inhibitor, the concentration can take any value, including much lower than 10 mM. Apparently, the glucose-induced liquid flow does not use the pores responsible for the streaming potential. In this case, the effect is manifested when a nutrient arrives with its transporter. If such transporters are distributed nonuniformly along the height villus, one can find the differences in thickness of the unstirred layers. Such effects also have been revealed (Metelsky, 2004a). The maximal values of the unstirred layer thickness are determined with monosaccharide glucose and disaccharide sucrose, and the smallest one with the amino acids leucine and alanine. Perhaps, these observations reflect the topography of the corresponding transporters distribution along the villus height (Fig. 14). If so, the excellent prediction (problem #4) formulated€ in Barry, Diamond, 1984 “Transport sites along the villus must differ as a function of solute concentration and uptake rate and may differ intrinsically for different solutes, causing unstirred layers thickness to vary with solute identity, concentration, and uptake rate. The arrangement of transport sites along the villus may constitute an unexplored principle of intestinal organization” is confirmed by the above facts. Now, one may conclude that this new principle of intestinal organization was really revealed (Metelsky, 1987, 2007a, 2007b, 2007c). Now, we can finally answer the question raised at the beginning of the chapter: how do pectin and guar gum reduce the rate of nutrient absorption in the GI tract followed by an increase in the unstirred layer thickness? In reality, with an increase in layer thickness, the value of responses to glucose addition decreases. It is caused by an increase in the diffusional resistance equal to D/δ and/or a decrease in diffusional

124€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

Enzyme-transporting ensemble for gly-ala

Enzyme-transporting ensemble for glucose

Unstirred layer thickness by glucose

Unstirred layer border

Unstirred layer thickness by Gly-Ala

Intestinal mucosa section Fig. 14. Determination of unstirred layer thickness near real mucosal surface of small intestine by short circuit current changes on addition of nutrients absorbed via Na+-dependent pathway.

permeability of the unstirred layer for glucose molecules while they approach the surface mucosa, where D is the diffusion constant for glucose and δ is the layer thickness. Apparently, pectin and guar gum produce a film on the mucosa that results in the increase of the diffusion resistance. As a result, the absorption of any substance should indeed decrease. But the main point resulting from this brief consideration of properties of unstirred layer, apparently, is that this rather abstract concept is already part of clinical gastroenterology: thickness of unstirred layers is artificially (due to introduction of pectin and guar gum) increased to reduce absorption of substances.

Chapter 8. Regulation of sodium transport in a small intestine€€€€€125

Chapter 8. Regulation of sodium transport in a small intestine 8.1. Active sodium transport in the absence of nutrients (basal SCC) After mounting a preparation of rat small intestine in the experimental chamber and a switching on the voltage clamp device, we can record (Metelsky, 1981) a relatively high SCC flowing through the preparation, which gradually decreases to the a quasistationary level with time (~20-30 minutes at 26°С) at a speed of 2.0-12.5 μА/cm2*min. This level of the basal SCC can change later during the experiment but at a much lower rate. In view of the large speed of decrease of the initial SCC, it is complicated to study the influence of various factors on the basal SCC in this phase. Therefore, as a rule, addition of substances takes place after the basal SCC has reached the quasistationary level.

8.2. Control of active sodium transport in tight epithelia As it was already pointed out, epithelial tissues are rather complex systems from a functional and morphological viewpoint, with extremely complex multilevel multistage regulation. Many hormones and drugs affect transport processes in epithelial tissues. Because hormones and drugs affect, as a rule, both membranes of epithelial tissues, apical and basolateral (Janacek, 1975), and also because of the presence of a coupling between sodium transport and some nutrients, it is difficult to interpret results on the action of various substances. In this respect, one can classify regulatory mechanisms according to their functional feature (depending on what stage of sodium transport the substance acts). It has been shown above that electrogenic sodium transport through an enterocyte most likely consists of two various components, beginning in the brush border membrane: the mechanism of transport through the channels, similar to that in tight

126€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

epithelia, and sodium transport by means of the coupled transporters for the nutrient and sodium. Controlling sodium transport in an enterocyte for each of these two potentially differing systems separately was not considered. It is appropriate therefore to consider the regulation of sodium transport through channels of the apical membrane of tight epithelia.

8.2.1. Control by intracellular sodium There is a negative feedback between the rate of sodium transport through the apical membrane and its intracellular concentration: with an increase of the sodium content inside a cell, the permeability for sodium of the apical membrane decreases (Larsen, 1975; Chase, Al-Awqati, 1981; Turnheim, 1983). The reduction of the labeled amiloride (sodium channel blocker) binding with the apical membrane with an increase of the intracellular sodium concentration supports these data (Cuthbert, Shum, 1978).

8.2.2. Control by intracellular calcium It is found that ionophores A 23187 reduces the SCC, and with removal of sodium from a solution washing the internal surface of a frog skin, transepithelial sodium transport decreases sharply, but the absorption of calcium and the rate of calcium outflow from the epithelium preliminary loaded by this cation increases (Grinstein et al., 1978). The sequence of events goes in the following way. When increasing intracellular sodium concentration (for example, with application of an inhibitor of sodium pump -ouabain), the calcium content in a cell increases. This is possible because of the decrease of the calcium outflow rate resulting from the reduction of the rate of sodium-calcium exchange mechanism operating in the apical membrane. This causes a partial inactivation of the sodium flux through the apical membrane in such a manner that fluxes of sodium through both membranes are balanced again. The link between intracellular calcium and transepithelial permeability for water supports the regulatory role of calcium as well (Taylor, Windhager, 1979; Chase, Al-Awqati, 1981).

8.2.3. Control of the interaction of sodium with the surface of an apical membrane By changing the ionic force, the ionic composition and рН of a solution washing the external surface of a frog skin, charges of the dissociated carboxylic groups at the input of a sodium channel may be shielded, resulting in the modification of sodium transport (Biber, Mullen, 1980; Grinstein et al., 1978; Awayda et al., 2000).

Chapter 8. Regulation of sodium transport in a small intestine€€€€€127

8.2.4. Control of permeability of the single channel One can change the channel permeability in two ways: by acting on the regulatory center (see above) and by modifying the channel permeability itself. According to (Walton et al., 1975), some substance, for example, the antidiuretic hormone, interacting with adenylate cyclase on the basolateral surface of a urinary bladder result in an increase of the cell cyclic adenosine monophosphate, which diffuses to the opposite side of the cell. Here, cyclic adenosine monophosphate stimulates the phosphatase, which dephosphorylates some protein; this is the state of high permeability of a sodium channel (increase in the SCC). Protein kinase can phosphorylate this protein and switch the channel to a state of low permeability (reduction of the SCC). Although there is no evidence so far that kinases play a functional role in vivo, numerous possible phosphorylation sites were underlined in the secondary structure of the Na+/glucose cotransporter.€ Phosphorylation€ may be involved in the fine tuning of the cotransporter activity and may up- or down-regulate the transporter activity depending on the load (Parent, Wright, 1993).

8.2.5. Control of the number of channels Although the existence of such a regulation might still be uncertain, the presence of this regulation is firmly proved only for the case of aldosterone. It turns out that aldosterone increases the permeability of the epithelial cells of a toad urinary bladder by an increase in the number of channels (Cuthbert, Shum, 1976). One is inclined to think that other compounds act in a similar way (LeFevre, 1975).

8.2.6. Control by change of permeability for counterion At least on a frog skin (Ussing, 1971b), the rate by which chloride is capable to follow sodium can determine the rate of the active sodium transport (certainly, under the condition when a limiting stage is crossed by sodium of an apical membrane).

8.2.7. Dependence of sodium transport on cell volume Membrane water transport is an essential event, not only in the osmotic cell volume change but also in the subsequent cell volume regulation (Kida et al., 2005). The nature of this phenomenon is not quite clear (see also Chapter 3), but it is shown that when epithelial cells are swelling, the SCC increases. It is suggested that swelling promotes the opening of sodium channels (Ussing, 1971b); indirect evidences in favor of this

128€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

hypothesis are the data in Wright, Smulders, Totmey,1972;€ Barry, Diamond, 1984. It was shown that, in response to addition of 300 mM sucrose to mucosal solution for planar segment of rabbit gallbladder, the tissue electrical resistance (measure of the swelling of the leaky epithelium cells) increases 2 to 3 times. However, some authors have cast some doubt on the control of sodium transport by cell volume regulation.

8.3. Control of ion absorption in an enterocyte Controlling ion transport in an enterocyte is difficult to consider within the analysis of the non-coupled sodium channels control. Almost all data on regulation of ion transport are devoted to a receptor regulation. Moreover, even with the fact itself of involving receptor regulation for the processes occurring in an intestine, the issue is not unequivocally settled. Receptor control is proved to follow the functions of the GI tract: exocrine secretion (water, electrolytes, proteins, and glycoproteins), motility, and survival functions (tissue metabolism and renewal of cells). For processes of absorption of ions and nutrients, especially along a transcellular pathway, the existence of receptor regulation is insufficiently documented (Bonfils, 1980). Besides, as indicated above, sodium absorption is a rather complex process. It includes NaCl absorption and electrogenic sodium absorption (both coupled and not coupled). However, this is not taken into account by the experts on regulation, and when the conclusion is drown that this substance influences sodium absorption, this refers to electroneutral absorption of NaCl only. Because of this, the issue about receptor control of electrogenic sodium transport (both coupled and not coupled) remains open. Besides, problems in studies of neuro-hormonal regulation are worse than those listed above (Tapper, 1983; Turnberg, 1983). Without going into details, we shall briefly list only some of them. One of the difficulties is that in enteric nervous system, the line of demarcation between hormone and transmitter is not clear. Endocrine cells, diffusely distributed in a mucosa while entering into the circulatory system, may act as an hormone but may have a local action on epithelial and subepithelial cells. This is the case for, for example, gastrin, cholecystokinin, substance Р, and enteroglucagon (Turnberg, 1983). The number of substances known first as hormones and then as neurotransmitters continuously increases (Klimov, 1983; Ugolev, 1985; Krieger, 1983). Vaso-active intestinal peptide, substance Р, serotonin, enkephalins, and bombesin are already found in the enteric nervous system. Some nerve endings contain more than one type of peptides. It appears that the localization of some of these peptides is not limited to nerves only; they can be found also in paracrine cells. Besides, some neurotransmitters can avoid entering the circulatory system and carry out the role of endocrine regulators. It is difficult to interpret the substance effect on intestinal transport independently of other coexisting effects stimulating and inhibiting messengers. The effect of several substances together can be different from the sum of the effects of

Chapter 8. Regulation of sodium transport in a small intestine€€€€€129

each substance. Moreover, studying the action of hormones that are found in plasma under physiological conditions (for example, after eating) with the concentration, may prove to be inadequate, because their concentration in plasma reflects only the high local concentration of the hormone near the mucosa (Turnberg, 1983). A great number of neuro-endocrine and hormonal factors act on electrolyte absorption in the intestine. It is unlikely that all of them have a physiological value.

8.3.1. Neuro-endocrine control Noradrenaline increases electrolyte absorption (NaCl), acting through α2-receptors (Chang et al., 1982). In experiments in vivo, it was shown by addition of sympathomimetic (with indirect action) thiramin that endogenous noradrenaline influences intestinal transport. Low concentration of noradrenaline has an effect only in the presence of an inhibitor of monoamine oxidase—thiramin (Tapper et al., 1981). This effect becomes clear when we consider the high contents of the monoamine oxidase in a brush border membrane (Levine, Sjverdsma, 1962; Glenner et al., 1967), capable to utilize thiramin. In the intestinal mucosa, there are mechanisms of specific absorption and release of noradrenaline. Because ion transport does not change after sympathectomy (Tapper et al., 1981) and because antagonists of a2-adrenergic receptors do not change ion transport (Chang et al., 1982), the process of basal release of noradrenaline, apparently, does not affect the electrolyte transport. The effects of noradrenaline on transport, realized through β-receptors, are found in vivo (Morris, Turnberg, 1981) but are absent in vitro (Field, McColl, 1973). The inhibiting effect of dopamine on sodium absorption in rat jejunum is caused by inhibition of Na+K+ATPase of enterocytes because of the binding with D1-like receptors (Vieira-Coelho, Soares-da-Silva, 2001). Acetylcholine is critical in controlling epithelial ion transport and hence water movements for gut hydration (Hirota, McKay, 2006). Besides, acetylcholine, acting on presynaptic receptors, stimulates additional (to basal) release of noradrenaline (Tapper et al., 1978; Tapper, Lewand, 1980). Moreover, it has been found that presynaptic muscarine and nicotinic receptors of adrenergic neurons in the intestinal mucosa have opposite effects on the release of noradrenaline and on ion transport (Purves, 1976; Loffelholz, 1979;). Effects of noradrenaline, acetylcholine, and some other substances fall into a pattern of local adrenergic modulations of intestinal transport (Ugolev, 1978; Tapper, 1983). According to Tapper (1983), there are the receptors on the membrane of the peripheral neuron ending stimulating or inhibiting the release of noradrenaline. The stimulation of noradrenaline release is observed under the action of angiotensin II and agonists of nicotinic receptors, upon action of prostaglandins Е and аgonists of muscarine receptors. It is conceivable that noradrenaline can be released not along but together with another neurotransmitter. Moreover, аgonists of muscarine and

130€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

nicotinic receptors can reduce and/or increase acetylcholine release from the endings of cholinergic peripheral neurons (Dzieniszewski, Kilbinger, 1978; Manher, Gershon, 1979; Briggs, Cooper, 1982). However, the presence of inhibition by atropine of basal cholinergic secretion in vivo (Tidball, 1961; Morris, Turnberg, 1980) and absence of such effect of atropine on the preparations mounted in the Ussing chamber (Isaacs et al., 1976; Tapper et al., 1978) have not obtained a satisfactory explanation so far. Another possible mechanism of local modulation of intestinal transport is related to the presence in mucosa of entero-endocrine cells containing neuropeptides. It is agreed that neuropeptides, released from such cells, have a short life time (Tapper, 1983). They can participate in paracrine intercellular relationships and act on the cell targets that are in immediate proximity. Such neuropeptides can change either microcirculation, ionic transport, or villi movement. The release of neuropeptides from entero-endocrine cells can be modulated by efferent innervations of various neurons of the enteric nervous system (Lundberg et al., 1978; Larsson, 1981; Ahlman et al., 1981; Zinner et al., 1982). It is noteworthy that neuropeptides of entero-endocrine cells can modulate noradrenaline and acetylcholine neurons. How neuropeptides secreted into the intestinal lumen can modulate intestinal transport remains to be understood. Closed-type entero-endocrine cells do not have any contact with the luminal content and can respond to nervous and mechanical stimulus and to hormonal influences. Open-type cells are characterized by the presence of microvilli turned into the lumen and by basal-located secretory granules. An example of such cells is the entero-chromaffin cells containing serotonin (important intestinal secretogen, acting by release of calcium). Such cells, also named enteroreceptors, are capable to respond to food, osmotic, and mechanical stimulus (Leek, 1972).

8.3.2. Hormones and drugs We believe that many diseases may be associated with a violation of sodium-dependent absorption of nutrients. No wonder that, in the past few years, the attention of researchers was focused on the discovery of natural and synthetic compounds that may affect the absorption of nutrients in general and particularly on sodium-dependent absorption of nutrients. Metformin is an oral anti-diabetic drug in the biguanide class. It is the first-line drug of choice for the treatment of type 2 diabetes, particularly in overweight and obese people and those with normal kidney function. Evidence is also mounting for its efficacy in gestational diabetes, although safety concerns still preclude its widespread use in this setting. Metformin is an orally administered drug that lowers blood glucose and improves insulin sensitivity in patients with non-insulin-dependent diabetes. Although the antihyperglycemic effect of metformin has been extensively studied, its cellular mechanism(s) of action (including the effect on enterocyte) remains unclear. It

Chapter 8. Regulation of sodium transport in a small intestine€€€€€131

was shown that metformin markedly inhibited glucose-induced SCC (approximately 77%) after mucosal addition. In addition, metformin reduced the glucose-induced abundance of SGLT-1 in brush border membranes and increased those of GLUT2, concomitantly increasing the phosphorylation of intracellular AMP-activated protein kinase alpha2 subunit. But transmural glucose transport measured in vitro was increased by 22% under metformin. The authors believe that metformin slightly increases intestinal glucose absorption by inducing a re-distribution of glucose transporters in brush border membranes and this could constitute a peripheral signal contributing to the beneficial effect of metformin on glucose tolerance. (Sakar et al., 2010). Leptin (Greek leptos meaning thin) is a 16 kDa protein hormone that plays a key role in regulating energy intake and energy expenditure, including appetite and metabolism. Leptin itself was discovered in 1994 by Jeffrey M. Friedman and colleagues at the Rockefeller University through the study of mice (Zhang et al., 1994). It turned out that this hormone affects the nutrient-dependent absorption of sodium.€ It is known that L-glutamine is the primary metabolic fuel for enterocytes. Glutamine from the diet is transported into the absorptive cells by two sodium-dependent neutral amino acid transporters present at the apical membrane: ASCT2/SLC1A5 and B(0)AT1/SLC6A19. It was shown that leptin is secreted into the stomach lumen after a meal and modulates the transport of sugars after binding to its receptors located at the brush border of the enterocytes. In Ussing chambers, 10 mM L-glutamine absorption followed as Na+-induced SCC was inhibited by leptin in a dose-dependent manner (maximum inhibition at 10 nM; I(C50) = approximately 0.1 nM). The increase in ASCT2 and B(0)AT1 gene expression induced by 60-min incubation of the intestine with 10 mM L-glutamine was strongly reduced after a short preincubation period with leptin. Altogether, these data demonstrate that, in rat, leptin controls the active Gln entry through reduction of both B(0)AT1 and ASCT2 proteins traffic to the apical plasma membrane and modulation of their gene expression. The authors note that the slower action of leptin on the serosal side of mucosa seems indirect and is likely mediated by endogenous cholecystokinin (Ducroc et al., 2005; Ducroc et al., 2010). In this context, we can not mention the recently discovered regulatory loop. A positive regulatory control loop between gut leptin and fructose in which fructose triggers release of gastric leptin which, in turn up-regulates GLUT5 and concurrently modulates metabolic functions in the liver. This loop appears to be a new mechanism (possibly pathogenic) by which fructose consumption rapidly becomes highly lipogenic and deleterious. (Sakar et al., 2009). An increased expression of resistin-like molecule-beta, a gut-derived hormone, is observed in animal models of insulin resistance/obesity and intestinal inflammation. In studies with jejunal mucosa mounted in Ussing chamber, luminal resistin-like molecule-beta inhibited SGLT-1 activity in line with a diminished SGLT-1 abundance in brush border membranes. Furthermore, the potentiating effect of resistin-like moleculebeta on jejunal glucose uptake was associated with an increased abundance of GLUT2

132€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

at brush border membranes. The effects of resistin-like molecule-beta were associated with an increased amount of protein kinase C betaII in brush border membranes and an increased phosphorylation of AMP-activated protein kinase. The authors believe that the regulation of SGLT-1 and GLUT2 by resistin-like molecule-beta expands the role of gut hormones in short-term AMP-activated protein kinase /protein kinase C mediated control of energy balance (Krimi et al., 2009). Orexins, also called hypocretins, are the common names given to a pair of excitatory neuropeptide hormones that were simultaneously discovered by two groups of researchers in rat brains. The two related peptides (orexin-A and B or hypocretin-1 and -2), with approximately 50% sequence identity, are produced by cleavage of a single precursor protein. It is known that orexins are neuropeptides involved in energy homeostasis. The rapid and marked increase in SCC induced by luminal glucose was inhibited by 10 nmol/l orexin A or orexin B (53 and 59%, respectively). The response curves to orexin A and orexin B were not significantly different with half-maximal inhibitory concentrations at 0.9 and 0.4 nmol/l, respectively. On the one hand, orexin A-induced inhibition of SCC was reduced by the neuronal blocker tetrodotoxin€ and by a cholecystokinin receptor-2 antagonist, indicating the involvement of neuronal and endocrine cholecystokinin-releasing cells. Orexin B-preferring orexin receptor-1 pathway was not sensitive to tetrodotoxin or to cholecystokinin receptor antagonists, suggesting that orexin B may act directly on enterocytic orexin receptor-1. These distinct effects of orexin A and orexin B are consistent with the expression of orexin receptor-1 and cholecystokinin receptor-2 antagonist mRNA in the epithelial and nonepithelial tissues, respectively. The authors believe that this data delineate a new function for orexins as inhibitors of intestinal glucose absorption and provide a new basis for orexin-induced short-term control of energy homeostasis (Ducroc et al., 2007). Glucocorticoids inhibit the absorption of NaCl and stimulate secretion in a rabbit ileum (Sellin, Field, 1981). However, the three-hour delay in the effect of these hormones points to the fact that de novo synthesized specific proteins are involved in transport control. Aldosterone (steroid) increases sodium absorption in the colon tight epithelium and does not affect the small intestine (Dolman, Edmonds, 1975; Frizzel, Schultz, 1978). Angiotensin II in low concentration increases sodium absorption in small intestine and colon (Poat et al., 1976; Levens et al., 1977). The physiological role of this effect is unclear (Turnberg, 1983). Upon addition of amphotericin B from the mucosa side, the SCC increases in a doso-dependent manner with Km 7.4 μM (Vieira-Coelho, Soares-da-Silva, 2001). Diuretic and natriuretic effects of interleukin-1β and its inhibiting effects on glucose absorption are caused by dawn-regulation of Na+-K+ATPase in jejunum and kidneys (Kreydyeh, Al-Sadi, 2002). Thus, even from such a brief consideration of the problem, one can see that the control of electrogenic sodium absorption in the small intestine is insufficiently

Chapter 8. Regulation of sodium transport in a small intestine€€€€€133

studied. The control of not-coupled mechanism of sodium transport in enterocytes of a small intestine may be inferred assuming its similarity to sodium channels of tight epithelia, but on the control of the coupled electrogenic mechanism of sodium transport in vitro are yet scarce (Wright et al., 1997). This situation is due to the following circumstances. First, sodium absorption depends on a great number of factors, and some of them are not controlled in experiments. Therefore, an in vivo situation on this process can influence the intestine muscular activity, microcirculation, and villi movement (Tapper, 1983; Turnberg, 1983). The indirect evidence supporting these reasons is the differences in action of some substances under in vivo conditions, where all of the listed factors are present, and in vitro conditions, where some of them do not work. For example, gastrointestinal hormones such as gastrin, secretin, cholecystokinin, substance P, enteroglucagon (Turnberg, 1983), and GIP cause secretion of electrolytes in vivo and are inefficient in vitro, even in high concentration (Matuchansky et al., 1972; Hicks, Turnberg, 1974; Modligliani et al., 1976; Pansu et al., 1980). Second, the treatment of results on regulation is complicated, because the exact innervation of mucosa cells is unknown (Isaacs, Turnberg, 1977; Tapper, 1983; Oettinger, Tsybulevsky, 1993). Thirdly, so far, experts on regulation do not distinguish different the components of electrolyte absorption. It is difficult to find studies where the control of electrogenic and nonelectrogenic sodium absorption is discussed separately. Let’s consider the action of some classical transport inhibitors in more detail.

8.3.3. Inhibitors of transport 8.3.3.1. The ouabain Shortly after the influence of sodium on the transport of sugars (Riklis, Quastel, 1958) was discovered, it was shown (Csaky et al., 1961; Csaky, 1963) that cardiac glycosides (digitalis, ouabain) inhibit the transport of sugars and amino acids in the intestine. The subsequent studies have confirmed that many Na+-dependent transport processes are inhibited by these inhibitors (Schultz, Curran, 1970; Kushak, 1983). It has been found that the influence of ouabain on Na+-dependent processes is indirect and secondary in relation to its direct action on the mechanism of sodium removing from cells through a sodium pump. As a result, the sodium concentration inside the cells increases. When the difference of sodium concentrations on the brush border membrane becomes small, the active nutrient transport becomes impossible. It has been found that the addition of 0.2 mM of ouabain in a serosal solution within 15 minutes results in an almost full suppression of the SCC (in the absence of glucose) through a rabbit small intestine, and in all cases, the effect of ouabain has a

134€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

small time frame (Schultz, Zalusky, 1964a). Already in the first study devoted to the effects of sugars on the SCC (Schultz, Zalusky, 1964b), it was shown that ouabain in concentration of 0.5 mM prevents or inhibits the increase of the SCC caused by addition of actively transported sugar. It was concluded that sodium transport both in the presence and in the absence of sugar was carried out through the same ouabainsensitive mechanism. In a basolateral membrane, there are two mechanisms for sodium transport, which are both ouabain-sensitive (Schultz, Zalusky, 1964b). The omission of potassium from an external solution inhibits also sodium transport (Christinsen et al., 1969; Widdicombe et al., 1979). Ouabain has an effect when added in a serosal solution but does not have any influence from the opposite mucosal side (Tab. 19). It is common knowledge that sodium pumps are localized almost exclusively on the basolateral membrane (Dibone, Mills, 1978; Ernst, Mills, 1980) and that ouabain is practically unable to penetrate through a tight junction (Schultz, Zalusky, 1964a, 1964b). Ouabain in concentration of 0.33 mM prevents the development of the SCC response to glucose with an efficiency of 89 % (Metelsky, 1987). Direct determination of inhibiting effects of this glycoside on a basal SCC has confirmed that it is insignificant (~15 %). The effect of ouabain develops slowly, within 20 minutes (Schultz, Curran, 1970; Okada et al., 1977; Metelsky, 1987). This is evidence of the glycoside action mechanism mentioned above. In order for ouabain to manifest its action on sodium flux, the inhibitor should diffuse through tissues to the basolateral membrane and impair the functioning of the sodium pump (Fig. 1). After that, the cell sodium concentration should increase noticeably, and only after that, the effect of ouabain on transepithelial transport can be observed. Metelsky’s (1987) data agree qualitatively with the results obtained by Schultz & Zalusky (1964b) but are different quantitatively. In Metelsky (1987), the full reverse effect of glucose was observed only in two cases; in all others, ouabain did not decrease the basal SCC down to 0, nor did it for the SCC response to glucose. However, Metelsky’s (1987) data agree both with the results of Albus et al. (1979), where it is found that the potential difference induced by glucose, even an hour Table 19. Effect of addition of 0.33 mM ouabain* to serosal solution on responses of basal and glucose (10 mM)-stimulated short circuit current across rat small intestine (Metelsky, 1987) Short circuit current

Basal

Initial values Changes on 0.33 mM ouabain addition % of inhibition

19.75±2.75 (4)

Glucose (10 mM)- The effect of glucose can be stimulated prevented by ouabain, % 17.75±6.53 (4) 100 (4)

-2.88±0.51 (4)

-8.65±3.51 (4)

-88.98 ± 6.3 (4)

14.6

48.6

89

* - with the addition of ouabain, 5.6 mM KСl was omitted from serosal solution.

Chapter 8. Regulation of sodium transport in a small intestine€€€€€135

after exposition to ouabain, does not disappear completely (remains about 15 %), and the results of Okada et al. (1977) that show that long expositions (1 hour) of rat small intestine to 0.3 mM ouabain do not eliminate completely the spontaneous transmural potential difference, nor do they suppress the responses of this potential difference to glucose. One can point out a few possible reasons for such quantitative distinctions. In order to be active, ouabain should enter through narrow lateral intercellular spaces into which most of the surface of the basolateral membrane of an enterocyte is turned (Fig. 1). Depending on the state of that space (contraction or dilatation) and on the intensity of transcellular sodium transport, a more or less intensive water flux enter the intestinal lumen through this space, interfering with the entrance of ouabain through the same space. Dynamics of inhibition by ouabain depends also on by much the potential capacity of the sodium pump exceeds the potential capacity of the mechanism providing the entrance of sodium in the cell through a brush border membrane. If this ratio is large, even in an hour, one can not see any effect of ouabain on transport. On the contrary, if the ratio is small, already with the minimal inhibition of the pump, one can observe the reduction of the rate of transepithelial sodium transport. In essence, both hypotheses which have been proposed for an explanation of the differences in ouabain action under various conditions assume the dependence of the effect of ouabain the on intensity of sodium transport. Such observations are described. It is common knowledge that if the SCC through a frog skin is more than 20 μA/cm2, a small concentration of this glycoside is necessary for inhibition of sodium transport, and if the SSC is less than 10 μA/cm2, the transport inhibition is observed only when 50 % of the ouabain-sensitive sites are filled (Cala et al., 1978). Besides, divergences can be caused by species differences; it is common knowledge that identical preparations (with respect to other properties) of Nа+-K+ATPase from a medullary substance of a dog and rat kidney can be distinguished by its sensitivity to ouabain by a factor of 1000 (Periyasamy et al., 1983). At last, it is possible that both ouabain-sensitive and ouabain-resistant sodium pumps are present in the basolateral membrane of an enterocyte, as it is shown for a rabbit urinary bladder (Eaton et al., 1982).

8.3.3.2. Amiloride In nature, apparently, there are two basic types of sodium channels: tetrodotoxinesensitive in excitable cell membranes and amiloride (or phenamil, see below)-sensitive in electrically unexcitable membranes, in particular, in epithelial tissue (Metelsky, 1984b). The occurrence of amiloride-sensitive sodium channels (ENaC) is proved for several types of epithelial tissues, in particular for primate gastric mucosa (Tripathi, Rangachari, 1980) for the mammal colon (Tomson et al., 1982; Grubb, 1999; Bize, Horisberger, 2007), for frog skin and toad urinary bladder (Moreno, 1975; Zeiske,

136€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

1975; Lindemann, Van Dreissche, 1978), for horse small intestine (Cehak et al, 2009), for ovine fetal small intestine (Keller-Wood M et al, 2009), for the rat small intestine€ as in intact preparation (Metelsky, 1987a, 1989b, 2007a) as well in preparation after total proctocolectomies (Fukushima et al, 2005), and for cultured monolayers of the dog jejunum (Weng et al, 2005). It is clear that amiloride (or phenamil)-sensitive channels are widely distributed in nature. Also, they exist in the small intestine of mammals. However, data on the existence of sodium channels in enterocytes of a mammal small intestine similar to those in other types of epithelial tissues remains are scarce so far. As it was pointed out above, the existence of two mechanisms of electrogenic sodium transport (the active absorption not coupled with movement of nutrients due to a process similar to that in tight epithelia and the sodium absorption directly coupled with transport of nutrients) (Schultz, 1981) in the brush border membrane of an enterocyte has been proposed. This assumption is been based on experiments carried out on distal segments of a rabbit small intestine; the author points to the fact that it remains obscure whether a similar mechanism of sodium transport (not coupled) exists in more proximal segments of the intestine. If that assumption is true, one can try to carry out (by means of amiloride) pharmacological discrimination of these two postulated types of sodium transporters, as this has been done in studies of exited membranes (Khodorov, 1975). According to the assumption of Schultz (1981), amiloride considerably inhibits the basal SCC in preparations made both from distal and medial parts of an intestine (Metelsky, 1987a, 1989b). The effect of amiloride develops quickly and is completely reversible. Incomplete blocking of the basal SCC in neonatal murine intestine (Grubb, 1999) may be due to the fact that the concentration of added amiloride only slightly exceeds the inhibition constant for this diuretic. The analysis of the dynamics of several changes of basal SCC under the action of amiloride by a single response method gives a value for Ki of 0.1 mM (Metelsky, 1987, 1989b). Hence, with a concentration of the added amiloride equal to 0.1 mM, we should observe only 50 % inhibition; that is in a good agreement with the value of 31.4 % (Tab. 20). If pharmacological discrimination of sodium transporters really takes place, it would be expected that the amiloride inhibits the SCC and Table 20. Effect of 0.1 mM amiloride addition to mucosal solution on responses of basal and glucose (10 mM)-stimulated short circuit current across rat small intestine (Metelsky, 1987). Glucose, mM 0 10

SCC basal or glucosestimulated, µA/cm2 34.0 ± 2.75 (11) 40.1 ± 4.9 (11)

SCC changes on 0.1 mM amiloride addition Absolute values, µA/cm2 % -10.7 ± 4.1 (8) - 31.4 +12.4 ± 3.7 (10) + 30.9

Initial resistance - 32.28±3.76 Ohm*cm2; initial potential difference - 2.91±0.42 mV. Experiments have been carried out on specimens opened both along mesenteric and contramesenteric lines (Metelsky, 1987).

Chapter 8. Regulation of sodium transport in a small intestine€€€€€137

the basal SCC in the presence of glucose precisely by the same value. Unexpectedly, it turns out that the amiloride in the presence of glucose not only does not inhibit the SCC but stimulates it by 31% (Metelsky, 1987, 1989b). The effect of amiloride in this case is explained in the following way (Metelsky, 2007a). With blocking of the amiloride-sensitive sodium transport, the share of the electrochemical sodium gradient falling on its coupled transport increases. As this takes place, total sodium transport through the epithelium (coupled and not coupled) does not change. In the presence of glucose, the amiloride blocks part of sodium channels which have not been involved in coupled transport; therefore, the membrane resistance increases (Fromter, Gebler, 1977), and the potential difference on it increases. This results in an increase in the rate of sodium transport through the second (glucosedependent) mechanism. The opportunity for the existence of a reverse effect is based on these concepts: upon addition of glucose, the rate of sodium transport through not-coupled channels should decrease. Such effect has been observed (Kimmich, 1981). Owing to allowance for the latter effect, it was possible to find that the stoichiometry of the coupled sodium and glucose transport is equal not to 1:1 but to 2:1 (Kimmich, 1981). If our assumption is true and different effects of amiloride on SCC both basal and stimulated by glucose are caused by interactions with the same binding center (at the entrance of a sodium channel), its Ki for effects of inhibition and stimulation should be close. Indeed, additional increase in the SCC response to amiloride in the presence of glucose is characterized by a Ki equal to 25-100 μM (single response method) (Metelsky, 1987). The similarity of Ki for stimulating and inhibiting effects of amiloride is the best proof for the common mechanism of action of diuretic in these two cases (Metelsky, 2007a). Therefore, we conclude that the amiloride-sensitive transporter of sodium (most likely channels), similarly to sodium channels in other epithelia, is localized in enterocytes (Lindemann, Van Dreissche , 1978; Palmer et al., 1980; Tomson et al., 1982) and that these channels may be distinguished from other transporters of sodium in pharmacological studies. The supporting evidence for this conclusion was obtained in studies of electrogenic + Na absorption in rabbit ileum (Sellin et al, 1989) when the effects of phenamil (an amiloride analogue) was examined. In that view, electrogenic Na absorption, independent of either nutrients or other ions, occurs in the rabbit ileum. It was shown that 10-4 M phenamil inhibited mucosal-to-serosal Na flux, net Na flux, and SCC without significantly altering other fluxes. These results demonstrate that electrogenic Na absorption in rabbit ileum may be blocked by the amiloride analogue phenamil, suggesting that, in this epithelium, Na absorption may occur via Na channels in which the amiloride-binding site has been significantly altered. It was shown that the Na+/glucose and the Na+/phosphate cotransporters have been found to exhibit a Na+ leak or uncoupled Na+ current, that is, downhill or passive Na+ transport in the absence of nutrients, and€ have formed an impression that one possible mechanism for Na+ absorption across the apical membrane of the intestine is

138€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

via the Na+ uncoupled currents of transporters. However, this assumption is contrary to above results and data that ENaC and glucose-dependent sodium channels are regulated entirely by - different ways€ (see above and see Tab. 25) (Metelsky, 1987, Metelsky, 2007). Furthermore, amiloride is used to block the sodium channels in the jejunum of horses (Cehak et al., 2009) and primary cultures and subcultures of the normal mammalian (dog) jejunum form polarized epithelial monolayers with transepithelial Na+ absorption mediated in part by SGLT1 and ENaC (Weng et al., 2005).€

8.3.3.3. Phlorizin It is common knowledge that phlorizin in low concentration inhibits glucose transport in many types of cells (Schultz, Curran, 1970). In particular, it effectively prevents glucose transport from a mucosal solution in epitheliocytes of a small intestine (Kushak, 1983; Newey et al., 1959; Schultz, Curran, 1970; Rongione et al., 2001). This is explained by the presence on the coupled transporter of two binding centers for glucose and phenol located near to each other. Because of this construction of the binding site for phlorizin, phlorizin interacts with the transporter more effectively than glucose (Alvarado, 1967; Brot-Laroche, Alvarado, 1983). Moreover, the phlorizin in concentration of 0.5 mM quickly eliminates the SCC response to this monosaccharide; the SCC declines almost back to its initial level (Schultz, Zalusky, 1964b). The phlorizin does not affect the SCC in the absence of glucose, but if it is added before glucose, this glycoside is capable to prevent the development of the SCC response to glucose. Results of experiments with phlorizin are compelling evidence of the modern concept on the indissoluble link between the coupled glucose or sodium transport. Upon the stoppage of the coupled glucose and sodium transport, the transport of sodium or glucose stops too. Addition of phlorizin from a serosal solution does not influence the SCC. According to the above concept, the presence of a significant inhibiting effect of 0.1 mM phlorizin is shown upon its addition in the mucosal solution washing a preparation of rat small intestine (Fig. 15) (Metelsky, 1987). Moreover, for the first time, it is shown (Metelsky, 1987, 2007a) that phlorizin as an inhibitor of SCC responses to glucose is effective throughout the length of rat small intestine and within a wide range of temperatures. These data suggest that the mechanism of the coupled transport does not vary with the decrease of temperature and is identical in various segments of a small intestine. Besides, phlorizin effectively inhibits SCC responses to glucose in a reptile small intestine (turtle) (Metelsky, 1987). These facts allow us to think about the inhibiting effect of phlorizin as a common property of the mechanism of the coupled glucose and sodium transport, inherent to all parts of the small intestine and independent of the animal species and on physical factors

Chapter 8. Regulation of sodium transport in a small intestine€€€€€139

-Glucose -Phlorizin 5 μM/cm2

200 s

+ Glucose 10 mM

+ Phlorizin 0.1 mM

Fig. 15. Short circuit current response across rat small intestine to 10 mM glucose addition and an inhibiting effect of 0.1 mM phlorizin.

(for example, temperatures). However, in this study (Metelsky, 1987), unlike that of Schultz & Zalusky (1964b), phlorizin does not completely take away the stimulating effect of glucose, only 73 % at 26°С. It is believed that such distinctions are related to the concentration of this glycoside used in Schultz & Zalusky (1964b) and in Metelsky (1987), 0.5 and 0.1 mM, respectively. At the same time, the incomplete inhibition (80 %) of the transport of monosaccharides in the presence of phlorizin is observed also in chicken small intestine (Shehata et al., 1981). The magnitude of the phlorizin-inhibiting effect depends maybe on the condition of the animal (satiety or fasting) and, definitely, on temperature. With a temperature decreased down to 16°С, the phlorizin inhibiting effect increases up to 92.3 % (Tab. 21) (Metelsky, 1987). The SCC reduction upon addition of phlorizin in the presence of glucose down to a level lower than the initial one, which is characteristic for proximal and distal parts of the intestine, suggests that phlorizin influences the basal SCC. Indeed, it turns out that at 26°С, there is a small inhibiting effect of phlorizin on the basal SCC (see 9.4.2.). This result (Metelsky, 1987) contradicts that of Schultz & Zalusky (1964b)

140€€€€€Transport phenomena and membrane digestion in small intestinal mucosa Table 21. Effect of 0.1 mM phlorizin on glucose (10 mM)-stimulated short circuit current at 26°С and 16°С (Metelsky, 1987). Temperature 26°С 16°С а

Inhibition of SCC responses on 10 mM glucose addition, % 73.0 ± 5.8 (9) 92.3 ± 4.3 а (7)

p< 0.02

but agrees with data of Muflich & Widdas (1976) where the phlorizin-inhibiting effect on the basal SCC has been carefully studied. It appears that Кi in this case is equal to 39 μM. The estimation of Ki by the single response method for the inhibition of the SCC stimulated by glucose yields a value of 10-25 μM (Metelsky, 1987), which is in rather good agreement with the inhibition constant of glucose transport by phlorizin, equal to 20 μM (Brot-Laroche, Alvarado, 1983) and 5- 15 μM (Parent, Wright, 1993). In the paper by Muflich & Widdas (1976), the phlorizin-inhibiting effect on the basal SCC is explained by the possibility of sodium transport through a coupled transporter. The similarity of phlorizin inhibition constants for basal SCC, determined in Muflich & Widdas (1976) as being equal to 39 μM, and the Ki, determined in experiments (responses of the SCC) by Metelsky (1987), amounting to 20 μM, proves the validity of the assumption made: sodium can be transported with a low rate through the coupled transporter in the absence of glucose. A possible explanation of the small phlorizin-stimulating effect at 16°С will be considered later. The absence of an appreciable effect of phlorizin in a phase of fast on the decrease of the basal SCC agrees with the observations discussed above and simultaneously points out to the fact that this phase is not caused by the wash-out of some exogenic glucose from a binding site of the coupled transporter.

8.3.4. Link with energetics 8.3.4.1. Aerobic metabolism. Influence of oxygen access It is accepted that sodium-dependent transport processes can be provided with energy through both direct hydrolysis of ATP and coupling of these processes with a Na+dependent ouabain-sensitive ATPase (Parsons, 1978; Kushak, 1983). We shall follow the link of cell energetics with sodium transport. It turns out that mucosal oxygenation is a little more effective than serosal oxygenation (Metelsky, 1987). This corresponds to modern ideas about the dependence of the mechanism of transepithelial-transport power supply on oxidative phosphorylation and about the localization of such mechanism closer to a mucosal solution, i.e., in enterocytes. With serosal oxygenation, oxygen molecules diffusing to enterocytes

Chapter 8. Regulation of sodium transport in a small intestine€€€€€141

are partially spent by others (subepithelial) cells. This fact agrees with observations that bilateral oxygenation and mucosal oxygenation are equally effective. In reality, if mucosal oxygenation effectively covers the requirements of cells for oxygen, then the addition of serosal oxygenation cannot intensify these processes any more. There are studies describing the effects of increased partial pressure of oxygen on transport processes. According to (Ekkert, Ugolev, 1982), an increase in oxygen pressure by 6-20 cm of the water column considerably stimulates the active accumulation of glucose in a tissue because of the additional mechanism of respiration in the enterocyte. Because the connection between sodium and glucose transport is considered as indissoluble, the stimulating influence of oxygenation on the SCC under an increased pressure should be expected. However, this effect has not been observed (Metelsky, 1987). The creation of anoxia results in the immediate decrease of the basal SCC by 41 % (Tab. 22) (Metelsky, 1987). It is common knowledge that under anaerobic conditions (nitrogenation), the ATP content in cytosolе decreases (Diez-Sampedro et al., 2000). The SCC response amplitude on glucose under conditions of anoxia when the basal SCC has already reached its quasi-stationary level did not decrease but even tended to increase (Tab. 22) (Metelsky, 1987). These results are in a good agreement with data of other authors. In studies of the transmural potential in rat duodenal intestine, it has been found that after of a long anoxia, the transmural potential does not decrease by more than by 30-40 % (Okada et al., 1977). This fact is supported by observations that the mucosal permeability is increased under ischemia because of an increase in intercellular permeability (Drewe et al., 2001). Responses of the potential difference to glucose are also maintained. The basal SCC reduction through a rabbit small intestine under conditions of anoxia was observed in (Schultz, Zalusky, 1964a). In studies on the influence of anoxia on transport processes in a pig stomach mucosa, it has been found that the active transport of chloride and protons stops under those conditions, and there exists a significant component in active sodium transport which is stable to anaerobiosis (Forte, Machen, 1975). It is shown also (Metelsky, 1987) that the generation mechanism of the SCC response to glucose Table 22. Effect of anaerobiosis on basal and glucose (10 mM)-stimulated short circuit current across rat small intestine (Metelsky, 1987).

а

Experimental conditions

Basal SCC, µA/cm2

Oxigenation of serosal solution Anaerobiosis (nitrogenation of serosal and mucosal solution)

28.9 ± 3.8 (12)

SCC response on 10 mM glucose addition, µA/cm2 30.2 ± 6.7 (10)

16.65 ± 2.45a (12)

32.65 ± 5.75 (12)

p< 0.02

142€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

under conditions of anoxia does not change because it is inhibited by the usually used concentration of phlorizin. These facts suggest a possible stimulation of glucose transport by anoxia. It turns out that this effect has already been described (Faust, 1962). During a short (7 minutes) incubation of everted sacs from a rat jejunum under oxygen-free conditions, the diminution of glucose from the solution sharply increases. If the period of incubation is increased up to 30 minutes, the effect of additional accumulation disappears fully. The author considers the penetration of glucose into cells as an oxygen-independent process. The stage of an apparent accumulation of glucose in cells against its concentration gradient (for example, glucose dimerization) is only sensitive to oxygen. However, it is common knowledge that glucose in enterocytes is not only not polymerized but, on the contrary, is extremely quickly utilized under both aerobic and anaerobic conditions (Nicholls et al., 1983). Additional understanding on the influence of anoxia on sodium transport is provided by results obtained on a frog skin (Lapin et al., 1983). It has been found that under anaerobiosis, the unidirectional sodium transport through skin is inhibited, but the ionic composition of the cells does not change. The addition of ouabain during anoxia results in an increase in sodium concentration and reduction of the potassium concentration in cells. The conclusion was drown that the Na+-K+ pump responsible for ionic homeostasis can function because of glycolysis. When comparing the cited facts, it is possible to draw the assumption that under anaerobic conditions, the SCC responses to glucose are maintained because of intensive recycling of the first molecules that have entered an enterocyte. However, this contradicts the results of experiments with galactose, in which SCC responses are also maintained under anaerobiosis. It is common knowledge that galactose is barely metabolized in rat enterocytes (Barry et al., 1964).

8.3.4.2. Anaerobic metabolism The process capable to be an energy source under anaerobiosisе is glycolysis. Known inhibitors of glycolysis are monoiodide acetate and sodium fluoride. The latter inhibitor blocks the process of glycolysis during early stages. As expected, under conditions of anoxia, sodium fluoride inhibits both basal SCC and SCC responses to glucose. The effect of sodium fluoride is observed upon its addition in both mucosal and serosal solution, but in the former case, the effect of inhibition develops a little more quickly than in the latter one (Metelsky, 1987). This fact, apparently, supports the localization of the inhibitor-action target more closely to the preparation mucosal surface. With simultaneous addition of inhibitor in both solutions, the effect develops much more quickly than upon addition of sodium fluoride only in one of the solutions. When glycolysis inhibition occurs, many transport processes are inhibited. The decrease in

Chapter 8. Regulation of sodium transport in a small intestine€€€€€143

the basal SCC upon action of sodium fluoride is observed in (Schultz, Zalusky, 1964a). Barry, Dikstein, and Matthews (Barry et al., 1964) support the conclusion about the basic role of the pentose shunt and glycolysis in the maintenance of the responses of spontaneous transmural potential differences on rat small intestinal wall under conditions of anoxia. The decrease of potential difference responses on a bullfrog intestinal wall under the action of inhibitors of glycolysis (fluoride and iodideacetate) was observed in (Levin, 1966). The activity of SGLT1 in rat jejunum upon hypoxia is controlled at the post-translational level (Kles, Tappenden, 2002). This information is extremely important for the optimization of nutrient composition of mixes for enteral feeding. Thus, it may be inferred that the basal SCC significantly depends on the presence of oxygen. The SCC, stimulated by glucose, does not depend on the presence of oxygen and is maintained not because of the energy liberated upon utilization of the entered glucose but because of the process of glycolysis of some intracellular stores.

8.3.5. Neuro-endocrine control, theophillin, hormones It should be pointed out that the effects of substances from this group are very inconstant. These substances often affect different preparations in different ways. We failed to observe any significant and well-repeated effects of catecholamines and acetylcholine on basal and stimulated SCC with the use of a solution based on sodium sulphate (Metelsky, 1987). At the same time, significant changes of the SCC upon addition of acetylcholine and isadrine can be observed if chloride is the major anion in solution (Hirota, McKay, 2006). Moreover, even the small effects of acetylcholine and catecholamines, taking place at the beginning of our experiments with sulphate solution, eventually disappear (Metelsky, 1987). It is important to note that both basal SCC and the SCC stimulated by glucose remain at a high level even after effects of these compounds have stopped. Therefore, we conclude that small and labile effects of catecholamine and acetylcholine, observable sometimes at the beginning of experiments, are caused by their effects on the processes which are distinct from transcellular electrogenic sodium transport. The same can be said about serotonin. However, upon addition of serotonin in a mucosal solution, some temporary effects (Metelsky, 1987) are sometimes observed. Therefore, one is inclined to think that small temporary effects of serotonin also are caused by anion secretion (either chloride from subepithelial tissues or sulfate which does not penetrate through the brush border membrane as well as chloride); the receptors for serotonin are not found in rat small intestine epithelial cells (Gaginella et al., 1983). In epithelial cells, theophillin, increasing the contents of cyclic adenosine monophosphate in a cell, can both inhibit sodium transport coupled with chloride (Field, 1971; Frizzel et al., 1972) and stimulate secretion of anion (Field, 1971). As may be seen from data (Metelsky, 1987), the effect of theophillin on secretion develops, apparently,

144€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

more quickly than the inhibition of sodium absorption. Secreted anion in this case can be sulfate; indeed, it is found that theophillin in concentration of 10 mM increases the conductivity of mucosal border for anions (Naftalin, Simmons, 1979; Naftalin, Smith, 1984) and chloride (Schulzke et al., 1995). As this takes place, anions are transported in a direction opposite to sodium transport, causing an increase of the SCC. Nevertheless, it is more probable that the secreted anion is chloride, because upon a long exposition to theophillin or after its repeated additions, the effect disappears. This can be caused by an exhaustion of intracellular chloride (when using chlorideless solutions). It is not impossible that the disappearance of the theophillin effect is related with an exhaustion of intracellular cyclic adenosine monophosphate. It is common knowledge that catecholamines, acetylcholine, and theophillin act on transport processes through the mechanism regulating the level of cyclic adenosine monophosphate. On a rabbit small intestine, it is found that 5-10 mM theophillin results in a sharp increase in the SCC because of chloride secretion (Field et al., 1976). On the contrary, under the action of 50 μM of adrenaline, the SCC through an rabbit ileum declines quickly down to 0 (Field et al., 1976). In this case, adrenaline simultaneously stimulates electroneutral absorption of NaCl and increases bicarbonate secretion. We shall notice that the concentration of bicarbonate used by these authors is very high, up to 50 mM. Authors do not exclude the possibility that the effect of catecholamines is mediated by a cyclic adenosine monophosphate. In another study carried out on a rabbit small intestine, an increase in the SCC upon action of theophillin and permeating analogue of cyclic adenosine monophosphate were also observed; these effects were related to the secretion of anions (Field et al., 1963). When replacing chloride with sulfate, the SCC responses on cyclic adenosine monophosphate decreased by 75 %. Moreover, if the bicarbonate present in a solution (25 mM) is replaced with sulfate, cyclic adenosine monophosphate becomes inefficient, although SCC responses to glucose were observed. Upon simultaneous perfusion of a rat small intestine and liver, it has been found that the introduction of insulin in the portal vein results in a sharp increase in glucose and galactose absorption by means of the transporter SGLT1. The mechanism of this effect is complex. The signal is transmitted against the bloodstream from portal veins to the intestine by hepatoenteral cholinergic nerves. Cyclic adenosine monophosphate is an intracellular messenger. The agent implementing the link between muscarine receptors and intracellular concentration of cyclic adenosine monophosphate is prostaglandin Е2 (Stumpel et al., 2000). Noradrenaline and adrenaline in concentrations of 50 μM can reduce the SCC from 140 down to 40 μA/cm2 (Field, McColl, 1973). And it is again observed that upon removal of 25 mM bicarbonate from the solutions the SCC does not change upon action of catecholamine. In studies on the influence of permeating analogue of cyclic adenosine monophosphate on glucose and sodium transport through vesicles isolated from a brush border

Chapter 8. Regulation of sodium transport in a small intestine€€€€€145

of rat kidney, it was concluded that the Na+-dependent transporter does not depend on cyclic adenosine monophosphate (Kahn et al., 1984). Looking at the complete picture, we conclude that neuro-endocrine control of sodium transport, both basal and stimulated by glucose, is possibly absent (Metelsky, Dmitrieva, 1983; Metelsky, 1987). Effects of the studied substances at the beginning of experiments are caused, apparently, by their effects on anion transport (perhaps chloride). However, the effects can be observed with different experiments. The Na+dependent absorption of glutamin increases upon oral introduction of insulin-like growth factor to pigs (Alexander, Carey, 2002). Under in vivo conditions, the growth factor increases the absorption of Na+, Cl-, H2O, and glucose because of up-regulation of cotransporters of SGlT1 (Rongione et al., 2001). Glucagon 37, more so than glucagon 29, stimulates glucose absorption in the intestine. This effect is mediated through special receptors for glucagon 37 (Stumpel et al., 1998). With a combined use of glutamin and an inhibitor of prostaglandin synthesis (indomethacin) in animals with the experimental expressed atrophy of villi, the full stimulation of Na+ and Cl- absorption resulting from the increased expression of the Na+-dependent transporters of amino acids in crypts is observed. The transport system for neutral amino acids ASC is observed only in the apical membrane of crypts (Blikslager et al., 2001). In perfused rat intestine, prostaglandin Е2 sharply increases glucose and galactose absorption and does not affect fructose absorption (Scholtka et al., 1999). Prostaglandin Е2 acts directly on the enterocyte SGLT1, and cyclic adenosine monophosphate plays the role of a secondary messenger.

8.3.6. Influence of some other agents 8.3.6.1. Influence of ethylene diamine tetraacetate (EDTA) and calcium Data on various biological effects of calcium are rather extensive. It is difficult to cite processes or functions where calcium does not participate. One of principal causes for a general role of calcium in biological processes lies in the fact that it is part of extremely widespread animate chains: hormone (or other ligand)—cyclic adenosine monophosphate—calmodulin—calcium—protein kinase—phosphorylation—effect (Greengard, 1979; Donowitz, 1983; Powell, Fan, 1983; Galkin, 2007). In any case, the majority of the effects from calcium are connected with cyclic adenosine monophosphate. Calcium participates in the development of acetylcholine effects on NаСl absorption in the colon (Zimmerman et al., 1982) and in glucose transport (but not fructose) in rat small intestine (Manning et al., 1978). Calcium also plays a central role in the control of secretion in small intestine (Field, 1980) and in control of NаСl transport in a rabbit ileum (Donowitz, 1983, Powell, Fan, 1983).

146€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

Moreover, calcium plays an important role in tight junction opening and closing (Kottra, Fromter, 1983; Lacaz-Vieira, Marques, 2003). It is found that incubation in a calciumless medium containing ethylene diamine tetraacetate (EDTA) opens a tight junction in epithelial tissues (Sedar, Forte, 1964; Hays et al., 1965; Martinez-Palomo et al., 1980). The tight junction closure is a reaction of the external cells in which ions act as ligands between the negative fixed charges on membranes of the adjoining cells (Kottra, Fromter, 1983; Lacaz-Vieira, Marques, 2004). The picture of calcium action as modulator of cell tight junctions is complicated by the fact that tight junction opening is possible with an increase of intracellular calcium. This effect is caused by the change in anchoring protein of the cytoskeleton (Martinez-Palomo et al., 1980). The latter effect explains the increase in leakage through cell junctions under the action of adrenaline (Реcker, Taylor, 1975), acetylcholine (Bihler, Sawh, 1975) and theophillin (Mandel, 1975). For calcium transport in an enterocyte, there are several mechanisms localized both in apical and in basolateral membranes. Besides, calcium can passively go through tight junctions (Nellans, Kimbert, 1978). In enterocytes, calcium is transported through the brush border membrane by means of two mechanisms: Na+-dependent facilitated diffusion and simple diffusion (Van Os, Chijsen, 1983). The mechanism of simple diffusion of calcium along its gradient of electrochemical potential is also localized in the basolateral membrane. In the same membrane, there are two more mechanisms providing calcium excretion from a cell against the large electrochemical gradient: the calcium pump and the rheogenic (electrically nonequivalent) exchange of external sodium for internal calcium; the stoichiometry of the latter process is 3:1 (Van Os, Chijsen, 1983). Under certain conditions (regularly consuming inulin-type fructans), increased calcium absorption occurs principally in the human colon (Abrams et al., 2007). A change of the intracellular calcium concentration influences transport processes; when it increases, inhibition of electroneutral NaCl absorption and induction of electrogenic secretion of chloride (in mammals) occur (Powell, Fan, 1983; Rao, Field, 1983). On the contrary, it can be inferred that with a decrease of intracellular calcium, the NaCl absorption, apparently, increases. The removal of calcium from mucosal and serosal solutions will variously influence the intracellular calcium. Apparently, the removal of calcium from a mucosal solution does not affect the concentration of calcium in cytoplasm significantly. The enterocyte shape can be imagined as a cylinder with the ratio of its height to its diameter equal to ~6. Removal of calcium from a solution washing one of two end faces of the cylinder (serosal) will result in that calcium will passively leave cells through the apical membrane, perhaps, with high rate because of an increase in the area of the membrane (microvilli). However, most of the cytoplasm is turned aside a solution containing a normal amount of calcium which can get into the cell by simple diffusion. At the same time, with removal of calcium from a serosal solution, one would expect a reduction of its cell concentration. Through

Chapter 8. Regulation of sodium transport in a small intestine€€€€€147

one of the end faces of the cylinder, calcium will enter a cell, perhaps, with a high rate (effect of the increase in the area because of microvilli). However, calcium will leave the cylinder through lateral surfaces and the other end face by means of three mechanisms: exchange, pumping, and diffusion. As a result, its intracellular concentration should decrease. It is difficult to predict how the membrane potential on a basolateral membrane changes upon removal of serosal calcium because both the calcium pump and sodiumcalcium exchange are activated. However, it is believed that this will result in an acceleration of sodium input through the basolateral membrane and, hence, to an apparent decrease in efficiency of the sodium pump that should result in the reduction of the transcellular sodium transport rate. From such short theoretical consideration follows also that to achieve full removal of calcium from a cell by means of its removal from one of the solutions is impossible. To deprive a cell from calcium is difficult, even with bilateral removal of calcium. To reduce both the basal SCC and SCC response to glucose down to 0, it takes approximately 110 minutes of specimen perfusion with a calciumless solution containing EDTA (Metelsky, 1987). It is important to keep in mind that during the first minutes after bilateral removal of calcium, the preparation is can generate SCC responses to glucose with almost the normal amplitude, thus demonstrating the absence of any membrane damage up to this moment. The effect of bilateral removal of calcium is not caused by anions because it develops almost identically in solutions based on sodium chloride and sodium sulfate as major components. Therefore, we reach the conclusion that short-term removal of calcium from a mucosal solution will affect, most likely, the cell tight junction, and its removal from a serosal solution will affect the intracellular calcium concentration, resulting in a decrease of the transcellular sodium transport rate. It has long been known that cell tight junctions of a small intestine have a weak cationic selectivity because of the presence in the pores of fixed negative charges (Schultz et al., 1974). The fluid in lateral intercellular spaces is hypertonic in relation to washing solutions because water enters, along with sodium, through a tight junction into these spaces (Gebhardt, 1974; Stevens et al., 1982; Larsen, Mobjerg, 2006). From the existence of hypertonity of lateral intercellular space follows that the rate of water transport is limited by the permeability of such junctions. If we can imagine that water passes freely through intercellular spaces, its hypertonity would be impossible. With calcium removal from a mucosal solution, the tight junctions open (Kottra, Fromter, 1983), perhaps in such a manner that their selectivity remains the same or even increases because of the liberation of two negative charges on opposite surfaces of the one where calcium was bound. As a consequence, water fluxes and entrained sodium (solvent drug) through a tight junction should be increased, which should be observed as an increase in the SCC.

148€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

In all series of experiments with removal of mucosal calcium (Metelsky, 1987), basal and stimulated SCC increase. An additional increase in the stimulated SCC is just below that for the basal SCC because an increase in osmolarity of the mucosal solution (the addition of 10 mM glucose was not isotonic) and higher sodium concentration in intercellular space are likely. Owing to these effects, with tight junction opening grows not only the sodium flux from a mucosal solution into intercellular space but also the sodium flux in the opposite direction. In the presence of 0.2 mM EDTA, the effect is equal to 2.75 μA/cm2 or 16 %; when increasing the EDTA concentration up to 0.5 mM, this effect increases up to 7.65 μA/ cm2 (45 %); hence, the effect of EDTA is approximately directly proportional to its concentration (Tab. 23). With calcium removal from a serosal solution (based on sodium chloride as the major component) and addition of EDTA, the basal SCC and glucose-stimulated SCC increase (Metelsky, 1987). As this takes place, the effects of the modification of mucosal and serosal solutions during measurements of both basal and stimulated SCC are additive. Hence, the effect of modification of a serosal solution is not related to the effects in a cell tight junction. Apparently, such effect is the sum of the two effects discussed above: increase of the coupled electroneutral transport of sodium chloride and apparent reduction of the rate of the sodium pump functioning. The increase in rate of electroneutral sodium chloride absorption, as well as the increase in sodium transport Table 23. Effect of washing solutions modification – exclusion of calcium and addition of EDTA (ethylene diamine tetraacetate) – on basal and glucose (10 mM)-stimulated short circuit current across rat small intestine (Metelsky, 1987). Basal SCC Glucose-stimulated SCC Changes Changes Changes Changes under under under under Solu- EDTA SCC mucosal serosal mucosal serosal tion concen- Initial response, solution solution solution solution SCC, based tration, A, 2 modifica- modificamodifica- modificaµA/cm on mM µA/cm2 tion, tion, tion, tion, µA/cm2 µA/cm2 µA/cm2 µA/cm2 33.75 + 4.0 - 2.75 27.5 -1.65 ± 0.65 - 1.25 0 (2) (2) (2) (1) (3) (2) 16.85± 1.15 +2.75± 0.50 0 12.8± 1.0 (8) +1.40± 0.35 0 0.2 Na2SO4 (9) (9) (6) (8) (6) 17.0± 2.2 +7.65± 2.0 -2.2± 0.9 30.0± 4.15 +5.7± 1.75 +0.4± 2.4 0.5 (10) (7) (8) (7) (5) (6) 38.55± 8.2 7.55± 2.75 11.51±1.52 71.25± 17.35 5.85± 1.76 9.0± 3.05 NaCl 0.5 (7) (7) (6) (4) (3) (5)

Chapter 8. Regulation of sodium transport in a small intestine€€€€€149

upon glucose addition, results in an increase of the intercellular-fluid hypertonity (Fig. 1). The sodium pump increases its rate independently of how sodium ions have entered through the apical membrane (electroneutrally or electrogenically). An increase in hypertonity of the intercellular fluid results, as indicated above, in an increase in the SCC, perhaps, because of the additional sodium flux through the tight junction. For an experiment carried out in a solution based on sodium sulphate as the major component, in the presence of 0.5 mM EDTA there is only one small effect (13 %) with the modification of the serosal solution (Metelsky, 1987), related to the reduction of sodium transcellular transport. This effect is insignificant and disappears in the presence of glucose or when decreasing of the EDTA concentration down to 0.2 mM. Such a small (8 %) effect was observed in the absence of EDTA (Metelsky, 1987).

8.3.6.2. Carbodiimides Carbodiimides are blockers of carboxylic and phosphatic groups of proteins (Means, Feeney, 1971). The influence of a water-soluble carbodiimide (CMCD) on transport processes in an intestine has not been studied before. In Metelsky (1987, 1989b), a significant (61 %) inhibition of the basal SCC under the action of a 5 mM reagent (Tab. 24) was found. This is in agreement with data showing that the carboxylic group is localized at the entrance of amiloride-sensitive sodium channels (Zeiske, Lindemann, 1975). Addition of glucose on the background of the developed inhibiting effect of carbodiimide results in the development of a SCC response of the same value as that before the beginning of the exposition to the reagent. Addition of glucose after the second inefficient addition of carbodiimide also results in a response with a normal amplitude. From these observations, it may be inferred that the carboxylic group at the entry of the coupled transporter is either absent or more probably, inaccessible to water-soluble CMCD (Metelsky, 1987, 1989b). CMCD is the third factor (amiloride, anaerobiosis) variously influencing basal and stimulated SCC. Dicyclohexyl carbodiimide (DCCD) is the liposoluble carbodiimide acting through the hydrophobic phase of a membrane. The negative test in searching the blocking action of DCCD supports the absence of carboxylic groups essential for transport in the hydrophobic phase or their inaccessibility for DCCD (Metelsky, 1987).

Table 24. Effect of 5 mM CMCD (cyclo morpholinecarbodiimid) on basal short circuit current across rat small intestine (Metelsky, 1987). CMCD concentration, mM 0 5

Basal SCC, µA/cm2 13.0 ± 5.0 (3) 5.0 ± 2.5 (3)

Inhibition, % 0 61

150€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

8.3.6.3. Copper and p-hydroxymercuribenzoate The p-hydroxymercuribenzoate (PHMB) and p-chloromercuribenzoate (PCMB) are permeating reagents on sulfhydric groups. The PCMB inhibits glucose transport but increases sodium transport (Spooner, Edelman, 1976; Luger, Turnheim, 1981), modifying sulfhydric groups of the glucose transporter. It is shown (Metelsky, 1987, 2007a) that upon addition in a mucosal solution of p-hydroxymercuribenzoate in a concentration of 0.25-2.0 mM, the basal SCC slowly increases at first and then decreases slightly. In parallel, the SCC response amplitude on glucose decreases. Thus, 23 minutes after addition of a reagent, the magnitude of the responses decreases by 35-60 %, and 30-40 minutes after, SCC responses decrease down to zero, whereas the basal SCC remains at a relatively high level. Perhaps, in the presence of these reagents, sodium and glucose transports are uncoupled. It is common knowledge that copper is capable of reacting with sulfhydric groups also (Ferreira et al., 1979). The effect of addition in a mucosal solution of 4-10 mM copper sulfate agrees closely with that of p-hydroxymercuribenzoate but develops much more quickly; this might be due to the larger concentration of copper used. Here, the stimulation effect of the SCC by nutrients disappears for 11-13 minutes; it is important to note that the basal SCC does not changed under such conditions (Metelsky, 1987a, 1989b, 2007a). These data are consistent with the results of Hilger et al, 2008; Pirch et al, 2003, where it was shown that the hydrophilic face of the transporter is accessible to bulky sulfhydryl reagents (fluorescein-5-maleimide), indicating an aqueous pathway between the site of substrate binding and the cytoplasm.

8.3.6.4. Guanidine and urea Many derivatives of guanidine have stimulating or inhibiting effects on active sodium transport in tight epithelia (Cuthbert, 1976, Metelsky, 1989b). The guanidine groups are available in molecules of widely used sodium channel blockers of exited and unexcitable membranes, such as tetrodotoxin, amiloride, and triamterene. Moreover, upon action of guanidine, the active sodium transport through tight epithelia can be both stimulated and inhibited, and the mechanism of stimulating effect of guanidine remains obscure (Cuthbert, 1976). In some studies (Metelsky, 1987), 5 mM of guanidine causes an increase in both the basal SCC and, to a greater extent, the glucose-stimulated SCC. In many respects, the urea molecule is similar to the guanidine molecule, but it does not have quaternary nitrogen. Because urea does not influence the SCC, we reach the conclusion that the quaternary nitrogen in guanidine plays an important role. The stimulating effect of guanidine on basal sodium transport suggests an organization of brush border sodium channels similar to that of tight epithelia (Metelsky, 1984a, 2007a).

Chapter 8. Regulation of sodium transport in a small intestine€€€€€151

8.3.6.5. Papaverine – the first chemical uncoupler of the coupled sodium and glucose transport in the rat small intestine? It has long been known that the coupled transport of ions and nutrients, taking place in the small intestine of any animals, can be studied by means of two equivalent approaches: from the viewpoint of biochemistry and from the viewpoint of biophysics, in particular in electrophysiology. In biochemical studies, the absorption of nutrients and the impact of the corresponding ions on this process are determined, and in electrophysiologal studies, the currents generated during the motion of ions through membranes are determined, as well as the impact of the corresponding nutrients on this process. The biochemistry and electrophysiology (biophysics) of the coupled transport of nutrients are different areas of knowledge, which seldom directly interact with each other. As already mentioned earlier, A.M. Ugolev was the first to begin carrying out a regular comparison of data from the two mentioned areas of study in 1979 within one laboratory (Laboratories of Nutrition Physiology, Institute of Physiology, USSR Academy of Sciences). In this laboratory, during comparisons of biochemical and electrophysiological data concerning Na+-dependent glucose absorption, the temperature dissociation of the coupled transport was discovered in 1983 (Metelsky et al., 1983). It has been proposed that, together with physical factors of dissociation of the coupled transport of ions and nutrients (temperature), a chemical uncoupler of this process can exist. It appears that the alkaloid of opium (papaverine, used basically as spasmolytic) has the same action as that of the temperature on the coupled sodium and glucose transport. Papaverine does not affect the glucose-dependent sodium transport and, at the same time, completely blocks glucose absorption in rat small intestine (Metelsky, 2001). So, owing to a line of investigation developed by A.M. Ugolev (the regular comparison of data on biochemistry and biophysics of absorption), the first uncoupler of the coupled sodium transport and glucose might have been discovered.

8.3.7. Effects of some other drugs It is appropriate to illustrate briefly the applicability of the SCC technique to pharmacological studies, without any analysis in each concrete case. Drugs of different classes have been tested: activator CNS, ganglionic blocker, analgetic, stimulant of uterus smooth muscles contraction, barbiturate, vasodilator, and spasmolytics (Metelsky, 1987, 1989b). It was found that strychnine, arfonad (trimetaphan camsilat), dimedrol, isoptin, and papaverine did not affect the basal nor the stimulated SCC. Drotaverine and nembutal did not affect the basal SCC. Benzohexamethonium and dihydroergotoxin slightly reversibly reduced the basal SCC, and benzohexamethonium, drotaverine, and nembutal slowly and irreversibly reduced the SCC stimulated by glucose.

152€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

Cytochalasin Е inhibits the functioning of Na+-glucose cotransporters (SGLT1) by an indirect manner because of the cell cytoskeleton destruction. Moreover, at the same time, the transport of amino acid–phenylalanine is also inhibited. Cytochalasin, apparently, modifies the structure of cytoskeleton proteins (Diez-Sampedro et al., 2000). At least, some antibiotics (erythromycin) are capable to inhibit indirectly or even directly (Bastidas et al., 1992) the Na+-dependent transport of some nutrients (L-threonine) (Navarro et al., 1992). Therefore, one can reach the following conclusions. First, the effects of drugs on the basal and stimulated SCC are independent; the substance can affect one of the two components of the SCC (may be irreversibly) and not the other. Secondly, the stimulated SCC is more sensitive to action of drugs than the basal one. Thirdly, it can be assumed that if benzohexamethonium, drotaverine, or nembutal had to be taken per os, we would recommend to take them on an empty stomach (in this case, they do not influence absorption or affects it reversibly) but not while or after eating (in this case, they irreversibly inhibit sodium absorption stimulated by nutrients).

8.4. Final remarks The control of transport processes in intestines both under normal and pathologic conditions is very complex. This is an insufficiently studied process in which local neurons of the intestine and neurons of CNS (through neurotransmitters and local action of intestinal hormones) participate. For example, the most important effectors of secretion are submucosal neurons, as well as serotonin, vasoactive intestinal polypeptide, acetylcholine, substance Р, and neurotensin (Holtug et al., 1996) and also histamine and prostaglandin Е2 (Larsen et al., 2001). It is common knowledge that in rabbit intestines, there is an electrogenic sodium absorption due to the special ion channels, independent of both nutrients and other ions (Sellin et al., 1989). The analogue of amiloride (phenamil) inhibits the basal SCC, and both the mucosal-serosal and total fluxes of sodium. At the same time, the functioning of other transport systems does not changed. Phenamil does not prevent the SCC responses to glucose. Hence, this analogue of amiloride inhibits the basal sodium transport and does not affect the sodium transport coupled with nutrients (Sellin et al., 1989). For the first time, a study of the control of the two SCC components taken separately has revealed that sodium transport, both coupled and not coupled, may be regulated by various factors. The most important conclusion of this analysis, apparently, lies in the fact that these two transport components are controlled by different mechanisms (Metelsky, 1989b). The basal sodium transport is stimulated by guanidine and (for the first 25 minutes) by p-hydroxymercuribenzoate and inhibited by amiloride, ouabain, sodium fluoride, and anaerobiosis (Tab. 25).

Chapter 8. Regulation of sodium transport in a small intestine€€€€€153

Table 25. Characteristics of basal and glucose (10 mM)-stimulated short circuit current across rat small intestine (Metelsky, 1987). SCC component Basal

Glucose-stimulated

Stimulants Guanidine, PHMB* (p-hydroymercuri benzoat) Amiloride

Inhibitors Anaerobiosis, Sodium fluoride, Amiloride, Ouabain, cyclo morpholinecarbodiimid Sodium fluoride*, Phlorizin, Nembutal*, NO-SPA*, PHMB*(p-hydroymercuri benzoat), cuprous sulfate*, Ouabain

* - irreversible action.

Sodium transport stimulated by glucose is inhibited by phlorizin, ouabain, sodium fluoride, copper, and p-hydroxymercuribenzoate. Na+/glucose cotransporter 1 (SGLT1) protein levels in brush border membrane vesicles were lower for lipopolysaccharide (an endotoxin causing sepsis)-treated animals than in control animals (Amador et al., 2007). Differences in the pharmacology of the two types of sodium transporters are revealed as well from experiments with a pharmaceutical substance. Pharmaceutical substances can influence the two types of electrogenic sodium transports in enterocytes in different ways. Amphiphilic amines (lidocaine, procaine, and tolicaine) considerably inhibit the active absorption of methyl a-D-glucoside and L-leucine (Na+-dependent transport) but do not affect the absorption of D-fructose (facilitated diffusion) and 2-deoxy-D-glucose (a passive diffusion). Inhibition occurs because of changes in Kt, but at the same time, Аmax does not change significantly (Strugala et al., 2000). It turns out that the neuro-endocrine control of both types of sodium transporters is absent in vitro. Sodium channels of exited membranes are poorly subjected to neuro-endocrine control (Khodorov, 1975); therefore, with respect to a poor control, sodium channels of exited and epithelial membranes are rather similar. The basal sodium transport is considerably suppressed under conditions of anaerobiosis, whereas the coupled transport does not depend on anaerobiosis; it is energized by glycolysis. Perhaps, during glycolysis, exogenous glucose, which is transported, is not utilized but rather endogenous glucose, which was in the cell earlier in the form of stores. Perhaps, a short-term preservation of active glucose absorption upon anoxia has a physiologic significance during blood supply disturbance of villi followed by the beginning of functioning of the arteriovenous shunt. It is important to keep in mind that by means of the SCC technique, one can test not only activity of transcellular processes but also the state of cell tight junctions. We have reached the same conclusion while studying osmotic phenomena.

154€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

Chapter 9. Molecular mechanisms of the coupled transport of glucose€€€€€155

Chapter 9. Molecular mechanisms of the coupled transport of glucose In GIT, carbohydrates are degraded into simple sugars or monosaccharides (e.g., glucose) and disaccharides (e.g. maltose). Pancreatic amylase breaks down carbohydrates into oligosaccharides. Brush border enzymes take over further processing of the resulting molecules. The most important brush border enzymes are dextrinase and glucoamylase which further break down oligosaccharides. Other brush border enzymes are maltase, sucrase, and lactase. Before discussing molecular models of coupled transport, it is appropriate briefly to familiarize oneself with molecular models of facilitated glucose transport through a membrane of erythrocytes. It is necessary, on one hand, because the given area, as a rule, is not familiar to researchers studying active transport of nutrients in epithelial tissues. On the other hand, in enterocyte systems for both active and facilitated transport, glucose is present. It is not inconceivable that systems of facilitated glucose transport in a basolateral membrane of an enterocyte are very similar to those in a plasmatic membrane of an erythrocyte (Brot-Laroche, Alvarado, 1983), which would be evidence in favor of Ugolev’s (1985) theory of functional blocks.

9.1. Mechanisms of facilitated glucose transport through plasmatic membranes The active transport of substances through plasmatic membranes has much in common with facilitated transport. Both processes are characterized by the presence of a high concentration of a substrate, both have stereoselectivity, both are inhibited by a low concentration of specific inhibitors, and both have high sensitivity to рН and temperature. The main distinction of an active transport system consists in its coupling with an energy source, enabling the transport of substances against its own gradient of electrochemical potential. Thus, having any model of facilitated glucose transport and adding to it Na+-dependent coupling with any energy source, we get the model

156€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

of coupling of sodium and glucose transport. Later it will be apparent that the basic models of coupling have been developed just by this means. First we will consider models of facilitated glucose diffusion that were developed before the models of sugar coupled transport. The first theoretically developed class of models could be characterized as having a motionless barrier of permeability and a mobile specific center for glucose binding. The first model of facilitated transport was a model (Widdas, 1952) in which the membrane of a placenta cell has a mobile carrier capable of selectively absorbing glucose on the external border of the membrane/water interface. The process of a glucose molecule adsorption proceeds equally effectively from both sides of the membrane, and the mobile carrier is capable of moving across a membrane due to fluctuations. The speed at which a carrier moves does not depend on whether it is loaded with glucose. The author has described the process of glucose binding with a carrier by Langmuir adsorption isotherm dependence (Markin, Chizmadgev, 1974), which in the areas of enzyme kinetics and transport processes is known as Michaelis-Menten kinetics. A similar model based on experiments studying glucose diffusion through a membrane of erythrocytes has been offered (LeFevre, LeFevre, 1952; LeFevre, 1975), in which it has been proposed that the membranous component having only one binding site for a substrate is capable of moving through a membrane linearly, or being reoriented in it (i.e., rotating). As a result of rotation of the carrier, having a diameter approximately equal to the thickness of a membrane, by 180o the binding site of glucose moves from one side of a membrane to another. Hypotheses of mobile carriers explain such experimental facts as the presence of a net flux of a substrate along a gradient of its concentration, the saturation effect of transport rate on a high concentration of a substrate, and competition between substrates. These hypotheses were also able to predict some facts found out later. Another class of models postulates a motionless binding site and a mobile gate in a pore or channel. There have been various updates to this proposed mechanism, in particular the studies by Holman (1980), Patlak (1956; 1957), and Vidaver (1966). In some models, there are small displacements of the binding site and the limiting barrier localized in the channel (Singer, 1974; Deves, Krupka, 1978; Klingenberg, 1981). The latter models propose two physically different centers localized in separate membrane proteins, but as a result of conformational relationships between proteins, at any moment within a water phase only one center is in contact. Unlike the above models where the binding site of glucose alternately is emerged or on one or other side of a membrane, there are models in which at any moment both sites on the opposite sides of a membrane are accessible. The model of “hemiport”€(Stein, 1969) postulates the presence of two types of components with a binding site for a substrate. Such components can be either external or on the internal side of a membrane and can move independently from each other in the plane of a membrane. The component outside interacts randomly with the component inside, forming a

Chapter 9. Molecular mechanisms of the coupled transport of glucose€€€€€157

temporary complex. During these moments of interaction, the bound molecule of a substrate can pass from one component to another, thus crossing the membrane. According to the model proposed by Lieb and Stein (1970), the transporter consists of a tetramere of cooperating subunits penetrating a membrane, each of them having a substrate binding center. On one pair of subunits (exterior) the centers face the outside. On another pair (in the middle) both centers face the center of membranes. Transport occurs on coordinated conformational transitions during which orientations of the centers directed outside and inside a membrane reverse their orientation. A similar model, “internal hemiport,” is offered by LeFevre (1975). This model postulates full independence of conformational transitions of monomers. The model proposed by Naftalin (1970) is similar to the model of single-file diffusion which was put forward by Heckmann (1972) and Markin & Chizmadgev (1974), but postulates a double line of centers binding a substrate inside of a single pore. Though these mechanisms are distinguished in detail, they share a common property: the existence of a few substrate binding centers penetrating a membrane from one side up to another. We emphasize that work of some molecular mechanisms cursorily examined above is not easy to visualize. Nevertheless, there are experimental data in favor of each of the considered models, and all of them explain well features of facilitated transport of substances through plasmatic membranes of erythrocytes, hepatocytes, cross-striated muscle fibers, cells of fatty and nerve tissue, and blood-brain barriers (LeFevre, 1975). In light of recent data and concepts, one can draw the following conclusions (Carruthers et al., 2009; Wright et al., 2011). The facilitative glucose transporters belong to the Major Facilitator Superfamily (MFS) of transporter proteins. Crystal structures of three members of this family have been obtained. These are the Lactose Permease (Abramson et al., 2003), Glycerol-3-phosphate (Huang et al., 2003), and Oxalate transporters (Hirai et al., 2003). From these structures, homology structural and functional models for the GLUTs have been proposed. The recent advances in understanding of GLUT function has been reviewed by Carruthers et al. (Carruthers et al., 2009). GLUT1 is the first equilibrative glucose transporter (facilitated duffusion) to be identified, purified, and cloned. GLUT1 is a polytopic, membrane-spanning protein that is one of 13 members of the human equilibrative glucose transport protein family (including GLUT2). This transporter presents a water-filled channel, whereas human GLUT1 is a strongly hydrothobic protein comprising 492 amino acids forming 12 hydrophobic, membrane-spanning α-helices.€ Two fundamently different models have been suggested for protein- mediated sugar transport, the simple carrier and fixed-site transporter models. The classic model for glucose transport [the simple carrier, the alternating conformer carrier, the mobile carrier, or the iso-uni-uni model for glucose transport (Lieb, Stein, 1970; Widdas, 1952)] proposes that the transporter exposes one sugar-binding site (an import site or exit one) at any instant and that conversion of the import to the

158€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

export site through a substrat-binding-induced conformational change is the central catalytic step mediating substrate translocation (Carruthers et al., 2009). Alternatively, the fixed-site or two-site transport mechanism describes a transporter that presents sugar import and sugar export sites simultaneously. Sugars bind at the site and then dissociate into a central cavity that is sufficiently large to allow sugars to bypass each other en route to the trans-binding site (Carruthers et al., 2009). The authors (Carruthers et al., 2009) conclude that some experimental data (GLUT1 ligand binding) are compatible with the fixed-site transport mechanism, although simple-carrier behavior is observed under special circumstances. The development of models of facilitated transport of sugars through plasmatic membranes has great importance for many areas of biophysics, biochemistry and physiology, as it stimulated the study of transport processes in cells and on cellular layers on the molecular level. Ideas for mechanisms of active transport originated from molecular models of facilitated diffusion. So, models of the functioning of active transport of potassium and sodium are based on ideas of carriers and channels (Levitt, 1980). It is apparent that the ideas developed for facilitated transport of glucose should affect the development of theoretical concepts in the field of molecular mechanisms of the coupled transport of nutrients.

9.2. Molecular mechanisms (models) of Na+-dependent transport The deep similarities between the processes of active and facilitated transport of substances were mentioned above. Furthermore, it is known that the most studied system of facilitated transport for nonelectrolytes is that for transport of monosaccharides in erythrocytes. More than a dozen various models of this process have been developed (Widdas, 1954; Patlak, 1957; LeFevre, 1975), and the basic models—carriers and channels—were put forward as hypotheses and were developed in detail in the mid-1900s (Widdas, 1954; Patlak, 1957). Clearly, the first molecular model of coupled glucose and sodium transport (Bihler et al., 1962; Crane, 1965) became the model of a mobile carrier of glucose supplied by a sodium “energizer.” According to this model, already mentioned earlier (Fig. 2), in an apical membrane there is the mobile carrier (like valinomicin (Page & DiCera, 2006)), which exhibits binding sites for glucose and sodium on an external or internal surface of a membrane. Upon the coupled glucose transport the ternary complex is formed: a carrier, sodium, and glucose. According to this hypothesis, the affinity of a carrier to glucose increases with sodium concentration in a solution. The model of a Na+-dependent glucose carrier (Crane, 1965) is sometimes designated as a hypothesis of sodium gradient, but it should be pointed out that such a term is not absolutely accurate. The “sodium gradient” is the indication only of an energy source for active transport of a nutrient, which in cells is only two—macroergic phosphates and transmembrane electrochemical gradients of

Chapter 9. Molecular mechanisms of the coupled transport of glucose€€€€€159

ions—but not of the concrete molecular mechanism. With the framework of any energizer, ionic or macroergic, there may exist a few mechanisms of coupling—a rotating carrier, pore, etc. (see 9.1)—but not the only model of a mobile carrier. The model of mobile carriers stimulated a wave of experimental studies and has attracted the attention of researchers for some decades. It has predicted such fundamental facts as the stimulating influence of nutrients (for example, glucose) on sodium transport and a causal relationship of value of the maximal gradient of glucose concentration between a cell and environment with a gradient of electrochemical potential of sodium. Besides, the achievements in studying molecular mechanisms of coupled transport in small intestines stimulated successful research and study of similar systems in other animals. In the light of what we have stated above, it is quite natural that the model of the common channel for glucose and sodium put forward in 1980 became the next molecular model of the coupled transporter (Hopfer, Groseclose, 1980) (Fig. 2). It is curious that here authors started with “enzymatic” inferences, but it is evident that the model of a pore for facilitated glucose transport with fluctuating gates has served as a prototype of such a model (Patlak, 1957; Vidaver, 1966). Hopfer & Groseclose (1980) called attention to their experiments (carried out on the isolated brush-border membranes of a small intestine and kidneys of a rabbit), in which the stage of translocation of glucose and sodium through a membrane occurred much more quickly than that of sodium dissociation. They reached the conclusion that translocation of glucose and sodium across a membrane is likely carried out by a channel, but not by a carrier. The channel transporter one can envision as a transverse pore in a membrane which is constantly closed. Once the sodium from outside is adsorbed on the gate mechanism of the transporter, adsorption on it of glucose molecules is possible. Then “the barrier of permeability” fluctuates in space in such a manner that glucose molecules and sodium appear from the inside from “a barrier of permeability” (Fig. 2). It is vital to note that the substrate first bound with the transporter must be the first to dissociate in intracellular space. The channel transporter explains the following features of the studied system: (a) linear dependence of rate of glucose transport on sodium concentration that points to a stoichiometry of glucose and sodium transport equal to 1:1; (b) independence of a seeming affinity of the transporter for glucose on sodium concentration; (c) a changing parity of glucose and sodium fluxes which, depending on the ratio of concentration of these two substrates, can be both more and less than 1; (d) a stimulation and an inhibition of transport of one substrate with concentration of another one. The model of the common channel transporter has undergone further developments in study (Kessler, Semenza, 1983). The additional argument in favor of that type of transporter proves to be its stable chemical and functional asymmetry. The Na+-dependent binding of phlorizin with the transporter (see Chapter 8) strongly depends on a potential difference on a membrane, pointing to the presence of a negative charge on gate mechanism of the transporter. The probability of translocation of

160€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

a partially occupied transporter is negligible. The role of the gate mechanism (or “a barrier of permeability”) is carried out by the ionized carboxyl group. In the structure of the transporter is a polypeptide chain with a molecular weight of 72 KDа; an aminoacid group participates in the binding of glucose, and carboxyl and phenyl groups participate in the binding of sodium. Upon considering the available data, it may appear that the number of models of coupling are much more than the two major ones considered above. In addition to the models mentioned earlier, there are those by Schultz & Curran (1970), Alvarado & Robinson (1979), Parent et al., (Parent et al., 1992b),€ Loo et al. (Loo et al., 2006), Csaky (1963b), Kimmich (1970) and Kimmich & Randles (1975). However, the first four of these are kinetic versions of the model of a mobile carrier (Crane, 1962), differing from that only in detail. Csaky (1963b), Kimmich (1970), and Kimmich & Randles (1975) present a model in which active glucose transport through an apical membrane occurs as a result of energy sources alternative to a gradient of electrochemical potential of sodium, or due to water fluxes, or due to special Na+-K+ATPase. However, as one of the authors has actually disputed this hypothesis (Kimmich, 1981), we shall not consider it. Both the model of common carriers and the model of a common channel imply serial binding and releasing of ligands for sodium and glucose and transfer of both ligands along the same pathway. From this viewpoint, all indicated earlier models can be characterized as common serial transporters (Metelsky, 1987, 2007a).

9.3. The predictions of serial transporter models (the common carrier and channel) A common transporter model predicts that by stopping any way of transport of one substrate (for example, glucose), transport of another substrate (sodium) should also stop. As this occurs, three cases are possible. (1) The stage of glucose adsorption on the transporter is inhibited; hence, the formation of a ternary complex in all models does not occur and therefore transition to a following stage—the translocation of a complex through a membrane—proves to be impossible. (2) Glucose on the transporter is adsorbed, but the stage of translocation through a membrane is sharply inhibited. Formation of a ternary complex is possible, but as its translocation across a membrane is complicated, glucose on the internal side of a membrane does not appear. (3) Stages of adsorption and translocation are possible, but glucose does not dissociate in intracellular space. In one-channel models, transition of the transporter in an initial state and also the following cycles becomes impossible. For carrier models it means that the carrier will fulfill one full cycle; in so doing, sodium should be transferred into intracellular space. Glucose will travel on a carrier to an internal surface of a membrane and back. In this case, the stage of dissociation of glucose in an external solution also becomes impossible or will be slowed. In actuality, it is difficult to imagine conditions

Chapter 9. Molecular mechanisms of the coupled transport of glucose€€€€€161

when dissociation of glucose from the transporter on one side of a membrane became impossible, and on another one to be unchanged. But the occurrence of such circulation of glucose in a membrane is unequivocally discriminated in the experiment. In this case the stimulating glucose effect will be maintained indefinitely (or, at least, for a long time) after its removal from a mucosal solution. Any attempts to observe such an effect have not met with success. Thus, irrespective of the reason, the termination of the coupled glucose transport should result in the termination of the coupled sodium transport. Such inference of common serial transporter models is supported by experiments using a specific inhibitor of the coupled glucose transport—phlorizin. According to data (Metelsky, 1987, 2005c), in the presence of 0.1 mM of phlorizin in a mucosal solution, active glucose transport stops almost completely and the stimulating glucose effect on the active sodium transport measured by the SCC technique is offset.

9.4. The parallel multipathway cotransporter model Apart from the two basic models mentioned above (carrier and channel), one more model of a coupled cotransporter for Na+ and glucose has been developed (Metelsky et al., 1983; Ugolev, Metelsky, 1984b, 1990; Metelsky, 1985, 1990b, 1992, 2007a; Metelsky, Ugolev, 1988; Ugolev, Kuzmina, 1993)—the parallel two-pathway cotransporter model (Fig. 16).€ Principles of the work of that transporter, in a membrane of an enterocyte

Fig. 16. Theoretical model for a two-channel co-transporter for Na+ and glucose with a gate mechanism.

162€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

of two parallel cooperating pathways (for Na+ and glucose) and the gate protein located on membrane surfaces exist. This protein binds glucose on an input in the transport system (maybe after the binding of Na+ with the corresponding site of the cotransporter) that results in activation of a sodium pathway. In a certain stage of movement of Na+ on its transporter, the glucose transporter on which the gate protein of the glucose molecule was originally fixed is activated. Through this glucose transporter glucose is transported. Previously, this model was called the parallel multi-channel model (Metelsky, 1987). But when taking into account that the operation mechanism of coupled cotransporters is still unknown and given the results of the study (Loo et al, 1993) where it was shown that the turnover of Na+/glucose cotransporters is 57/sec (at least those that express in oocytes), it would be more correct to call it the parallel multi-pathway cotransporter model. Indeed, the only new rational kernel of this concept is the adoption of the parallel paths of transport of sodium and glucose. Therefore, the term multi-channel model was replaced with a more neutral term multi-pathway model (Metelsky, 2009a). This model is based on the study of temperature dependences of intestinal transport processes.

9.4.1. The influence of temperature on sodium and glucose transport There are two fundamentally different specific mechanism of sodium transport in enterocytes, amiloride-dependent and nutrient-dependent ones (see 8.4. and Tab. 25), which may be measured as the basal SCC and the nutrient-dependent SCC, respectively. The basal SCC is observed in all ranges of the studied temperatures; it is characterized by a rather low value of Q10 = 1.76 ± 0.12 (Tab. 26). These results correspond closely to the data obtained from a rabbit small intestine, with Q 10 = 1.6 (Curran et al., 1967), and from a frog skin (Lagerapetz, Skyttl, 1979), with Q 10 = 1.76-1.84, through which the SCC is almost completely caused by active sodium transport through amiloridesensitive channels. Low Q 10, apparently, is the common property of all types of sodium channels; it is common knowledge that Q 10 for sodium channels in exited membranes is also low: 1.3–1.8 (Khodorov, 1975). Table 26. Temperature coefficients (Q10) for basal and glucose (10 mM)-stimulated short circuit current (Metelsky, 1987). SCC component Basal SCC (without nutrients) SCC response on 10 mM glucose addition а

p < 0.001

Q10 1.76 ± 0.12 (19) 2.51 ± 0.20а (17)

Chapter 9. Molecular mechanisms of the coupled transport of glucose€€€€€163

SCC responses to glucose also are observed in the temperature range of 7–38 оС, but Q 10 for this effect is considerably above the Q 10 for the basal SCC and is equal to 2.5 (Metelsky, 1987a). The established significant distinctions in Q 10 for basal and stimulated SCC are not unexpected, since these components of the SCC are caused by sodium transport through two various systems (see Chapter 8) (Metelsky, 1987a, 1989b, 2007a). It is common knowledge that Q 10 for the stimulating effect of nutrients is rather high. So, Q 10 for the stimulating glucose effect on the SCC through a preparation of a rabbit small intestine, washed by a solution based on sodium chloride as a major component, is equal to€ 3-7 (Parent, Wright, 1993), 2.0 (Smith, 1966) or 4.9 (Schultz, Zalusky, 1964b). In later studies the distinctions in Q 10 for processes of not-induced sodium transport and for sodium transport stimulated by a nutrient (alanine or glucose) (Curran et al., 1967) have been found. The difference between values of Q 10 for the SСC response to glucose in Metelsky (1987) and in Schultz and Zalusky (1964b) can be caused by the fact that in Schultz and Zalusky (1964b) the addition of glucose was made non-isotonically. The Q 10 for the osmotic response should be insignificant, and therefore its contribution at low temperatures should be increased. In actuality, if glucose is added non-isotonically the SCC response to glucose “disappears,” already at 10–16 °С (Metelsky, 1987). Thus, temperature very strongly influences the process of an induction of sodium transport by glucose (SCC value response) through the preparation of an intestine which is being mounted in the experimental chamber. However, relative rate of development of effect (a) (see Chapter 6) is kept constant in a wide range of temperatures—(2-З) *10-2 s-1. Before chambering, the small intestine inside the animal had a sufficiently high temperature; then, during preparation following any traditional techniques, its specimen undergoes deep cooling (Danilevskaya, 1987). The high sensitivity of sodium transport, stimulated by glucose, to temperature allows one to assume that procedure of cooling of a specimen during its preparation is not indifferent for it. It was found (Metelsky, 1987) that short incubation of a preparation in warm or in cold (Tab. 27) essentially influences the value of stimulating glucose effect on the SCC.

Table 27. Effect of excluding the stage of intestine specimen preparation, carried out usually in cold, on glucose stimulated short circuit current (Metelsky, 1987). Preincubation conditions In warmth In cold а

p < 0.05. For details, see text.

Electric resistance, Ohm*cm2 11.30 ± 0.78 (8) 13.62 ± 1.34 (8)

SCC response on 10 mM glucose addition, µA/cm2 51.5 ± 12.0 (9) 24.5 ± 4.5 а (11)

164€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

It is vital to note that change of a stimulated SCC more than twice is not caused by essential changes of resistance of a preparation. It points to the fact that at low temperatures the properties of a preparation as a whole do not deteriorate; the mechanism of influence of low temperatures is finer. These results are unexpected enough and, perhaps, at a later time it will be necessary to reconsider critically some concepts concerning work with living tissues. Using a procedure of concentration dependences in a wide temperature range (10, 16, 26 and 38 °С) (Metelsky, 1987) a relative persistence of a transport constant Kt was revealed; it changes in narrow limits of 3.0–4.1 mM (Tab. 28). The conclusion about persistence of Kt with temperature has been supported by an independent technique, by determination of Kt from the single response method (Metelsky, 1987b). Though the SCC response to glucose depends very strongly on temperature, relative independence of Kt in this case is surprising. From this viewpoint the glucose-dependent transporter is similar to the majority of enzymes. It is common knowledge that the maximal rate of enzymatic reactions changes with temperature and the Michaelis constant for its remains constant. Perhaps the persistence of Kt with temperature points to the fact that Kt characterizes the process of glucose adsorption on certain sites of the transporter which should depend on temperature only slightly. Unlike Kt, the maximal SCC response to glucose depends on temperature very strongly, and in that the deep analogy between enzymatic and transport processes is manifested too. As indicated above, with temperature decreases in enzymatic processes just the maximal rate of reaction decreases, but not Kt. At all studied temperatures intensity of passive glucose transport in enterocytes is rather close (Thomson, Dietshy, 1980; Metelsky et al., 1983; Metelsky, 1987a), and Q10 for this process proves to be equal to 1.2. Unlike passive transport, active glucose transport stops at 16 °С. Unfortunately, data on the temperature dependence of values of stimulating glucose effect on the SCC or on sodium transport through a small intestine are insufficient. Usually the magnitudes of Q 10 cited for such process invariably prove to be very high, for example, nearly 5 (Curran et al., 1967) or€ 3-7 (Parent, Wright, 1993) . Apparently, calculation of Q 10 for active glucose transport in a wide range of temperatures is incorrect, as: 1) temperature dependence is not described by Table 28. Kinetic parameters of glucose-stimulated short circuit current determined by the method of double reciprocal coordinates (Metelsky, 1987). Temperature, °С

Transport constant, Kt, mM

37 26 16 10

4.1 4.0 3.4 3.0

Maximal stimulating effect of glucose on SCC, Amax, µA/cm2 90.0 34.7 7.5 1.3

Chapter 9. Molecular mechanisms of the coupled transport of glucose€€€€€165

an exponent; 2) active glucose transport stops at 16 °С. Depending on the range of temperatures used for measurements, one can obtain various values for Q 10. So, it is evident that calculation of Q 10 in the temperature range 16–26°С will give a Q10 value as large as is wished. The comparison of temperature dependences of active transport and stimulating glucose effect has been carried out more carefully (Metelsky et al., 1983; Ugolev, Metelsky, 1984b, 1990; Metelsky, 1985, 1990b, 1992; Metelsky, Ugolev, 1988) (Fig. 17). Curves 1 and 2 (Fig. 17) at high temperatures correspond to those between glucose and sodium transport of a rigid stoichiometry. The presence of coupling between glucose

A, μA/cm2

[Glucose], mM

60

24

40

16

20

8 1 2

0

10

20

30

40

T0С

Fig. 17. Temperature-dependences of glucose (10 mM)-stimulating short circuit current across a segment of rat small intestine (curve 1) and active glucose accumulation in the intestinal tissue (curve 2). The coordinates scale was chosen in such a way so that both curves coincide in the high-temperatures area.

166€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

and sodium transport could be inferred by a course of these curves. Hence, contrary to predictions (see 9.3) of modern common transporter models of coupled transport (common carriers and channels) with a temperature decrease to 16°С, conditions are created in which active glucose transport disappears completely, but the inducing glucose effect on active sodium transport remains. Hence, active sodium transport and active glucose transport at temperatures below 16°С are dissociated. As glucose at low temperatures does not permeate enterocytes, it stimulates sodium transport from an external surface of an apical membrane. Therefore, one is inclined to think about the superficial mechanism of an induction of sodium transport by glucose when the phenomenon of an induction itself is caused by the binding of glucose with some nearby receptor of the sodium transporter. The presence of a stimulating glucose effect upon its binding with an external surface of an apical membrane points out that sodium transport through a brush border in the absence of glucose proceeds at a rate slower than the maximal rate and that a stage limiting transcellular active sodium transport is the stage of crossing by sodium of a brush border. In actuality, if we assume that a limiting stage is the crossing of a basolateral membrane, it is difficult to imagine the mechanism according to which the acceleration of a fast stage of crossing an apical membrane could result in acceleration of active transcellular sodium transport. The conclusion that crossing an unstirred layer and an apical membrane is a limiting stage of all processes is consistent with the conclusion obtained earlier about activation of special transport systems in a brush-border membrane for sodium as glucose molecules approach them. As has already been mentioned, substances absorbed from an intestinal lumen in an active Na+-dependent manner are capable, on their addition to a mucosal solution, of stimulating the SCC and active sodium transport. The indicated observation has served as a basis for development of a new method for studying the coupled transport of nutrients and ions. Any restrictions to the application of that technique are absent, and at present it is considered as the assay which can always be applied. The termination of glucose transport at a temperature of 16°С can be demonstrated and from dependence of Kt on temperature. Because of the presence of active glucose transport at temperatures above 26°С, superficial glucose concentration is a little below that of sugar concentration in a bulk solution; therefore, the somewhat overestimated value of Kt can be measured. If this is the case then a decrease in Kt below 16°С should stop. However, distinctions in Kt at temperatures of 16° and 10°С are unreliable. Thus, with the termination of active glucose transport Kt changes insignificantly, suggesting the validity of the estimation made earlier that active glucose transport influences glucose concentrations near a brush-border membrane only slightly. The established procedure of temperature-dependence analysis is plotting a curve using coordinates (Arrhenius plot): the logarithm of effect magnitude—reciprocal temperature (Ugolev, Kuzmina, 1993). The Arrhenius plot for stimulating glucose effect on the SCC is linear in all ranges of studied temperatures and has the activation

Chapter 9. Molecular mechanisms of the coupled transport of glucose€€€€€167

energy 22 kcal/mol (Metelsky, 1987). On the contrary, the Arrhenius plot for active glucose transport is essentially nonlinear. The break on this plot is observed at 25–26°С; above this point the activation energy is 22 kcal/mol, and below it is 49.74 kcal/mol. These data are in agreement with those where, on a rather different biological system (Xenopus oocytes where the cloned human transporter (hSGLT1) was expressed by the number of functional hSGLT1 proteins), it was shown that activation energy for this cotransporter is equal to 26± 4 kcal/mol (Parent, Wright, 1993, Loo, Zeuthen, Chandy et al, 1996). The values of activation energy sharply contrast both with the activation energy for the diffusion of sodium in water (Hille, 2001), which is 4.2 kcal/mol, and that for glucose diffusion (see above), which is 5.6 kcal/mol, as well as with the activation energy for the diffusion of water permeability coefficient on the human epithelial cell line (Kida et al., 2005). Observed (Metelsky, 1987) values of activation energy are rather high, but it has long been known that for transport processes through membranes the value of activation energy is 80 kcal/mol (Kotyk, Janacek, 1977). Proceeding from high (in comparison with diffusion) values of activation energy (21.95 and 49.74 kcal/mol), we consider that the energy barrier to sodium and glucose transport is localized at a level of passage of a brush-border membrane and it is a limiting stage of processes of coupled transport. The functioning of transporters depends on the fluidity of a brush-border membrane. So, by feeding rats retinyl palmitate it was possible to increase the Na+-dependent transport of glucose, seemingly due just to an increase in fluidity (Tomimatsu, Horie, 2001). The break on the Arrhenius plot at 26°С is characteristic of the phase state of lipids of a enterocyte brush-border membrane of a rat (Schachter, Shinitzky, 1977; Brasitus, 1983). As a matter of fact, there are a few causes of nonlinearity of the Arrhenius plot (Urrry et al., 1984): 1) existence of a number of barriers with various activation energy instead of one barrier; 2) the dynamic nature of the transporter in consequence of which distribution of channel states depends on temperature; 3) temperature dependence of a dynamic transporter itself; thereby the movement of side chains of transporter proteins depends on a state of the nearest lipids; 4) it is not inconceivable that as temperature decreases an activation energy of some stage of transport (for example, the termination of transport because of narrowing the channel) sharply increases, an opportunity which was never observed. As transport processes in an intestinal epithelium strongly depend on a status of tight cell junctions, the break on the plot theoretically can be caused by its opening or closing at a certain temperature. However, two facts contradict that opportunity. First, the break is observed only on the plot for transport of glucose, but not sodium. Secondly, it has long been known that the activation energy for tight cell junctions of a leaky epithelium does not exceed 3.2–4.0 kcal/mol (Gonzalez-Mariscal et al., 1984). The second hypothesis conflicts with the fact that all known channels are characterized by a minor number of states and allow ions to pass using the all-or-nothing principle. We could not choose between the third and fourth opportunities of the termination of

168€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

transport, namely between closing of the transporter because of lipid phase transfer and because of narrowing of the glucose transporter. As was pointed out earlier, the issue regarding stoichiometry of the coupled transport of nutrients and sodium in epithelial tissues remains open because of its contradictory nature.€ Although some researchers believe that, at least for the system where the cloned SGLT1 expressed in oocytas, it was well established that the stoichiometry is 2 Na+/1 glucose (Mackenzie et al, 1998).€ Some inconsistency is partly due to the fact that the same coupled transporter can operate with various stoichiometry under different conditions; however, we shall not analyze here whole numbers of stoichiometry for any nutrients except glucose. We will give only one example of evaluation of a stoichiometry of glucose and sodium transport through a brush-border membrane. As indicated above, the stoichiometry of glucose and sodium transport at 37°С may vary (Kimmich, 1981, 1983; Meinild et al., 1998; Diez-Sampedro et al., 2001). At 22°С, the stoichiometric relationship of transport (glucose: sodium) proves to be a non-whole number and is distinguished from that found earlier (1:3.2) (Wright et al., 1983), or 0.31. It should be pointed out that in most cases a fractional number (not whole) of stoichiometry of the transport of sodium and glucose is measured, but then for some reason they are truncated to whole values (Parent et al., 1992; Diez-Sampedro et al., 2001), apparently under the assumption that the stoichiometric measurements were not quite precise. As observed in (Metelsky, 1987; Ugolev, Metelsky, 1990), the stoichiometry of glucose and sodium transport decreases with a decline in temperature, from 0.5 down to 0. At 22°С it is equal to 0.37. That agrees closely with the value of the coefficient 0.31 which was found by Wright et al. (1983). Contrary to the predictions of common serial transporter models, it was found that upon a decrease in temperature to 16°С active glucose transport completely stops, but the stimulating glucose effect on active sodium transport remains to the full extent. Data related to an opportunity of dissociation of active transport of glucose and its inducing effect demand revision of the basic models of coupled transport. Interpreting the data that have been obtained may be useful to the hypothesis about parallel pathways of sodium and glucose transport through a membrane. Four basic types of parallel models are possible: a glucose carrier and a sodium carrier; a glucose channel and a sodium carrier; a glucose carrier and a sodium channel; a glucose channel and a sodium channel. Carriers of sodium ions in biomembranes have not been found until now, but on the contrary, transport mechanisms for sodium in epitheliocytes are identified as channels with increasing frequency (Eldrup et al., 1980; Sariban-Sohraby et al., 1984; Hunter et al., 1984; Bize, Horisberger, 2007). Therefore, the first two models are improbable. As a matter of fact, a range of parallel models for co-transport of sodium and glucose is much more than two, since the group of models of the facilitated glucose transport through plasmatic membranes is great (see above). The coupled transporter of glucose and sodium is an integral membrane protein (Kessler, Semenza, 1983). The structure of a bacterial homology (vSGLT from

Chapter 9. Molecular mechanisms of the coupled transport of glucose€€€€€169

Vibrio Parahaemolyticus) of the Na+/glucose cotransporter has been solved to atomic resolution (Faham et al.,€ 2008). Freeze-fracture electronmicroscopy indicated that the protein is a monomer and is not part of a multimeric complex (Zampighi et al., 1995). Because the flip-flop mechanism for integral proteins is a rare occurrence, the “diffusing” and “rotating” carriers should be excluded from consideration. Therefore, the model of a sodium-glucose transporter as presented on Fig. 18 is more accurate (Metelsky et al., 1983; Ugolev, Kuzmina, 1983; Ugolev, Metelsky, 1990; Metelsky, 1992, 2007a). It is characterized by the presence of two parallel cooperating pathways—one for sodium and one for glucose—and the superficial gate mechanism binding glucose on an input in the transport system. The transport cycle of this model is characterized by the following states: (1) in the beginning glucose and sodium transporters are not active; (2) the sodium transporter is activated with the binding of glucose to the allosteric center on the gate mechanism, similar to the control of sodium permeability by acetylcholine, and sodium moves from an extracellular into an intracellular fluid; (3) at the specific stage of movement of sodium on its transporter takes place an allosteric activation of the glucose transporter; (4) the glucose molecule originally attached to the gate mechanism is transported through the activated glucose transporter; (5) a dissociation of glucose from that is accompanied by a deactivation of a sodium transporter. A reactivation of a sodium transporter occurs with the binding of a glucose molecule to an allosteric center. From that model follows that the rate of glucose and sodium transport in a certain range depends on sodium concentration in a mucosal solution. The offered scheme takes into account the properties of all previous models, including the allosteric model (Alvarado, Robinson, 1979). On the other hand, such a parallel two-pathway cotransporter model found an unexpected strong support (Faham et al., 2008). In this study (Fig 1B, 1C), where the structure of hSGLT1 viewed in the membrane plane and from the intracellular side was presented, one can clearly see that bounded galactose and Na+ in the middle of the cotransporter are separated from one another by 2-3 helices! Then, authors note that structural alignment with LeuT revealed a possible Na+-binding site at the intersection of TM2 and TM9, ~10 Å away from the substrate binding site. This is quite a large distance. According to Hille (Hille, 2001) the cross-section of the sodium channel is 3 Å x 5 Å. Hence, at a distance of 10 Å, two channels like sodium one could be placed. Authors (Faham et al., 2008) suggest that the intracellular exit pathway appears as a large hydrophilic cavity blocked by the intracellular gate residue (Y263 on TM7E). The last Fig. 4 shows that the studied cotransporter€ work according to a one-pathway model (see above), which is not consistent with Fig. 1B and 1C, where it is clearly visible that Na+ and glucose in the center of the core€ are spatially separated. In our view, there are direct evidence in favor of the two-pathway parallel model, such as Na+ and galactose entering into a large hydrophilic cavity together then being transferred via two differ rent pathway.

170€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

Glucose

Na+

a

b

d

c

e

Fig. 18. A conceptual model of glucose and sodium transport in a plasma cell membrane. On the left – Na+-channel; on the right – glucose channel; top – gate mechanism; open pentagons – glucose molecules; filled circles – sodium ions. a – initial transporter state (both channels are closed); b – after glucose binding with its allosteric center of the gate mechanism, the sodium channel opens; c – during sodium movement along its channel, an allosteric activation of the glucose channel takes place; d – a glucose molecule is transferred from the gate mechanism through its activated channel; e – the site release on the gate mechanism results in closing the sodium and glucose channels.

Chapter 9. Molecular mechanisms of the coupled transport of glucose€€€€€171

The feature of the offered cotransporter is the separation of a stage of opening of a sodium transporter upon binding of a glucose molecule with a gate mechanism from a stage of energization of a glucose molecule transfer along its transporter. From this feature follows the attribute which distinguishes it from models discussed earlier, which had a common carrier and transporter. The multi-pathway model predicts what even under such states of a cell when the coupled glucose and sodium transport stops, addition of glucose in a mucosal solution will result in opening of a sodium transporter. For the purpose of verification of this prediction, experiments were carried out on preparations in which respiration and glycolysis were suppressed (Ugolev, Metelsky, 1990).

9.4.2. Evidence for a multi-pathway model Let us consider the experiments by Ugolev and Metelsky (1990) in greater detail. The initial characteristics of segments of a rat small intestine are the following: the resistance of a preparation is equal to 28.8 ± 4.0 Ohm*cm2, a spontaneous transmural potential difference to 2.2± 0.4 mV, and a value of a response to 10 mM of glucose to 27.0 ±5.0 μA/cm2. Under conditions of anaerobiosis and simultaneous blocking of glycolysis by 0.1% sodium fluoride, the stimulating effect of glucose is gradually reduced to 0 (Fig. 19B, a middle curve). At the same time, the effect of 0.1 mM of phlorizin in the presence of glucose in parallel also disappears. The resistance of a preparation decreases and becomes equal to 19.06 ± 3.0 Ohm*cm2. If after that to hold on the preparation a potential difference of +3.2 mV and to add in a mucosal solution 10 mM of glucose, one can register an increase in current through the preparation (Fig. 19B, upper curve). It is particularly remarkable that 0.1 mM of phlorizin eliminates such a glucose effect (compare with Fig. 15). On a holding potential of -3.2 mV (a minus in a mucosal solution) the current will flow through the preparation in the opposite direction. An addition of 10 mM of glucose to a mucosal solution under such conditions also results in an increase in the absolute value of the current. Just as in the previous case, the 0.1 mM of phlorizin eliminates the increase in such opposite directed current caused by the application of glucose (Fig. 19B, the bottom curve). If on a preparation to hold again a potential equal to 0, one may be convinced that responses of a current to glucose and the effect of phlorizin have disappeared. If on preparation to give a holding potential (positive or negative) not equal to 0, one can again see on it responses to the glucose eliminated by phlorizin. Hence, the effect of a potential difference on a direction and magnitude of the SCC response to glucose is characterized by: (i) reversibility and (ii) reproducibility of results. Thus, one can see that the glucose-sensitive current through a preparation is related by linear dependence with value of the potential difference generated on a preparation (Ugolev, Metelsky, 1990). The voltage-current characteristic of a deenergizied prepa-

172€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

А

+

Glycine

B

-

Glucose

+

+4.4 mV

Phlorizin

+

+3.2 mV

10 µA/cm2

20 µA /cm2

+1.2 mV

200 s

200 s

-0.2 mV

-6.9 mV -3.2 mV Fig. 19. On the left (panel A) – effect of 40 mM glycine on current across a specimen of the rat small intestine treated by 0.1% NaF under anaerobic conditions upon holding +4.35, +1.15, or –6.85 mV (plus in mucosal solution) on it. On the right (panel B) – effect of 10 mM glucose and 0.1 mM phlorizin on current across a specimen of the rat small intestine treated by 0.1% NaF under anaerobic conditions upon holding +3.2, –0.2, or –3.2 mV on it.

ration are linear, with its slope corresponding to resistance 2000 Ohm*cm2. It should be pointed out that the relative value of responses of a current (the ratio of value of a response of a current on glucose to absolute value of a current) is small ~1%. But since such glucose effect is completely eliminated by usually used concentration of high specific inhibitor phlorizin, it is recorded reliably enough. Responses of opposite directed currents to glucose can be eliminated also by isotonic omission of glucose from a mucosal solution. Hence, it has been established (Ugolev, Metelsky, 1990; Metelsky, 2007a) that on suppression in a preparation of the processes of respiration and glycolysis in response to addition of glucose in a mucosal solution, the absolute value of the current passing through it increases irrespective of a sign of a holding potential. In this case active

Chapter 9. Molecular mechanisms of the coupled transport of glucose€€€€€173

transport processes in cells stop, and the preparation represents a passive element. It is apparent that the zero current through a preparation will not change with switching off of resistance Rgl—the resistance of glucose (glycine)-dependent sodium transporters. Specific responses of a preparation to glucose nevertheless can be revealed, holding on it a positive or negative potential difference. A small part (~1%) of the total current passing through a preparation is caused by sodium transport through epitheliocytes. Switching on of resistance Rgl caused by opening in an apical membrane of an additional pathway, sensitive to glucose, with holding any potential difference between mucosal and serosal solutions (except for 0 mV) should result in increase in absolute value of current flow (Ugolev, Metelsky, 1990). If the resistance of an apical membrane is higher than that of a basolateral membrane, then the voltage-current characteristic reflects, apparently, properties just of the apical membrane. In this case the value of resistance 2 kOhm сm2, characterizing sodium transporters sensitive to glucose, is consistent with the resistance of galactose-sensitive channels in an apical membrane of an enterocyte—1.7 kOhm сm2—measured on an intact preparation (Gunter-Smith et al., 1982). The agreement of values of resistance of the glucose-sensitive sodium transporters, determined by Ugolev & Metelsky (1990) and by Gunter-Smith et al. (1982), and blocking of glucose-sensitive sodium conductivity by phlorizin unequivocally suggests that responses of an electric current to glucose (Fig. 19B) are caused by sodium transport through a cotransporter. It is little wonder that the voltage-current characteristic of sodium pathways is linear- i.e., there is a symmetry between forvard and reverse Na+- glucose transport. By building an epitheliocyte amiloride-sensitive sodium channel in a bilayer lipid membrane, it has been found that the voltage-current characteristic of a single channel is in actuality linear in a wide range of voltage (± 60 mV) (Sariban-Sohraby et al., 1984). On the other hand, it was shown (by using the inside-out membrane patches from Xenopus laevis oocytes) that there is an asymmetry in sugar kinetics and specificity between forward and reverse modes at work of SGLT1 (Eskandari et al., 2005). Authors conclude that, under physiological conditions, the transporter is poised to accumulate sugar efficiently in the enterocyte. All molecular machines, and in particular, transport ones, work in reverse. We will illustrate this point by giving two examples. From sheep reticulocytes were obtained vesicles in usual orientation and turned inside out (Weigensberg et al., 1982). It was found that a coupled Na+-dependent transporter for glycine is capable of transferring through a membrane both sodium and glycine in both directions. It is common knowledge that a sodium pump is reversible and capable of both hydrolizing and synthesizing ATP. It was found that after reversing the action of a sodium pump, the ion (sodium and potassium) currents through it can flow in an opposite direction with stoichiometry, characteristic for currents in a direct direction, i.e., 3:2 (DeWeer, Rakowski, 1984). In accordance with the preceding it was found that work of a sodium transporter of a coupled cotransporter is also reversible (Ugolev, Metelsky, 1990, Metelsky, 2007a),

174€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

and fluxes of sodium in both directions are governed by the gate mechanism located on an external surface of a membrane. The idea about the cooperating transporters located side by side proves to be unoriginal. It has been described already as the well-founded two-channel (sodium and potassium) model for an explanation of the mechanism of functioning of a sodium pump (Levitt, 1980). From the multi-pathway parallel model, it follows that as in the molecule of phlorizin one glucose molecule is included under certain conditions one would expect that phlorizin would manifest a stimulating effect. Such conditions have been found (Metelsky, 1987, 2005с). At high temperatures (26°С and above), phlorizin inhibits a basal SCC due to the termination of sodium transport (sliding) through a cotransporter at 6.33% (Tab. 29). At low temperatures the inhibition is replaced by stimulation, apparently due to interaction of phlorizin with the center on the gate mechanism. On the average, such an effect is insignificant, equal to 6.07%. However, if the stimulating action of phlorizin is counted out from its inhibiting action, then the effect is equal to a considerable value: 6.33% + 6.07% = 12.4%. It is vital to note that Ki for phlorizin determined in the same experiment with stimulation of the basal SCC and on inhibition of the SCC response to the glucose were distinguished as 22 μM and 4 μM (Metelsky, 1987). Hence, these effects of phlorizin are caused by its interaction with different binding sites. Upon the binding of phlorizin with the site on the gate mechanism, the gates open and sodium transport increases. As would be expected during transfer of glucose (in this case of phlorizin) from the gate mechanism on an input of the glucose transporter, the sodium transporter is closed, resulting in decrease of the stimulated SCC.

9.4.3. Structure of a multi-pathway cotransporter 9.4.3.1. The gate mechanism There are four types of ion channels that depend on a stimulus to control their opening and closing. They are classified as follows (Fuller, Shields, 2006): (a) A calcium channel controlled by a ligand; its opening is due to the energy of the ligand binding; Table 29. Effect of 0.1 mM phlorizin on basal short circuit current at 26°С and 16°С (Metelsky, 1987). Temperature 26°С 16°С а

p< 0.02

Effect on basal SCC, % - 6.33 ± 2.90 (9) + 6.07 ± 3.51 а (7)

Chapter 9. Molecular mechanisms of the coupled transport of glucose€€€€€175

(b) A channel controlled by phosphorylation; its opening is due to attachment to a high-energy phosphate; (c) An electrically controlled channel; its opening is due to change of a membrane electric potential difference; (d) A mechanically controlled channel; its opening is due to stretching of or pressure on a cell membrane and a cytoskeleton. The basic characteristic of a parallel multi-pathway cotransporter is the transporter with a gate mechanism. In an enterocyte, as well as in any cell, there should be transporters that change their conformation in response to certain stimuli, including changes in an electric field, transmitters or nutrients. Such conformation changes are usually termed “gate mechanisms” as, while opening or closing the transporter, they control the movement of ions. In the case of coupled transport, fluxes of sodium and glucose through an apical membrane are interdependent. Sodium energizes glucose transport along its transporter, and glucose controls a gate process of a sodium transporter. It is unclear how the mechanism of linking between glucose and sodium transporters operates, but the following considerations can be applied to the gate mechanisms of a sodium transporter. It has been earlier shown that the opening of a sodium transporter occurs in the absence of glucose transport along its transporter. This method of control, unlike the gate mechanism, which is controlled by an electric field (as in the case of ion channels of excited membranes), should not depend on the direction of movement of ions through the channel. Following this prediction, it has been found that the gate mechanism can open the transporter for a sodium flux from outside to inside a cell, and in the opposite direction (Metelsky, 1987). What is the location of gate mechanisms? According to data gathered by Metelsky, Roshchina, and Ugolev (Metelsky et al., 1983; Ugolev, Metelsky, 1990), glucose transport completely stops at 16°С. Apparently, as this occurs, glucose cannot enter into its transporter. Due to simple diffusion, glucose cannot enter an internal surface of a membrane, since the permeability of a lipid bilayer for glucose is 1.1*10-10 cm/s (Brunner et al., 1980). Hence, the gate mechanism of a sodium transporter controlled by glucose is on an external surface of an apical membrane. Other restrictions on possible models of a cotransporter are imposed due to the issue of an energy source for the gate process. Experiments with the preparations of an intestine processed by sodium fluoride under conditions of anaerobiosis show that, as well as in the case of excited membranes, high-energy compounds or special solutions are not required for processes of opening and closing the transporter. Upon addition of glucose in a solution washing such a “de-energized” preparation, the transmembrane electric field of an apical membrane also, apparently, changes insignificantly, and consequently cannot serve as an energy source for the gate process as it occurs in sodium transporters of membranes of excitable cells. The possible energy source for that process is the energy released from the binding of a glucose molecule with the corresponding center on the gate mechanism. Such a

176€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

mechanism can be named by a sensor or a receptor of glucose. It should be noted that chemically controlled ion transporters are not peculiar to sodium-glucose transporters of enterocytes. The sodium channels controlled by acetylcholine are located in postsynaptic membranes. It is appropriate to compare the basic properties of the gate mechanisms of multipathway parallel models with the gate mechanisms described in the study of Kessler & Semenza (1983). The behavior of the gate mechanisms (Kessler, Semenza, 1983) is determined, basically, by a value and sign of a potential difference on a membrane. If the potential difference of a membrane is positive or is equal to 0, such a mechanism has one conformation (inaccessible to phlorizin); on negative potentials (the minus inside of a cell), the conformation of a gate changes in such a manner that the ability of the transporter to bind phlorizin and glucose sharply increase (Kessler, Semenza, 1983). In the authors’ view, that property of the transporter is well explained by the assumption about the presence inside of a common channel with a mobile negative charge. The charge (carboxylic group) on positive potential and on a potential difference equal to 0 is localized on an internal surface of a membrane; on negative potential difference, it is localized on an external surface. On the contrary, the conformation of the gate mechanism considered seems not to be affected significantly by a potential difference (Metelsky et al., 1983; Ugolev, Metelsky, 1990; Metelsky, 1992, 2007a), since on generation on an epithelium (and, hence, on both membranes) positive or negative potential sodium ions are transported through the transporter with the same efficiency. A location of the negative charge (Kessler, Semenza, 1983) (like the ionized carboxylic group) on a receptor of glucose is unlikely. The foregoing data (Chapter 8) regarding a water-soluble carbodiimide (CMCD)—the modifier of carboxylic and phosphate groups—indicate that CMCD blocks only basal sodium channels and does not affect glucose-dependent sodium transporters. The groups bearing some positive charge are more likely necessary for the binding of glucose. According to the multi-pathway parallel models, regardless of a potential difference on a membrane, the receptor of glucose on the gate mechanism is localized on an external surface of a membrane. Thus, the properties of the gate mechanisms described in previous studies (Kessler, Semenza, 1983; Metelsky et al., 1983; Ugolev, Metelsky, 1990; Metelsky, 1992, 2007a) are dramatically distinguished. It is not unlikely that the properties of the gate mechanism described in the study of Kessler and Semenza (1983) are actually properties of two gate mechanisms—mechanisms for a glucose transporter and a sodium transporter (ENaC). To demonstrate that the carboxylic group can play the role of gate mechanism by helping to substrate raking up, researchers should first demonstrate the dependence on a sign of potential—not only the ability of a channel to bind phlorizin, but also the availability of the carboxylic group to corresponding reagents (for example, by CMCD). However, such an experiment has not been carried out.

Chapter 9. Molecular mechanisms of the coupled transport of glucose€€€€€177

9.4.3.2. The sodium channel Let us consider more properties of sodium channels, which may be useful for understanding the Na+-glucose cotransporter. One of the basic characteristics of any ion channel is its ability to pass one ion and not to pass others. By what factors is such selectivity determined? Ionic radii of monovalent cations Li Rb> K> Na> Li. For small anions the attraction to a cation prevails, and we have the selectivity sequence Li> Na> K> Rb> Cs, characteristic of sodium channels of excitable membranes. All XI sequences predicted by Eisenman are found in various systems. The concept of a field force is the convenient characteristic of the binding centers. The center with a larger charge, smaller radius, greater number of dipoles, and greater dipole moment possesses a greater field force. Study of the concept of permeability of epithelial membranes was possible by means of the field equation (Fuchs et al., 1977, Palmer, 1982). The difficulties are related to the morphological complexity of epithelial cells, namely, the presence of two membranes that have rather different properties. In this case, sodium channels sensitive to amiloride have been characterized, but regular study of the selectivity of channels has not been carried out. The study by Okada et al. (1975) performed on the enterocytes of a duodenum is the exception. It was found that the ratio of permeability of an apical membrane for potassium and sodium is equal to 0.07. In several studies, the influence of ionic replacements on the rate constant of ion influx into a cell was investigated. So, the ratio of rate constant for transmural fluxes of lithium and sodium through an apical membrane of a colon epithelium proves to be equal to 0.75 (Sarracino, Dawson,

178€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

1979). It was concluded that lithium penetrates an apical membrane as well as does sodium, and the lowered rate constant for transmural lithium transport is followed by lowered affinity of a sodium pump to this cation. Hence, in the case of amiloride-sensitive channels of epitheliocytes, a sequence of permeability for sodium channels is Na> Li> K and this sequence is consistent with the data (Bize, Horisberger, 2007). The existence of selectivity sequences distinct from the XI sequences of Eisenman is improbable, for this type of channel matches X or XI sequences of Eisenman. The selectivity of a membrane of Ranvier’s node of a nerve fiber also is described by the XI sequences of Eisenman. Until now, the problem of selectivity of glucose-sensitive sodium channels has not been posed in scientific literature; consequently, corresponding data are absent. Suitable approaches for studying the permeability and selectivity of this type of channel have not been developed. Actively transported sodium perhaps passes through cell junctions that can have selectivity distinct from the selectivity of the glucose-sensitive sodium channels, and that fact complicates the situation. Therefore, one cannot make any strict conclusions about the selectivity of glucose-sensitive channels. However, from the results of experiments on the sensitivity of the coupled transporter for sugars and amino acids to the replacement of sodium by other cations, one can try to estimate the selectivity of rate constant of sodium flux along its channels. At 37°С, there is a rigid stoichiometry between sodium and glucose transport. The replacement of sodium by a poorly penetrating cation rate of nutrient transport through the parallel channel will decrease in accordance with the reduction of permeability of the channel for this cation. Hence, on change in rate of Na+-dependent nutrient transport in response to replacement of ions, the permeability of a sodium channel for that cation may be estimated. In such an inference, the impairing influence of cation selectivity of the Na+-K+-ATPase site in intracellular solution is neglected. However, if a sequence of selectivity of this site coincides with or is similar to a sequence of selectivity for a glucose-sensitive sodium channel, these distinctions will be minimal. To support these arguments, we will first analyze the data gathered by Schultz, Fuisz, and Curran (1966). Fig. 5 shows the data on accumulation of 3-О-methyl glucose in rabbit ileum enterocytes in the presence in a mucosal solution of sodium, choline, and potassium. Setting the sugar accumulation in the presence of sodium as 1.0, the accumulation of glucose in the presence of choline and potassium will equal 0.14 and 0.04, respectively. One can conclude, then, that a sequence of selectivity for the ion channel is Na> choline> K. These data are consistent with those of Parent, Wright, 1993, where it was shown that cation substitution by Li, K, or choline significantly reduce sugar transport. If for formation of a corresponding multi-pathway cotransporter it is enough of a combination of the same sodium transporters with any glucose or amino acid transporters, then the same sequence should be obtained with glycine. Indeed, such is the case. Processing the same way the data presented on Fig. 3 from the quoted study gives the ratio of rate transport constant of sodium, choline and po-

Chapter 9. Molecular mechanisms of the coupled transport of glucose€€€€€179

tassium 1: 0.12: 0.04. These results, on one hand, confirm that the structure of glucose and amino acid coupled transporters includes the same sodium transporter, and on the other hand, have been found to bear witness that upon reduction of rate transport constant through the nutrient transporter one can evaluate the permeability of the ion transporter. With the replacement of sodium by lithium, the rate of glucose transport is great enough, and the transport process is inhibited by both phlorizin and by other actively transportable sugars. Thus, one can conclude that a probable sequence of selectivity for a nutrient-sensitive sodium transporter is Na> Li> K (as for an amiloride-sensitive sodium channel), which corresponds to X sequence of Eisenman. This conclusion is supported by results (Metelsky, 1987) on full replacement of sodium in solutions washing a preparation with potassium. It has been found that SCC responses to glucose under such conditions almost completely disappear. The detection and careful study of the phenomenon of high selectivity of ion channels in in excitable membranes have led to the concept of selectivity filters of channels. In such a way it was possible to explain all aggregates of experimental data on studying the selectivity of channels. As indicated earlier, there are convincing arguments to assume a high selectivity for sodium channels in epithelial cells. Therefore, we can raise the question: What is the arrangement of the selectivity filter in ion channels? A very wide water pore will poorly discriminate small ions with the same charge, since such ions have in a pore enough space to move while remaining in water with the usual properties and without interacting with pore walls. A sequence of selectivity for such a channel should coincide with a sequence of this ion mobility in water (Cs> Rb> K> Na> Li); in this case, the ratio of selectivity for the two outermost members is equal to two. Such a sequence has not been observed in any known types of ion channels in plasmatic membranes; in all found sequences, the ratio of selectivity even for the next members is considerably higher. So, the ratios of permeability for potassium and sodium in exited and epithelial membranes equal, respectively, 0.086 and 0.040.07 (see above). Therefore, the sodium channel should be narrow enough to partially dehydrate passing ions and interact with them directly. The opened channel will have the maximum permeability, while the narrow part of the channel (or transporter) should have the minimal length in several Å. Other parts of the channel (or transporter) can be much wider. It is unclear whether the putative selectivity filter of ENaC presents hydrophilic side chains or backbone carbonyl O atoms to line the pore and generate selectivity (Page & DiCera, 2006). The narrow selectivity filter makes the selection of ions according to the geometrical factor (size). The further selection occurs, apparently, due to Eisenman’s factor—interactions of a cation with the center which has the strong field—and is localized, possibly, in a narrow part of the channel. Only with such an arrangement can the center with a strong field effectively perform its function.

180€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

In the majority of the channels found previously, the role of the anion center with a strong field is carried out by a carboxylic group. The experiments with CMCD (Metelsky, 1987) described earlier point out the presence of carboxylic (or phosphatic) groups in an entrance (accessible to water-soluble reagent CMCD) of sodium channels. However, in the case of glucose-dependent sodium transporters, one cannot find such groups either in an entrance or in deeper parts of the transporter (experiments with a liposoluble carbodiimide, DCCD). Perhaps the selectivity filter of a glucose-dependent sodium transporter is not accessible to water-soluble reagent CMCD. Therefore, the concept of sodium channels (or transporters) is basic. In particular, the assumption about sodium channels supplied by a gate mechanism is implicit in the mathematical theory of the SCC response, which underlies the single response method (see 6.1.). It is common knowledge that in some types of ion channels, for example, sodium channels of exited membranes, the neuroendocrine control is absent (Khodorov, 1975). Hence, if we are looking only at channels, but not carriers, then studying the regulation of sodium transport should reveal it. The problem of the presence of the receptor control for the absorption of nutrients proceeding by Na +-dependent manner has not been resolved until now. The study carried out by Metelsky (2007a) points to an absence of such control for the coupled cotransporters. To what extent absorption in an intestine is controlled is not clear. This is mainly due to the fact that the difficulties of studying the regulation of transport processes in an intestinal epithelium are often underestimated. The following are a few factors capable of affecting transport processes. 1. The mechanism of microcirculation, which is present in intestinal walls. It reduces the thickness of an unstirred layer of fluid in tissue and removes the toxic products given off by metabolism. As a rule, in studies on regulation, this parameter is not controlled. 2. The movement of villi, which cannot be controlled. This movement can perhaps reduce the thickness of an unstirred layer on both sides of an epithelial sheet. 3. The several components of sodium transport, as indicated above, though in studies of regulation this point has been ignored. It has been proposed that regulation of any components of sodium transport is the same. We have tried to take into account this latter circumstance and have shown that these components are controlled absolutely variously; this fact should be taken into account in subsequent studies. There is no reason to be surprised that neuro-endocrine control for sodium channels of an enterocyte is absent. Indeed, the enterocyte moves along villi at a very high speed, about 1 μm/hour. Therefore, if at some moment of time the enterocyte is opposite the nerve ending and could be controlled, it will already be far from that ending in only a few hours. Besides this, it is common knowledge that the molecules of most of the membrane receptors consist of two parts at least. One of them (external) serves for binding of a hormone (ligand), and the second, less polar part serves for its anchoring in a lipid

Chapter 9. Molecular mechanisms of the coupled transport of glucose€€€€€181

bilayer and for the transfer of the accepted signal inside of a cell. It was shown that the main role in binding a hormone is played by the polysaccharide chains of a receptor molecule. The cells treated by enzymes, splitting polysaccharide chains, become tolerant to hormones. Therefore, we assume that the absence of receptor control for the coupled co-transport and distinctions of data on receptor control of the transport phenomena in an intestine, in general, can be caused by uncontrollable contact of a mucosa with a powerful pool of the various enzymes usually presenting in an intestinal lumen and splitting carbohydrates. There are at least two types of sodium transporters in an enterocyte apical membrane—one that opens in the presence of nutrients and one that is nutrient-independent. Apparently, that fact will influence various areas of interdisciplinary sciences, and, in particular, on gastroenterological pharmacology. The opening of sodium transporters in the presence of glucose, glycine, alanine and disaccharides is the first observable stage of functioning of the coupled cotransporters, after which sodium transport energizes transport of a corresponding nutrient against a gradient of its electrochemical potential. Properties of sodium channels of enterocytes, their arrangement, and evidence in favor of their existence were discussed insufficiently. Here we have tried to fill the gap in our knowledge of this area. We would like to argue in favor of the existence of sodium channels in epithelial membranes and consider their properties, taking into account the well-developed “channellogy” of exited membranes. We shall discuss the properties of each type of sodium channel (nutrient-independent and nutrient-dependent) separately as much as possible. The idea of specific channels in plasmatic membranes was proposed for the first time, it seems, in 1935 (Osterhout, 1935). Later, more evidence was obtained in favor of this hypothesis, and now one can study single channels (Eldrup et al., 1980; SaribanSohraby et al., 1984; Hunter et al., 1984). Sodium channels have been found in one of the most studied epithelia—frog skin (Fuchs et al., 1977). Scientists, as a matter of fact, present evidence in favor of the channel, which was used earlier as one of the basic arguments in favor of channels in exited membranes, namely, high rate of functioning. Subsequently, similar study has been carried out on the other tight epithelium—a rabbit colon. In this case also, electrodiffusion of sodium through specific channels has been demonstrated. Later, more direct evidence was obtained in favor of the idea that transporters carrying out ion transport in epithelial cells are channels. So, by patch-clamp technique the functioning of a single potassium channel in an apical membrane of epitheliocytes of collecting tubes of a medullary substance of rabbit kidney was recorded (Hunter et al., 1984). By building fragments of an apical membrane in a bilayer plain membrane, it was demonstrated that amiloride-sensitive sodium transporters of a culture of epithelial cells are also channels (Sariban-Sohraby et al., 1984). It turns out that in both cases the ion channels work according to the all-or-nothing principle. Both types of sodium

182€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

channels found in an epithelium of a skin and a colon are sensitive to amiloride. It was found that the sodium flux inhibited by amiloride exists in a small intestine epithelium as well. One is inclined to think that, as in the above examples, such intestinal flux is caused by functioning of sodium channels in an apical membrane. It is appropriate to compare the basic properties of sodium channels in exited and epithelial membranes. So, for example, on a surface of membranes of the former type, the density of channels is equal to 13-400 1/μm2, and in epithelial tissues -50-300 1/ μm2 (Cuthbert, Shum, 1976). Hence, in exited and epithelial membranes, approximately the same number of molecules of proteins participates in selective sodium transport. Values of conductivity of the single open channel for sodium in both types of membranes, estimated on noise measurement, agree amazingly closely and are equal to 2*106 ion/s. Taking into account that gradients of electrochemical potential of sodium ions through an exited and epithelial membrane are approximately equal, it is believed that equality in conductivities points to the functional and molecular similarity of the organization of two types of sodium channels. In the present-day view, ion channels are considered as the water pores of the atomic sizes formed by macromolecules of integral proteins. The conduction of a membrane to ions changes as a result of discrete transitions between the open and closed states of single channels having a stick-slip nature (the all-or-nothing principle). It should be pointed out that there are few data on the number of glucose cotransporters in any cell. A long time ago, it was found that the competitive glucose transport inhibitor phlorizin binds with a cell surface in the amount 2000 1/ μm2 (Almera, Stirling, 1984). According to Loo et al., 1993, in Xenopus oocytes where the cloned human transporter (hSGLT1) was expressed, the number of functional hSGLT1 proteins was 104/µm2. However, it was then found that there are three components involved in binding phlorizin with a membrane surface. We have accepted that a small fraction of bound inhibitor—5% or 100 1/ μm2—accounts for specific binding of phlorizin with glucose transporters that, certainly, gives overestimated evaluation. A single open sodium channel passes 2∙106 ions/s. Let us accept the area of sodium channel cross-section in epithelial tissue to be equal to that in exited membranes, which is known to amount to 3Å *5 Å (Hille, 2001). Such a huge frequency of transfer approximately corresponds to the frequency of collision of hydrated sodium ions with the entrance of the channels. As well as in the case of exited membranes, these estimations support the idea that the transport pathway is a water pore or channel. Although the structure of the channel and the mechanism of its functioning are unknown, we can functionally reconstruct such a mechanism. So, one of the most comprehensive models in the case of exited membranes (Hille, 2001), and perhaps epithelial membranes, is the rather narrow pore in which ions move by jumps along a linear sequence of the centers of weak binding. A filling of the channel is

Chapter 9. Molecular mechanisms of the coupled transport of glucose€€€€€183

determined by a simple competition for the limited number of the binding centers or by mutual repulsion.

9.5. Final remarks An important conclusion about constancy of transport constant for glucose (Kt), derived from the analysis of the behavior of single SCC responses at various temperatures, conforms to the results obtained on measurements of concentration dependences. Contrary to predictions of common transporter models of the coupled glucose and sodium transport (the common carrier and channel), it has been found that with decreases in temperature the stoichiometry of sodium and glucose transport can vary, and at temperatures below 16°С one can observe sodium transport induced by glucose in the absence of active transport of glucose. The data obtained regarding an opportunity of dissociation of active transport and inducing effect of glucose have allowed us to offer a multi-pathway model of the coupled cotransporter of glucose and sodium. The model is characterized by the presence of two interacting pathways located side by side (for glucose and for sodium) and a superficial gate protein mechanism binding glucose on an input in a transport system resulting in the opening of a sodium pathway. The multi-pathway model is proved in experiments on the preparations processed by sodium fluoride (glycolysis inhibitor) under conditions of anoxia. It has been found that, as this occurs, electric current through the sodium transporter of the multi-pathway cotransporter can flow in both directions. Both these sodium fluxes can be blocked by phlorizin or gate mechanism. Final proofs in favor of the multi-pathway model have been obtained by detection for phlorizin not only on the center on binding with which sodium transport through a cotransporter is blocked (entrance of the transporter), but also the center on binding with which sodium transport through a cotransporter increases (the gate mechanism). The important indirect evidence in favor of the multi-pathway model is the fact that the glucose-dependent transporter of sodium demonstrates properties not of a carrier, but of the channel or transporter whose properties agree closely with that of sodium channels in exited membranes. A sodium transporter of a glucose cotransporter represents, apparently, a rather narrow pore with a selectivity filter (Ugolev, Metelsky, 1990). A strong evidence for our model was obtained when studying the crystal structure of a sodium galactose transporter€ (Faham et al, 2008). In this study, it was shown that there was a possible Na+-binding site at the intersection of TM2 and TM9, ~10 Å away from the substrate binding site. One is inclined to think that such a large distance between Na+ and substrate binding sites favors our multi-pathway model and is against the common channel model. Really, the size of Na+ channel is equal to 3*5 Å (Hille, 2001) or 4 Å for gramicidine one (Page & DiCera, 2006). Hence, at a distance of 10 Å, one can fit two such channels, for example, one for sodium and

184€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

one for glucose. In addition, it is difficult to imagine the functioning of a common channel for sodium and glucose of the size of at least 10 Å. Found in Faham et al, 2008, two Na+ binding sites on the coupled transporter are probably elements of sodium transporter selectivity filter. In Na+ symporters, it is clear that Na+ binding involves complete dehydration of the ion, thus yielding high selectivity and affinity (Page & DiCera, 2006).€

Chapter 10. Hydrolysis-dependent disaccharide transport€€€€€185

Chapter 10. HYDROLYSIS-DEPENDENT DISACCHARIDE TRANSPORT Earlier we discussed mechanisms of nutrient monomer transport. However, it is agreed that as a result of cavital digestion, oligo- and polymers of nutrients are formed. Therefore, we shall consider maltose, consisting of two glucose molecules, as one of the best studied dimers as regards the mechanisms of assimilation (from the viewpoint of absorption). Maltose, or malt sugar, is a disaccharide formed from two units of glucose joined with an α (1→4) linkage. It is the second member of an important biochemical series of glucose chains. The addition of another glucose unit yields maltotriose; further additions will produce dextrins (also called maltodextrins) and eventually starch (glucose polymer). Maltose can be broken down into two glucose molecules by hydrolysis. In living organisms, the enzyme maltase can achieve this very rapidly. Discussion about a hydrolysis-dependent disaccharide transport has not only practical, but also great theoretical, significance, since understanding mechanisms of monomer absorption can lead to understanding the absorption of dimers.

10.1. The mechanisms offered for an explanation of the phenoÂ�menon The phenomenon of hydrolysis-dependent disaccharide transport was discovered in the 1950s and ‘60s. It was found that hydrolysis of a disaccharide of sucrose (glucose + fructose) occurs on the surfaces of an enterocyte membrane where an output in fluid of formed monomers is complicated; as a result, the liberated glucose and fructose diffuse in cells (Miller, Crane, 1961). Later these authors, in essence, refused that hypothesis of “local concentrations” formulated in this work, believing that the glucose which has been liberated from sucrose is transported in an enterocyte more easily than free glucose. This resulted in the formulation of a hypothesis of “kinetic preferences” of liberated glucose (Crane, 1977). The essence of that phenomenon is that rate of glucose absorption from a solution containing free glucose is less than that

186€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

from a solution containing an equimolar (on glucose) concentration of a disaccharide. The same group of researchers in studies of glucose accumulation in intestinal tissue demonstrated the full additivity of a mucosal solution saturated with mono- and disaccharides (Malathi et al., 1973). Based on these experiments, they reached the conclusion that the mechanism of disaccharidase-dependent transport is additional to the mechanism of transport of free monosaccharides but is strongly distinguished from it. Disaccharidase-dependent transport does not depend on sodium, and in the absence of sodium in a mucosal solution the liberated glucose does not mix with a pool of free glucose in this solution, and can enter the transport system directly (Ramaswamy et al., 1974). The studies by Ramaswamy et al. (1974) give results of experiments with the replacement of sodium on choline and the influence of such replacement on glucose accumulation in small-intestine tissue in the presence of a mucosal solution of 30 mM of disaccharide (maltose, sucrose) or 30 mM of glucose. It was found that on transport of free glucose in a tissue, 20.5 mM of glucose is accumulated, and with removal of sodium the contents of glucose decrease to 0.83 mM. In other words, with removal of sodium an accumulation of glucose reduces by 96%. In the case of sucrose these values are equal to 16.9 and 8.4 mM, respectively. Upon incubation in the presence of maltose, the removal of sodium results in reduction in glucose accumulation in a tissue from 23.3 to 8.5 mM, or by 66%. In actuality, it can be seen that in the case of functioning of hydrolysis-dependent transport in the absence of sodium, glucose accumulates in a tissue by a factor of ten more than that in the case of functioning of the usual system for monosaccharide transport. On the strength of these data, the independence of disaccharidase-dependent transport from sodium can be concluded. It is our opinion that these experiments suggest there are only two components of maltose glucose transport—the greater part is Na+-dependent, and the smaller part is Na+-independent. The data obtained unequivocally point to the dependence of the transport of free and liberated glucose on sodium. One question that arises is this: Why is free glucose transport inhibited by 96% with the removal of sodium, while maltose glucose is inhibited only by 66%? One of the reasons may be direct entry into enterocytes of non-hydrolyzed disaccharides (see below); however, such opportunity in the cited study is not discussed. On the strength of these data, the conclusion about independence of disaccharidase-dependent sodium transport is made, without any discussion of the reasons of the presence in this type of glucose transport of rather significant components, depending on sodium. In fact, authors unreasonably avoid discussing the effects of sodium found out in their studies, considering it, apparently, as some artifact. In the following study carried out on brush-border vesicles of a guinea pig small intestine, the previously discussed results have been confirmed (Ramaswamy et al., 1976). With removal of sodium, an accumulation of glucose (appeared in the hydrolysis of maltose) in vesicles decreased by more than a factor of three, but again authors insist

Chapter 10. Hydrolysis-dependent disaccharide transport€€€€€187

that disaccharidase-dependent transport does not depend on sodium. A third part of the amount of glucose determined in vesicles is caused by direct penetration through a membrane of non-hydrolyzed sucrose due to passive diffusion (compare above: maltose transport on a third does not depend on sodium). The following conclusions were reached in summarizing the article of this group of researchers: (1) there are kinetic preferences for the transport of disaccharidase glucose over free glucose; (2) disaccharidase transport does not depend on sodium; (3) the glucose molecules liberated from maltose, sucrose, and trehalose move through a membrane independently of each other; and 4) glucose liberated upon hydrolysis does not mix with a pool of free glucose (Crane, 1977). Another group of researchers in the middle of the 1970s has published data on hydrolysis-dependent transport of the glucose liberated upon hydrolysis of phlorizin (phloretin + glucose), caused by brush border disaccharidases (Hanke, Diedrich, 1974; Diedrich et al., 1975). On this model they have confirmed a hypothesis about kinetic preferences of phlorizin glucose and about the existence of a special transport system for the monomers liberated during hydrolysis. Authors pointed out that glucose split from phloretin enters enterocytes by way of a mechanism that is distinct from a phlorizin-inhibited Na+-dependent mechanism of transport of free glucose. Conclusions made by these authors were used as powerful arguments in favor of the existence of disaccharidase-dependent transport (Crane, 1977). The views of the authors, however, have changed (Warden et al., 1980), essentially, to the following: glucose split from phloretin is transported by a small part of the usual glucose transporters which have avoided blockade by phlorizin—a competitive inhibitor of transport of sugars—and due to the mechanism of passive diffusion. The phlorizin is hydrolyzed in an unstirred layer near places of input for glucose. The existence of a special system of transport (similar to the one discussed above, a disaccharidase-dependent system) is not necessary to explain the results that were obtained. An important insight into transport mechanisms has been gained through research of preparations of an intestine, perfused both through a lumen and through a vascular system (Parsons, Prichard, 1965). The basic properties of transport mechanisms for free glucose and for the glucose liberated upon hydrolysis of maltose prove to be surprisingly similar kinetically, being strongly inhibited with the application of phlorizin and the removal of sodium. Maltose glucose does not have any kinetic preferences and was transported slightly slower than free glucose. In later study, mechanisms of transport for free and maltose glucose have been examined more comprehensively (Parsons, Prichard, 1971). These results may be summarized as follows: the maltose is first hydrolyzed, and then the two released glucoses are transported. Molecules of free and maltose glucoses enter the same pool, which is localized equally easily on or near a brush border, or intracellularly. The rate of transcellular glucose transport is determined by its concentration in this pool. The authors emphasize the following essential stages of process: interaction of maltose with the free binding site on a surface

188€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

of a membrane; hydrolysis of a dimer to two glucose monomers; and binding of these monomers with two centers of the transport system located in the immediate vicinity (each monomer can then either cross a membrane or dissociate from the transporter and re-enter a solution).

10.2. The dependence on sodium of transport of the glucose liberated as a result of hydrolysis Experiments in humans have demonstrated the identical sensitivity of absorption of free and maltose glucose to the removal of sodium (Sandle, Lobley, 1982). The authors agree with the opinion of the previous group of researchers that the rate of maltose hydrolysis is higher than the transport rate of liberated monomers. A high glucose concentration in the presence of a saturating concentration of maltose (Malathi et al., 1973) results in additional increase in absorption of glucose. However, here (Malathi et al., 1973) the effect is insignificant (14%) and can be explained by passive glucose transport in enterocytes. In contrast to Malathi et al. (1973) and according to Parsons & Prichard (1971), glucose inhibits hydrolysis-dependent transport of maltose. This effect is apparently caused by inhibition of maltase activity. The primary factor determining rate of absorption of maltose glucose is glucose concentration near a brush border. The conclusion that has been reached is that maltose glucose is absorbed through the usual system of transport for free glucose. The existence of a special disaccharidase-dependent system is not evident. These conclusions were confirmed by Sandle et al. (1983). Therefore, in studies of hydrolysis-dependent transport there have been various conclusions. On one hand, there are conclusions of the former group (Crane, 1977) that hydrolysis-dependent transport is carried out by a special transport system not dependent on sodium, and liberated glucose does not leave in a solution. On the other hand, data of other groups of researchers (Parsons, Prichard, 1971; Warden et al., 1980; Sandle, Lobley, 1982) suggest that maltose glucose is transported through the usual system of transport for monosaccharides. Contradictions between conclusions of the former and other groups are rather essential. The phenomenon of additional increase of glucose transport upon addition of glucose in a solution containing a saturating concentration of maltose, according to Malathi et al. (1973), is caused by the existence of two independent mechanisms of transport: one for free monosaccharides and one for disaccharide monosaccharides. Full additivity of the effects of maltose and glucose has prevented these authors from achieving any compromise and recognizing that a Na+-dependent component of hydrolysis-dependent transport is caused by the contribution of the usual transport system, and a Na+-independent one is caused by the contribution of disaccharidasedependent transport.

Chapter 10. Hydrolysis-dependent disaccharide transport€€€€€189

According to Sandle & Lobley (1982), the effect of additivity is caused by the presence of two independent transport mechanisms: the usual Na+-dependent one and the mechanism of simple diffusion. Hence, for an explanation of additional effects produced by a saturating concentration of maltose and glucose, it is not necessary to postulate the existence of an additional disaccharidase-dependent transport. Some researchers (Malathi et al., 1973; Ugolev, Smirnova, 1977; Crane, 1977) posit that disaccharide glucose cannot mix with free glucose and, hence, cannot leave in a perfusion solution. Other investigators believe that maltose glucose fills a membrane’s unstirred layer of a fluid (Parsons, Prichard, 1965; Parsons, Prichard, 1971; Warden et al., 1980; Sandle, Lobley, 1982). It is difficult to explain divergences in the results of these researchers. They are not caused by using a high concentration of disaccharides, as in Sandle, Lobley (1982; 1983), and Warwick (1983), as well as in (Malathi et al., 1973; Ramaswamy et al., 1974) using a high concentration of substrates also. However, in the first study (Sandle, Lobley, 1982; Sandle et al., 1983), unlike the second (Malathi et al., 1973; Ramaswamy et al., 1974), addition of a high concentration of substrates is carried out in such a way that the total osmolarity of solutions remains constant. All four groups of researchers used a different techniques; therefore, observed divergences did not result from one group using a completely different method. Such contradictions, perhaps, are not followed by species differences, since the experiments carried out (as well as in (Malathi et al., 1973; Ramaswamy et al., 1974)) on a hamster small intestine (Alvarado et al., 1984) have not confirmed the existence of a special system of disaccharidase-dependent transport. The cornerstone of the argument of researchers of the former group in favor of peculiar properties of disaccharidase-dependent transport is its independence of sodium (Crane, 1977). Earlier we had the opportunity to be convinced that such independence is rather relative. Nevertheless, as this conclusion was repeatedly quoted, let us suppose that such independence of sodium actually exists, and we will try to imagine what may be the causes of such principal distinctions between the views of researchers from the former and other groups. It is conceivable that under certain conditions such a divergence might be caused by recirculation of sodium (Alvarado, 1976; Brot-Laroche, Alvarado, 1983; Larsen, Mobjerg, 2006). Sodium is actively transported from an intestinal lumen through the cells into lateral intercellular space, from which it can partly (through a tight junction between cells) leave in a mucosal fluid. For this reason, removal of sodium from a mucosal solution does not guarantee its absence in the immediate proximity of an enterocyte brush border. Owing to that effect, even in studies that depend on sodium, it may be inferred that such dependence is weak or even absent. However, the effect of recirculation alone cannot explain the seeming independence of the transport process of sodium (Larsen, Mobjerg, 2006), since with the removal of sodium from the incubating medium, the transport of sucrose glucose into brush-border vesicles (where the effect of recirculation is impossible) decreases more than by a factor of three (see above).

190€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

On the external surface of a brush-border membrane, a large enough pool of sodium—4.8 mM—is localized (Faust et al., 1968; Brot-Laroche, Alvarado, 1983; Robinson, VanMelle, 1985). Therefore, the exception of sodium from a medium in which vesicles were incubated cannot guarantee the complete absence of sodium on an input in the transport system. Additional experiments are necessary to prove the independence of such a transport process of sodium. Hence, the situation in the area of exploration of hydrolysis-dependent transport can be summarized in the following way. Most researchers conclude that for an explanation of results of experiments with such classical objects as maltose and phlorizin, the use of a hypothesis concerning a special disaccharidase-dependent transport is not necessary. However, though the viewpoint of the former group of investigators about the existence of special disaccharidase-dependent transporters is not supported by the experimental data, it has not yet been disproved and consequently should be taken into consideration. Properties of hydrolysis-dependent transport are rather contradictory, as, according to data of the majority of researchers, it depends on sodium. It remains obscure where the liberated monomers enter: one author posits that they enter in an unstirred layer, and others are inclined to believe in direct transfer of monomers from an output of a hydrolytic system on an entrance of a transport one. Therefore, for the final resolution of the issue, additional studies are necessary.

10.3. The enzyme-transport ensemble The concept of an enzyme-transport ensemble was successful and viable enough that it was used for ten years to describe the coupling of the processes of hydrolysis and transport (Ugolev, 1972, 1977, 1985, 1989; Ugolev, Smirnova, 1977; Metelsky, 2008). Close integration within one membrane or even within one ensemble of hydrolizing enzyme and a transporter suggests the presence between them of allosteric interactions. Enzymes and carriers constantly or periodically come into contact with each other (Ugolev, 1986). Thus far, it has been difficult to imagine the mechanism of formation of a complex of an enzyme and a carrier in the formation of which sodium ions take part. It can be assumed that if the formation of a complex occurs periodically, its dissociation will occur after the transition of the transported molecule from the enzyme on the binding center of a carrier (Ugolev, 1972). According to the model of enzymetransport ensemble, the monomers released as a result of membrane hydrolysis of dimers are transferred directly from the enzyme on the binding site of the transport system without an exit in a water medium. At the same time, not all carriers are in contact with the enzymes responsible for the final stages of hydrolysis. One can suppose that in parallel with the carriers interacting with enzymes, there is a pool of carriers participating in the transport of free monomers (Ugolev, 1972). The following hypothesis was advanced: with highly

Chapter 10. Hydrolysis-dependent disaccharide transport€€€€€191

functional loading of carriers by various nutrients, their redistribution between various enzyme-transport ensembles occurs. However, it should be pointed out that the attempts of authors to avoid contradictions within the framework of this hypothesis have not been successful. So, in one of the first publications concerning enzyme-transport ensembles (Ugolev, 1972) the issue about the dependence of its functioning on sodium was not raised at all. On the contrary, in others of Ugolev’s publications, the work of that ensemble is considered Na+-independent, and even the participation of a special ATPase for energization of hydrolysis-dependent transport (Ugolev, 1977) is proposed. As indicated above, the question of independence of hydrolysis-dependent transport from sodium is the point at issue. To prove the existence of coupling between sodium and nutrient transport, it is necessary to be convinced of the following: (1) the rate of nutrient transport increases in the presence of sodium; (2) the rate of sodium transport increases when a nutrient is added. It has been discovered that sodium and glucose transport, as well as sodium and amino-acid transport are carried out by the coupled cotransporter. From this viewpoint the situation with disaccharides, in particular with maltose, is distinguished from that with monosaccharides. The point at issue was the dependence of disaccharide transport on sodium, but now that maltose transport is known to be Na+dependent, it is beyond question. The question of the ability of maltose to stimulate sodium transport in enterocytes, however, still remains open. As indicated above, such experiments are a missing part in the proof of the presence or absence of coupling between the transport of maltose and sodium. In view of the fact that the link between the transport of maltose and sodium is, perhaps, bilateral, there should exist not only a stimulating effect of sodium on the transport of maltose and glucose (the effect which is used in all studies), but also a stimulating effect of maltose on active sodium transport. It should be pointed out that in the study of Kohn et al. (1968), in which the influence of maltose (among other nutrients) and glucose on a potential difference were studied using the small intestinal wall of a rat, the effects of maltose and glucose were found to be very similar. The authors have not made any conclusions concerning a hydrolysis-dependent transport mechanism, as the interpretation of potential difference changes (unlike the SCC one) on such a morphologically complex object as the small intestine involves difficulties. The registration of a stimulating effect of a low enough concentration of maltose on sodium transport was possible in a study by Metelsky (1986). Thereby the proof of existence of coupling between the transport of maltose and sodium is complete. From the first registered effects of maltose on the SCC, its large similarity on all parameters with SCC responses to glucose has engaged us, in particular on relative rates of development effects (Tab. 30). The similarity of parameters is characteristic for SCC responses to maltose and glucose registered one after another. However, the similarity of SCC responses to glucose and maltose extends further. Both glucose and maltose are inefficient from

192€€€€€Transport phenomena and membrane digestion in small intestinal mucosa Table 30. Effect of addition of fairly high glucose and maltose concentrations on relative initial rate of short circuit current response (α) across rat small intestine (Metelsky, 1987). Sugar concentration Glucose, mM Maltose, mM 10 5 20

Relative initial rate of SCC response development, α*102 s-1 2.39 ± 0.38 (5) 2.70 ± 0.67 (5) 2.27 ± 0.33 (5)

10 40

2.12 ± 0.30 (5) 2.48 ± 0.52 (5)

20

2.42 ± 0.50 (5)

a serosal solution, suggesting that the mechanism of stimulation of SCC in the presence of maltose is essentially asymmetric. This is in agreement with the localization of disaccharidases almost exclusively in an enterocyte brush-border membrane (Sacktor, Wu, 1971). The effects of both sugars are equally easy to reverse; hence, the energy of the binding of maltose with certain centers of coupled maltose transporters is insignificant. Maintenance of the SCC stimulated by maltose for a long time at the same level points to the fact that during the coupled transport of maltose and sodium there are no irreversible impairments in cells and tissues. The time dependences of SCC responses to maltose and glucose in rat intestines are absolutely the same; they are parallel, increasing and decreasing simultaneously (Metelsky, 1986, 1987). From here it may be inferred that in mechanisms of SCC stimulation in the presence of both sugars, there is one common stage or process. In actuality, if such a stage is absent, parallel change over a wide range of SCC responses to maltose and glucose even in one experiment would be extremely improbable. Nevertheless, the similarity, or even identity of dynamics of development of SCC responses to maltose and glucose in rats is the most surprising to us. Since it has long been known that maltose consists of two glucose molecules, such identity can have two explanations. Theoretically, it is possible for stimulation of sodium transport due to maltose binding with two adjoining binding “sites” of the coupled transporter for monosaccharides to control the work of a sodium transporter. The effect may be the same whether two glucose molecules or only one maltose molecule has bonded with such a double “site.” In this case parameters of the SCC response to 5 mM of maltose and 10 mM of glucose must also be very close. The latter explanation, in our opinion, is more realistic. It is common knowledge that rate of maltose hydrolysis by corresponding enzymes is very high, considerably above the transport rate of the liberated monomers. Therefore any maltose molecule that has entered some premembrane layer will be immediately hydrolyzed, creating in

Chapter 10. Hydrolysis-dependent disaccharide transport€€€€€193

this area a concentration of the glucose approximately equal to a double concentration of the added maltose. In previous studies, there have been indications in favor of high rate of maltase reactions (McMichael et al., 1967). Moreover, the direct measurement of rate of hydrolysis of maltose and glucose transport rate has shown that these values are distinguished by a factor of three (Parsons, Prichard, 1971). Two basic inhibitors of transport processes in enterocytes equally affect SCC responses to maltose and glucose: glycosides—ouabain (an inhibitor of active sodium transport)—and phlorizin (an inhibitor of active transport of glucose) (Metelsky, 1986). The identical sensitivity of maltose and glucose to ouabain points to the fact that energization of additional sodium flux (comparative basal one) in the presence of a disaccharide is the same as in the presence of a monosaccharide, and it is due to functioning of a sodium pump localized in a basolateral membrane. Further, the maltose is added from a mucosal solution and ouabain from a serosal one. As tight cell junctions are poorly permeable for molecules like maltose and ouabain, the effects of these substances are caused, apparently, by their interaction with the opposite sides of an enterocyte, and the induced sodium flux passes through two opposite membranes. In other words, sodium transport induced by maltose proceeds transcellularly. Sodium transport induced by glucose, it has long been known, also proceeds transcellularly. Identical concentrations of ouabain cause identical effects; hence, energization is needed for the same stage of the mechanism of sodium transport stimulation. The sensitivity of the stimulating effects of maltose to phlorizin points to the fact that the interaction of this disaccharide with the sodium cotransporter can be regulated to some extent. The fact that the same concentration of phlorizin (0.1 mM) can to the same extent suppress a SCC response to maltose and glucose means that the inhibition is followed by binding of a glycoside molecule with the same center of a cotransporter. This conclusion is supported by experiments in which the phlorizin effectively inhibits the “combined” SCC response to the simultaneous addition of maltose and glucose (Metelsky, 1986, 1987). The phlorizin seems to affect a stage common for mechanisms of activation of sodium transport in the presence of maltose and glucose. This stage is not necessarily the same one which is implied from the identical timing of SCC responses to maltose and glucose. Hence, a mechanism of stimulation of sodium transport in the presence of maltose compares closely to that for glucose; moreover, for these two mechanisms there are necessarily common stages that assume their close spatial proximity. Experiments with change of temperature have also been found to bear witness to this point (Metelsky, 1987). The SCC responses to maltose and glucose are fast enough in parallel (temperature factors for them are similar) are decline with temperature decrease. Temperature factors Q10 for SCC responses to maltose and glucose are very close, equal to 2.45 ± 0.30 (n=4) and 2.31 ± 0.26 (n=4), respectively. Hence, SCC responses to maltose are observed in the same temperature range as are responses to glucose.

194€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

Furthermore, one can record the stimulating effect of maltose at low enough temperatures (Metelsky, 1986, 1987). It has long been known that the phase state of membrane lipids changes in a range of 10 to 37°С (Ivkov, Berestovsky, 1982). As phase transformations of lipids depend on their structure, then from the identical behavior of SCC responses to mono- and disaccharides it may be inferred that cotransporters for maltose and glucose are localized in very close domains or even in the same lipid domain. The dependence of the stimulating effects of maltose and glucose on their concentration have hyperbolic character (Metelsky, 1986), and kinetic parameters (Kt, Amax) of these two effects proves to be equal, respectively, to 4.8 (in translation for glucose concentration) and 4.35 mM and 71.4 and 67.6 μA/cm2. The value of Kt for maltase (Metelsky, 1986) equals 2.4 mM, corresponding closely to the value of Kt for maltase—isoamylase purified from disaccharidases and peptidases, isolated from a pig brush border. Kt for this enzyme at рН 6.7 equals 1.7 mM (Sørensen et al., 1982). It is significant that the similarity and even equality of kinetic parameters of transport for both free and maltose glucose were observed by many researchers using different techniques and on different objects (Ugolev, Smirnova, 1977). In Metelsky’s (1986) study, the correlation of SCC responses upon the addition of equimolar (relative to glucose) concentrations of maltose and glucose (85 of pair effects) was examined. In this case, the following factors were varied: temperature (from 10 up to 37oC), segments of the small intestine (proximal, medial, distal), the time from the start of experiment (from 10 minutes to 4 hours), state of animals (satiety, fasting), localization of the section of preparation (mesenteric or contramesenteric border), composition of Ringer’s solution (on a basis of NaCl or Na2SO4), concentration of sugars (glucose from 1 up to 10 mM, and correspondingly, maltose from 0.5 up to 5 mM), and concentration of ouabain (0 mm – 83 pairs, 0.5 mM – 2 pairs). The SCC response amplitude to maltose varied from 1.8 up to 102.0 μA/cm2. The coefficient of correlation equals 0.992, and the equation of linear regress is Y = 0.1 + 1.05*Х. The stimulating effects of glucose and maltose on the SCC closely resemble each other. Practically identical sets of values of effects result when these saccharides are taken with concentrations 2:1. This fact suggests that in the case of maltose the effect develops as a result of glucose liberated during hydrolysis. To elucidate the role of hydrolysis in the effect of stimulation of the SCC by maltose, such an effect was compared (Metelsky, 1986) to that for three disaccharides consisting of two monosaccharides, causing separately the stimulating effect (glucose and galactose), but hydrolyzed with differing rates—easily hydrolyzed maltose (glucose + glucose), poorly hydrolyzed (in adult rats) lactose (glucose + galactose), and not hydrolyzed in rat intestine cellobiose (glucose + glucose) (Alvarez, Sas, 1961). It can be inferred that if adsorption of non-hydrolyzed disaccharide is of importance all three disaccharides will give approximately identical effects. If the stage of hydrolysis is of importance, one would expect that cellobiose will not produce an effect, and the effect of lactose will be not so large in comparison to the effect of maltose.

Chapter 10. Hydrolysis-dependent disaccharide transport€€€€€195

Metelsky’s (1986, 2007a) data have been found to bear witness to the latter viewpoint. It was revealed that cellobiose does not result in the stimulation of sodium transport up to high enough concentration when its minute stimulating effect is caused, apparently, by the presence of free glucose. The stimulating effect of 10 mM of lactose is less than the effect produced by a simultaneous addition in a solution of 10 mM of glucose and 10 mM of galactose by a factor of ten. The additions of 10 mM of maltose and 20 mM of glucose increase sodium transport to the same extent. Hence, the more easily and more quickly the disaccharide is hydrolyzed by intestinal enzymes, the greater its effects on the SCC. For easily hydrolyzed disaccharides, the SCC value response can be estimated, proceeding from the SCC response value to an equimolar mix of appropriate monomers. From these experiments the extremely important conclusion has been drawn (Prichard, 1971; Warden et al., 1980; Sandle, Lobley, 1982; Parsons et al., 1985; Metelsky, 1986) that observable effects are preceded by a stage of hydrolysis of a disaccharide. Maltose glucose liberated by hydrolysis, seemingly, is chemically identical to molecules of free glucose; at any rate, the binding centers of the transporter do not distinguish them. This conclusion follows from the equality of effects of 10 mM of maltose and 20 mM of glucose. Thus, maltose has been found to stimulate sodium transport due to the liberated glucose molecules (Metelsky, 1986). The range of possible models of hydrolysis-dependent transport is essentially limited, if free glucose can change the sodium transport stimulated by maltose. To find out if this is the case, SCC responses upon addition of 10 mM of glucose and 5 mM of maltose in a mucosal solution containing already 10 mM of glucose or 5 mM of maltose, respectively, were compared (Tab. 31) (Metelsky, 1986). In this case it was found that the value of relative stimulating effects of the maltose (to glucose) in the presence of 5 mM of maltose (0.95) and in the absence of sugars (0.90), Table 31. Short circuit current responses across rat small intestine on 10 mM glucose and 5 mM maltose additions, measured in the presence of 10 mM glucose and 5 mM maltose (Metelsky, 1986). Initial solution contains a nutrient Addition SCC response on addition, µA/cm2 Relative SCC response on maltose addition

Absence of a nutrient

Glucose 10 mM

Glucose Maltose Glucose 10 mM 5 mM 10 mM 22.25±5.89 19.95±7.20 4.67±0.63а (10) (10) (8) 0.90

Maltose 5 mM 1.64±0.40 (9)

0.35

Maltose 5 mM Glucose 10 mM 4.85±1.15 (8)

Maltose 5 mM 4.6*

0.95

p < 0.001 versus 5 mM maltose in the presence of 10 mM glucose. * calculated by assuming that Km for maltose is greater by 1 than Km for glucose and equal to 6.

а

196€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

decreases by a factor of 2.6–2.7 in the presence of glucose. So, in any additivity between the effects of maltose and glucose were not revealed, although a sharp reduction in the relative SCC response amplitude on maltose in the presence of glucose was evident. What is the cause of the depression of relative responses to maltose in the presence of glucose? One can concede that there are two effects. First, the reaction of maltose hydrolysis is inhibited by a product, glucose. However, apparently, such effect should not be significant, since a very high glucose concentration (224 mM) inhibits maltase activity by only 44% (Sandle, Lobley, 1982). More probable is the following explanation (Metelsky, 1986, 1987). It has long been known that the active maltase center consists of two sites (each of them binds the monomer only) between which is a catalytic center (Klesov, 1984). Binding of the maltose with the active center of the maltase is possible when both sites are free. In a solution that contains glucose, the part of the site is occupied and that complicates adsorption of maltose and results in a sharp decrease in its stimulating effect. Hence, in studies of the influence of maltose and glucose on active sodium transport, some sort of competition has been found between them (Parsons, Prichard, 1971). These results suggest that, at least in rats, molecules of free and maltose glucose enter the same hydrolysis-transport system from the very beginning. If this is the case, the competitive inhibitor of glucose transport, phlorizin, in the same concentration should inhibit equally both the stimulating effects of glucose and maltose separately and the stimulating effects of a mix of these sugars. That prediction has been confirmed (Metelsky, 1986), and this is in agreement with the results on inhibition of transport of free and maltose glucose by phlorizin (Parsons, Prichard, 1965, 1971). However, there is an alternative opinion. A number of researchers suppose that glucose that has been liberated as a result of hydrolysis of a dimer at first enters an unstirred layer of a fluid near a brush-border membrane and then enters a transport system (Prichard, 1969; Hanke, Diedrich, 1974; Warden et al., 1980; Sandle, Lobley, 1982; Gruzdkov, 1986). Against such, one can adduce the following arguments. As the rate of maltose hydrolysis much more exceeds the transport rate of the released monomers (Parsons, Prichard, 1971; Sandle, Lobley, 1982), all maltose which reaches a membrane surface should be immediately hydrolyzed, and its part inevitably should leave in a mucosal solution in spite of the fact that the unstirred layer possesses some “locking” properties (Winne, 1973; Wilson, Dietschy, 1974). The ratio of SCC responses upon addition of equimolar concentrations of maltose and glucose under all conditions is equal to 0.95 (the equation of linear regress is Y = 0.1 + 1.05*Х) (Metelsky, 1986, 1987), and, hence, losses of maltose from an unstirred layer do not exceed 5%. The efficacy of the unstirred layer in reflecting the liberated glucose is difficult to explain without introducing some special mechanisms. So, one is inclined to think that near a membrane surface are local osmotic water fluxes. The contribution of such small local osmotic fluxes should decrease in the presence of significant transepithelial fluxes of

Chapter 10. Hydrolysis-dependent disaccharide transport€€€€€197

the water directed perpendicularly to the membrane surface. Powerful transepithelial water fluxes should influence variously the stimulating effects of maltose and glucose, because in this case the distribution of glucose concentration in a direction perpendicular to the intestinal surfaces will have different structures. In the presence of 50 mM of mannitol in a serosal solution, the water flux through a preparation is directed from a mucosal solution into a serosal one. As a consequence, the sugar is «pressed» to the brush-border membrane, causing the membrane concentration to increase (Tab. 32) and resulting in an insignificant increase in the amplitude of SCC responses to maltose and glucose. On the contrary, in the presence of 100 mM of mannitol in a mucosal solution, water is transported in an opposite direction, from a serosal solution to a mucosal one; sugars in the unstirred layer are depleted, resulting in a reduction of the amplitude of SCC responses to maltose and glucose. But of even greater importance is the fact that the ratio of stimulating effects of maltose and glucose is independent of the existence and direction of transport of a fluid. Hence, maltose glucose produced from hydrolysis enters the transport system directly, rather than leaving in a mucosal fluid. In other words, we have come to the concept of hydrolysis-transport or enzyme-transport ensemble (Ugolev, 1972; Ugolev, Smirnova, 1977; Metelsky, 1986, 2007a). Moreover, practical equality of SCC responses upon addition of maltose and of the double glucose concentration in a wide range of experimental conditions points to the absence of special separate systems of transport for monosaccharides, at least in rats. The transport of free glucose is carried out through its “slipping” through the hydrolysis-transport ensemble for maltose, which depends on sodium. Within the framework of these concepts, the suppressing effect of phlorizin is, apparently, due to the fact that the property of blocking the glucose transporter of the parallel multi-pathway transporter does not belong to phlorizin itself, but its aglycon—phloretin (Diedrich, 1968).

Table 32. Effect of osmotic pressure gradient on short circuit current responses on sugars across rat small intestine (Metelsky, 1986).

а b

Direction and magnitude of osmotic pressure gradient (mannitol), mM

SCC response on 10 mM glucose addition, µA/cm2

SCC response on 5 mM maltose addition, µA/cm2

0 - 50 (serosa) + 100 (mucosa)

20.15 ± 1.81 b (6) 22.3 ± 4.90 (3) 13.3 ± 1.45 (3)

20.15 ± 2.00 а (6) 22.3 ± 4.80 (3) 13.0 ± 1.15 (3)

p < 0.02 versus 5 mM maltose in the presence of 100 mM mannitol. p < 0.02 versus 10 mM glucose in the presence of 100 mM mannitol.

Ratio of SCC responses on maltose to those on glucose, % 100 100 97.7

198€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

The multi-pathway cotransporter for sodium and glucose has the gate mechanism. As it has been found (at least in small laboratory animals) that the same transporter is also a hydrolysis-transport ensemble, it can be assumed that the gate mechanism of a cotransporter , perhaps, participates in the hydrolysis of disaccharides (Metelsky, 1986, 1987). Association in one block of the hydrolysis-dependent transporter and of the transporter for free monosaccharides has some advantages from the viewpoint of its efficiency; the hydrolysis-transport ensemble is loaded more uniformly: in actuality, it functions when either glucose or maltose is present in an intestinal lumen.

10.4. Final remarks Transport of two substances may be characterized as coupled if mutual stimulation of transport of one substance in the presence of another is proved. It has been found that in the presence of sodium maltose transport increases considerably. Upon registration of SCC responses to maltose, it has been found that sodium transport is stimulated in the presence of this disaccharide, and, thereby, it is proved that for maltose and sodium transport there should exist a coupled cotransporter . Regular comparison of SCC responses to maltose and glucose, carried out in a wide range of experimental conditions, has revealed their equality provided that the concentration of mono- and disaccharides are present in the ratio 1:2. Also it was found that the the easier disaccharide is hydrolyzed by intestinal enzymes, the higher its stimulating effect on the SCC. Summarizing all the experimental data, we conclude that a separate independent system of transport for sodium and glucose, at least in rats, does not exist; that glucose is transported through an Na+-dependent hydrolysis-transport ensemble; and that the gate mechanism of the parallel multi-pathway coupled transporter for sodium and glucose participates in the hydrolysis of disaccharides. The resolution of the issue of hydrolysis-dependent transport within the framework of the parallel multi-pathway cotransporter carrying out transport of monomers and dimers allow us to explain a number of well-known facts.

Chapter 11. Transport systems for amino acids€€€€€199

Chapter 11. Transport systems for amino acids The overwhelming majority of amino acids is transported by means of Na+-dependent transporters (Holtug et al., 1996; Broer, 2008); hence, in studying that phenomenon one can use electrophysiological techniques. It should be pointed out that there is not one amino-acid transport that could be studied so fully as glucose transport. An example of glucose transport carefully analyzed above has allowed us, in our opinion, to get a fuller understanding of sugar transporters as a whole. Aimed to reach the same results in the case of amino acids, we have concentrated on the properties of the transport system for the most studied amino acid—glycine.

11.1. Attempts to classify the transporters of amino acids As the number of natural, actively transported amino acids considerably exceeds the number of actively transported sugars, and as their molecules are very distinct in terms of chemical properties, it is no wonder that there exist for their transport several types of transporters with mutually overlapping specificity. However, a generally accepted classification of such transporters, apparently, has not existed until now. Already the first description of separate transport systems for cationic amino acids evidences some ability of that system to transport neutral amino acids. It has been found that some neutral amino acids can not only inhibit, but also stimulate, the transport system for cationic amino acids (Hagihira et al., 1961). At a later time the assumption was made that neutral amino acids cross a brushborder membrane due to two mechanisms: the Na+-independent carrier working at a high rate and having a low affinity to a substrate, and the Na+-dependent carrier functioning at a low rate and having a high affinity to a substrate (Munck, Schultz, 1969а, 1969b; Munck, 1983). However, the majority of research supports the idea that in a rabbit small intestine there are three types of carriers for amino acids (Curran et al., 1967; Christinsen et al., 1969; Munck, Schultz, 1969а; Paterson et al., 1981). Carrier 1. This is the basic carrier for neutral amino acids. It transports glycine, proline, leucine, valine, methionine, and serine, but does not transport sarcosine, as-

200€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

partate, glutamate, lysine, ornithine, or arginine. In the presence of 140 mM of sodium, it is characterized by high affinity to a substrate and by high rate of functioning; in the absence of sodium, it has low affinity and it is capable of transferring neutral amino acids at a high rate. Carrier 2. Both in the presence and in the absence of sodium, it has high affinity for neutral and cationic (lysine, ornithine, arginine, and some other) amino acids and functions at a low rate. Carrier 3. In the presence of sodium, it has low affinity to a substrate and transfers neutral amino acids at a high rate. In the absence of sodium, it is characterized by low affinity to a substrate and transports only lysine at a high rate, but does not transport neutral amino acids. Some years later a more refined version of this idea appeared. For transport of neutral amino acids through a brush-border membrane of intestines there are two Na+-dependent systems (system ATB (0) and system B (0, +)) and one Na+-independent system (АТВ (0, +)) (Hatanaka et al., 2002; Hatanaka et al., 2004; Broer, 2008a). The absorption of neutral amino acids is mostly attributable to the functioning of system ATB (0). The system of transport of amino acids designated as АТВ (0) (or ASCT2) is similar to system B (0) in functional characteristics (Avissar et al., 2001). It may seem that the elucidation of mechanisms of amino-acid transport and their classification is relevant only to an academic investigation and does not have practical application. However, this is not so. Knowledge about the mechanisms of transport of amino acids is beginning to be demanded by medical practice. So, a medically relevant line of investigation is the use of natural transporters for the delivery of medical products in an organism. For example, system ATB (0,+), which transports glycine and derivatives of aspartate and glutamate, is capable of transferring valacyclovir, an ether of acyclovir (antiviral preparation from group of analogues of nucleosides) and valine. The ability of the indicated system to transport valacyclovir is comparable to that for the peptide transporter PEPT1 (Hatanaka et al., 2004). Moreover, PEPT1 can transport midodrine (oral drug for orthostatic hypotension) and contribute to the high bioavailability of this drug (Tsuda et al., 2006). Anticonvulsive preparations such as gabapentin and pregabalin are absorbed in the intestines. The absorption of gabapentin is carried out by means of system b0,+, and pregabalin by means of systems B0 and B0,+ for the transport of amino acids (Piyapolrungroj et al., 2001). Imine acid transport by a separate transport system was described for the first time using a hamster intestine. That carrier transports glycine, N-mono-, di-and trimethyl glycine, proline, alanine, valine, leucine, taurine, and serine and does not transports β-alanine (Hagihira et al., 1962). The transport of imine acids and neutral amino acids has been studied more carefully using preparations of a rat small intestine (Munck, 1983). In 1964 it was found that glycine, methionine and proline share a common

Chapter 11. Transport systems for amino acids€€€€€201

transport mechanism. In addition, glycine and proline are transported by one more, additional methionine-insensitive system (Newey, Smyth, 1964b). In an intestine there seems to be one more Na+-dependent transport system for anion amino acids (Schultz et al., 1970, Munck, 1981). However, in view of the fact that in an intestine glutamic and aspartic acids are quickly transaminated, studying the transport of such amino acids is extremely complicated (Neame, Wiseman, 1957). In particular, it is unclear how neutral amino acids share this transport system (Munck, 1981). However, it has been found that Hg2+ can block completely by a dose-dependent manner transport of L-glutamin and L-threonine in a brush-border membrane; as a result, it is now possible to study a seeming diffusion of L-amino acids (Fan et al., 2001). Hence, one can recognize that epithelial cells possess extremely complex systems of active Na+-dependent transport of amino acids. Such systems have organ and species specificity. So, for example, in kidney proximal tubules and in tumoral cells there is an additional mechanism for transport of neutral amino acids (Christensen, 1964; Dantzler, Silbernage, 1976; McNamara et al., 1976). The most characteristic feature of the transport of amino acids in an enterocyte is high-degree doubling of its systems that, apparently, elevates both the reliability of its functioning and its adaptability to various diets. For example, glycine, as a neutral amino acid, can enter enterocytes by means of carriers of three types and, at the same time, it shares the corresponding mechanism for transport of imine acids. Similarly, L-alanine is absorbed in a Na+-dependent manner by means of two systems—A and ASC (Medina et al., 2000; Broer, 2008a). It should be pointed out that the presence of several types of carriers for certain amino acids and the absence of specific inhibitors of transport (like phlorizin in the case of glucose) creates certain difficulties in studying molecular mechanisms of amino-acid transport. We emphasize that transport of amino acids existing in the form of zwitterion through an apical surface of a human small intestine can be carried out in symport with H +; the concentration gradient of H+ through an apical membrane may be considered an essential driving force for this process (Thwaites, Stevens, 1999) (see also Chapter 12). Apparently, we should recognize that the classification of epithelial amino acid transport systems is a difficult task, and this work is far from being finished. According to (Broer, 2008a), the identification of most epithelial amino acid transporters over the past 15 years allows the definition of these disorders at the molecular level and provides a clear picture of the functional cooperation between transporters in the apical and basolateral membranes of mammalian epithelial cells. Transport of amino acids across the apical membrane not only makes use of sodium-dependent symporters, but also uses the proton-motive force and the gradient of other amino acids to efficiently absorb amino acids from the lumen. In the basolateral membrane, antiporters cooperate with facilitators to release amino acids without depleting cells of valuable nutrients. With very few exceptions, individual amino acids are transported

202€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

by more than one transporter, providing backup capacity for absorption in the case of mutational inactivation of a transport system.

11.2. Mutual inhibition between sugars and amino acids in their transport Mutual inhibition between sugars and amino acids in their transport is a rather contradictory phenomenon (Schultz, Curran, 1970; Munck, 1972a; Kimmich, 1973). This type of inhibition was observed in intestines of the dog, guinea pig, hamster (Hindmars et al., 1966; Robinson, Alvarado, 1971), rabbit (Chez et al., 1966; Frizzel, Schultz, 1971; Robinson, Alvarado, 1971) and rat (Reiser, Christiansen, 1969; Bihler, Sawh, 1975; Sauer et al., 1983); in the isolated enterocytes of a chicken small intestine (Kimmich, Randles, 1973; Tucker, Kimmich, 1973); and in a preparation of vesicles from isolated brushborder membranes of the rat (Murer et al., 1975). In the guinea pig, rabbit, and rat, such inhibition was observed both in measurements of total transepithelial transport and in studies of accumulation of amino acids in an epithelium under steady conditions (Munck, 1981). However, in the rabbit and rat, such inhibition of fluxes entering through a brush-border membrane is insignificant (Chez et al., 1966; Munck, 1981). On the contrary, studying the input of sugars and amino acids through a luminal membrane of a guinea-pig small intestine has confirmed the existence of the phenomenon of mutual inhibition (Munck, 1980), but in this case the value of effects was equal only to 15–20% (Alvarado, 1966; Chez et al., 1966; Munck, 1980). The mutual inhibition of transepithelial mucose-serosa transport (Frizzel, Schultz, 1971), of uptake by the vesicles isolated from brush-border membranes (Murer et al., 1975), and of the flux entering through a brush-border membrane (Munck, 1980) depend on sodium. As it has been found using a bullfrog small intestine and rabbit small intestine (Rose, Schultz, 1971; White, Armstrong, 1971), saturated concentrations of sugars and amino acids cause a depolarization of a membrane by 15 mV, which results in 18% inhibition of potential sensitive cation transport (Frizzel, Schultz, 1972). To explain inhibiting interaction between sugars and amino acids, various hypotheses were offered: reduction of the cellular ATP level (Newey, Smyth, 1964a; Kimmich, Randles, 1973; Sauer et al., 1983; Yang et al., 1999), an increase in intracellular activity of sodium (Semenza, 1971), negative allosteric influence of sugars on the binding center for amino acids, and vice versa (Alvarado, 1968), as well as depolarization of a luminal membrane (Murer et al., 1975; Schultz, 1976; Munck, 1980). The first and second hypotheses cannot explain the inhibition of initial rates of fluxes entering through a brush-border membrane (Alvarado, 1968; Munck, 1980). Besides, the latter hypothesis contradicts some experimental data (Koopman, Schultz, 1969; Lee, Armstrong, 1972). The third hypothesis conflicts with the fact that until now there has been found no direct compelling evidence for the presence of allosteric interaction between sugars

Chapter 11. Transport systems for amino acids€€€€€203

and amino acids (Munck, 1981). The latter hypothesis has been supported by experimental evidence (Rose, Schultz, 1971; White, Armstrong, 1971) and seems to account for all the data. The depolarization of a brush-border membrane will inhibit an entering flux of a nutrient and stimulate its outflowing flux, decreasing the total flux through a luminal membrane (Munck, 1981). It will cause reduction of nutrient accumulation in epithelia and decrease in rate of an output of a nutrient through a basolateral membrane. As a result of these processes, the rates of all stages of nutrient transepithelial transport from a mucose to a serose will decrease. Somewhat contradictory results on the interaction of a monosaccharide-amino acid have been obtained upon free perfusion of a human intestine (Cook, 1971, 1972a, 1972b). In one study it appeared that glycine absorption was inhibited by glucose, in another study it was shown that glucose did not affect the absorption of methionine, and in a third study absorption of glycyl-glycine was inhibited in the presence of glucose. In the first and third studies the total absorption of water was significantly inhibited upon the addition of glucose, and in the second one the rate of water absorption increased in response to the addition of glucose. These results have raised questions regarding possible convection effects on a transepithelial water flux (Lifson et al., 1972). Results of experiments also testify in favor of such opportunity. The water flux induced by glucose can (due to convection effects) influence a transport of amino acids that can be wrongly interpreted, as result of competitive relations (Munck, 1968, 1983). In a frog small intestine, perfused through a lumen and through vessels, the inhibiting influence of amino acids on an exit of a monosaccharide (methylglucoside) through a basolateral membrane was revealed. Such an effect has been demonstrated for leucine, phenylalanine, isoleucine, tyrosine, valine, norleucine, and cycloleucine. Methionine and alanine prove to be inefficient as inhibitors, and leucine did not affect the transport of 3-О-methyl-D-glucose (Boyd, 1977, 1979). The inhibiting effect of leucine on the transport of a-methylglucoside through a basolateral membrane is characterized by saturation with a high concentration of leucine in an intestinal lumen. It is unknown whether a-methyl glucoside renders similar inhibiting effects on amino acids. So we believe mutual inhibition between sugars and amino acids in their transport is firmly established fact.

11.3. The two-pathway transporter for glycine Earlier it has been found (Metelsky et al., 1983; Kessler, Semenza, 1983; Kоnо, 1984; Metelsky, 1987; Ugolev, Metelsky, 1990) that the basic details in the transporter for sodium and glucose are the transmembrane transporters. However, according to Kessler, Semenza, (1983) and Kono (1984), the corresponding transporter consists of a common channel for sodium and glucose. The study by Metelsky, Roshchina, and

204€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

Ugolev (1983) presented evidence (see Chapter 9) that the coupled transporter for nutrients and sodium consists of cooperating transporters for nutrients and sodium located side by side (Ugolev, Metelsky, 1984b, 1990; Metelsky, 1990b, 1992; Ugolev, Kuzmina, 1993). The situation with glycine closely parallels that of glucose. In actuality, according to Kushak (1983), an active transport of glycine through a brush-border membrane of enterocytes processed by 0.1% sodium fluoride under conditions of an anoxia should stop completely. Nevertheless, under such conditions glycine is capable of inducing an additional sodium flux through the transporter in both directions (Ugolev, Metelsky, 1990). Unfortunately, any specific inhibitor of glycine transport that does not allow any nonspecific effects of this amino acid is absent. At the same time, there is a deep similarity between the results of experiments with glycine and glucose. So, for observations of effects of these nutrients a concentration of glycine and glucose usually used in experiments on intact preparations was used. The effects of both nutrients are easily reversible. Increase in a current (distinct from 0) through a preparation in response to addition of glucose or glycine is observed regardless of its direction (Fig. 19). Their voltage-current characteristics are linear, and its slope in both cases is approximately identical and corresponds to resistance 2 and 1.2 kΩсm2. It is important to note that sodium transport through a glycine-dependent transporter can be carried out directly and in the opposite direction (Ugolev, Metelsky, 1990; Metelsky, 2007a). Hence, the coupled cotransporter for sodium and glycine can carry out glycine-dependent sodium transport in the absence of active glycine transport. Thus, dissociation of active transport and a stimulating effect of glycine was revealed. In the case of glucose, finding the phenomenon of dissociation of active nutrient transport and its stimulating effect on sodium transport resulted in the development of a multi-pathway parallel cotransporter model (Metelsky et al., 1983; Ugolev, Metelsky, 1990; Metelsky, 1992, 2007a). Apparently, the Na+-dependent cotransporter for glycine is arranged in the same manner as the Na+-glucose transporter—parallel cooperating transporters for glycine and sodium located side by side and gate mechanisms (Fig. 18). The similarity of resistance of sodium transporters for glucose and glycine cotransporters has been found to bear witness to the neighbours of product of n*g (n = density of transporters on unit of the area, g = conductivity of the single transporter) for glucose and glycine cotransporters. The similarity of values of the maximal density of a current through sodium transporters of glucose and glycine cotransporters at 26°С—67.6 and 76.3 μA/cm2, respectively—also suggests the identity of the organization of two transporters. So, when transport of glycine and glucose through its own transporters is stopped, both nutrients keep their ability to open the coupled sodium transporters; at that point, sodium transport along its transporters in both directions occurs with identical efficiency (Ugolev, Metelsky, 1990). Hence, the basic properties of the coupled transporters for glucose and glycine prove to be rather similar. The issue of whether

Chapter 11. Transport systems for amino acids€€€€€205

the coupled transporters for various nutrients are capable of influencing each other has been unresolved until now.

11.3.1. The electrophysiological effects of glycine It has been found earlier that glycine can stimulate a potential difference (Okada et al., 1968; Levin, 1966) and the SCC (Schultz, Zalusky, 1965) through the small intestinal wall of various animals. The form of SCC responses to glycine and glucose is qualitatively the same (Metelsky, 1987, 1992). It has been found that glycine and alanine stimulate active sodium transport in the intestines of mammals (rat) (Metelsky, 1987, 1992). This circumstance, together with the fact of the presence in the intestine of Na+-dependent active absorption of glycine, is a necessary and sufficient condition for the existence of coupling between glycine and sodium transport through an apical membrane of an enterocyte. The similarity between SCC responses to glycine and glucose points to that as well. Both types of responses are developed quickly enough if nutrients are added in a mucosal (but not serosal) solution. Responses can be easily repeated and are easily reversible. There is no lag period for responses; the second response can be demonstrated any time after the beginning of the wash-out of the first one. To receive an appreciable effect of glycine, it is necessary to take it in high enough concentration (in comparison with glucose), which perhaps points to a lower affinity of the coupled cotransporter to glycine. On the contrary, distinctions in rates of wash-out of effects, both absolute and relative, for glycine and glucose are less significant. However, if the phase of response development to glucose always looks like a curve without discontinuity, the response development to glycine in approximately 25% of cases can be described by a curve with a discontinuity. According to Metelsky (1987), the SCC response value rises with an increase in the concentration of glycine in a mucosal solution. It is important to note that the stimulating effect of glycine depends on its concentration nonlinearly. With an increase of glycine concentration from 5 up to 10 mM, the SCC response value almost doubles, from 9.55 up to 15.15 μA/cm2 (Tab. 33). With a further increase in glycine concentration, the growth rate of its stimulating effect is slowed. So, with 20 mM of glycine, the SCC value response is equal to 29.3 μA/cm2. With a double concentration of glycine (40 mM), the SCC response value to glycine equals 40.35 μA/cm2. Processing of these results by a procedure of double reciprocal coordinates gives values of Kt and Аmax equal to 40 mM and 76.3 μA/cm2, respectively. The value of a transport constant determined by a procedure of concentration dependences (Metelsky, 1987) accords well with the data obtained in biochemical studies. Thus, the transport constants for active transport of glycine through a small intestinal wall of a rat, hamster and human are equal to 34–43 mM (Finch, Hird, 1960;

206€€€€€Transport phenomena and membrane digestion in small intestinal mucosa Table 33. Short circuit current response amplitudes and kinetic parameters of co-transporter for glycine in relation to its concentration (DGly = 4*10-6 cm/s) (Metelsky, 1987). Glycine SCC concentra response, A, tion, mM µA/cm2 5.0 7.5 10.0 15.0 20.0 40.0

9.55±0.99 (8) 11.50±2.50 (3) 15.15± 1.39 (21) 22.05±4.86 (11) 29.30±3.57 (21) 40.35±6.27 (17)

Relative initial rate of SCC response development, α*102 s-1 1.98±0.32 (8) 2.51±0.13 (3) 1.91±0.18 (17) 2.21±0.20 (9) 1.65±0.18 (17) 1.47±0.19 (13)

Relative initial rate of SCC response wash-out, β*102 s-1 1.32±0.16 (8) 1.54±0.20 (3) 1.21±0.17 (16) 1.31±0.17 (9) 0.83±0.10 (17) 0.64±0.07 (16)

22.3±12.2

Maximal SCC response, Amax, µA/cm2 52.0±24.0

27.1±8.0

53.1±17.7

39.0±16.2

74.2±25.4

50.2±17.3

95.8±33.0

48.8±13.6

100.8±23.5

77.6±19.3

118.6±22.8

Transport constant, Kt, mM

Hellier et al., 1972; Wisemann, 1973; Munck, 1981), and for the transporter of imine acids, 37 mM (Munck, 1981). The value of a transport constant calculated according to the SCC single response method to 40 mM of glycine proves to be equal to 77.6 ± 19.3 mM, and to 10 mM of glycine, 39.0 ± 16.2 mm (Tab. 33). The value of a transport constant determined from a single response to 10 mM of glycine (39.0 ± 16.2 mM) corresponds closely to the value of the constant determined from concentration dependence (40 mM). Hence, the single response method can be applied to a value estimate of Kt for glycine as well. The origins of dependence of Kt for glycine from its concentration remain obscure, but one is inclined to think that with an increase in the concentration of this amino acid in glycine transport through an enterocyte brush border, other types of transporters with higher Kt (see below) or deeper cells begin to participate.

11.3.2. Link between stimulating effects of glycine and glucose on the SCC The SCC increases in response to the addition of glucose or glycine to a mucosal solution (Schultz, Zalusky, 1964b; Metelsky, 1992). Thus, by adding various nutrients to a mucosal solution and calculating the value of SCC change, one can estimate the

Chapter 11. Transport systems for amino acids€€€€€207

quantity of coupled transporters for a corresponding nutrient (Kohn et al., 1968; Syme, Levin, 1976; Smith et al., 1981; Metelsky, 1987). In studies conducted by Metelsky (1987; 1992), a preparation was tested by recording on it the pairs of SCC responses to 10 mM of glucose and 40 mM of glycine. Such responses varied in magnitude by more than a factor of ten, although a good correlation (r = 0.995) between SCC changes in response to the addition of glucose and glycine was observed, and the equation of linear regression looks like Y = -1.1 + 0.68X. In the following series of experiments (Tab. 34) (Metelsky, 1987, 1992), the total value of SCC response to 10 mM of glucose + 10 mM of glycine was recorded. The registration of the total response began at the addition of glycine or glucose. The value of total SCC change in the beginning increases and decreases slightly by the end of the experiment. The relative value of stimulating effects of glycine and glucose (in comparison with value of total effect) remains constant, (i. e., the percentage composition of the total response during the experiment does not change). The value of SCC changes on the total addition of two nutrients does not depend on the order in which they are added (Metelsky, 1987, 1992); on 10 mM of glucose it is equal to 58.5 μA/cm2, and on 10 mM of glycine in the presence of glucose result in an additional increase by 6.7 μА/cm2. The value of SCC changes upon the addition of 10 mM of glycine equals 14.7 μA/cm2, and the addition of 10 mM of glucose in the presence of this amino acid result in an additional increase in SCC of 47.0 μA/cm2. Hence, the value of the effect of glycine on the background of glucose decreases by a factor of two, and the value of SCC change on glucose in the presence of glycine decreases by 19% (Tab. 34). Hence, there is a link between SCC changes on glucose and glycine. It is of interest to find out just how strong is this link and whether there are any conditions under which it could be broken. In experiments in situ it has been established (Metelsky, 1987, 1992) (Tab. 35) that in control segments (preincubation without nutrients) of an intestine, the value of effects of glycine and glucose are equal to 13.8 and 23.7 μA/cm2 (ratio 0.58), respectively. In the Table 34. Changes in short circuit current on glucose and glycine additions across rat small intestine measured in the presence of glycine and glucose in mucosal solution, respectively (Metelsky, 1987). 1st addition Glucose, 10 mM Glycine, 10 mM

2nd addition

Glycine, 10 mM Glucose, 10 mM

Changes in SCC responses on nutrient addition, µA/cm2 58.5 ± 10.4 (16) 6.7 ± 1.5 (16) 14.7 ± 3.2 (15) 47.0 ± 3.2 (15)

Changes in SCC responses on both additions, µA/cm2 69.9 ± 11.7 (16) 62.2 ± 12.8 (15)

208€€€€€Transport phenomena and membrane digestion in small intestinal mucosa Table 35. Effect of preincubation of rat small intestinal segments in situ in the presence of 1.5% glycine and 5% of glucose on responses of short circuit current through preparations isolated from the corresponding intestinal segments upon addition of 10 mM glucose and 10 mM glycine (Metelsky, 1987). Nutrient and its concentration in 0 (control) preincubation solution Addition to Glucose, Glycine, experimental using 10 mM 10 mM chamber 23.7±8.4 13.8±3.9 SCC response, µA/cm2 (3) (3) Ratio of SCC responses to glycine to those to 0.58 glucose Changes in the ratio of SCC responses (ratio in 1.0 control taken as unit)

Glycine, 1.5%

Glucose, 5%

Glucose, 10 mM

Glycine, 10 mM

Glucose, 10 mM

Glycine, 10 mM

37.7±13.7 (3)

15.7±8.3 (3)

21.3±10.8 24.3±11.1 (3) (3)

0.42

1.14

0.72

2.0

segments preincubated with glycine, distinctions between these effects tend to increase 15.7 and 37.7 μA/cm2, respectively (ratio 0.42). On the contrary, for the preparations preincubated with glucose, the value of the effects of glycine and glucose become almost identical—24.3 and 21.3 μA/cm2, respectively (ratio 1.14). The ratio of stimulating effects of glycine and glucose in control preparations are taken as a unit; then after the treatment of preparations by glycine this ratio decreases by a factor of 1.4; and after treatment by glucose it increases by a factor of two. It is significant that in both cases the change in this ratio is achieved mainly due to an increase in SCC response to addition of the nutrient which was initially absent in an preincubation medium (Metelsky, 1987). The following data were obtained in studying the influence of long incubation of preparations in a Ussing chamber in the presence of 10 mM of glucose or 20 mM of glycine on SCC response to extra addition of glucose or glycine (Metelsky, 1987, 1992) (Fig. 20). The ratio of SCC changes over a long period of time on the extra addition of glycine or glucose in a solution containing 20 mM of glycine in the beginning of the experiment are equal to 0.27; in 45 minutes this ratio tends to increase some (0.31); in the next 45 minutes the ratio of responses remains almost constant (0.28). However, after the fourth cycle (135 minutes after application in a solution of 20 mM of glycine), this ratio starts to decrease (0.23) until at the eighth cycle (315 minutes) it becomes close to 0. Other experiments in which the background solution constantly presents 10 mM of glucose were also carried. The results are consistent with those mentioned above (Metelsky, 1987, 1992).

Chapter 11. Transport systems for amino acids€€€€€209

(N) Responses ratio 0.3

0.2

0.1

0

90

180

270

360, min

Fig. 20. Time dependence of relative values (N) of SCC responses on glycine (open circles, n = 5) and glucose (filled circles, n = 6). In the former case (open circles), the mucosal solution contains 20 mM glycine throughout the experiment. SCC responses on glycine and glucose were recorded against the background of 20 mM glycine and N = Aglycine/Aglucose. In the latter case (filled circles), SCC responses on 20 mM glucose and 40 mM glycine were recorded against the background of 10 mM glucose and N = Aglucose/Aglycine.

From the studies above, it follows that the maximal values of SCC changes in response to glucose and glycine (the maximal current density Amax through glucose and glycine transporters) are close: 67.6 and 76.3 μA/cm2, respectively; the electric resistances of sodium transporters of glucose transporter agree closely with that for glycine (Ugolev, Metelsky, 1990). It should be pointed out that affinity of values for the maximal stimulating effects of glycine and glucose on potential-dependent fluorescence of the dye added in suspension of villi of a brush border of a rabbit small intestine was marked already (Schell et al., 1983). However, any discussion of the possible causes of such similarity in this article is absent. If it is granted that the stoichiometry of transport of glucose-sodium and glycinesodium through corresponding transporters are identical, this suggests that the structure of glycine and glucose transporters includes sodium transporters of the same type. If this is the case, then the maximal currents for glycine and glucose transporters should agree for each preparation separately. In actuality, correlation of SCC changes

210€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

on glucose and glycine has been described above. Moreover, since Kt for glucose is equal to 4.3 mM (procedure of double reciprocal coordinates) (Metelsky, 1987, 1992) in the presence of 10 mM of glucose the value of its stimulating effect is equal (10 mM /(10 mM + 4.3 mM)) to ~70% from the maximal (Chapter 6). Kt for glycine is equal to 40 mM; therefore, in the presence of 40 mM of glycine the value of its stimulating effect is equal to 50% (40 mM /(40 mM + 40 mM)) from the maximal. If the structure of glucose and glycine transporters in actuality includes the same type of sodium transporters, then the maximal values of effects of sugar and amino acid should be equal. It follows here from immediately that the theoretical slope of the straight line describing the correlation of glycine and glucose effects should be equal to 50%/70% = 0.71. The latter value is consistent with the coefficient of linear regression 0.68 for SCC responses to these nutrients (see above).

11.3.3. Hypothesis about the parallel multi-pathway cotransporter for nutrients Finishing his fundamental review on amino acid transport across mammalian intestinal and renal epithelia and discussing the issue about how glycine, proline, and the β-amino acids cross the basolateral membrane, the author (Broer, 2008a)€ raises some very interesting high-priority and reasonable issues without the solving of which it is difficult to understand this matter: “Do epithelial transporters form complexes in the membrane? Are they held in place by scaffolding proteins?” Scaffoldings are an important family of scaffolding proteins that assemble a variety of cellulases into the so-called cellulosome, a microbial extracellular nanomachine for cellulose adhesion and degradation. These proteins anchor the microbial cell to cellulose substrates, which makes their connecting region likely to be subjected to mechanical stress. Scaffoldings are noncatalytic structural proteins of the cellulosome, a multienzyme, cell-surface complex required for adhesion and degradation of crystalline cellulose, a particularly recalcitrant substrate. Scaffolding proteins act as a molecular Lego, binding a number of cellulases through its type I cohesin (cohesin I) modules to spatiotemporally regulate the efficiency of the entire enzymatic cascade (Valbuena et al, 2009). Therefore, we consider the issues raised above (Broer, 2008a) to be very accurate and timely. And in turn, we can confirm there importance by the following facts and considerations. The presence of a strong link between SCC changes on glycine and glucose in each preparation points to the fact that glucose and glycine transporters may be incorporated in one quaternary structure (perhaps with the help of scaffolding-like or tetraspaninlike proteins) with the same type of sodium transporters. This is rather surprising because the transport system for glycine (as for other amino acids) is pretty much

Chapter 11. Transport systems for amino acids€€€€€211

duplicated. So, there are four different transporters for glycine, proline/hydroxyproline, namely, 1) a common transporter for all three amino acids in the kidney, 2) a common transporter for all three amino acids in the intestine, 3) a specialized transporter for glycine, and 4) a specialized transporter for proline/hydroxyproline. It should be mentioned that a significant fraction of proline and glycine transport in both kidney and intestine is mediated by the neutral amino acid transporter B0AT1 (SLC6A19). As a result, five transporters contribute to the transport of these three amino acids. The common transporter for all three amino acids in the intestine is the proton amino acid transporter PAT1 (Broer, 2008a). By taking into account the existence of a strong relationship between Na+-dependent transport of glycine and glucose (see above), one can offer two types of such transporters: two separate two-pathway transporters (sodium-glucose and sodium-glycine). There are direct evidence in favor of the two-pathway parallel model, such as Na+ and galactose entering into a large hydrophilic cavity together then being transferred via two differ rent pathway (see 9.4.1.) (Faham et al, 2008) or the three-pathway transporter (sodium-glucose-glycine). Because the idea of spatiotemporally complexes of transporters (used by Ugolev (Ugolev, 1972) and discussed so far (Broer, 2008a)) has received strong support in the form of detection of scaffoldings, hypothesis of the existence of quaternary structures of transporters does not seem quite incredible. Therefore, the scaffolding protein PDZK1 provides spatiotemporal coupling of cAMP transporter to CFTR chloride channel function in the gut epithelia (Li et al, 2007). The latter, three-transporter type of transporters seems more probable. If all three transporters (for Na+, glucose and glycine) can unite in a single structure, then there should be “competitive relations” between SCC changes on glycine and glucose for sodium ions passing through its transporter. That conclusion is supported by experiments using the addition of glucose and glycine on backgrounds of glycine and glucose, respectively (Metelsky, 1987, 1992) (Tab. 34). On a background of glucose a response to glycine decreases by 8.0 μA/cm2, and on a background of glycine a response to glucose decreases by a similar value (11.5 μA/cm2); but, at the same time, the first responses to 10 mM of glycine are less by a factor of four than responses to 10 mM of glucose! It is not likely that development of the response to the second addition is inhibited because of a depolarization of a brush-border membrane followed by the first one, since the depolarization of a membrane in this case is insignificant (Оkadа et al., 1977; Оkadа, 1979). It is also unlikely that the work of sodium transporters of the glycine transporter with the rate 20% (10mM/(10 mM + 40 mM)) (Chapter 6) of the maximal one can cause the same changes of a membrane potential or of a chemical gradient of sodium as switching on of sodium transporters of the glucose transporter for the near maximal rate (70%). However, switching on of the smaller part of sodium transporters of the glycine transporter (20%) and the greater part of sodium transporters of the glucose transporter (70%) suppresses the SCC response to the addition, respectively, of glucose and glycine with a close efficiency. Hence,

212€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

the competition between glucose and glycine occurs for sodium passing through the common transporter or for interaction with the sodium transporter. The above viewpoint is in agreement with the universally accepted one that the competition between amino acids and sugars in their transport occurs because of a competition for a gradient of electrochemical potential of sodium on a brush-border membrane or even for a membrane potential only (Rose, Schultz, 1971; White, Armstrong, 1971). In actuality, sodium transport through its transporter will depend on the value of its gradient of electrochemical potential. The time-dependences of SCC responses to the addition of glucose and glycine are the same: they are increased and decreased simultaneously (Metelsky, 1987). It is difficult to explain these data if we suppose that the induction of additional sodium transport in the presence of glycine or glucose occurs due to two separate two-transporter molecular machines. However, if the work of glucose, glycine, and sodium transporters is probably coupled (maybe due to scaffolding-like protein) as is the case of coupling of cAMP transporter to CFTR chloride channel function in the gut epithelia (Li, 2007), the above data are easily explainable (Metelsky, 1990b, 2007a). In actuality, if permeability of the common sodium transporter for some reason increases or decreases with time, it will result in a corresponding simultaneous increase or decrease in SCC changes on glycine and glucose. It should be mentioned that a significant fraction of proline and glycine transport in intestine is mediated by the neutral amino acid transporter B0AT1 (SLC6A19) (Broer, 2008b). Na+ -dependent transport of glucose in intestine is mediated by SGLT1. Therefore we must assume that both the transporter B0AT1 and SGLT1 during their operation in the small intestine must somehow interact (may be due to scaffoldings- like protein?). It would not be surprising if it turns out that the enzymes formed a quaternary structure with scaffoldings can influence each other’s work. This is a risky assumption, it is contrary to some known facts. But as it was pointed above some transporters may be united in such spatiotemporal complexes (Li, 2007) But if we admit that this assumption is correct, we can assume that in the case of prolonged transportation of one nutrient, transport of another one can be stimulated. This prediction is supported by experiments in situ (Metelsky, 1987, 1992). The preincubation of intestine segments with glycine result in substantial growth of SCC changes (through a preparation obtained from that intestine segment) on glucose. Similarly, upon preincubation of intestine segments with glucose, SCC responses to glycine (Tab. 35) are considerably increased. These results can be explained in the following way (Metelsky, 1987, 1992, 2007a). Cycling of transporter proteins between intracellular storages and a plasmatic membrane seems to be a widespread process (Dahl et al., 1981; Flagg-Newton et al., 1981; Loo et al., 1983). This way the transport of glucose transporters from a Golgi complex on the adipocyte membrane transition under the action of insulin was revealed (Kоnо et al., 1982; Cushman et al., 1984; Kono, 1984). Additional sodium transporters can appear in an apical

Chapter 11. Transport systems for amino acids€€€€€213

membrane of enterocytes under the action of some agents such as the antidiuretic hormone (Lewis, 1983). One is inclined to think that such a cycle occurs for all transporters of a multi-pathway cotransporter —for glucose, glycine, and sodium. In actuality, it is a well-known phenomenon of up-regulation of glucose transport in response to the increased loading by carbohydrates (Philpott et al., 1992; Kellet, Brot-Laroche, 2005). Upon preincubation with glycine, part of the glycine transporter degrades (Metelsky, 1992); the “damaged” glycine transporter can remain in the structure of the multi-pathway transporter or dissociate from it. The glucose transporter of the transporter meanwhile is not used, and therefore remains intact. With use of such “damaged” glycine transporters, a constant rate of glycine transport is maintained due to delivery of new transporters to a brush-border membrane. Owing to fusion with a brush-border membrane of additional transporters some time after the beginning of preincubation with glycine, the SCC response to glucose increases. Apparently, in vitro the nutrient-dependent sodium transport can be “damaged” in the process of use (Metelsky, 1987, 1992). When in a mucosal solution glucose is ever present, the part of the multi-pathway transporter carrying out transport of glucose constantly works, and the part carrying out transport of glycine remains inactive (Fig. 20). Testing the state of these multi-pathway transporter components by adding an additional quantity of glucose or glycine to a solution has revealed that, in actuality, the value of an additional stimulating glucose effect relative to a stimulating effect of glycine gradually decreases down to zero. It is significant that the disappearance of such relative effect of glucose is not caused by an exhaustion of power resources of a cell, since responses to glycine at this time are maintained.

11.4. Final remarks So, a stimulating effect of glycine on active sodium transport has been analyzed and compared to a stimulating glucose effect. It was shown that in cells with suppressed processes of respiration and glycolysis, the dissociation of active transport and stimulating effect of glycine on the SCC can be observed. By using these data, the cotransporter model (as in the case with glucose, see 9.4.3) was proposed, and its validity was demonstrated. This model consist of transporters located side by side for glycine and sodium and gate mechanisms. If the parallel model of Na+-dependent transport is valid and at each Na+-dependent nutrient transporter, regardless of its localization in the tissues and its specificity in the architecture of the core of the cotransporter protein, these models must be similar. Surprisingly the architecture of the core of the Na+/glucose cotransporter protein is similar to that of the leucine transporter (LeuT) from the family of Na+-dependent neurotransmitter; members of the family include Na+/glycine, Na+/Cl-/γ-amino-butyric acid (GABA), and Na+/serotonin cotransporters.

214€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

In addition, the Na+-coupled benzyl-hydantoin transporter, Mhp1, from Microbacterium liquefacients, also shows a core structure similar to that of the sugar and neurotransmitter transporters (Weyland et al., 2008).

Chapter 12. Mechanisms of peptide transport in the small intestine€€€€€215

Chapter 12. MECHANISMS OF PEPTIDE TRANSPORT IN THE SMALL INTESTINE Peptides (from the Greek πεπτίδια, «small digestibles») are short polymers formed from the linking, in a defined order, of α-amino acids. In GIT, peptides are degraded into amino acids. Chemical breakdown begins in the stomach and continues in the small intestine. Proteolytic enzymes, including trypsin and chymotrypsin, are secreted by the pancreas and cleave proteins into smaller peptides. Carboxypeptidase, which is a pancreatic brush border enzyme, splits one amino acid at a time. Aminopeptidase and dipeptidase free the end amino acid products. The intestinal proton-coupled€ oligopeptide transporter PEPT1 mediates the transport of all possible di- and tripeptides but not that of free amino acids (Boll et al. 1994; Fei et al. 1994; Liang et al. 1995; Mackenzie et al. 1996; Daniel, 2004).€ PEPT1 (gene SLC15A1) is the prototype member of the proton-coupled oligopeptide transporters superfamilly (Daniel, 2004) and is expressed mainly in the brush-border membrane of enterocytes, renal proximal tubular cells, and bile duct epithelial cells (Daniel & Kottra, 2004).€ Transport by hPEPT1 is electrogenic, proton-coupled, and voltagedependent (Mackenzie et al. 1996). Membrane topology model of human hPEPT1 may be described in the following way (Liang et al. 1995): this isoform is composed of 708 amino acid residues and predicted to contain 12 membrane-spanning domains, with a large extracellular loop between the transmembrane regions 9 and 10 and with amino and carboxy termini facing the cytosol. Essential knowledge about the molecular mechanisms of H+/dipeptide transport was obtained€ in studies€ of heterologous expression systems (e.g., PEPT1 expressed in Xenopus oocytes). The most important results can be summarized as follows (SalaRabanal et al, 2006): (1) the activity of PEPT1 is electrogenic, with negative membrane voltages increasing dipeptide transport; (2) negative membrane voltages increase the affinity of PEPT1 for protons and increase the rate of reorientation of the transporter from inward-facing to outward-facing conformations; (3) PEPT1 does not interact with Na+ ions, and transport activity is independent of Na+; (4) the kinetic characteristics of H+/dipeptide cotransport are very similar to those for Na+/glucose cotransport; (5)

216€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

there is an inhibition of dipeptide transport by PEPT1 at low pH, and this has been proposed as a competition/interference between protons and the substrate; and (6) the turnover number of the human PEPT1 (for a typical substrate glycylsarcosine) is 135/s. The discovery of membrane digestion (Ugolev, 1960a, 1960b, 1972) raises the question as to how the products of its hydrolysis are absorbed. It is now universally accepted that the absorption, for example, of dipeptides, is carried out by a specific mechanism; of importance is the fact that it is carried out sometimes more effectively than the absorption of the sum of corresponding amino acids. Some data point to faster absorption from a mix of monomers in comparison with absorption of a corresponding dipeptide (Ugolev, Kushak, 1966; Ugolev, 1972; Matthews, 1975; Matthews, Рауnе, 1980). The absorption of nutrients, in particular amino acids and dipeptides, can be carried out coupled with sodium or a proton (Matthews, 1975; Matthews, Рауnе, 1980; Hoshi, Нimucai, 1982). In both cases, on addition of such nutrients in a mucosal (washing a mucosa) solution through an epithelial sheet, an additional electric current should flow which can more conveniently be recorded by the SCC technique. In studies (Metelsky, 1989a, 1990a) where values of SCC change in response to the addition of a nutrient in a mucosal solution were used as the characteristic of such nutrient absorption, it was found that depending on the рН of the mucosal solution the SCC response to the addition of dipeptide (or efficiency of dipeptide absorption) can be more or less than the SCC response to an equimolar mix of corresponding amino acids (or efficiency of absorption from an amino-acids mix).

12.1. The ph-dependence of peptide effects on the SCC In a рН range of 5.5 to 8.5, the following characteristics (Metelsky, 1989a) have been studied: (1) SCC responses to dipeptides, (2) SCC responses to an equimolar mix of corresponding (for this dipeptide) amino acids, and (3) SCC responses to individual amino acids. According to the universally accepted views (Ugolev, Kushak, 1966; Ugolev, 1972; Matthews, 1975; Matthews, Рауnе, 1980), at рН 8.5 SCC responses to 10 mM of glycyl-L-leucine, glycyl-DL-methionine, or glycyl-L-alanine are higher then SCC responses to a mix of amino acids (Tab. 36). According to the concept of more effective transport of dipeptides (Ugolev, 1972; Matthews, 1975; Matthews, Рауnе, 1980), the same correlation should occur at any рН. However, it was unexpectedly found that at рН 5.5 SCC responses to a mix of amino acids are a little higher then SCC responses to dipeptides (Tab. 36). The observed phenomenon is characteristic of not only glycine-containing dipeptides, but also of DL-alanyl-DL-asparagine (Tab. 36) and can be demonstrated in each experiment. The SCC responses to nutrient monomers depend on рН changes in a different manner (Tab. 37) (Metelsky, 1987, 1989a). With an increase in рН, responses to DL-methionine and glycine decrease, responses to glucose and L-leucine remain

Chapter 12. Mechanisms of peptide transport in the small intestine€€€€€217

Table 36. Responses of short circuit current (mA/cm2) across rat small intestine on addition of dipeptides or an equimolar mixture of corresponding amino acids at different pH values (Metelsky, 1989a). Nutrients 10 mM Glycine + 10 mM DL-Methionine 10 mM Glycil-DL-Methionine 10 mM Glycine + 10 mM L-Leucine 10 mM Glycil-L-Leucine 10 mM Glycine + 10 mM L-Alanine 10 mM Glycil-L-Alanine 20 mM Glycine + 20 mM DL-Leucine 20 mM Glycil-DL-Leucine 20 mM DL-Alanine + 20 mM DL-Asparagin 20 mM DL-Alanil-DL-Asparagin 5 mM Glycine + 5 mM L-Tyrosine 5 mM Glycil-L-Tyrosine

SCC response, A, µA/cm2 рН = 8.5 рН = 5.5 8.8 ± 3.0 (6) 13.8± 3.3 (7) 11.5 ± 2.1 (9) 7.6 ± 1.2 (10) 10.3 ± 2.7 (5) 11.0 ± 3.5 (8) 15.1 ± 4.8 (6) 9.5 ± 1.8 (10) 15.0 ± 1.9 (5) 14.6 ± 2.4 (4) 21.3 ± 7.7 (7) 13.4 ± 1.8 (5) 17.0 ± 6.5 (3) 12.5 ± 6.0 (4) 17.5 ± 2.5 (4) 7.8 ± 0.3 (4) 15.4 ± 3.2 (3) 8.6 ± 1.6 (4)

constant, and responses to L-alanine and L-histidine tend to increase. At рН 5.5 omitting sodium from washing solutions results in a considerable decrease in SCC responses to amino acids (Hoshi, Нimucai, 1982). Unexpectedly, we found (Metelsky, 1987, 1989a) that at рН 8.5, after omitting sodium, SCC responses to amino acids change its sign on opposite (Tab. 37). Responses to glucose remain, on averTable 37. Effect of sodium omission from washing solutions on responses of short circuit current across rat small intestine upon 10mM nutrient monomers addition to mucosal solutions at different pH values (Metelsky, 1989a). Nutrient D-Glucose Glycine L-Alanine L-Leucine DL-Methionine L-Hystidine

рН 5.5 8.5 5.5 8.5 5.5 8.5 5.5 8.5 5.5 8.5 5.5 8.5

SCC response, A, µA/cm2 + Na+ - Na+ 20.5 ± 4.4 (18) 1.4 ± 0.3 (9) 19.5 ± 3.4 (18) 0.4 ± 0.6 (9) 6.9 ± 1.0 (18) 1.0 ± 0.5 (8) 3.9 ± 0.8 (18) -1.4 ± 0.7 (8) 9.0 ± 0.9 (4) 1.7 ± 0.3 (8) 12.0 ± 1.6 (5) -0.1 ± 0.7 (8) 10.9 ± 2.0 (5) 3.3 ± 0.7 (4) 11.3 ± 2.6 (5) -2.9 ± 1.6 (3) 11.4 ± 2.1(6) 6.2 ± 2.2 (4) 2.0 (2) 2.85 (2)

218€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

age, positive (0.4 ± 0.6 μA/cm2), though responses in the opposite direction were sometimes observed (Metelsky, 2007a). In studies of pH-dependence of effects of various dipeptides, it was found that unlike glycyl-DL-methionine, glycyl-L-leucine, and glycyl-L-alanine, SCC responses to glycyl-glycine and carnosine (β-alanil-L-histidine) with рН tend to reduce. With the omission of sodium from solutions (рН 8.5), SCC responses to glycyl-L-alanine (р <0.1) and glycyl-L-leucine (р <0.05) decrease, but nevertheless remain at a high level. Of importance is the fact that with an increase in рН SCC responses to two indicated dipeptides tend to reduce (Metelsky, 1989a). These results are consistent with the data which were obtained by using radiotracer uptake and electrophysiological methods on hPEPT1s expressed in Xenopus oocytes (Sala-Rabanal et al, 2006), where it was shown that Gly-Sar-evoked currents were unaffected by external Na+ and were significantly attenuated at pH 7.5.

12.2. Final remarks In studies of the effect of sodium removal from washing solutions on SCC responses upon addition in a mucosal solution of dipeptides, amino acids, and glucose at acid (5.5) and alkaline (8.5) рН, it was possible to observe some important effects (Metelsky, 1989a). In the study quoted above the variability of the relative efficiency of dipeptide transport has obtained a satisfactory interpretation. The researcher (Metelsky, 1987, 1989a) working at рН 8.5 will observe greater efficiency of dipeptide transport, since SCC responses to dipeptides are higher than SCC responses to a mix of appropriate amino acids. On the contrary, at рН 5.5 the reverse situation takes place—SCC responses to a mix of amino acids are higher than SCC responses to corresponding dipeptides. Thus, the researcher working at рН 5.5 will find that the absorption from a mix of amino acids is more effective than that from a solution of dipeptides. From the available data (Metelsky, 1989a) it follows that at neutral рН, the proportion between efficiency of absorption from a solution with dipeptide and from a solution containing a mix of amino acids can be any. Hence, the widespread approach to determining dipeptide transport efficiency by comparing (at neutral рН) rates of absorption from a solution with a mix of amino acids and from a solution containing corresponding dipeptide only is incorrect. Some researchers (Rubino et al., 1971; Ugolev, 1972; Matthews, 1975; Matthews, Рауnе, 1980; Hoshi, Нimucai, 1982; Kushak, Basova, 1988) have considered that transport of dipeptides through an apical membrane of enterocytes may proceed by two pathways: as the transport of intact dipeptides coupled with a proton (Ihara et al., 2000; Sala-Rabanal et al, 2006) and as the transport of the monomers (amino acids)

Chapter 12. Mechanisms of peptide transport in the small intestine€€€€€219

which have formed as a result of membrane hydrolysis of dipeptides, coupled with sodium. According to Winckler et al. (1999), Gly-Glu and Gly-Sar in rat intestines are transported by means of the transporter similar to the РерТ transporter which makes a significant contribution to the absorption of amino acids. The kinetic model of the mechanism of H+–oligopeptide cotransport was improved (Sala-Rabanal et al, 2006) with the assumptions that ligand binding to hPEPT1 is ordered, with H+ binding before the substrate, and cotransport is a series of conformational changes induced by ligand (H+ and dipeptide) binding and membrane voltage. Under such conditions, the authors were able to estimate the turnover rate for this Gly-Sar transporter; it was equal to 130 s−1. After omitting sodium, the рН-dependence of stimulating effect of dipeptides considerably changes (Metelsky, 1989a). It was found that the Na+-dependent component of a response increases with рН and the H+-dependent component decreases. The driving force for the H+-dependent component of dipeptide transport of glycyl-sarcosine is the gradient of a proton on a brush-border membrane (Scharrer et al., 1999; Sala-Rabanal et al, 2006). These observations are in agreement with the data obtained in studies on the stimulating effect of dipeptide on transmural potential difference through a toad small intestinal wall (Аbе et al., 1987). It is common knowledge that aminopeptidase activity of an enterocyte brush border reaches its maximum at рН ≈8 (Robinson, Shaw, 1960; Ugolev, 1972). Therefore, we€ with (Аbе et al., 1987) believe that increasing the Na+-dependent component of stimulating effect of easily hydrolyzed dipeptides with рН is caused by membrane digestion (Metelsky, 1989a, 2007a). If this is the case, then the stimulating effect of poorly hydrolyzed dipeptides with рН should behave like well hydrolyzed dipeptides in a solution without sodium (i.e., should decrease). Such is indeed the case: SCC responses to glycyl-glycine and carnosine decreased with рН. In a study by Аbе, Hoshi, and Tajama (1987), the stimulating effect of glycyl-glycine with рН slightly increased, and in a study by Metelsky (1989a) it slightly decreased. Such distinctions, perhaps, are caused by the fact that in a toad small intestine glycylglycine is hydrolyzed easier than in a rat small intestine. It is agreed that transport of monomers is basically Na+-dependent (Hoshi, Нimucai, 1982) though some dependence on рН is peculiar also (Аbе et al., 1987; Diaz et al., 2000). In Metelsky (1989a), modest influence of рН on SCC responses to glucose (Tab. 37) is shown as well. The clearly defined pH-dependence of the stimulating effect of L-alanine, L-leucine, glycine in the absence of sodium was detected also, which points to the fact that participation of protons in energization of aminoacid transport is possible. Besides, in a study by Metelsky (1989a), it was discovered that at рН 8.5 in a sodium-less medium in the presence of amino acids, the SCC is directed not in the usual direction (from a mucosal solution to serosal), but in the opposite one. We believe (Metelsky, 2007a) that such a current can be generated by potassium ions. Potassium absorption is due not only due to diffusion through a para-

220€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

cellular pathway, but also to the active transport mechanism through a transcellular pathway (Inagaki et al., 2002). Such an assumption is in agreement with observations concerning decrease in intracellular activity of potassium in the presence of glucose (Lee, Armstrong, 1972) and with increase in an output of potassium from isolated enterocytes in the presence of amino acids (Hoshi, Нimucai, 1982). Apparently, the potassium leaving isolated enterocytes under conditions of an intact epithelial sheet is capable of generating an electric current through it. Perhaps the potassium component of a stimulating effect of amino acids revealed by authors cited above has a physiological value (Metelsky, 2007a).

Chapter 13. Gerontological aspects of absorption and membrane digestion ...€€€€€221

Chapter 13. Gerontological aspects of absorption and membrane digestion in the small intestine It has long been known that aging is accompanied by changes of function in the GI tract, in particular, of absorption and membrane digestion (Valenkevich, Ugolev, 1984; Drozdowski, Thomson, 2006). However, data regarding the influence of aging on absorption of nutrients and thickness of unstirred layer of a fluid near an intestinal surface are contradictory (Penzes, 1974a, 1974b; Penzes, Boross, 1974; Hollander, Dadufaza, 1983; Holt et al., 1984; Navab, Winter, 1988; Drozdowski et al., 2003). One of the possible reasons for these contradictions is that in the overwhelming majority of studies the conclusions about increase or decrease in Na+-dependent absorption of a nutrient were drawn without consideration of kinetic parameters of absorption. At the given concentration of nutrient reduction in rate of its absorption may be caused both by reduction of number of corresponding transporters in an enterocyte membrane (Аmax) and by decrease of transporter affinity to this nutrient (Metelsky, 1992, 2004a).

13.1. Kinetic parameters of Na+-dependent absorption of nutrients in young and old animals It turns out that initial electrophysiological parameters of intestine preparations of young and old rats have already been distinguished (Metelsky, 2004a). So, specific electric resistances of intestinal walls in young and old animals are significantly distinguished: 26.0 ± 0.8 and 12.2 ± 2.7 Ohm*cm (p <0.001), while initial potential differences on an intestinal wall of young and old rats are distinguished only slightly: 1.77 ± 0.36 and 1.54 ± 0.4 mV, respectively. The basal SCC in young and old rats is equal to 24.0 ± 2.95 and 23.6 ± 3.1 μA/cm2, respectively. The results obtained on measurements of A, a, and β (see Chapter 6) are presented in Tab. 38. In old animals, the amplitude of SCC response to nutrients tends to be more than that in young animals, and for L-leucine such a distinction is significant (p <0.02). In

222€€€€€Transport phenomena and membrane digestion in small intestinal mucosa Table 38. Parameters of short circuit current responses across small intestine in old (22 months) and young (4.5 months) rats on nutrient addition to mucosal solution (Metelsky, 2004a).

Nutrient, mM

D-Glucose, 10 mM Maltose, 5 mM L-Alanine, 10 mM L-Leucine, 10 mM Gly-Lα-Ala, 2 mM D-Glucose, 10 mM Maltose, 5 mM Sucrose, 10 mM L-Alanine, 10 mM L-Leucine, 10 mM Gly-Lα-Ala, 2 mM а

Relative initial rate of SCC response development, α*102 s-1 Old animals 45.2 ± 12.1 (5) 3.17 ± 0.63 (5) 44.7 ± 14.2 (5) 2.60 ± 0.47 (5) 38.7 ± 9.5 (5) 1.42 ± 0.19 (5) 1.97 ± 0.30 (5) 22.0 ± 2.66а (5) 25.6 ± 8.4 (5) 1.16 ± 0.23 (5) Young animals 20.0 ± 3.8 (13) 2.13 ± 0.17 (13) 22.7± 4. 0 (13) 2.09 ± 0.19 (13) 17.1 ± 3.1 (13) 1.34 ± 0.16 (13) 18.5 ± 3.6 (13) 1.72 ± 0.22 (13) 12.6 ± 1.98 (13) 2.47 ± 0.27 (13) 14.7 ± 2.35 (13) 1.45 ± 0.15 (13)

SCC response, A, µA/cm2

Relative initial rate of SCC response wash-out, β*102 s-1 0.96 ± 0.09 (5) 0.73 ± 0.14 (5) 0.55 ± 0.09 (5) 0.51 ± 0.09 (5) 0.59 ± 0.14 (5) 0.83 ± 0.11 (13) 0.80 ± 0.12 (13) 0.82 ± 0.11 (13) 0.63 ± 0.07 (13) 0.59 ± 0.06 (13) 0.75 ± 0.09 (13)

p < 0.02 versus young animals. For details, see text.

old rats, the relative initial rate of response development α is maximal for glucose, and in young ones is maximal for L-leucine. The relative initial rate of wash-out of the SCC response β to glucose is maximal both in young and old animals. The parameters α and β are much less variable (root-sum-square uncertainty/arithmetic mean) from experiment to experiment than values of responses (A); this fact has engaged our attention. The kinetic parameters and values of thickness of unstirred layers of a fluid (δ) calculated according to our equations (see Chapter 6) are shown in Tabs 38-40. According to data gathered in experiments in rats (Penzes, 1974a; Metelsky, 2004a), the maximal rates of absorption (Amax) for L-alanine and L-leucine increase considerably with age (Tab. 39). As this occurs, according to Penzes (1974a), Kt for alanine with aging increases from 7.2 to 35.5 mM. According to Metelsky (2004a) in young and old rats these parameters are equal to 16.8 and 17.5 mM, respectively (i.e., they do not change). Maximal and minimal values of Kt for leucine in Penzes (1974a) are equal to 18.2 and 2.6 mM. According to Metelsky (2004a), magnitudes of Kt for leucine are also in the same range—10.4 and 11.6 mM. However, while Kt for indicated amino acids in the Penzes (1974a) study increases considerably with age, in the Metelsky (2004a) study Kt for alanine and leucine remains constant (Tab. 40). The maximal values of response (Аmax) to glucose, as that to amino acids, are considerably higher in old rats than those in young rats (Tab. 39). Kt for glucose varies only slightly with aging (Tab. 40).

Chapter 13. Gerontological aspects of absorption and membrane digestion ...€€€€€223

Table 39. Maximal short circuit current responses (Amax) across small intestine of old (22 months) and young (4.5 months) rats on nutrient addition to mucosal solution (Metelsky, 2004a). Amax, µA/cm2 Old animals Young animals а

D-Glucose 10 mM 101.0 ± 15.5 b (5) 51.1 ± 9.7 (13)

Maltose 5 mM 99.0 ± 28.0 (5) 62.4 ± 14.3 (13)

Sucrose 10 mM

91.3 ± 19.9 (13)

L-Alanine 10 mM 103.0 ± 24.0 а (5) 48.4 ± 8.9 (13)

L-Leucine 10 mM 45.5 ± 7.5 а (5) 24.6 ± 3.6 (13)

Gly-La-Ala 2 mM 82.0 ± 15.5 (5) 60.8 ± 10.1 (13)

p < 0.05 versus young animals; b p < 0.02 versus young animals.

Table 40. Transport constants (Kt) for nutrients in old (22 months) and young (4.5 months) rats (Metelsky, 2004a). Kt, mM Old animals Young animals

D-Glucose 10 mM 14.9 ± 2.8 (5) 17.6 ± 2.9 (13)

Maltose 5 mM 12.7 ± 2.3 (5) 16.6 ± 6.2 (13)

Sucrose 10 mM

41.8 ± 5.9 (13)

L-Alanine 10 mM 17.5 ± 2.5 (5) 16.8 ± 1.8 (13)

L-Leucine 10 mM 10.4 ± 0.9 (5) 11.6 ± 1.8 (13)

Gly-La-Ala 2 mM 13.7 ± 3.5 (5) 6.2 ± 1.0 (13)

Unlike monomers, upon absorption of dimers (maltose and glycyl-Lα-alanine) significant increase in Аmax with aging is not revealed, but such tendency is maintained in this case. As this takes place, the affinity of enzyme-transport ensemble for glycyl-Lα-alanine tends to decline, and for maltose to increase. The greatest values of Аmax and Kt for sucrose (in young rats) are 91.3 μA/cm2 and 41.8 mM, respectively. Data on the effect of aging on the thickness of the unstirred layer of a fluid near an intestine surface are also contradictory. In Metelsky (2004a; 2007b; 2007c) (Tab. 41), Table 41. Unstirred layer thickness (δ) at mucosa surface of rat intestine, calculated from short circuit current responses to nutrients in young (4.5 months) and old (22 months) rats (Metelsky, 2004a). Rats Young animals Old animals

D-Glucose 10 mM 324 ± 15 (13) 273 ± 13 (13)

Maltose 5 mM 263 ± 20 а (13) 260 ± 22 (13)

Sucrose 10 mM 315 ± 21 (13)

L-Alanine 10 mM 287 ± 15 (13) 310 ± 21 (13)

L-Leucine 10 mM 240 ± 12 c,d (13) 265 ± 18 (13)

p < 0.05; b p < 0.01; c p < 0.001 versus glucose; d p < 0.05 versus alanine.

а

Gly-La-Ala 2 mM 258 ± 17 b (13) 293 ± 28 (13)

224€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

according to the data of Hollander & Dadufaza (1983), the layer thickness determined by glucose decreases with rat age. On the contrary, the layer thickness determined with amino acids and dipeptide tends to increase with aging, which is in agreement with results provided by Thomson (1979). In the Penzes (1974a) study, the layer thickness decreases from 318 to 268 μm with age. The values obtained by Metelsky (2004a) (Tab. 41) are in good agreement with those given above.

13.2. Final remarks The electric resistance of an intestinal wall decreases more than twice with aging. The major contribution to electric resistance of an intestinal wall brings the resistance of an epithelial sheet (Ugolev et al., 2001; Metelsky, 2007a). As resistance of epithelial tissues is determined mainly by resistance of its tight junctions, and the height of villi does not vary or decrease with aging (Penzes, Boross, 1974; Hollander, Dadufaza, 1983), we reach the conclusion that with aging tight junctions between enterocytes become more permeable (Weber et al., 2002; Drozdowski, Thomson, 2006). It is agreed that thickness of the unstirred layer is commensurate with the height of villi (Metelsky, 1987). Data have been published showing that with aging this parameter increases in a rabbit (Penzes, Boross, 1974; Navab, Winter, 1988) and in a human (Holt et al., 1984), or decreases (Hohn et al., 1978; Hollander, Dadufaza, 1983), or remains constant in rats (Penzes, Boross, 1974) and in humans (Lipski et al., 1992). As it follows from Metelsky’s (2004a) study, δ determined by glucose decreases, determined by maltose tends to decrease, and determined by amino acids and a dipeptide tends to increase with age. Such distinctions can be caused by the fact that nutrient molecules of the latter group, unlike molecules of sugars at neutral рН, exist in the form of zwitterion. It is common knowledge that microvilli of intestines are covered by a glycocalix having an electric charge (Komissarchik, Ugolev, 1970; Ugolev, 1972). However, as the height of villi in a rat intestine does not change with age (Holt et al., 1984), distinctions between δ (measured on absorption of nutrients) for animals of the former and latter groups with aging are caused, apparently, by changing of the electric charge of glycocalix and/or of an apical membrane. Ability of an intestine to absorb nutrient monomers (glucose, amino acids) with aging increases by approximately 100%, and ability to utilize dimers (a maltose, dipeptide) from an intestinal lumen tends to increase by 30–50% (Tab. 39). This points to the fact that in an enterocyte membrane of old animals the number of transporters for each of the analyzed nutrients sharply increases. As this occurs, the affinity of transporters for glucose, maltose, alanine, and leucine practically does not change with aging. While in old rats the coefficient of correlation Kcorr between a transport constant Kt and maximal response Аmax for all five nutrients is equal to 0.796, in young ani-

Chapter 13. Gerontological aspects of absorption and membrane digestion ...€€€€€225

mals such a correlation is absent (Kcorr = 0.022). Hence, the fewer transporters for a given nutrient there are in an enterocyte membrane of an old animal, the more their affinity to this nutrient. Perhaps that observation reflects some adaptable changes in an animal with aging. Thus, in a rat small intestine, there are essential functional changes with aging. The ability to absorb nutrient monomers increases, and that to absorb dimers tends to increase, probably, because of€ a rise in the number of transporters. Data gathered in experiments using glucose (Metelsky, 2004a) are consistent with this (Thompson et al., 1988). As both groups of animals (Metelsky, 2004a) obtained the same rations, this means that an old rat adapted thus for the raised concentration of monomers in an intestinal lumen. It has long been known that monomers may be formed at cavital digestion. Hence, additional transporters in a brush-border membrane appear, apparently, in response to increase in a level of cavital digestion, resulting in an increase in nutrient monomer concentration in an intestinal lumen. Upon aging, the ratio between cavital and membrane digestion shifts in favor of the former, though at the same time membrane digestion tends to increase its intensity (disaccharide and dipeptide). So, in studies in young and old rats, it is shown (Metelsky, 2004a) that the maximal SCC response through a small intestinal wall upon addition of monomers of the nutrients, characterizing the number of transporters for nutrients and sodium in a brush-border membrane, in an old animal is approximately twice that in a young one, with similar affinity of transporters to transported molecules. In the case of absorption of glycyl-La-alanine, the tendency to increase in a transport constant for this dipeptide with aging points to change of properties (decrease of affinity) of digestivetransport ensemble. It can be inferred that the GI tract of old animals is adapted in such a manner that cavital digestion starts to play a greater role. With aging, thickness of the unstirred layer of the fluid adjoining an intestinal mucosa, determined on SCC responses to glucose, decreased.

226€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

Chapter 14. Intestinal absorption at satiety, fasting and refeeding€€€€€227

Chapter 14. Intestinal absorption at satiety, fasting and refeeding The finding of physiological distinctions among such fundamental states of an organism as fasting, satiety, and refeeding has until now been a problem. This problem is closely related to the food behavior and appetite whose regulation is carried out with participation of CNS, humoral factors, and also some environmental factors (for example, temperature). A few basic hypotheses have been suggested for eating behavior: glucose-static, amino acid-static, metabolic, lipostatic, etc. (Ugolev, 1972; Lytle, 1977; Ugolev, Timofeeva, 1989). It is believed that blood levels of glucose and amino acids mainly determine the balance between two processes, namely: (a) an entrance of these nutrients from the intestine, and (b) their catabolism and excretion from the organism. The daily production of glucose due to gluconeogenesis averaged 10–20% of total glucose content in an organism (Sokoloff, Fitzgerald, 1977). By means of the above approach one can try to solve the problem of whether any distinctions exist between states of satiety, refeeding, and fasting on a level of one of the first stages of assimilation of a food—mechanisms of membrane digestion and absorption in a rat small intestine. To achieve a state of refeeding, fasting rats were fed boiled low-fat beef for 30 minutes, then food debris was removed, and in two hours the animals was taken for the experiment (Metelsky, 2005a). During the 30 minutes the rat could eat meat in the amount of 4% of its weight. For two days of starvation the weight of rats decreased by ~17%, and for five days by ~23%. In studies of initial electric parameters (Metelsky, 2005a), the electric resistance of preparations from medial segments after two-day starvation tends to decrease, and after five-day starvation this parameter significantly decreases by 38% from 30.9 ± 1.4 down to 19.2 ± 2.5 Ohm*cm2 in comparison with a condition of satiety (Tab. 42). The data are in agreement with the observation that upon starvation the structure and transport function of intestines are sharply changed (atrophy); in this case, permeability of an intestinal wall for electrolits and macromolecules increases (Yang et al.,

228€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

1999; Ferraris, Carey, 2000). It is of interest that after refeeding following two- and five-day starvation (Tab. 42), the electric resistance is not restored to its initial level. The electric resistance of distal segments after five-day starvation decreases by 59% from 27.8 ± 4.9 down to 12.4 ± 2.3 Ohm сm2, which is in agreement with a study by Ferraris and Carey (2000). Similarly, as in the previous case, the electric resistance magnitude does was not restored after refeeding (Tab. 42). The basal SCC through preparations of medial segments after two- and five-day starvation significantly decreases, from 95.8 ±Â€8.0 (satiety) down to 12.8 ±Â€5.5 and 48.7 ±Â€18.5 μA/cm2, respectively (Metelsky, 2005a). On refeeding after two-day starvation, the basal SCC (unlike electric resistance) has had time to be restored almost up to the satiety level, and after five-day starvation considerably exceeds that—139.0 ± 17.5 μA/ cm2 (p <0.05) (Tab. 42). On distal segments such changes of the basal SCC manifested as tendency only. On medial segments, refeeding after two- and five-day starvation resulted in significant increase in basal SCC (Tab. 42) from 12.8 ± 5.5 up to 80.3 ± 10.8 μA/cm2 and from 48.7 ± 18.5 up to 139.0 ± 17.5 μA/cm2, respectively. There were no significant changes of the SCC in response to addition of nutrients (except for dipeptide glycyl-La-alanine) on medial segments (Tab. 43). Only after two-day starvation SCC responses to glycyl-La-alanine significantly increase from 10.2 ± 2.5 (satiety) up to 17.8 ± 2.4 μA/cm2. With an increase in the duration of starvation from two to five days, SCC responses to dipeptide decrease (practically to a level of satiety) from 17.8 ± 2.4 down to 9.2 ± 3.0 μA/cm2. Hence, the dependence of digestion and absorption rates of dipeptide on the duration of starvation is revealed. A new effect has been discovered on medial segments (Metelsky, 2005a, 2007a). Preliminary experiments have shown the following. If on the same preparation Table 42. Initial electric parameters (resistance – R and basal short circuit current – SCCb) of isolated segments of rat small intestine (Metelsky, 2005a) at satiety, fasting, and refeeding. Segment of small intestine Medial

Distal а

Electric parameters R, Ohm*cm2 SCCb, µA/cm2 R, Ohm*cm2 SCCb, µA/cm2

Fasting 2 days

Fasting 5 days

Refeeding 2 days

Refeeding 5 days

Satiety

25.0±5.4 (7) 12.8±5.5 b (7) 20.0±2.9 (4) 106.3±52.7 (4)

19.2±2.5 b (8) 48.7±18.5a (8) 12.4±2.3а (4) 98.8±29.7 (4)

25.2±4.4 (8) 80.3±10.8d (9) 20.2±2.3 (6) 119.2±32.4 (6)

19.9±2.1b (8) 139.0±17.5a,c (8) 14.7±1.5а (4) 135.0±59.2 (4)

30.9±1.4 (48) 95.8±8.0 (47) 27.8±4.9 (4) 156.2±84.2 (4)

p < 0.05, bp < 0.01 versus satiety. cp < 0.01, dp < 0.001 versus fasting.

Chapter 14. Intestinal absorption at satiety, fasting and refeeding€€€€€229

Table 43. Short circuit current responses (mA/cm2) across isolated medial segments of rat small intestine on different nutrients (Metelsky, 2005a). Nutrient, concentration Glucose, 10 mM Leucine, 10 mM Gly-Ala, 10 mM a

Fasting 2days 5 days 18.0 ± 3.9 21.8 ± 7.1 (7) (8) 10.9 ± 3.7 11.0 ± 3.9 (6) (8) 17.8 ± 2.4а 9.2 ± 3.0 b (8) (6)

Refeeding 2 days 5 days 22.2 ± 2.4 40.9 ± 7.4 (6) (8) 11.5 ± 2.1 17.8 ± 2.6 (4) (8) 9.9 ± 2.9 11.9 ± 1.3 (6) (8)

Satiety 28.0 ± 4.0 (44) 11.7 ± 2.9 (4) 10.2 ± 2.5 (7)

p < 0.05 versus satiety; b p < 0.05 versus 2-day long fasting.

two serial SCC responses to the same nutrient are registered, then the second response can be distinguished (by greater or smaller value) from the first one by ~10%. Unexpectedly, it turns out that on medial segments after refeeding (twoday starvation) the situation is dramatically changed—the second response to the same nutrient (a monosaccharide, amino acid, dipeptide) is always more than the first one, and in the case of glycyl-La-alanine such distinctions are significant (an increase by a factor of 3.4, from 9.9 ± 2.9 up to 33.4 ± 9.7 μA/cm2 (p <0.05)). Thus, for the first time the effect of increase (“buildup”) of SCC response amplitude on the same nutrient was revealed. Perhaps the choice of medial segments for revealing distinctions in absorption between states of satiety, fasting, and refeeding was not optimal. Indeed, it turns out that responses to all studied nutrients in distal segments are much less than in medial segments, which is consistent with results on glucose and valine absorption (Levin et al., 1983) and with the existence in the small intestines of a proximo-distal gradient of absorption and digestion (Ugolev, 1972). In actuality, more certain results on distal segments were obtained after refeeding (Tab. 44). It turns out that states of satiety, fasting and refeeding are sharply distinguished. On distal segments after transition from a satiety state to a fasting state (five days), SCC responses to dipeptide increased from 1.8 ± 0.30 to 5.3 ± 1.4 μA/cm2 (Tab. 44), which is consistent with the dynamics of SCC responses to dipeptide after starvation (two-day) on medial segments (increase from 10.2 ± 2.5 up to 17.8 ± 2.4 μA/ cm2)(Tab. 43). After five-day refeeding (in the cases of glycine and dipeptide, and after two-day refeeding also) SCC responses to all studied nutrients increase significantly in comparison to a state of satiety (Metelsky, 2005a). After refeeding in rats starved for five days, SCC responses to glucose increase by a factor of 4.4 (Tab. 44) from 5.9 ± 2.6 to 25.9 ± 5.9 μA/cm2. We emphasize that the effect of “buildup” of serial SCC responses through a wall of distal segments of an intestine upon addition of any nutrients is absent.

230€€€€€Transport phenomena and membrane digestion in small intestinal mucosa Table 44. Responses of short circuit current (mA/cm2) across isolated distal segments of rat small intestine on different nutrients (Metelsky, 2005a). Nutrient, concentration Glucose, 10 mM Glycine, 10 mM Leucine, 10 mM Gly-Ala, 10 mM a

Fasting 2 days 5 days 9.7 ± 5.5 5.9 ± 2.6 (4) (4) 2.8 ± 0.80 3.8 ± 1.5 (4) (4) 7.4 ± 3.2 9.8 ± 4.0 (4) (4) 6.8 ± 3.2 5.3 ±1.4a (4) (4)

Refeeding 2 days 5 days 11.9 ± 3.8 25.9 ± 5.9 b,d (6) (4) 6.2 ± 0.9 c 3.4 ± 0.9 a (6) (4) 9.0 ± 2.3 19.4 ± 4.2 c (6) (4) 6.4 ± 1.6 a 7.85 ±1.35 b (6) (4)

Satiety 4.2 ± 0.80 (4) 0.9 ± 0.3 (4) 4.3 ± 1.2 (4) 1.8 ± 0.30 (4)

p < 0.05; b p < 0.02; c p < 0.01 versus satiety; d p < 0.02 versus fasting.

14.1. Final remarks After starvation, the decrease of electric resistance, determined basically by the resistance of tight cell junctions (Ugolev et al., 2001; Metelsky, 2007a), has rather resistant character, as after refeeding it is not restored. This fact is consistent with observations that permeability (inversely proportional to measured resistance) is determined by a state of tight junctions (Thomson et al., 2002). The surprising thing is that after five-day starvation, decline of electric resistance in medial and distal segments (by 38 and 59%, respectively) considerably exceeds body weight loss (by 23%); it points to the fact that changes in the GI tract have specific character not proportional to weight. Apparently, the organism compensates for the loss of plastic materials, first of all, due to the GI tract. In actuality, it has been found that on starvation small intestinal mucosa atrophy was observed (Ugolev, 1972) and in enterocytes were detected vacuoles which were identified with raised autophagy (Ugolev, Timofeeva, 1989). On medial segments the basal SCC, unlike electric resistance, is practically restored after refeeding up to the values observable in a state of satiety. It is consistent with finding that after two-day starvation the height of the villus, the cellular surface and mitotic index of enterocytes are decreased, but after refeeding these parameters are restored up to a reference value (Yamauchi, Tarachai, 2000). It is common knowledge that after feeding of fasting rats absorption increases (Ugolev, Timofeeva, 1989). After refeeding of rats starved for five days, the glucose absorption in distal segments of an intestine also increases. Moreover, in both periods of fasting, irrespective of localization of an experimental segment (medial or distal), the observed increase of absorption is manifested as the tendency of SCC responses on all studied nutrients (except for dipeptide on two-day fasting) to increment. This

Chapter 14. Intestinal absorption at satiety, fasting and refeeding€€€€€231

fact is in agreement with observations that on refeeding activity of Na+-K+ATPase in rats jejunum increases (Lucas-Teixeira et al., 2000). We did not manage to find any distinctions in absorption of nutrients between satiety and fasting states, the only exception being dipeptide glycyl-La-alanine. This fact is consistent with observations that upon malnutrition, despite atrophic changes in mucosa, expression of a gene of Н+/peptide cotransporter PepT1 in a rat small intestine considerably increases (Ihara et al., 2000). However, there are distinctions (at least, for distal segments) between a satiety state and a state of refeeding. After refeeding, the level of absorption of nutrients exceeds that in a state of satiety by a few times. The surprising thing is that such an effect is not observed on medial segments. Perhaps it is followed by much lower rates of absorption of nutrients in distal segments in comparison with medial segments occurring in a satiety state. Data (Metelsky, 2005a) are the direct evidence for the role of distal segments of a small intestine as “reserve zones” for digestion and absorption. It is suggested that glucose absorption is determined by a sodium gradient on a brush border of an enterocyte which, in turn, depends on the activity of Na+K+ATPase, localized in a basolateral membrane (Ugolev, 1972; Philpott et al., 1992). In this case, there should be a correlation between rate of glucose absorption and activity of Na+-K+ATPase. The analogue of active sodium transport rate is the basal SCC, and the analogue of nutrient absorption rate is SCC response to nutrients. In medial segments of the intestine for all five studied states of animals, the coefficient of correlation between the indicated parameters for glucose is equal to 0.92, and that for leucine is equal to 0.81(Metelsky, 2005a). One is inclined to think that the value of the basal SCC is the measure of a reserve (some, perhaps, latent, not manifested in the given state) capacity of SCC to respond to glucose. In actuality, rates of absorption of nutrients are maximal and similar for medial and distal segments after five-day refeeding (Tab. 43 and 44); at the same time, the basal SCC on five-day refeeding is practically identical for both segments of an intestine (Tab. 42). It is known that starvation results in a shift of distribution of activity of enzyme-transport ensembles in distal direction (Ugolev, 1989). In medial segments on refeeding after two-day starvation, the rate of dipeptide absorption tends to reduce; the similar effect occurs a lesser extent in distal segments. Perhaps it is caused by specificity of food offered to animals on refeeding—a protein food (meat). Occurrence in an intestinal cavity of protein molecules and their small fragments results in full loading and the subsequent exhaustion of enzyme-transport ensembles (Ugolev, 1972; Metelsky, 1987) for dipeptides, in particular for glycyl-Laalanine (see 11.3.3). As this takes place, the effect of “buildup” of neighboring responses to dipeptide in medial segments of an intestine after refeeding of rats, starved for two days, perhaps reflects an increase in the number of enzyme-transport ensembles for glycyl-La-alanine in an enterocyte brush border in response to the first addition of dipeptide. The phenomenon of substrate stimulations of absorption (Philpott et al.,

232€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

1992) has been known for a long time. However, any attempts to observe such an effect of “buildup” on distal segments have not met with success. Thus, it is found that medial and distal segments of a rat small intestine respond to changes in the food status in various ways (Metelsky, 2005a). On medial segments, the values of the basal SCC in states of fasting and of subsequent refeeding are significantly distinguished. The active sodium absorption after feeding increases by a factor 3 to 6, depending on duration of starvation, and at the same time its electric resistance practically does not change (Tab. 42). Two states of satiety were elucidated (Metelsky, 2005a): when the rats were in a cage with the meal the whole time, and after refeeding following a five-day starvation, absorption of nutrients in the latter state was much higher, at least in the distal small intestine. After long fasting the permeability of tight cell junctions appears to increase. Fast nutrient adaptation (~30 minutes) of enzyme-transport ensembles upon addition of a substrate-effect “buildup” of neighboring SCC responses is revealed (Metelsky, 2005a). It is possible that increased nutrients (glucose and amino acids) entering mucosa after the fifth day on refeeding play a role as a primary signal for change of animal behavior, which has been found to bear witness in favor of glucose-static and amino-acid-static hypotheses of food behavior. There is good reason to believe that inhibition of food intake on intake of monosaccharides followed by appearance of signals from the GI tract arising in response to orosensory perception result in stretching of the stomach and nutrients binding with their receptors in the small intestine. Signals emanating from the oral cavity elicit the full range of physiological process that occurs as food passes through the GI tract. This raises the possibility that they aid in tailoring the response to the varying characteristics of foods ingested during each eating event. Along with further identification of the range of responses, there is a need to better characterize their importance and potential for manipulation for intended purposes, such as appetite and feeding regulation (Mattes, 2006). In response to stimuli from the small intestine, the hormones of satiety, including glucagon-like peptide-1 and amylin (Feinle et al., 2002), are liberated, and a gastric emptying and intestinal transit are retarded. As this occurs, the probability for nutrients to be absorbed is raised (Feinle et al., 2002).

Chapter 15. Clinical study of absorption and membrane digestion€€€€€233

Chapter 15. Clinical study of absorption and membrane digestion The modern views on mechanisms of Na+-dependent absorption and digestion in animals under various physiological states (young and old, satiety and fasting, etc.) represent a wide range of theoretical and practical interest. However, this book would be incomplete if we did not make any attempt to outline the earlier stages of application of the approaches described above in clinical study. In clinical investigation for an estimation of absorption a number of approaches are used (Loginov, Parfenov, 2000; Valenkevich, Yakhontova, 2001; Mukhina et al., 2003), including loading and tolerance tests and the technique of jejunoperfusion (Parfenov, 1987a, 1987b). But, apparently, in view of the complexity of realization the latter approach is rarely used. The determination of absorption and membrane digestion of nutrients in humans, in general, and Na+-dependent absorption, in particular, is elaborated methodically insufficiently. These facts are an obstacle to both modern clinical diagnostics of malabsorption and development of correcting diets. The SCC technique for human biopsies was elaborated for the first time in 1995 (Schulzke et al., 1995). Finding of SCC responses to nutrients in this case indicated that they are absorbed through human mucosa in an the Na+-dependent manner (Alexander, Carey, 2000; Green et al., 2000; Kroesen et al., 2002; Metelsky, Zvenigorodskaya, 2003; Metelsky, Parfenov, 2003a; Metelsky et al., 2003). At the same time, as indicated above, SCC value response characterizes rate of absorption of a nutrient (Metelsky, 2007a). Moscow City, Russia is globally the fifth scientific center since 2003 where biopsy specimens obtained using the procedure of gastrointestinoscopy have been used for SCC investigations (Metelsky, Parfenov, 2003a; Metelsky et al., 2003; Metelsky, Zvenigorodskaya, 2003). Following Pratha et al. (1998), the area of a study surface (the aperture of the chamber) of the preparations in Metelsky & Zvenigorodskaya (2003) is equal to 2–4 mm2, and the preparation keeps its viability for at least two hours. After staying in the Ussing chamber, biopsies morphologically do not change, with the exception of edge damage (see above) (Reims et al., 1997; Larsen et al., 2001; Metelsky et al., 2007).

234€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

15.1. Absence of an influence of essential amino acid on the SCC It is common knowledge that the essential amino acids (leucine among them) enter a human organism in an exogenous way; at that, they are absorbed by the small intestine using different mechanisms, including Na+-dependent ones. The absence of Na+-dependent absorption of the essential amino acid leucine in a patient suffering from polyvalent allergy, depression, and bulimia (Metelsky, Parfenov, 2004) is described below. The patient, 44 years old (height 169 cm, body weight 84 kg), was under examination in the hospital (Central Research Institute of Gastroenterology, Moscow) for the purpose of finding a possible link between polyvalent allergy to many medicines and foods and a pathology of the digestive apparatus. Upon examination by a psychiatrist, the patient was diagnosed with neurotic depression with frustration of sleep, hypochondriacal depression, and bulimic syndromes. Clinical trials and instrumental diagnostics, duodenogastroscopy with a biopsy specimen, were carried out. Sodium-dependent absorption of nutrients in the biopsy specimen was determined by the SCC technique. The rate of Na+-dependent absorption of maltose proved to equal 2.5, sucrose 3.0, lactose 1.3, glucose 2.8, alanine 1.5, glycine 1.0, glycyl-glycine 0.5, and leucine 0 μA/cm2. The contents of all amino acids in plasma were normal, and leucine was below normal. The lowered concentration of leucine in plasma was described on starvation, kwashiorkor, hyperinsulinism, after extensive operative interventions on the abdominal cavity, Huntington’s chorea, hepatic encephalopathy and burn disease (fourth day) (Tietz, 1995). The reason of impairment of active absorption of leucine in this patient was not established. You may anticipate that the selective malabsorption of this amino acid resulted in allergic and psychological abnormalities. Thus, for the first time, the absence of Na+-dependent absorption of the essential amino acid leucine was revealed.

15.2. Absorption of nutrients, coupled with sodium, in the small intestine of patients with irritable bowel syndrome and celiac disease The primary functions of the gastrointestinal tract have traditionally been perceived to be limited to the digestion and absorption of nutrients and electrolytes, and to water homeostasis. A more attentive analysis of the anatomic and functional arrangement of the gastrointestinal tract, however, suggests that another extremely important function of this organ is its ability to regulate the trafficking of macromolecules and ions between the environment and the host through a barrier mechanism (Fasano, Shea-Donohue, 2005). When the finely tuned trafficking of macromolecules is deregulated in genetically susceptible individuals, both intestinal and extraintestinal autoimmune disorders can

Chapter 15. Clinical study of absorption and membrane digestion€€€€€235

occur. This new paradigm subverts traditional theories underlying the development of autoimmunity, which are based on molecular mimicry and/or the bystander effect, and suggests that the autoimmune process can be arrested if the interplay between genes and environmental triggers is prevented by reestablishing intestinal barrier function. Understanding the role of the intestinal barrier in the pathogenesis of gastrointestinal disease is an area of translational research that encompasses many fields and is currently receiving a great deal of attention (Fasano, Shea-Donohue, 2005). In patients with irritable bowel syndrome the electric resistance of a biopsy specimen is equal to 27.8 ±Â€3.8 Ohm сm2, and with celiac disease to 19.9 8 Ohm€сm2 (Metelsky, Parfenov, 2003a, Metelsky, Neiman, 2004). This is consistent with observations that with celiac disease electric resistance decreases by 56% (Schulzke et al., 1995). In patients with celiac disease, SCC responses to monomers of sugar (glucose) and amino acids (alanine, glycine, leucine) (Metelsky, Parfenov, 2003a; Metelsky et al., 2003) were registered. SCC responses upon addition of dimers of nutrients were observed: sugars (maltose and sucrose), and dipeptide glycyl-glycine, that, apparently, point to the presence in an apical membrane of enterocytes of enzyme-transport ensembles. In patients with irritable bowel syndrome, SCC responses to glucose, alanine, glycine and leucine, and also on maltose, sucrose and on glycyl-glycine (Metelsky et al., 2003) have been measured. As indicated above, presence of SCC responses to studied nutrients points to the fact that they are absorbed through mucosa in a Na+dependent manner. As this takes place, the SCC value response characterizes rate of absorption of a nutrient. In patients from both groups spectrums of absorption are similar (Metelsky, Parfenov, 2003a); among dimers the greatest is absorption of maltose, and among monomers the greatest is amino acids. However, in patients with celiac disease, glycine demonstrates the maximal rate of absorption, and in patients with irritable bowel syndrome, it is leucine. In both groups of patients the rate of glucose absorption is minimal. Moreover, the glucose absorption in patients with irritable bowel syndrome and celiac disease proves to be considerably below that found in healthy volunteers (Larsen et al., 2001). The question arises as to whether kinetic parameters of transporters (the constant describing the affinity of a transporter to nutrient Kt, and maximal absorption ability Аmax) in humans are different from those in animals. This problem was resolved using the single response method (see Chapter 6). In a person with celiac disease (Metelsky, Parfenov, 2003b), constants of transport (Kt) are as follows: for D-glucose, 15.5; for L-alanine, 33.8; for glycine, 36.2; for glycyl-glycine, 42.8; for L-leucine, 43.6; for maltose, 47.4; and for sucrose, 92.2 mM. These values are similar to those for young rats (Metelsky, 2004a). The maximal transporting abilities in a biopsy specimen (Аmax) are as follows: for D-glucose, 3.32; for L-alanine, 11.4; for glycine, 13.9; for glycyl-glycine, 52.8; for L-leucine, 11.3; for maltose, 10.5; and for sucrose, 18.7 μA/cm2. These are essentially less than those in young rats (Metelsky, 2004a). It is conceivable that lowered (in comparison with rats) maximal transporting ability of glucose, alanine, leucine,

236€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

maltose, and sucrose in patients with celiac disease are for the first time elucidated as an objective manifestation of a syndrome of malabsorption peculiar to this disease (Loginov, Parfenov, 2000). Thus, for the first time on each biopsy specimen of a small intestine of patients with celiac disease, it was possible to determine the kinetic constants of Na+-dependent absorption for seven nutrients, owing to this fact one can treat parameters of absorption in humans in terms of molecular mechanisms. It is likely that in a patient with celiac disease the properties of transporters (Kt) are not changed, and the number of corresponding cotransporters (Amax) in mucosa are decreased.

15.3. Use of SCC technique on ischemic diseases of the digestive tract As indicated above (see Chapter 13), aging is accompanied by change in functions of the GI tract, in particular absorption and membrane digestion (Valenkevich, Ugolev, 1984). Data pertaining to the influence of an abdominal ischemia syndrome, which often accompanies aging, on the absorption of nutrients are insufficient and contradictory. In electrophysiological studies, the electric resistance of an intestine preparation is the integral characteristic of its epithelium barrier function and the measure of its morphological integrity and permeability (Schulzke et al., 1995; Metelsky, 2007a). In various studies (Metelsky, Zvenigorodskaya, 2003, 2004; Metelsky, Neiman, 2004; Metelsky et al., 2006), the electric resistance of a preparation from senior patients and from patients with irritable bowel syndrome proves to be equal to 26.3 ± 8.9 Ohm€сm2 and 27.8 ± 3.8 Ohm€сm2, respectively, and in young rats it was equal to 26.0 ± 0.8 Ohm€сm2 (Metelsky, 2004a). The potential difference on a mucosa epithelium in senior patients proves to be equal to 0.97 ± 0.12 mV and was less than that found in rats (1.78 ± 0.36 mV) (p <0.05) (Metelsky, 2004a). At the same time, the value of the basal SCC on irritable bowel syndrome (24.02 ± 1.93 μA/cm2) was equal that (24.0 ± 2.95 μA/cm2) for rats (Metelsky, 2004a) and (because of elimination in the cited study of a component of SCC caused by active chloride transport), it is consistent with the value of a basal SCC obtained in studying biopsies of a human duodenum (Larsen et al., 2001). In other words, electric characteristics of biopsies of patients (Metelsky, Zvenigorodskaya, 2004; Metelsky et al., 2006) are consistent with those obtained from animals. This latter circumstance indirectly supports the fact of the absence of any morphological changes of mucosa with the presence of irritable bowel syndrome. For the first time it was possible to observe a situation (Metelsky, Zvenigorodskaya, 2004; Metelsky et al., 2006) in which€ SCC responses to 10 mM of glucose were considerably lower then SCC responses to 5 mM of maltose (p <0.05); usually, they are approximately equal (see Chapter 10). Moreover, responses to glucose in patients

Chapter 15. Clinical study of absorption and membrane digestion€€€€€237

with irritable bowel syndrome (Metelsky, Zvenigorodskaya, 2004) were below (p <0.05) those in healthy volunteers—172 ± 22.1 μA/cm2 (Larsen et al., 2001). In senior patients the malabsorption (Na+-dependent) of amino acids (glycine and alanine) is revealed, giving good grounds for the prescription of glycine to such a patient; it may also be necessary to consider the problem of prescribing alanine to such a patient also. For the first time the obtained spectrum of absorption in patients for 11 nutrients is presented on Fig. 21 (Metelsky et al., 2006). SCC response, µA/cm2 6

5

4

3

2

1

0 1

2

3

4

5

6

7

8

9

10

11

Control Abdominal ischemia Fig. 21. A nutrient absorption profile studied in two groups of subjects: group I (n = 8; patients with circulation disorders, diagnosed by any of the known methods) – blue columns; group II (n = 4, controls, suffering from chronic gastritis without circulation abnormalities) – yellow columns.

238€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

15.4. New experimental opportunities for the development of clinical nutrition and nutritional supplements Measuring or monitoring certain human organism parameters is necessary for the elaboration of problems of nutritional supports to patients and creations of enriched foodstuffs (Tutelian et al., 2003). However, widespread control of composition and weights of a diet does not mean control over intake of nutrients in an organism (Metelsky, 2003a; Metelsky, Zvenigorodskaya, 2004). Control of the contents of nutrients in blood flow is not effective, as nutrients go through the portal vein after absorption in the intestines directly to the liver where they are exposed to significant changes. A balanced laboratory examination in which the differences in the contents of some substances in the diet and in stool are measured is practically not used. The most adequate approach which could form a scientific basis for dietotherapy is a measurement of the spectrum of nutrient absorption on patient biopsies by SCC technique in vitro (Metelsky, 2003a, 2003b, 2004b; Metelsky, Zvenigorodskaya, 2004). One additional example (see 15.3) can be given to illustrate this issue. It is found that in jejunum biopsies of five patients with an abdominal ischemia syndrome (Metelsky, Zvenigorodskaya, 2003), Na+-dependent absorption of maltose proceeds at the rate 3.75 ± 0.97, sucrose€ at 2.14 ± 0.47, glucose at 2.11 ± 0.52, alanine at 1.35 ± 0.49, glycine at 0.37 ± 0.1, leucine at 2.68 ± 1.21, and glycyl-glycine at 0.99 ± 0.42 μA/ cm2. In comparison with control patients and patients from the group with irritable bowel syndrome, the absorption of glycine is lowered approximately by a factor of 10 while the absorption of glycine from a solution containing glycyl-glycine in such patients is still great. This example is a way in which the offered approach can contribute to the scientific resolution of the issues of dietotherapy. The scientific approach to the development of such problems is impossible without an exact knowledge about the state of absorption and membrane digestion, and how in that group of patients (for which such diet is elaborated) various components of the diet in the GI tract are absorbed (Metelsky, 2003a, 2003b). That technique has been successfully applied in veterinary science for finding approaches for the rational organization of pathogenetic therapy by including in composition of rehydration mixes of essential amino acid in optimum combinations and concentration (Polyakov, Danilevskaya, 1989; Danilevskaya, 1987, 1989). This approach outlined the way for the estimation of influence of components of a diet on one of the basic functions of the intestine—nutrient absorption.

Chapter 15. Clinical study of absorption and membrane digestion€€€€€239

15.5. Final remarks and prospects Earlier we could be convinced that while using new investigative techniques for studying absorption and membrane digestion, the SCC technique in particular, progress was made toward the resolution of serious theoretical and applied problem of gastroenterology. Now the SCC technique assists in clinical investigation also (Mall et al., 2000; Pratha et al., 2000; Metelsky, 2007a). The presented examples, apparently, have been found to bear witness to the application of the SCC technique for estimating the efficiency of Na+-dependent transport processes, and of states of membrane digestion in small intestine, at least, in some groups of patients. Application of the described technique for clinical investigation can give a medical and biological basis for developing enriched foodstuffs both for one patient and for specific groups in the population. Also, the SCC technique can be used for testing already known drugs and for the development of new drugs. Thus, the preliminary results allow the clinician to pose new problems. For example, is the absence of Na+-dependent absorption of essential amino acids in patient (44 years old) intestine (see above) relate to her allergic and psycho-emotional abnormalities? Should a phenomenon of decrease in mucosa electric resistance by a factor of 1.5 shown in patients with celiac disease be included in the list of obligatory laboratory examinations required for diagnosing that disease? What role can malabsorption (Na+-dependent) of some nutrients (for example, glucose) play in patients with celiac disease and with irritable bowel syndrome? It is significant that an observed decrease in glucose absorption is due to a reduction in the number of cotransporter Amax, and at the same time the cotransporter molecules remain unchanged inasmuch as the affinity of cotransporters to glucose (Kt) does not change. In patients with an abdominal ischemia syndrome, Na+-dependent absorption of amino acid glycine and, perhaps, alanine has been found to be disturbed. In actuality, for such patients (as a rule, they are older people), glycine is often prescribed, although this is practically not absorbed. Perhaps it is necessary to prescribe glycine to such patients, not in the form of free amino acid, but in the form of dipeptide glycyl-glycine (which is hydrolyzed in the intestine on two glycine molecules) since their absorption is impaired to a much lesser degree. Many diseases are followed by abnormalities of absorption in the small intestine. Therefore, the issue of the role of any methods of diagnostics of malabsorption in the GI tract will be raised. Determination of Na+-dependent nutrient absorption (the most specific and fast mechanism of absorption) by the SCC technique is at the same time the only one ensuring the recording of process under online conditions.

240€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

Chapter 16. Conclusions€€€€€241

Chapter 16. CONCLUSIONS The SCC technique is a simple, convenient, rather effective and powerful study tool. The validity and reliability of the results obtained by this approach have considerably grown as a result of the modifications described earlier and the theoretical analysis carried out of the bases of SCC technique.

16.1. Osmotic responses and transtissue transport of water The SCC technique is successfully used for studying cellular mechanisms of transport. However, for studying cellular-tissue mechanisms of transport, this method, at least regularly, has not been used until now. For a long time the basic discussion in this area was in regard to the causes of the osmotic response of electrophysiological parameters (transmural potential differences and the SCC): change of paracellular pathway resistance, the basic contribution in which brings resistance of cell junctions, or change of EMF of this pathway (Fig. 1) (Pidot, Diamond, 1964; Smyth, Wright, 1964, 1966; Armstrong et al., 1975; Garcia-Diez, Corcia, 1977). In the elementary case this resistance is determined, basically, by a degree of tight cell junction opening. The physiological analogue of EMF of paracellular pathway is the driving force of a water flux through the cell junctions, moving sodium ions in a direction opposite to its active transport. The discussed problem, thus, is reduced to a issue what determines the osmotic response: resistance of tight cell junction (and hence its selectivity) or rate of water transport through it. It turns out that depending on a state of a intestinal wall epithelium which may exist only two, the pivotal role can play any of mentioned factors. With small resistance of a preparation (less than 22 Ohm*cm2) the value of the maximal osmotic response is determined by selectivity of tight junctions (by its resistance), and with greater resistance (more than 23 Ohm*cm2) – by the value of a water flux through cell junctions (i.e. its EMF). Dynamics of development and wash-out of a response to addition of osmotically active substances and nutrients are determined by the rate of diffusion of nutrient molecules through an unstirred layer of a fluid to a surface of a brush-border membrane.

242€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

As the membrane surface is approached, nutrient molecules are adsorbed on a certain binding site of gate mechanisms of the coupled cotransporter , resulting in opening in a membrane of additional sodium transporters. This stage is similar to the control of sodium permeability of exited membranes by acetylcholine. Besides, by the single response method one can determine the thickness of an unstirred layer of a fluid near a brush-border membrane surface.

16.2. Intestinal absorption: a stage of the approach of nutrients from bulk to a mucosa surface In order for nutrients from a bulk (chymus) to reach an intestine surface where membrane digestion and absorption occur, they must overcome a so-called unstirred layer of fluid (Metelsky, 1987, 2007c). The concept of unstirred layers distinguishes two parts in the diffusion barrier: a step that can be somehow controlled (e.g., by vigorously stirring the solution)—substrate supply from the bulk to unstirred layer boundary—and the step that cannot be influenced by the experimenter, determined by simple diffusion through the unstirred layer to the membrane. The existence of unstirred layers is a general but poorly studied phenomenon, which may be due to the ordering of the adjacent water layer, which resembles the formation of hydration shell around dissolved molecules and ions (Metelsky, 2007b, 2007c). Usually the thickness of an unstirred layer of fluid is measured with a response of electric parameters of an intestinal wall to the addition of impermeant sugar (as a rule, mannitol) in a solution washing a mucosa. Sometimes the thickness of the unstirred layer is measured on the SCC responses through a small intestinal wall on actively absorbed nutrients (sugar, amino acid, dipeptides). As this occurs, it remains unclear whether the thickness of the unstirred layer determined with nutrients will be changed with a variation of physiological states of an animal, and in what degree the values of thickness of the unstirred layer depend on characteristics of the nutrient itself. It is agreed that thickness of the unstirred layer near an internal surface of an intestine reflects the morphology of its surface and is commensurate with the height of the villi (Metelsky, 1981, 2007c; Gusev et al., 1983). The thickness of the unstirred layer near the mucosa surface is the integral characteristic of absorption determined by the dynamics of the approach of a nutrient to an intestinal surface. It is determined by the rate of diffusion of a nutrient, by the geometry of a surface mucosa, by an electric charge of a nutrient, etc. The thickness of an unstirred layer depends on the substance used. Perhaps that effect is caused by the topography of distribution of corresponding enzyme-transport ensembles along villus height (Fig. 14). If so, the excellent prediction of Barry and Diamond (Future prospects # 4) “Transport sites along the villus must differ as a function of solute concentration and uptake rate and may differ intrinsically for different solutes, causing unstirred layers thick-

Chapter 16. Conclusions€€€€€243

ness to vary with solute identity, concentration, and uptake rate. The arrangement of transport sites along the villus may constitute an unexplored principle of intestinal organization” (Barry, Diamond, 1984) is confirmed now. One may conclude that this new principle of intestinal organization was really revealed (Metelsky, 1987a, 2007a, 2007b, 2007c).

16.3. Application of the single response method for a wide spectrum of nutrients and inhibitors of transport. The underestimated approach The express procedure offered (Metelsky, Dmitrieva, 1987; Metelsky, 1987, 2004a, 2007a) for an estimation of kinetic parameters and thickness of an unstirred layer of a fluid on the single SCC response to some substance proves to be applicable for all tested nutrients. Moreover, the single response method can be applied not only for the nutrients stimulating the SCC, but also for inhibitors of absorption (phlorizin, amiloride). In spite of the fact that the Kt measured by this method varies (for various substances) by a factor of thousand, the single response method allows for the estimation of the Kt of a substance, irrespective of the value of such parameters and of the nature of added substances. The procedure of determination of a transport constant from the single SCC response has certain advantages compared to a procedure of determination of Kt from concentration dependences: the time taken to complete an analysis and material expenses (both reactants, and animals) are much less (Fig. 12). Also, the single response method unlike a procedure of concentration dependences allows us to study changes of Kt and Аmax during the experiment on one preparation. Owing to such a method, it was possible to explain an increase in efficiency of inhibition of SCC responses to glucose by phlorizin as temperature decreased. Development of the single response method has practically transformed the SCC technique into a new, extremely powerful, informative and reliable method of research which has been underestimated until now (from the viewpoint of its rare use).

16.4. Parallel multi-pathway cotransporter – transporters with gate mechanisms A strong evidence for our model was obtained when studying the crystal structure of a sodium galactose transporter€ (Faham et al, 2008). In this study, it was shown that there was a possible Na+-binding site ~10 Å away from the substrate binding site. One is inclined to think that such a large distance between Na+ and substrate binding sites favors our two-pathway model and is against the common one-pathway model

244€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

(see Fig. 4). Really, the size of Na+ channel is equal to 3*5 Å (Hille, 2001). Hence, at a distance of 10 Å, one can fit two such channels or transporters, for example, one for sodium and one for glucose. In addition, it is difficult to imagine the functioning of a common one-pathway channel for sodium and glucose of the size of at least 10 Å, the binding sites in the transporter core of which are separated by one or two helices (Faham et al, 2008, Fig.1B, 1C). According to the parallel multi-pathway model, the structure of the coupled transporter necessarily includes a sodium transporter (Figs 18). This will allow for an understanding of a large set of the facts. So, it was explained the phenomenon of dissociation of active glucose transport and of stimulating glucose effect on sodium transport, identity of characteristics of sodium transport induced by glucose and glycine, ability of a preparation for long time (up to 12 hours) to response to glucose, etc. However, apparently, for many researchers concept about glucose-dependent sodium transporters proves to be unusual and consequently we have tried to strengthen a line of evidences. What data has been found to bear witness in favor of the transporters? Earlier near resemblance between sodium channels of exited membranes and sodium transporters in enterocytes (both amiloride-sensitive, and a nutrient-dependent) was mentioned. Such similarity extends on all basic properties of sodium channels: (1) close values of density of channels; (2) identical carrying capacity of the channel; (3) presence of the selectivity filter; (4) presence of a gate mechanism. We consider the analogy between the properties of sodium transporters in exited and epithelial membranes as direct evidence for the channel nature as amiloridesensitive and glucose-dependent sodium transporters. Furthermore, one can bring a number of indirect suggestions in favor of channels or transporters (instead of carriers), summarized from various source of information. The sodium-glucose transporter has a stable chemical and functional asymmetry (Kessler, Semenza, 1983). The presence of such types of asymmetry in multi-pathway parallel model resulted from presence of the gate mechanism on an external surface of an apical membrane. The rough estimates (Metelsky, 1987) of number of collisions of hydrated sodium ions with a lumen of gramicidin channel with radius 2 Å (Page & DiCera, 2006) according to the known expression for molecular physics n*v*s/4, (where n is concentration of ions in 1 cm3, v is speed of ions, s is the area of lumen) gives the value 6*107 s-1 that is consistent with the value of carrying capacity of the gramicidin channel, equal to 6*107 s-1 (Lauger, 1980). In the case of the gramicidin channel, the solid angle under which ions can collide with an input of the channel is equal to ~2π. Known carriers of ions nonactin and valinomicin (Page & DiCera, 2006), have the maximal rate of a turnover equal to 1*104 and 3*104 s-1, respectively (Lauger, 1980). The classical gramicidin channel (Page & DiCera, 2006) has a carrying capacity of 6*107 s-1.

Chapter 16. Conclusions€€€€€245

From Parsegian’s study (1969) it may be inferred that the channel should be much more effective than the charged carrier. According to Metelsky’s (1987) data, the activation energy of the glucose-dependent transporter for sodium is constant in a wide range of temperatures where phase transformations of lipids are observed (Ivkov, Berestovsky, 1982). Such property of the transporter is consistent with properties of channels but not carriers. At last, on direct study of functioning of the single ion transporters by a technique of their buildup in bilayer membrane and by patch-clamp technique, channels, but not carriers, were found exclusively (Sariban-Sohraby et al., 1984; Matteson, Dentsch, 1984; Hunter et al., 1984). The microscopic study of chicken coprodeum (a part of gut after a ureter output) by a freeze-etching technique suggests the presence of channels (Eldrup et al., 1980). As the authors believe, they managed for the first time to see amiloride-sensitive sodium channels. All evidences taken together with those considered earlier suggest that in the cases of both the coupled transporter and amiloride-sensitive one, sodium transport is carried out through channels. But after (Loo et al, 1993) where it was shown that the turnover rate of Na+/glucose cotransport is 57/s, four orders of magnitude lower than the turnover rate for channels, we should be probably more cautious and use the term transporter instead of the term channel. The concept about glucose and sodium transporters incorporated in one structure proves to be very fruitful and allows us to explain a phenomenon of hydrolysis-dependent coupled disaccharide transport also. Any changes of the parallel multi-pathway model are not required. It turns out that the gate mechanism has the property of maltase subunits. That fact that transport of both free glucose and glucose formed as a result of hydrolysis (at least, in rats) is carried out by the same molecular mechanism—the parallel multi-pathway cotransporter —revised our understanding of disaccharide transport. However, it offers a clearer view of a number of well-known facts. It is agreed that glucose leaves an enterocyte through a basolateral membrane by means of the facilitated transporter, most likely, the special channel (Parsons, Prichard, 1971) blocked by phloretin. According to the parallel multi-pathway model, the structure of the coupled transporter for sodium and glucose also includes the glucose transporter. As both types of glucose transporters GLUT2 and SGLT1 are synthesized in the same cell, it may be naturally inferred that these transporters have similar properties, and both may be blocked by phlorizin. Disaccharidases on a basolateral membrane are absent; therefore, the glucose transporter is open for phloretin action and cannot be blocked by phlorizin. On the contrary, the input in the glucose transporter localized in an apical membrane is closed by the gate mechanism or by a hydrolizing subunit, and consequently the hydrolysis-transport ensemble is blocked by phlorizin only. In the evolved view, glucose-transport activity and maltase activity of enterocytes are closely interlinked. In those segments of an intestine where maltase activity is

246€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

maximal, it should be maximal glucose-transport activity and, on the contrary, in those segments of an intestine where maltase activity is minimal, it should be minimal glucose-transport activity (rats). In other words, distribution maltase and glucosetransport activity along a small intestine should be identical. In actuality, it is common knowledge that hydrolysis-dependent transport of maltose and transport of free glucose have an identical profile of distribution along a small intestine (Ugolev, 1972). This fact is a trivial consequence of the concept of the uniform hydrolysis-transport ensemble. The saccharase-isomaltase complex was first built in a bilayer lipid membrane by Storelli et al. (1972). It turns out that the permeability of a membrane for the hydrolyzed monosaccharides has grown considerably; however, active transport of sugars was absent. Unfortunately, that study provided no information on changes of membrane permeability to sodium. One is inclined to think that sodium permeability of a membrane at building in it of saccharase-isomaltase complex has changed slightly. Such a result is quite explicable from our viewpoint. Eventually, as interlink between sodium and nutrient transporters, perhaps, is labile enough (see Chapters 8 and 9) one can observe not only inducing glucose effect in the absence of its transport, but also the facilitated transport of monosaccharides in the absence of sodium transport. Therefore, we consider that during isolation of such a complex the sodium transporters of the coupled cotransporter were lost or were irreversibly damaged. Fruitfulness of concepts about a parallel multi-pathway cotransporter has been confirmed in a series of studies with glycine where for the first time it was possible to observe a number of the important phenomena. Hence, the phenomenon of cross stimulation of a SCC was revealed, when on long preincubation of the intestine with glycine (glucose), SCC responses to glucose (glycine) were increased. The similar effect of an induction was observed in studies of transport of amino acids through a membrane (plasmalemma) of alga Chlorella vulgaris (Sauer et al., 1983). It turns out that treatment of cells by 13 mM of glucose or its nonmetabolizing analogue induces the formation of two systems of transport of amino acids: one for the transport of both arginine and lysine, the other for carrying out the transport of glycine, proline, alanine and serine. During a long preincubation (4–5 hours) of preparations with a nutrient, it was possible to observe use-dependence effects for the first time. It turns out that the more functional loading on a sodium transporter, the more likely it fails to operate; in other words, the life span of “details” of the molecular machine of a parallel multi-pathway cotransporter depends on intensity of their use. On the strength of these data the concept that sodium transporter plays a role of the universal block for the transport of nutrients through its specific transporters is put forth. Association of a sodium transporter (may be in Golgi complex, may be in apical membrane) with various sets of transporters for nutrients result in formation of parallel multi-pathway cotransporters with various specificity.

Chapter 16. Conclusions€€€€€247

The fruitful idea concerning a sufficiently labile group of transporters incorporated in one quaternary structure was first documented by A. Ugolev (1967). The notion of universality laid in the mechanism of a parallel multi-pathway cotransporter is extremely attractive and, certainly, it turns out that some analogue in the literature already is available. According to model (Robinson, Alvarado, 1979), sugar and amino acids mutually inhibit transport of each other as various carriers are located side by side or even are incorporated in a uniform complex, forming a “multifunctional carrier.” Owing to such a mechanism, transport of one substrate allosterically inhibits the neighboring carrier for other substrate. At a later time this model was modified—sugar and amino acids have competed for a gradient of electrochemical potential. Sodium, sugar and amino acids contact with “multifunctional carrier” in the ratio 1:1:1. However, within the framework of model of carriers the existence of such “multifunctional carrier” is difficult to imagine and, perhaps, it was one of the reasons why this model has not obtained wide recognition and has not mentioned at all in reviews of one of its authors (Brot-Laroche, Alvarado, 1983). On the contrary, the concept about “multifunctional transporter” in the form of multi-pathway parallel model (Metelsky et al., 1983; Ugolev, Metelsky, 1990b; Metelsky, 2007a) is devoid of contradictions inherent in concept about “multifunctional carrier,” and as we were convinced above, has a significant predictive force. Do epithelial transporters of nutrients form complexes in the membrane (Broer, 2008a)? Are they held in place by scaffolding proteins (Broer, 2008a)? It is possible. Scaffolding protein PDZK1 provides spatiotemporal coupling of cAMP transporter to CFTR chloride channel function in the gut epithelia (Li et al, 2007). Scaffolding proteins act as a molecular Lego, binding a number of cellulases through its type I cohesin (cohesin I) modules to spatiotemporally regulate the efficiency of the entire enzymatic cascade (Valbuena et al., 2009). The presence of a strong link between SCC changes on glycine and glucose in each preparation points to the fact that glucose and glycine transporters may be incorporated in one quaternary structure (perhaps with the help of scaffoldings-like or tetraspanins-like proteins) with the same type of sodium transporters. The studies on the architecture of the Na+/glucose cotransporter protein (Faham et al, 2008) and of the leucine transporter (LeuT) from the family of Na+-dependent neurotransmitter (members of the family include the Na+/glycine, Na+/Cl-/γ-amino-butyric acid (GABA), and the Na+/serotonin cotransporters of the Na+), coupled benzyl-hydantoin transporter, Mhp1, from Microbacterium liquefacients, also show that they have similar core structures (Weyland et al., 2008). These studies had a major impact on our understanding of the coupled transport of Na+ and nutrients. Our understanding of the molecular mechanisms of coupled transport processes would have made a big leap,€ if a study like that of Faham et al, 2008 of the crystal structure of such quaternary complexes of proteins were possible to hold at least for such two-way transporters like cAMP transporter with CFTR chloride channel (Li, 2007). But the problem is that the crys-

248€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

tallization of such a spatiotemporally complex of transporters existing together only with scaffoldings-like molecules bounded to each other through rather weak forces may be more difficult task.€ Views developed here fall into the more common concept of blocks (Ugolev, 1985; Ivashkin, Minasyan, Ugolev, 1990). According to this concept, evolution and adaptation in animate nature are more likely achieved not due to “invention” and synthesis of new molecular mechanisms, but due to more economical use of previous elementary blocks like transporters, enzymes, etc. The system gets new qualities when existing earlier types of elementary blocks combine in new ways. Developed views about the existence of multi-pathway parallel cotransporters, which transporter composition can change, as a matter of fact, are one more suggestion of that attractive concept. Thus, the hypothesis of a parallel multi-pathway cotransporter for Na+-dependent transport of nutrients allows us to explain all data available today, including that gathered by Kellet & Brot-Laroche (2005).

16.5. The SCC technique is the only one that can enable online estimations of vector properties of mucosa biopsies from the human gastrointestinal tract The biopsies obtained by gastrointestinoscopy are the most valuable material for diagnostics, but practically 95% are used for morphological and electron-microscope investigations only. It becomes gradually apparent that such a situation is unacceptable. First, morphological and electron-microscopic changes are followed by functional changes typical for the given disease, though this is a point at issue. The functional changes, resulting in or resulting from the disease (revealed, in particular, by the SCC technique), have possibly arisen earlier, and morphological changes of a tissue are recorded later. Secondly, except for freezing techniques, the obtained biopsy specimen from the GI tract is almost always placed in formalin at once. Then the “poisoned” (from the viewpoint of the physiologist) specimen takes to the further chemical treatment. And then the conclusions about impairments of morphology, structure and ultrastructure of such a rather inadequate preparation are made. Sometimes biopsies without any treatment are studied using biochemistry, and in this case the studied piece of a mucosal tissue is considered the amorphous catalyst of some biochemical reactions. As this takes place, the fact that such a biopsy specimen has vector properties (i.e., it is capable of transferring nutrients from a mucosal side to serosal one) and that the function of membrane digestion is localized exclusively on a mucosal surface are often overlooked. Now, apparently, the intact biopsy should be studied from the viewpoint of its (inherent) vector properties. Therefore, use of the investigative techniques of gut biopsies, in which they are mounted (in the form of

Chapter 16. Conclusions€€€€€249

a spread-out sheet) between two halves of the experimental Ussing chamber, shows considerable promise. Now only several dozen studies concerning transport and absorption processes in intestine biopsies carried out using the SCC technique have been published. Such an effort for gastroenterologists is a novelty. To them, it is necessary to become accustomed to the terminology in this area of knowledge, to understand the borders of the opportunities offered by this method. It is necessary to recognize that both parties—clinicists and researchers (biophysicists, physiologists)—so far grope only for closer mutual contacts. Nevertheless, the preliminary data presented here testify to such an approach’s potential. Malabsorption of essential amino acid—leucine in patient, suffering from allergy and having some psychological impairments is revealed. The question arises: Is the malabsorption the consequence or the cause of her allergy and/or neural disorder. Further studies should respond to this question. The same concerns the revealed malabsorption of nutrients (glucose) and changes of electric resistance of a mucosa on celiac disease. In our clinical study, the malabsorption on celiac disease cited in every monography was documented. Clinical study on elderly patients with an abdominal ischemia syndrome, on the one hand, has confirmed the truth of prescription to them of glycine (but this amino acid is absorbed in such patients very slowly). The standard approach to clinical nutrition is as follows: a lack of some substance is observed in the blood of a patient. This substance is then added to the diet to compensate for its lack/shortage in the patient. Such an approach, in actuality, works successfully when it is necessary to normalize sharply changed parameters of blood (reanimation, for example). One is inclined to think, however, that it is an incorrect method to be used generally. It is necessary to control absorption (Na+-dependent or Na+-independent) of the given substance in the GI tract. Now in the clinicodiagnostic laboratory there is a method suitable for researching the spectrum of absorption (Fig. 22) and membrane digestion of a population. It is important to keep in mind that such a technique is the only one allowing us to record online dynamics of absorption. With its help one can: (a) diagnose impairments of molecular mechanisms of absorption; (b) select optimal dietotherapy for the patient; and (c) test drugs, both new and those already on the market. Demonstration of the opportunities of the SCC technique using several examples (see Chapter 15), in our opinion, convincingly points to its high prospects for the purposes of diagnosing and studying various pathologies, as well as for a scientific resolution of the issues of clinical nutrition and enriched foodstuffs.

250€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

Acknowledgements For the help in our studies, and also in the preparation of the manuscript, I am sincerely grateful to all my colleagues at the Laboratory of Nutrition Physiology, Institute of Physiology, Russian Academy of Sciences, St. Petersburg, as well as to Prof. V. A. Metelskaya and Prof. V. P. Mineyev. I am grateful to Dr. D. D. F. Loo for very valuable comments. Miss Anne Devismes kindly corrected the English.

Abbreviations and designations€€€€€251

Abbreviations and designations Re—resistance of epithelial sheet Rse—resistance of subepithelial tissues Rа—resistance of apical membrane of enterocyte Rbl—resistance of basolateral membrane of enterocyte Rvs—resistance of vascular system Rsh—resistance of enterocyte shunting or of paracellular pathway EMF—electromotive force Еа—electromotive force of apical membrane Еbl—electromotive force of basolateral membrane Еsh—electromotive force of pathway shunting enterocytes SCC—short circuit current Vн—rate of response development of short circuit current upon addition of substance Vо—speed of wash-out of response of short circuit current α—relative rate of response development of a short circuit current β—relative speed of wash-out of response of short circuit current τ—time constant A—value of response of short circuit current Аmax—maximal value of response of short circuit current Kt—transport (or Michaelis) constant for transport of nutrient δ—thickness of unstirred layer of fluid near a surface C—concentration GI—gastrointestinal PCMB—p-chlormercuri benzoate PHMB—p-hydroymercuri benzoate CMCD—cyclo morpholinecarbodiimid ATP—adenosine triphosphate cAMP—cyclic adenosine monophosphate CNS—central nervous system EDTA—ethylene diamine tetraacetate DCCD—dicyclohexyl carbodiimid

252€€€€€Transport phenomena and membrane digestion in small intestinal mucosa

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