Opioid µ, δ and κ Receptors for Endorphins

Opioid , , and  Receptors for Endorphins Daniel J. J. Carr1 and J. Edwin Blalock2,* 1

Department of Microbiology and Immunology, LSU Medical Center, New Orleans, LA 70112-1393, USA 2 Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, AL 35294-0005, USA * corresponding author tel: 205-934-6439, fax: 205-934-1446, e-mail: [email protected] DOI: 10.1006/rwcy.2000.23005.

SUMMARY The three major types of opioid receptors (, , and ) are members of the DRY (Asp-Arg-Tyr)-containing subfamily of seven transmembrane spanning receptors. Opioid receptors on cells of the immune system are virtually identical to those on neuronal cells. The activation of opioid receptors in the CNS leads to analgesia while activation of opioid receptors on immune cells can enhance or suppress immune function depending on the target cell and immune parameter measured.

BACKGROUND

Discovery The molecular characterization of opioid receptors has been investigated for nearly 25 years. However, the activities of these receptors, as manifested in the effects of opioid compounds (e.g. opium, of which the main active ingredient is morphine), have been known for at least 6000 years, since the time of the Sumerians (4000 BC). The discovery of endogenous opioid peptides, including the enkephalins (met- and leuenkephalin) (Hughes et al., 1975), the endorphins

(, , and ) (Bradbury et al., 1976; Cox et al., 1976), dynorphin (Goldstein et al., 1979), and endomorphin (Zadina et al., 1997), suggested the existence of multiple types of receptors (termed opioid receptors) for these natural ligands. Evidence for multiple types of opioid receptors was obtained using congeners of morphine in spinal studies in dogs (Gilbert and Martin, 1976; Martin et al., 1976). Originally identified in the early 1970s (Pert and Snyder, 1973; Simon et al., 1973; Terenius, 1973), today, three main types of opioid receptors have been defined and cloned: , , and  opioid receptors with pharmacologically distinct subtypes for  (1 and 2),  (1, 2, and 3), and  (1 and 2) (Pasternak, 1993). Two other receptors including " (specific for endorphin) and  receptors were originally described as opioid receptors but have since been redefined as nonopioid (Simon, 1991). All of these receptors have been identified and characterized on cells of the immune system (Garza and Carr, 1997). Another opioid-like receptor, referred to as the nociceptive receptor (orphan opioid receptor), originally described in human brainstem (Mollereau et al., 1994) was also found in mouse spleen T and B lymphocytes, where it was first coupled to a physiological role (Halford et al., 1995). This review focuses on the immune cell-derived opioid receptors, comparing their physicochemical properties with those found in the

2212 Daniel J. J. Carr and J. Edwin Blalock nervous system as well as defining their role in the immune system.

Structure The three major types of opioid receptors are members of the (DRY)-containing subfamily of seven transmembrane spanning receptors. There is 60% amino acid identity between each type of opioid receptor with the membrane-spanning regions (transmembrane I±VII) and the intracellular loops connecting these segments being highly conserved between receptor types. Studies indicate that ligands (agonists and antagonists) to these receptors bind to different regions of the extracellular domain and such interaction can be greatly influenced by the transmembrane segments (predominantly TM II, III, and VI) (Kong et al., 1993, 1994; Surratt et al., 1994). Also, changes in one amino acid in the TM IV spanning region has been shown to alter opioid antagonist to agonist activity (Claude et al., 1996). Since the amino acid sequences of the neuronal- and some immunederived receptors are nearly identical, it is predicted that a similar relationship between agonist/antagonist-binding domains and the influence of the transmembrane spanning regions will be found in the immune-derived opioid receptors. However, immunederived opioid receptor-binding domains according to some investigators may be distinct since binding or biochemical characteristics of these sites are not characteristic of neuronal opioid sites (e.g. Stefano et al., 1992; Makman et al., 1995).

Main activities and pathophysiological roles The primary function associated with neuronal opioid receptors is the control of the sensation of pain either through receptors located spinally (1, 1, and 2) or supraspinally (2, 2, 3, and 1) (Pasternak, 1993). Within the immune system, opioid receptors found on immune cells may augment or suppress immune function depending on the cell type and stimulation (Carr, 1991). However, alkaloid opioid ligands (e.g. morphine and fentanyl) are potent immunosuppressive compounds affecting the immune system primarily by indirect pathways ligating to receptors found within the CNS and activating secondary systems (including the adrenergic pathway and the hypothalamuspituitary-adrenal axis) (Carr et al., 1996). Other functions of immune-derived opioid receptors may pertain to the response to infectious pathogens. As an

example,  opioid receptors bound to -selective opioid ligands have been found to reduce significantly monocytotropic HIV-1 SF162 strain replication in microglia-enriched cultures (Chao et al., 1996).

GENE

Accession numbers The  (L06322, L11065),  (L11064), and  (L22455, L20684) opioid receptors have been cloned from neuronal tissue (Evans et al., 1992; Kieffer et al., 1992; Li et al., 1993; Thompson et al., 1993; Wang et al., 1993; Yasuda et al., 1993).

PROTEIN

Accession numbers Protein Information Resource: Human  opioid receptor: 2135858 Human  opioid receptor: 631277 Human  opioid receptor: 2134989

Sequence The neuronal opioid receptors are composed of between 370 and 389 amino acids encoded by mRNAs ranging in size from 1.9 to > 10.0 kb (Carr et al., 1996). Both  (Figure 1a; Sedqi et al., 1996) and  (Figure 1b; Belkowski et al., 1995) receptor full-length cDNAs (predicted to be 372±400 amino acids in length) have been identified in thymocytes or a thymoma cell line. However, only a partial sequence (441 bp) of a  opioid receptor has been identified by RT-PCR in peripheral blood mononuclear cells (Chuang et al., 1995) and rat peritoneal macrophages (Figure 1c; 721 bp) (Sedqi et al., 1995). All immune cell-derived receptor sequences identified thus far are nearly identical ( 99% homology) with the receptors in the nervous system.

Description of protein By a variety of techniques, the neuronal opioid receptors were observed to range in size from 40 to 65 kDa (Simonds, 1988; Loh and Smith, 1990; Wollemann, 1990). The data concerning the biochemical properties of these receptors may be limited by the uncertain specificities of some of the antireceptor antisera.

Opioid , , and  Receptors for Endorphins 2213 Figure 1 (a) Deduced amino acid sequence of the  opioid receptor cloned from activated murine thymocytes as reported by Sedqi et al. (1996). Bold letters indicate changes from the published rodent brain  opioid receptor. (b) Deduced amino acid sequence of the  opioid receptor cloned from R1.1 thymoma cell line as reported by Alicea et al. (1998). Bold letters indicate changes from the published rodent brain  opioid receptor. (c) Deduced amino acid sequence of the  opioid receptor cloned from adherent peritoneal macrophages as reported by Sedqi et al. (1995). Bold letters indicate changes from the published rodent brain  opioid receptor. (a) MELVPSARAELQSSPLVNLSDAFPSAFPSA GANASGSPGARSASSLALAIAITV LYSAVC AVGLLGNVLVMFGIVRYTKLKTATNIYIFN LALADALATSTI PFQSAKYLMETWPFGELL CKAVLSIDYYNMFTSIFTLTMMSVDRYIAV CHPVKALDFRTPAKAKLIQ ICIWVLASGVG VPIMVMAVTQPRDGAVVCMLQFPSPSWYWD TVTKICVFLFAFVVPILIITVCYGLMLLRL RSVRLLSGSKEKDRSLRRITRMVLVVVGAF VVCWAPIHIFVIVWTLVDINRRDPLVVAAL HLCIALGYANSSLNPVLYAFLDENFKRCFR QLCRTPCGRQEPGSLRRPRQATTRERVTAC TPSDGPGGGAAA

30 60 90 120 150 180 210 240 270 300 330 360 372

(b) MESPIQIFRGNPGPTCSPSACLLPDSSSWF PDWAESNSDGSVGSENQQLESAHISPAIPV IITAVNSVVFVVGLVGDSLVMFVIIRIYTK MKTATDIYIFDLALANALVTTTMPFQSAVY LMDSWPFGNVLCKIVISINYYDMFTSIFTL TMMSVNRYIAVCHPVKALNFRTPLKAKIID ICIWLL ASSVGISAIVLGGT KVRENVNVIE CSLQFPNNEYSWWNLFMKICVFV FAFVIPV LIIIVCYTLMILRLKSVRVLSGSREKNRDL RRITKLVLVVVAVFIICWTPIHIFILVEAL GSTSHSTAALSSYYFCIALGYTDSSLDPVL YAFLNEDFKRCFRNFCFPIKMRMERQSTDR DTVQNPASMRNVGGMDKPV

30 60 90 120 150 180 210 240 260 290 320 350 369

labeled proteins migrating at 70, 46, and 31 kDa from brain tissue, while a major protein species migrating at 31 kDa was labeled from spleen tissue. The 31 kDa species was thought to be a degradative form of the mature protein. Subsequent analysis of immune cellderived , , and  opioid receptors determined the size to be nearly identical to that of the neuronal receptors (Carr, 1991).

Relevant homologies and species differences Opioid receptors from immune cells are virtually identical ( 99% homology) to those on neuronal cells. The various opioid receptor types (, , ) show about 60% homology.

Affinity for ligand(s) The identification of the types of opioid receptors has been greatly facilitated by the design and synthesis of opioid ligands selective for the types of receptors to which they bind (Table 1). Similar to cell-associated neuronal opioid receptors, the cloned neuronal opioid receptors expressed in PC-12 cells showed highaffinity binding to ligands ranging from 0.2 to 3.0 nM (Raynor et al., 1994). Opioid receptors found on cells of the immune system display a modestly reduced affinity for their ligands ranging from 20 to 900 nM, depending on the ligand and receptor (Garza and Carr, 1997). For example,  receptors display affinities ranging from 4.1 to 65.0 nM (Garza and Carr, 1997), whereas a unique alkaloid-specific 3 opioid receptor found on granulocytes has a Kd of 44 nM (Makman et al., 1995). One investigation reported the IC50 for an immunoaffinity-purified opioid receptor isolated from mouse spleen preparations to be approximately 700 nM, suggesting a loss in affinity upon purification (Carr et al., 1990).

(c) MGTWPFGTILCKIV ISIDYYNMFTSIFTLC TMSVDRYIAVCHPVKA LDFRTPRNAKIVNV CNWILSSAIGLPVMFMATTKYRQGSIDCTL TFSHPTWYWQNLLKICVFIFAFIMPILIIT VCYALMILRLKSVRMLSGSKEKNRDLRRITR MVLVVVAVFIVCWTPIHIYVIIKALITIPE TTFQTVSWHFCIALGYTDSCLDPVLYAFLN

30 60 90 120 150 180 210

Using a site-directed acylating agent derived from fentanyl (known as superfit) that is highly selective for  opioid receptors, a comparison of the mouse brain cell- and spleen cell-derived  opioid receptor. Superfit

Cell types and tissues expressing the receptor Within the immune system, there is some disagreement as to the population of cells that express opioid receptors. To this end, Table 2 and Table 3 summarize the evidence for the presence of opioid receptors on primary cells of the immune system (Table 2) and cell lines derived from cells of the immune system (Table 3) based on pharmacological (radioreceptor

2214 Daniel J. J. Carr and J. Edwin Blalock Table 1 Commercially available selective opioid agonists/antagonists  Opioid receptor ligands

 Opioid receptor ligands

 Opioid receptor ligands

DADLE (agonist)

Bremazocine (agonist)

DAMGO (agonist)

DPDPE (agonist)

U-50488 (agonist)

Endomorphin 1 (agonist)

SNC 80 (agonist)

U-69593 (agonist)

Endomorphin 2 (agonist)

DSLET (agonist)

ICI-199,441 (agonist)

Fentanyl citrate (agonist)

SNC121 (agonist)

Nor-binaltorphimine (antagonist)

-Funaltrexamine (antagonist)

Superfit (affinity label)

DIPPA (antagonist)

Naloxonazine (antagonist)

Naltrindole (antagonist)

Cyprodime HBr (antagonist)

ICI-174,864 (antagonist) BNTX (antagonist) Naltriben (antagonist)

Table 2 Evidence for the presence of opioid receptors on primary cells of the immune systema Cell type

 Receptor

 Receptor

 Receptor

Mouse T lymphocyte

B, M

±

B

Mouse B lymphocyte

B

±

B

Mouse thymocyte

±

±

P, M

Mouse splenocyte

B, M

B

B

Human PBLs

M

M

±

Human T lymphocyte

P, B

M

±

Human B lymphocyte

B

±

±

Human granulocyte

±

±

P, M

Human monocyte

±

M

M

Monkey PBLs

M

M

M

Human microglia

±

M

±

Rat macrophage

±

±

M

a

Evidence for the existence of the receptors is defined using pharmacological (P), biochemical (B), or molecular biology (M) approaches (Alicea et al., 1998; Chao et al., 1996; Gaveriaux et al., 1995; Miller, 1996; Roy et al., 1992; Wick et al., 1996) or as reviewed by Carr (1991), Carr et al. (1996). ±, Suggests either a lack of detection or that the analysis has not yet been determined.

assays), biochemical (affinity labeling), or molecular biology techniques (cloning or RT-PCR studies).

Regulation of receptor expression The expression of  and  opioid receptors on immune cells is reportedly induced by the activation of cells by IL-1 in the case of the thymocyte  receptor

(Roy et al., 1992) or mitogen (concanavalin A) in the case of the mouse T lymphocyte  opioid receptor (Miller, 1996). Furthermore, the activation of leukocytes also leads to the production of endogenous opioid peptides (Blalock, 1989). Since the leukocytederived opioid peptides have also been shown to be functionally active (Blalock, 1989), there is reason to believe that autocrine regulation of receptor expression may occur as well.

Opioid , , and  Receptors for Endorphins 2215 Table 3 systema

Evidence for the presence of opioid receptors on cell lines derived from cells of the immune

Cell type

 Receptor

 Receptor

 Receptor

M M

P ±

± ±

M

±

±

B

P, B

±

P, M

±

M M B, M

M ± M

M ± ±

M

M

M

±

Mouse T cell lines EL-4 11.10 Mouse B cell line CH27 Mouse macrophage cell line P388d1 Mouse R1.1 thymoma Human T cell lines CEMx174 HSB2 MOLT-4 Human B cell line EBV-transformed Human monocyte cell line U937

±

a

Evidence for the existence of the receptors is defined using pharmacological (P), biochemical (B), or molecular biology (M) approaches (Gaveriaux et al., 1995; Chao et al., 1996; Wick et al., 1996; Alicea et al., 1998) or as reviewed by Carr (1991), Carr et al. (1996). ±, Suggests either a lack of detection or that the analysis has not yet been determined.

SIGNAL TRANSDUCTION

Cytoplasmic signaling cascades Neuronal opioid receptors modify a variety of signaling cascades including cAMP through the activation of Gi, increases in GTPase activity, phosphatidylinositol turnover, mobilization of Ca2+, and K+ channel activity (Childers, 1991; Chen and Yu, 1994). In a similar fashion, immune cell-derived opioid receptors are coupled to a Gi protein and influence K+ channel conductance and calcium mobilization (Carr, 1991). In addition, the endogenous opioid peptide -endorphin has been shown to modify CD3 phosphorylation following phorbol ester stimulation, either increasing or decreasing phosphorylation of the CD3 chain depending on the concentration of the peptide (Kavelaars et al., 1990). These results suggest that the endorphins may act as a governor on T cell activation depending on the local concentration of endogenous opioid peptide. Specifically, endorphins at mid-picomolar levels may

augment T cell activation through the increase in phosphorylation of the CD3 complex intracellular tyrosine-activation motifs (ITAMs) and presumably the activation of the inositol trisphosphate (IP3) cascade via ZAP-70, whereas at femtomolar levels the endorphins would suppress T cell activation by reducing phosphorylation.

DOWNSTREAM GENE ACTIVATION

Transcription factors activated The success in transfecting Jurkat T cells (which do not express opioid receptors, Gaveriaux et al., 1995) with a functional  opioid receptor (Sharp et al., 1996) allowed for the identification of potential transcriptional regulatory elements involved in opioid modulation of immune function. Previous studies reported the augmentation of IL-2 production by activated T cells stimulated with endogenous opioid peptides

2216 Daniel J. J. Carr and J. Edwin Blalock (Carr, 1991). In an elegant study, reporter gene constructs were used to map deltorphin ( selective agonist)-elicited augmentation of IL-2 production by  opioid receptor-transfected Jurkat T cells to the AP-1- and NF-AT/AP-1-binding site (Hedin et al., 1997). This effect was apparently independent of calcineurin and unrelated to the elevation in [Ca2+ i ] but required pertussis toxin-sensitive G protein. Since the NF-AT/AP-1 complex is involved in the induction of a number of cytokine genes (Rao, 1994) and endogenous opioid peptides modify the production of a number of cytokines (Peterson et al., 1998), it is quite possible that the NF-AT/AP-1 complex is involved. In addition, since leukocyte activation is primarily mediated by cytokines, it is highly probable that opioid receptor promoters possess binding domains for cytokine responsive elements. As an example, the  opioid receptor promoter possesses a NF-IL6 domain (Min et al., 1994).

Genes induced IL-2.

BIOLOGICAL CONSEQUENCES OF ACTIVATING OR INHIBITING RECEPTOR AND PATHOPHYSIOLOGY

Unique biological effects of activating the receptors The response to opioid receptor activation depends on the location of the receptor, the type of receptor, and the level of activation of the cell population. Endogenous opioid peptides can either enhance or suppress immune function depending in part on the state of target cell activation and the immune parameter (antibody production, natural killer activity, cytokine synthesis) measured. Likewise, peripheral blood mononuclear cells from individuals can respond differently (sometimes completely opposite of one another) to opioid ligands evident in both human and mouse populations. However, the administration of opioid alkaloids (i.e. morphine, heroin, or fentanyl) tends to elicit a significant suppression of immune function primarily by opioid receptors found in the mesencephalon (Shavit et al., 1986; Weber and Pert, 1989). The activation of the `central' opioid receptors elicits the activation of neuroendocrine pathways.

The hypothalamic-pituitary-adrenal axis results in the production of adrenal steroids such as glucocorticoids which suppress immune responses in part by preventing translocation of NFB to the nucleus (Baldwin, 1996). Alternatively, morphine may activate the sympathetic/parasympathetic arm of the autonomic nervous system known to innervate lymph nodes and spleen (Felten et al., 1987) and modify immune function through the release of monoamines (e.g. catecholamines) (Carr and Serou, 1995). Other studies suggest that endogenous opioid peptides may supplement antimicrobial drugs or local immune reactivity against viral infections. Studies have suggested that met-enkephalin suppresses influenza virus infection in mice through the effects on natural killer cells and cytotoxic T lymphocytes (Burger et al., 1995). Another study has found that met-enkephalin synergizes with azidothymidine in blocking feline leukemia virus replication (Specter et al., 1994). It has also been reported that endogenous opioids induce the synthesis of novel fentanyl derivatives that possess analgesic activity in the absence of opioid immunosuppression (Carr and Serou, 1995).

Phenotypes of receptor knockouts and receptor overexpression mice Mu opioid receptor (MOR) knockout mice have been developed and tested for immune deviation in the presence and absence of the clinically relevant, prototypic  ligand morphine. MOR knockout mice exhibit normal immunological endpoints including natural killer activity, antibody production, and mitogeninduced lymphocyte proliferation (Gaveriaux-Ruff et al., 1998). However, bone marrow cells from MOR knockout mice exhibit an altered pattern of early hematopoiesis (Tian et al., 1997). In addition, treatment with morphine had no effect on immune parameters assayed in MOR knockout mice, but significantly suppressed selectively measured immune parameters (e.g. natural killer cell activity) and induced lymphoid organ atrophy in wild-type mice (Gaveriaux-Ruff et al., 1998). These results suggest that the absence of the  opioid receptor has no detrimental effect on immunocompetence per se, but is directly responsible for the immunomodulatory effects of exogenous morphine. Accordingly, modification of immune responses to antigen or microbial pathogens by endogenous opioid peptides does not necessarily involve the activation of  opioid receptors on immune cells.

Opioid , , and  Receptors for Endorphins 2217

THERAPEUTIC UTILITY

Effects of inhibitors (antibodies) to receptors The classical pharmacological definition of the existence of opioid receptors on cells of the immune system has come from the ability of opioid antagonists (competitive or noncompetitive) to block the immunomodulatory effects in a stereospecific manner (Sibinga and Goldstein, 1988) (see Table 1). Antibodies have also been generated against opioid receptors that recognize proteins expressed on cells of the immune system. One such antibody was found to possess agonist activity and to recognize a putative  class opioid receptor on mouse leukocytes (Carr et al., 1990). A second antibody was generated against the predicted N-terminal sequence of a  opioid receptor and was found to act as a noncompetitive selective antagonist recognizing  opioid receptors on U937 cells (Buchner et al., 1997). However, the use of antibodies is more apt to focus on characterizing the structural properties of the cloned receptor (e.g. mapping ligand-binding domains) rather than using such antibodies to antagonize tolerance or chemical dependence.

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