Molecular pharmacology of the opioid receptors

Pergsmon

Olfi3-7258(95)02011-H

Pharmac. 77th~~Vol. 68, No. 3, pp. 343-364, 1995 Copyright 0 1995 Elsevier Science Inc. Printed in Great Britain. All rights reserved 0163-7258195 $29.00

Associate Editor: M. KIMURA

MOLECULAR PHARMACOLOGY THE OPIOID RECEPTORS MASAMICHI

SATOH* and MASABUMI

OF

MINAMI

Department of Molecular Pharmacology, Faculty of Pharmaceutical Sciences, Kyoto University, Kyoto 606-01, Japan Abstract - Opioid receptors are the primary sites of actions of opiates and endogenous opioid peptides, which have a wide variety of pharmacological and physiological effects. The opioid receptors are classified into at least three subtypes, p, 6, and x, and their cDNAs have been cloned. In this review, we describe the molecular cloning of opioid receptor gene family and studies of the structure-function relationships, modes of coupling to second messenger systems, pharmacological effects of antisense oligonucleotides, and anatomical distribution of opioid receptor mRNAs.

Keywoads - Opioid receptors, molecular cloning, receptor subtypes, _ second messenger - systems, . in situ hybridization, morphine.

CONTENTS 1. Introduction 2. Molecular Cloning of the Opioid Receptor Gene Family 2.1. Molecular cloning of p-, a-, x-, and other related opioid receptors 2.2, Structural characteristics of opioid receptors 3. Molecular Pharmacology of the Ouioid Receotors 3.1. Pharmacology of zoned opidid receptois 3.1.1. ,e-Opioid receptors 3.1.2. &Opioid receptors 3.1.3. x-Opioid receptors 3.2. Relationship between the structure and function of opioid receptors 3.2.1. Subtype-specific ligand binding 3.2.2. Effects of sodium ions upon agonist and antagonist bindings 3.2.3. Coupling to the second messenger systems 3.3. Second messenger systems coupled to opioid receptors 3.3.1. Adenylate cyclase 3.3.2. Ca* + channels 3.3.3. K+ channels 3.3.4. Phospholipase C 3.4. Pharmacological studies with antisense oligonucleotides to opioid receptors 4. Distribution of Opioid Receptor mRNAs 4. I. Opioid receptor expression in the central nervous system 4.1-l. p-Opioid receptors 4.1.2. d-Opioid receptors 4.1.3. x-Opioid receptors *Corresponding

344 344 344 345 345 345 345 348 348 349 349 351 351 351 351 352 352 352 353 353 353 353 355 355

author.

Abbreviatibns-DADLE,

[D-Ala*, DLeu5lenkephalin; DAMGO, [D-Ala2, MePhe4, Gly-oP]enkephalin; DPDPE, [D-Pen*, D-Penslenkephalin; DRG, dorsal root ganglia; DSLET, [D-Ser2, Leus, Thr6]enkephalin; PAG, periaqu uctal gray; PPTA, preprotachykinin A; SP, substance P; U50,488H, (f)-trans-3,4-dichloroN-methyl-N-[ -( 1-pyrrolidinyl)cyclohexyl] benzeneacetamide; U69,593, (+)-(5a!,7o1,8/3)-N-methyl-N[7-( 1-pyrroli ” inyl)- 1-oxaspiro[4,5]dec-8-yl benzeneacetamide; X-OR, X-opioid receptor. 343

344

M. Satoh and M. Minami 4.2. Opioid receptor expression in the dorsal root ganglia 4.2.1. In situ hybridization histochemistry of p-, a-, and x-opioid receptors 4.2.2. Coexistence of opioid receptors with substance P 5. Concluding Remarks References

356 356 358 359 359

1, INTRODUCTION Opiates, such as morphine, and endogenous opioid peptides are considered to exert their pharmacological and physiological effects on target tissues through binding to cell surface receptors termed opioid receptors. In the early 197Os,opioid receptors were demonstrated in the brain by binding studies using radiolabeled opioid ligands (Pert and Snyder, 1973; Simon et al. , 1973; Terenius, 1973). Several types of opioid receptors had been postulated, since nalorphine did not necessarily antagonize different narcotic analgesics to the same extent (Veatch et al., 1964; Cox and Weinstock, 1964), and the first definitive pharmacological evidence supporting this notion was reported by Martin and colleagues (Martin et al., 1976; Gilbert and Martin, 1976). They proposed that there were three types of opioid receptors (“opiate receptors” at that time) based on the pharmacological actions of various opiates and their derivatives in chronic spinal dogs, and named them p for the morphine group, x for the ketocyclazocine group, and cr for N-allylnormetazocine (SKF10047). In addition to these three types of receptors, Lord et al. (1977) found a high-affinity receptor for enkephalins in the mouse vas deferens and named it the b-receptor. Furthermore, an e-receptor was proposed as the binding site for fl-endorphin in the rat vas deferens (Schulz et al., 1979). Several investigators have isolated and purified opioid receptor proteins from cell membranes since their existence was confirmed (for a review, see Simon and Gioannini, 1993). For example, we purified a 58 kDa protein that selectively bound to p ligands, such as [D-Ala*, MePhe4, GlyoP]enkephalin (DAMGO), and functionally coupled to pertussis toxin-sensitive G-protein (Gi and G,) in reconstituted phospholipid vesicles (Ueda et al., 1988). However, none of these attempts revealed the structures of the proteins that were pharmacologically defined as opioid receptors. Evans et al. (1992) and Kieffer et al. (1992) have identified the structure of the d-opioid receptor without purifying the protein. Thereafter, cDNAs of the p- and x-opioid receptors were cloned. Cloning the cDNAs of p-, 6- and x-opioid receptors have allowed the clarification of the regulation of synthesis and anatomical distributions of these receptors, as well as their biochemical and pharmacological properties. This article basically reviews the progress in opioid research using the cloned opioid receptor cDNAs.

2. MOLECULAR 2.1.

Molecular

CLONING

OF THE OPIOID

RECEPTOR

GENE

FAMILY

Cloning of p-, 6-, x-, and Other Related Opioid Receptors

Evans et al. (1992) and Kieffer et al. (1992) cloned the cDNA encoding mouse b-opioid receptor form NG108-15 cells by expression cloning with radiolabeled &opioid ligands. Thereafter, CL-and x-opioid receptor cDNAs were cloned based upon their homology to the cloned 8-opioid receptor. Rat p-opioid receptor cDNA was cloned by Chen et al. (1993), Fukuda et al. (1993), Thompson et al. (1993), Wang et al. (1993), and Minami et al. (1994). Min et al. (1994) cloned the mouse CL-opioidreceptor gene. Furthermore, alternative splicing of CL-opioidreceptor mRNA has been suggested, although it appeared not to affect the ligand binding and coupling to second messenger systems (Zimprich et al., 1994; Bare et al., 1994). Mouse (Yasuda et al., 1993) and rat (Li et al., 1993; Meng et al., 1993; Minami et al., 1993b; Nishi et al., 1993) cDNAs encoding the x-opioid receptor have been cloned. A rat &opioid receptor cDNA has been cloned (Fukuda et ul., 1993). Other receptor cDNAs thought to belong to this gene family have been cloned. Several investigators have cloned the same rat cDNA (Bunzow et al., 1994; Chen et al., 1994; Fukuda et al., 1994; Wang, et al., 1994a; Wick et al., 1994). In the present review, this clone is called X-opioid receptor (X-OR), referring to the paperTublished by Wang et al. George and colleagues have cloned a cDNA that may encode the e-receptor, from a human genomic library (for a review, see Uhl et al. , 1994). The amino acid sequence

345

Opioid receptors

of this receptor is 36-39% identical with those of the p-, 6-, and x-opioid receptors. Although the expressed gene product binds to /3-endorphin and has some pharmacological characteristics of the e-receptor, the submicromolar affinity for P-endorphin is much lower than the nanomolar affinity of the e-receptor determined by most pharmacological experiments using the rat vas deferens. Human cDNAs for p-, 6-, and x-opioid receptors have been described (Wang et al., 1994b; Knapp et al., 1994; Mansson et al. , 1994). The human p-, 6-, and x-opioid receptor genes are located on chromosomes 6q24-25 (Wang et al., 1994b), 1~34.3-36.1 (Befort et al., 1994), and 8q11.2 (Yasuda et al., 1994), respectively. A human cDNA corresponding to rat X-OR has also been cloned (Mollereau et al. , 1994). 2.2. Structural

Characteristics

of Opioid Receptors

Hydrophobicity analyses of the deduced amino acid sequences of the cloned opioid receptors have indicated that these receptors have seven putative transmembrane helices characteristic of the G-protein coupled receptor family. A proposed model of the membrane topography of the rat p-opioid receptor is shown in Fig. 1. In Fig. 2, amino acid sequences of rat p-, 6-, and x-opioid receptors are compared. Overall, the amino acid sequences of these receptors are about 60% identical to one another (Table 1). Higher identities are found in the transmembrane regions (73-76% of those identified) and intracellular regions (63-66% of those identified). Conversely, the extracellular regions are considerably divergent (34-40% of those identified). Among the other G-protein coupled receptors, somatostatin ,receptors are the most similar to these opioid receptors, having 34-42% identity in amino acid sequences overall. Furthermore, AT iA and ATla angiotensin II receptors, formylmethionylleucylphenylalanine receptor, NPY I and NPYJ neuropeptide Y receptors and interleukin8 receptor have 20-30% identity. There are potential N-linked glycosylation sites in the N-terminal domains of the p-, 6-, and xopioid receptors and X-OR (Table 2). The long form of X-OR also has a putative glycosylation site in the second, extracellular loop. Two conserved cysteine residues, which are thought to be involved in disulfide bonding, are found in the first and second extracellular loops of these four receptors. A cysteine residue in the C-terminal domain, which is a potential site for palmitoylation, is conserved and the amino acid sequence from the start of the C-terminal intracellular domain to this cysteine residue is highly conserved across these receptors. This suggests that this region constructs a fourth intracellular loop that plays an important role in coupling to second messenger systems.

3. MOLECULAR

PHARMACOLOGY

3.1. Pharmacology

OF THE OPIOID

RECEPTORS

of Cloned Opioid Receptors

3.1.1. p-Opioid Receptors The cloned IL-opioid receptor has high affinity to morphine, naloxone and [D-Ala*, D-Leu5] enkephalin (DADLE). Furthermore, the p-selective ligand DAMGO (Kosterlitz and Paterson, 1981), but not the &selective ligand [D-Pen*, D-PetQ]enkephalin (DPDPE) (Mosberg et al., 1983) or the x-selective ligands (f)-trans-3,4-dichloro-N-methyl-N-[2-(l-pyrrolidinyl)cyclohexyl] benzeneacetamide (U50,488H) (von Voigtlander et al., 1983) and (+)-(5cr,7o,8~)-iV-methyl-N-[7-(l-pyrrolidinyl)-loxaspiro[4,5]dec-8-yl benzeneacetamide (U69,593) (Lahti et al., 1985), binds to the receptor with a high affinity, confirming that the cloned receptor is of the p-type (Table 3). The CL-opioidreceptors are further classified into pl- and F2-subtypes (Pasternak and Wood, 1986). The former are characterized by their high affinity for naloxonazine (irreversible binding) and some opioid ligands, such as morphine and DADLE (Kd = 0.1-2 nM). They are thought to be involved in several opioid effects, such as supraspinal analgesia, prolactin release, decrease in acetylcholine turnover, and the induction of catalepsy (Pastemak and Wood, 1986, Pasternak, 1988). The latter are characterized by a negligible affinity for naloxonazine and lower affinity for morphine and DADLE (& = 5-50 nM). They are thought to be involved in respiratory depression, decreased dopamine turnover, and the delayed gastrointestinal tract transit induced by opioids. Thompson et al. (1993) have classified the cloned p-opioid receptor as the p2-subtype due to its relatively low affinity for morphine and DADLE (Ki = 6-8 nM). On the other hand, the high affinity of the same cloned CL-opioidreceptor for naloxonazine

Fig. 1. Proposed model for the membrane topography of the rat p-opioid receptor. 3ranched and zig-zag structures show the potential sites for N-linked glycosylation and palmitoylation, respectively. Cysteine residues in the first and second extracellular loops are considered to be invoked in disulfide bonding.

CELLULAR

EXTRACELLULAR

347

348

M. Satoh and M. Minami Table 1. Amino Acid Identities Among the

Rat p-, 6-, and x-Opioid Receptors Amino acid identity

PI6 Extracellular regions N-terminal First loop Second loop Third loop Transmembrane (TM) regions TM1 TM2 TM3 TM4 TM5 TM6 TM7 Intracellular regions First loop Second loop Third loop C-terminal (before palm.) C-terminal (after palm.) Total

CL/X

%

%

6/x %

34 25 72 42 18

36 33 67 35 18

40 31 72 54 11

76 69 100 82 45 79 77 86

73 62 84 91 32 79 73 95

74 62 84 91 55 75 68 90

63 90 91 87 82 27

66 100 91 87 91 31

63 90 95 83 82 21

58

59

61

Palm., conserved cystein residue for potential palmitoylation.

suggests that the cloned p-receptor corresponds to the pl-subtype (Chen et al., 1993; Raynor et al., 1994; Wang et al., 1993). 3.1.2.

&Opioid Receptors

The cloned b-opioid receptor has high affinity for DADLE and the &opioid selective ligand DPDPE, but not the p-selective ligand DAMGG or the x-selective ligands U50,488H and U69,593, confirming that the cloned receptor is of the B-type (Table 3). The existence of d-opioid receptor subtypes has been suggested by behavioral and binding studies (Portoghese et al., 1992). DPDPE, DADLE, [D-Ala*, Leu5, Cy#]enkephalin and 7-benzylidenenaltrexone bind to Al-sites with high affinity, while [D-Ser2, Leu5, Thtilenkephalin (DSLET), [D-Ala2]deltorphin II, naltrindole S-isothiocyanate and naltriben bind to b2-sites with high affinity. Although whether the cloned li-opioid receptor is 61 or 62 has not been defined due to a lack of sufficiently selective ligands, its higher affinity for 62 than for Sl ligands suggests that it is of the 62-subtype (Raynor et al., 1994). This assumption is supported by the finding that antisense oligonucleotides to the cloned b-opioid receptor suppress the analgesic effects of the 62, but not those of the 61 agonist (see Section 3.4). 3.1.3.

x-Opioid Receptors

The cloned x-opioid receptor has high affinity for dynorphin A (l-17), which is supposed to be an endogenous x-opioid agonist (Chavkin et al., 1982), and the x-selective ligands U50,488H and U69,593, but not the p-selective ligand DAMGO or the &selective ligand DPDPE, confirming that the cloned receptor is of the x-type (Table 3). The x-opioid receptors have been classified into xland x2-subtypes (Zukin et al., 1988). Although most x-opioid ligands, including dynorphin A (l- 17), U50,488H, ethylketocyclazocine, bremazocine, and tifluadom, bind to both sites, U69,593 selectively binds to the xl site with high affinity. In contrast, there are no selective ligands for the x2 site yet. In addition to these two x sites, Clark et al. (1989) proposed a x3 site, which has high affinity to naloxone benzoylhydrazone, but no affinity for U50,488H. Since the cloned x-opioid receptor binds with high affinity to U69,593, as well as dynorphin A (l-17), U50,488H, ethylketocyclazocine, bremazocine, and tifluadom (Raynor et al. , 1994), the cloned receptor corresponds to the x l-subtype.

Opioid receptors

349

Table 2. Properties of Opioid Receptors Cloned cDNA

human

human mouse rat

mouse rat

X-OR2

x-OR

&OR

u-OR’

human mouse rat -

human rat X-0RL2 X-0RS2 (rat)

Splice variants

/L-OR P-OR!? (human, rat)

Structural information

400 a.a.4 (human a-OR) 392 a.a. (human p-ORS) 398 a.a. (rat p-OR, mouse) 391 a.a. (rat p-ORS)

372 a.a. (human, mouse, rat)

380 a.a. (human, mouse, rat)

370 a.a. (human) 395 a.a. (rat X-ORL) 367 a.a. (rat X-ORS)

Chromosomal localization

6q24-25 (human)

1~34.3-36.1 (human)

8qll.2

-

Putative glycosylation sites

5 (human, rat) 4 (mouse)

2 (human, mouse, rat)

2 (human, mouse, rat)

3 (human, rat X-ORS) 4 (rat X-ORL)

Putative palmitoylation

yes (human, mouse, rat)

yes (human, mouse, rat)

yes (human, mouse, rat)

yes (human, rat)

Signal transduction

CAMP 1 Ca2+ channel 1 K+ channel t

CAMP 1 Ca2+ channel 1 K+ channel t

CAMP i Ca2+ channel 1 K+ channel t

CAMP 1? -

(human)

IOR, opioid receptors. 2Refers to the paper of Wang et al., 1994a. 3p-ORS, p-OR shorter form. ‘+a.a.. amino acid residues.

3.2. Relationship 3.2.1.

Between the Structure and Function of Opioid Receptors

Subtype-specific Ligand Binding

Pharmacological studies using various subtype-specific ligands have classified several subtypes or subclasses of opioid receptors. The molecular basis for how these ligands discriminate among the subtypes or subclasses of opioid receptors remains unknown and of considerable interest. Cloning the cDNAs for CL-,6-, and x-opioid receptors has allowed investigation into the structural basis for the subtype specificity of the opioid receptors in their ligand binding by molecular biological means. Chimeric and mutated opioid receptors are thought to be useful materials with which to investigate this issue, as demonstrated by their application to understanding adrenaline (Kobilka et al., 1988; Frielle et al., 1988), acetylcholine (Wess et aE., 1992), dopamine (McAllister et al., 1993), and tachykinin (Yokota et al., 1992; Gether et al., 1993) receptors.

Table 3. Aflnity (Ki) of Typical Opioid Ligands to c-, S-, and x-Opioid Receptors Bpressed in CHO Cells Ligand Morphine Naloxone DADLE DAMGO DPSPE Dynorphin A (1-17) U50,488H U69,593

a

6

x

1.4 3.9 6.4 0.87 >looo 120 >looo >lOOO

>looo 95 2.8 >looo 7.6 45 >looo >looo

163 16 514 >looo >looo 5.48 1.4 2.3

The calculated Ki (nM) values are obtained from displacement studies with [3H]DAMG0, [3H]DPDPE and [3H]U69,593 as subtypespecific radiolabeled ligands for p-, a-, and x-opioid receptors, respectively (unpublished data).

M. Satoh and M. Minami

350

P

DAMGO

3.46 f 0.84

Bremazocine 2.68

f 0.22

MDDD

DMMM

MDMM

DMDD

Low affinity

3.16 I!Z0.91

Low affinity

5.24 I?I0.86

4.71 f 0.66

0.95 f 0.17

1.35 + 0.18

2.29 + 0.19

Fig. 3. I
DAMGO is a highly selective p-opioid receptor ligand. A deletion of 64 N-terminal amino acids failed to affect the binding of DAMGO to the truncated p-opioid receptor (p65-398), indicating that the N-terminal of the ,u-opioid receptor is not involved in the p-selective binding of DAMGO (Surratt et al., 1994). Onogi et al. (1995) have demonstrated that the structure around the first extracellular loop, in which there are only 7 amino acid residues differing between p- and b-opioid receptors, is critical for DAMGO to distinguish between p- and &opioid receptors. They constructed several PIA chimeric receptors and found that all of those with the regions around the first extracellular loop of the p-opioid receptor bound to DAMGO with high affinity, whereas those having the first extracellular loop derived from the &opioid receptor lost the high affinity binding to DAMGO (Fig. 3). Naloxone is an opioid receptor antagonist with a high level of affinity for p-, a moderate level for x-, and relatively low affinity for b-opioid receptors (Table 2). The N-terminal truncated Cc-opioid receptor (p65-398) binds naloxone with a similar affinity to that of the wild-type (Surratt et al., 1994), suggesting that the N-terminal domain of the p-opioid receptor is not essential for binding naloxone, as well as DAMGO. On the other hand, through studies using the N-terminal truncated (x79-380) x-opioid receptor and a 6/x chimeric (61-69/x79-380) receptor, Kong etal. (1994b) found that the N-terminal of the x-opioid receptor is important for naloxone binding. These conflicting results concerning the recognition sites of naloxone between cc- and x-opioid receptors may be due to the size of the deletion. The truncated x-opioid and 6/x chimeric receptors lost not only the Nterminal extracellular domain, but also about 75% of the first transmembrane domain of the x-opioid receptor, whereas the truncated p-opioid receptor lost only the N-terminal extracellular domain. A difference in the recognition sites for agonists and antagonists was revealed in the x-opioid receptor, using chimeric 6/x- and x/&opioid receptors (Kong et al., 1994b). The 6/x chimeric receptor bound the x-selective agonist U69,593, but not the antagonist naloxone, whereas the x/6 chimeric (x1-781670-372) receptor bound naloxone, but not U69,593. These findings indicated that the recognition site for the x-selective agonist U69,593 does not locate in the N-terminal domain, unlike that for naloxone. Kong et al. (1994a) have reported that the second extracellular loop of x-opioid receptor is important for the binding of the x-selective agonist U50,488. However, Wang et al. (1994c) have revealed that the second extracellular loop of the x-opioid receptor is important for the high affinity binding of dynorphin A (l-17), but not that of U50,488. In contrast to the x-selective ligands, both the &selective agonist DPDPE and antagonist naltrindole bound to the x/S chimeric (x l-78/670-372), but not 6/x chimeric (6 1-69/x79-380), receptor, suggesting

Opioid receptors

351

that the N-terminal domain of the &opioid receptor is not necessary for binding either the selective agonist or antagonist (Kong et al., 1994b). 3.2.2.

Effect of Sodium Ions Upon Agonist and Antagonist Bindings

The affinity of opioid receptors for opioid agonists is decreased by sodium ions, whereas that for antagonists is rather enhanced (Pert and Snyder, 1974). The replacement of aspartic acid in the second transmembrane domain of the CL-and d-opioid receptors with asparagine reduces the affinity of agonist binding and the sodium effect (Surratt et al., 1994; Kong et al., 1993), indicating that this negatively charged residue is involved in the regulation of opioid agonist binding by sodium ions. Such regulation was much more remarkable in the binding of subtype-selective agonists such as DAMGO for the p-receptor and DPDPE, DSLET, and Met-enkephalin for the &receptor than non- or less-selective agonists, such as morphine, bremazocine, and buprenorphine. 3.2.3.

Coupling to the Second Messenger Systems

While both substitutions of aspartic acid with asparagine and glutamic acid in the second transmembrane domain of CL-opioidreceptor reduced the affinity for morphine to a similar extent, the difference in inhibitory coupling to adenylate cyclase was considerable between these two mutant receptors (Surratt et al. , 1994). Glutamic acid substitution retained the same level of adenylate cyclase inhibition as the wild-type, but asparagine substitution reduced the inhibition to less than the onefifth the wild-type level. These data suggest that a negative charge in the second transmembrane domain of the p-opioid receptor is necessary for efficient coupling between the morphine-stimulated receptor and G-protein. On the other hand, the substitution of aspartic acid with asparagine in the second transmembrane domain of the b-opioid receptor did not reduce the inhibitory effects of the b-selective agonist DSLET and the nonselective agonist bremazocine on adenylate cyclase (Kong et al. , 1993). 3.3. Second Messenger

Systems Coupled to Opioid Receptors

3.3.1. Adenylate Cyclase Coupling of the opioid receptors to the inhibitory system of adenylate cyclase has been studied in transformed cell lines and in brain membranes (for a review, see Childers, 1993). The mechanisms of the receptor coupling to adenylate cyclase have been studied mostly in NG108-15, which is a hybridoma cell line consisting of mouse neuroblastoma N18TG-2 and rat glioma C6Bu-1. Opioid receptors on NG108-15 cells were identified as being of the &type (Chang et al., 1981). In this cell line, opioids inhibit both basal and prostaglandin El-stimulated adenylate cyclase activity (Sharma et al., 1975). The human neuroblastoma cell line SK-N-SH and its subclone SH-SYSY express both p and &opioid receptors, which are coupled to the inhibitory system of adenylate cyclase (Yu et al., 1986, 1990). Pertussis toxin abolishes the inhibition of adenylate cyclase by opioids in NG108-15 cells (Bums et al., 1983), suggesting that Gi- (or G,,-) protein is coupled to these opioid receptors to exert their inhibitory effects. The coupling of p- and 6-opioid receptors to the inhibitory system of adenylate cyclase has been demonstrated in the brain using membrane preparations derived from several brain regions, including the striatum (Collier and Roy, 1974), thalamus (Childers, 1993), and periaqueductal gray (PAG) (Fedynyshyn and Lee, 1989). On the other hand, it was controversial as to whether or not x-opioid receptors were coupled to this inhibitory system. Although Polastron et al. (1990) have reported that adenylate cyclase is not inhibited by x-opioid agonists in the guinea pig cerebellum in which abundant x-opioid binding sites have been identified, the inhibitions of adenylate cyclase activity by x-agonists in the membrane preparations from the guinea pig cerebellum (Konkoy and Childers, 1989) and from the rat spinal cord (Attali et al., 1989) have been demonstrated. The cloned p-, 6- and x-opioid receptors expressed in COS or CHO cell lines are coupled to the inhibitory system of adenylate cyclase via pertussis toxin-sensitive G-proteins (Evans et al., 1992; Yasuda et al., 1993; Chen et al., 1993; Wang et al., 1993, Meng et al., 1993). Furthermore, Kaneko et al. (1994b) revealed that the x-opioid agonist U50,488H potentiated the increase in the intracellular CAMP concentration evoked by forskolin/3-isobutyl-1-methylxanthine using the Ximopus oocyte co-

352

M. Satoh and M. Minami

injected with RNAs encoding the x-opioid receptor and cystic fibrosis transmembrane conductance regulator, a Cl- channel protein activated by CAMP-dependent protein kinase. This effect was blocked by pertussis toxin, suggesting mediation via Gi- and/or Go-proteins. In this context, stimulation of adenylate cyclase through fly subunits of GicO,-protein has been reported (Federman et al., 1992). Adenylate cyclase Type II is stimulated by &-subunits. This kind of stimulation of adenylate cyclase by opioid agonists has been found in the rat olfactory bulb (Olianas and Onali, 1992; Onali and Olianas, 1991), where &opioid receptors (Mansour er al., 1987) and Type II adenylate cyclase (Feinstein et al., 1991) are abundantly expressed. 3 . 3 .2 . Ca2+ Channels One cellular event that underlies the effects of opioids to reduce cellular excitability and neurotransmitter release is the inhibition of voltage-dependent Ca2+ channels (for reviews, see North, 1986, 1993). The activation of CL-,6-, and x-opioid receptors reduces voltage-dependent Ca*+ currents in various preparations, including a human neuroblastoma cell line (Seward et al., 1991), dorsal root ganglion cells (Schroeder et al., 1991; Gross and MacDonald, 1987; MacDonald and Werz, 1986), and guinea pig submucosal neurons (Surprenant et al., 1990; Shen and Surprenant, 1990). This reduction of Ca2+ currents through opioid receptors is blocked by pertussis toxin (Seward et al., 1991; Surprenant et al., 1990), indicating the involvement of Gi- and/or Go-proteins. Hescheler et al. (1987) revealed that G, is more effective than Gi in reconstituting the inhibitory action of opioids on Caz+ channels in the cells pretreated with pertussis toxin. The cloned x-opioid receptor expressed in PC12 cells couples with the Ca2+ channel system to reduce Ca2+ conductance through N-type channels (Tallent et al., 1994). Functional coupling between the cloned x-opioid receptor and voltage-dependent Ca *+ channels has been identified in the X&opus oocyte system. The x-opioid agonist U50,488H inhibited Ca2+ channels in Xbzopus oocytes co-injected with in vitro transcribed mRNAs encoding the x-opioid receptor and c-wl-and P-subunits of the Ca2+ channel (Kaneko et al., 1994a). 3.3.3.

K+ Channels

Another cellular event, which is thought to be important for opioids to reduce cellular excitability and inhibit neurotransmitter release, is the membrane hyperpolarization caused by an increase in K+ conductance (for reviews, see North, 1986, 1993). The activation of p- and S-opioid receptors increases an inwardly rectifying K+ conductance in various preparations, including the locus coeruleus (North et ul., 1987), hippocampus (Wimpey and Chavkin, 1991), and submucosal plexus (North et al., 1987). The x-opioid receptor also increases K+ conductance in guinea pig substantia gelatinosa neurons (Grudt and Williams, 1993). These increases in K+ conductance caused by the activation of opioid receptors are sensitive to pertussis toxin, indicating mediation through Gi- and/or Go-proteins (Tatsumi et al., 1990). The increase in K+ conductance by the stimulation of opioid receptors has been confirmed using the Xenopus oocyte system, in which the cloned p- or x-opioid receptor and G-protein-activated K+ channel are co-expressed (Chen and Yu, 1994; Henry et al., 1994). These increases of K+ conductance were also blocked by pertussis toxin, 3.3.4.

Phospholipase

C

As described in Sections 3.3.2 and 3.3.3, activation of opioid receptors generally inhibits neuronal excitability through inhibiting Ca 2+ channels and activating K+ channels. However, opioids are frequently excitatory in vivo. Most of these effects are considered to be due to disinhibition mechanisms: for example, inhibition of GABAergic neurons. Attempts to reveal the direct coupling of opioid receptors to an excitatory system, such as phosphoinositide cascade, in NG108-15 and SK-N-SH cells have not succeeded (Yu and Sadee, 1986). However, opioids mobilize Ca*+ from the inositol 1,4,5trisphosphate-sensitive store in NGlOS-15 cells (Jin ef al., 1994). This mobilization of Ca2+ from internal stores was mediated by Gi- and/or Go-protein and depended upon the cell growth conditions. The cloned opioid receptors expressed on Xenopus oocytes can mediate the oscillatory Cl- current response (Minami et al., 1993; Miyamae et al., 1993b; Kaneko et al., 1994b). This type of oocyte

353

Opioid receptors

response is mediated by mobilizing Ca 2+ from internal stores via inositol phosphate formation (Nomura et ~1.) 1987). Gi- and/or Go-proteins are probably involved in this response in Xenopus oocytes expressing opioid receptors, since pertussis toxin blocked it (Kaneko et al., 1994b). The @y-subunits liberated by activating heterometic Gi- and G,-proteins can stimulate phospholipase C/32 (Camps et al., 1992; Katz et al., 1992). This mechanism probably contributes to the pertussis toxinsensitive activation of phosphoinositide metabolism evoked by the stimulation of opioid receptors. 3.4. Pharmacological

Studies with Antisense Oligonucleotides to Opioid Receptors

Several investigators have performed neurophatmacological studies using an antisense oligonucleotide strategy (for a review, see Wahlestedt, 1994). The basic principle of this strategy is to inhibit the synthesis of a specified protein by interference with the information flow from gene to protein. Selective pharmacological tools can then be designed to test pharmacological and biological hypotheses. Cloning and sequencing opioid receptor cDNAs have enabled the use of antisense approaches to elucidate receptor-mediated opioid pharmacology. An injection of antisense, but not mismatch, oligodeoxynucleotides directed against the cloned p-opioid receptor into the PAG of rats eliminated the analgesic effects of morphine injected into the same site (Rossi et al., 1994). Lai et al. (1994) has reported that the intracerebroventricular administration of antisense, but not sense, oligonucleotides for the cloned &opioid receptor selectively inhibited the antinociceptive effect of an intracerebroventricular injection of [D-Ala2, Glu4]deltorphin (classified as an opioid 62 agonist), without altering the antinociception produced by DPDPE, a putative 61 agonist. These findings suggested that the cloned 6 receptor is the same as that classified pharmacologically as 62. Moreover, an intrathecal injection of antisense, but not mismatch, oligonucleotide reduced the antinociceptive effect of both deltorphin II and DPDPE injected into the same site (Standifer et al., 1994). These 6 agonists appear to produce analgesia through the 62 receptors in the spinal cord (Mattia et al., 1992). Furthermore, an intrathecal injection of antisense, but not mismatch, oligonucleotides for the cloned x-opioid receptor selectively blocked the antinociceptive effect of U50,488H, a x-opioid selective agonist, without affecting the antinociceptive effects of p- and &opioid agonists (Chien et al., 1994).

4. DISTRIBUTION

OF OPIOID

4.1. Opioid Receptor Expression

RECEPTOR

mRNAs

in the Central Nervous System

4.1.1. p-Opioid Receptors The expression of CL-opioidreceptor mRNA in the rat brain has been revealed by in situ hybridization histochemistry (Delfs et al. , 1994; Mansour et al. , 1994b; Minami et al. , 1994). In the telencephalon, signals of p-opioid receptor mRNA have been found in the internal granular and glomerular layers of the olfactory bulb. Dense patchy signals of CL-opioid receptor mRNA have been detected in the caudate putamen and nucleus accumbens. Although the microelectrophoretic application of morphine to these areas reportedly inhibits the firing of neurons (Gayton and Bradley, 1976; McCarthy et al., 1977)) the contribution of these actions to the effects of systemic opiates on locomotion and analgesia remains unknown. The expression of the message is moderate to intense in the globus pallidus and ventral pallidus, and is intense in the medial septum and nucleus diagonal band. Cells expressing p-opioid receptor mRNA are disseminated in the hippocampus, where locally applied opiates and opioid peptides excite the pyramidal cells, probably through the disinhibition of tonically active inhibitory neurons such as GABAergic neurons (Zieglgansberger et al., 1979). The relatively few basket cells in the hippocampus inhibit pyramidal cell activity, probably by releasing y-aminobutyric acid (Curtis et al. , 1970).,~-Opioid receptor mRNA is moderately to intensely expressed in the amygdaloid complex, including the basolateral, medial, cortical, and central amygdaloid nuclei. The b-opioid receptor mRNA is also expressed in the preoptic area, being especially intense in the medial region. In the diencephalon, expression of the yopioid receptor mRNA is intense in the paraventricular, paratenial , me&dorsal, paracentral, centmmedial , centrolateral, ventmlateral, ventromedial, rhomboid,

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and posterior thalamic nuclei, and moderate in the lateroposterior thalamic nuclei. In the ventral posterolateral and ventral posteromedial thalamic nuclei, expression of p-opioid receptor mRNA is relatively weak in the anterior part, but intense in the posterior part. Signals of p-opioid receptor mRNA are very weak or undetectable in the anteroventral, anterodorsal, laterodorsal, reticular, and parafascicular thalamic nuclei. Anatomical and physiological studies indicate that neurons of the medial thalamic nuclei, which express high levels of g-opioid receptor mRNA, play a role in the transmission of nociceptive information (Albe-Fessard and Kruger, 1962; Kaelbar et al., 1975). Furthermore, the neurons in these nuclei are selectively depressed following the administration of morphine (Duggan and Hall, 1977; Nakahama et al., 1981). The expression of CL-opioid receptor mRNA is intense also in the ventrolateral and ventromedial thalamic nuclei, where morphine also depresses nociceptive responses in neurons (Nakahama et al., 1981). The levels of p-opioid receptor mRNA are high in the posterior thalamic nuclear group and the caudal part of the ventral posterolateral and ventral posteromedial thalamic nuclei, but low in the rostra1 part of those nuclei. These findings are consistent with the results of an electrophysiological study, which demonstrated that analgesic doses of intravenous morphine depress the discharges evoked by noxious stimulation in the posterior thalamic nuclear group and the caudal part of the ventrobasal thalamus, but not in the rostra1 part of the ventrobasal thalamus (Shigenaga and Inoki, 1976). In the lateral and medial geniculate nucleus, the expression of p-opioid receptor mRNA is intense in the dorsal parts and moderate in the ventral parts. The mRNA signals are moderate in the lateral, anterior, arcuate, supramammillary, and medial mammillary nuclei of the hypothalamus. The level of Cc-opioid receptor mRNA is high in the medial, but not the lateral, habenular nucleus. In the mesencephalon, p-opioid receptor mRNA is intensely expressed in the interpeduncular nucleus. In the midbrain raphe nuclei, p-opioid receptor mRNA is expressed intensely in the medial raphe nucleus and weakly in the dorsal raphe nucleus. The mRNA signals are moderate in the PAG and superior colliculus, and intense in the inferior colliculus. In the pons medulla, CL-opioidreceptor mRNA is intensely expressed in the locus coeruleus. This nucleus is composed almost entirely of noradrenergic neurons. Since the cell bodies of noradrenergic neurons that account for more than half of the not-adrenaline content of the brain are found in this nucleus (Amaral and Sinnamon, 1977), a considerable portion of the terminals, as well as cell bodies, of the noradrenergic neurons, which widely innervate many other brain regions, may have p-opioid receptors. Indeed, a p-selective agonist, DAMGO, reportedly inhibits the release of noradrenaline from slices of the rat cortex, hippocampus, and cerebellum (Werling et al., 1987). Opiate analgesics such as morphine have several side effects. Especially in humans, death from morphine poisoning is nearly always due to respiratory arrest (Jaffe and Martin, 1990). Respiratory depression by opiates, at least in part, is caused through their direct effect on the brain stem respiratory centers. Respiratory neurons are located in pontobulbar structures, especially in the nucleus solitaris, ambiguous nucleus, and parabrachial nucleus (Vibert et al. , 1976). The expression of p-opioid receptor mRNA is intense in these nuclei, suggesting that this receptor plays an critical role in respiratory depression by opiates. Moderate expression of the mRNA is observed widely in the pons medulla, including the raphe magnus, intermediate reticular, gigantocellular reticular, and lateral paragigantocellular nuclei. Supraspinal mechanisms, as well as spinal mechanisms, of the analgesic effects of opiates have been investigated vigorously. Opiates act on several regions of the lower brain stem to activate the descending pain inhibitory systems, which consist of descending noradrenergic and serotonergic neurons (Basbaum and Fields, 1984). Microinjections of morphine into the gigantocellular reticular nucleus (Takagi et al., 1977), lateral paragigantocellular nucleus, raphe magnus nucleus, and PAG (Satoh et al., 1983) produced analgesic effects in much lower doses than systemic injections, suggesting that the cells specific to the supraspinal mechanisms for the analgesic effects of opiates exist in these brain regions. The CL-opioid receptor mRNA apparently is not expressed in the cerebellum. In the spinal lumbar dorsal horn (Maekawa t al., 1994), expression of p-opioid receptor mRNA is intense in the superficial laminae (laminae I and II), where nociceptive C and A6 fibers of primary afferents principally terminate (Light and Perl, 1977) and where Met-enkephalin immunoreactive fibers are distributed (Merchenthaler et al., 1986). This suggests that the receptors play important roles in the modulation of nociceptive information at postsynaptic sites of the primary afferents. There are moderate signals in the neck of the dorsal horn (laminae V and VI), but weak ones in

Opioid receptors

355

laminae III and IV, where p-agonists fail to inhibit the nociceptive responses of multireceptive cells (Hope et al., 1990). In the lumbar ventral horn, there are moderate to intense signals of ,u-opioid receptor mRNA in laminae VII and VIII. Many neurons in lamina VIII project into the thalamus (Granum, 1986) or medullary reticular formation (Kevetter and Willis, 1982) and are involved in relaying nociceptive information. The IL-opioid receptor expressed in this lamina might function in modulating nociceptive information through spinothalamic or spinoreticular tracts. The expression of CL-opioid receptor mRNA is slight in lamina IX. The mRNA is moderately expressed in lamina X, neurons which receive nociceptive information and project to the lateral reticular nuclei, nucleus reticularis gigantocellularis, or nucleus reticularis paragigantocellularis (Nahin et al., 1984). 4.1.2.

S-Opioid Receptors

In situ hybridization histochemistry for &opioid receptor mRNA in the mouse brain has been performed by ,Mansour et ~1. (1993) and IeMoine et al. (1994). Bzdega et al. (1993) gave a preliminary description of the distribution of b-opioid receptor mRNA in the rat brain. In the telencephalon of the mouse brain, b-opioid receptor mRNA is expressed in the cerebral cortex, predominantly in layers II, III, V, and VI. Relatively high levels of &opioid receptor mRNA are expressed in the caudate putamen and olfactory tubercle. In the nucleus accumbens, the expression of the mRNA is intense in the ventral portions of the anterior shell, with lower expression in the core. There is no detectable’&opioid receptor mRNA in the globus and ventral pallidurn. 6-Opioid receptor mRNA is localized in the pyramidal cell layer of the hippocampus and in the granular cell layer of the dentate gyrus. In the amygdaloid complex, the expression of b-opioid receptor mRNA is intense in the lateral and basolateral amygdaloid nuclei, moderate in the cortical nucleus, and low or absent in the medial and central nuclei. The mRNA for the &opioid receptor is also expressed in the olfactory bulb, primarily in the internal granular cell layer. In the diencephalon of the mouse brain, there is little or no expression of 3-opioid receptor mRNA throughout the thalamus. In the hypothalamus, significant, but low density, signals of b-opioid receptor mRNA are found in the ventromedial nucleus. In various midbrain regions, such as the interpeduncular nucleus, substantia nigra, and periaqueductal grey, &opioid receptor mRNA is undetectable, whereas the level of the mRNA is high in the entopeduncular nucleus. The &opioid receptor mRNA is also expressed in the pontine nuclei, reticular tegmemnl pontine nucleus, trapezoid nucleus, and nucleus solitaris, but not in the dorsal, median, and linear raphe, parabrachial, medial vestibular, dorsotegmental, lateral reticular, cochlear, and olivary nuclei. The b-opioid receptor mRNA is undetectable in the mouse cerebellum. In the rat brain, b-opioid receptor mRNA is located in the inte 11granular cell layer of the olfactory bulb, cerebral cortex, hippocampal area, amygdala, hypothalamic ventromedial nucleus, pontine nuclei, anterior pituitary, and pineal glands, but not in the caudate (Bzdega et al., 1993), where the level of expression is relatively high in the mouse brain. However, the absence in the rat caudate is probably due to insufficient sensitivity. Indeed, we detected b-opioid receptor mRNA at relatively high levels in the caudate putamen by Northern blotting (Minami et al. , 1995) and by in situ hybridization histochemistry (unpublished data). In the rat spinal cord, the expression of b-opioid receptor mRNA is low to moderate throughout laminae I-VI. In the ventral horn, the signals are moderate in laminae VII and VIII and low in lamina X. In lamina IX, b-opioid receptor mRNA is intensely expressed on the cells that probably consist mostly of motoneurons (unpublished data). 4.1.3.

x-Opioid Receptors

The distribution of x-opioid receptor mRNA in the brain of the rat (Minami et al., 1993a; Mansour et al. , 1994a) and mouse (DePaoli et al. , 1994) has been revealed by in situ hybridization histochemistry. Xie et al. (1994) described the distribution of x-opioid receptor mRNA in the guinea pig brain. In the telencephalic regions of the rat brain, the expression of x-opioid receptor mRNA is intense in the caudate putamen, especially in the medial part, and nucleus accumbens, but weak in the globus pallidus. The:expression of x-opioid receptor mRNA is low to moderate in the septum. In the cortical region, the mRNA was intensely expressed in the layer VI of the parietal, temporal, and occipital

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M. Satoh and M. Minami

cortex, likewise in the endopiriform nucleus, claustrum, and olfactory tubercle. Dense signals are evident also in the basolateral, central, medial, and anterior cortical amygdaloid nuclei. In the olfactory bulb, there are a few positive cells in the internal plexiform layer and glomentlar layer. The expression of x-opioid receptor mRNA is intense in the preoptic area, which plays an important role in thermoregulation. The x-opioid receptor expressed in this area might mediate a fall in body temperature by the administration of x-opioid agonists such as U50,488H (Olson et al., 1992). In the rat diencephalon, x-opioid receptor mRNA is intensely expressed in the paraventricular and supraoptic hypothalamic nuclei. x-Opioid agonists such as U50,488H reportedly induce diuresis through inhibiting the release of the antidiuretic hormone vasopressin (Leander et al., 1985), which is synthesized in these hypothalamic nuclei. The x-opioid receptor mRNA is highly expressed in the dorsomedial and ventromedial hypothalamic nuclei, which are thought to play an important role in the regulation of feeding. These nuclei might mediate an increase in feeding after the administration of x-opioid agonists (Olson et al., 1992). In the thalamus, x-opioid receptor mRNA is intensely expressed in the paraventricular thalamic nucleus and parafascicular thalamic nucleus. In the mesencephalic regions of the rat brain, x-opioid receptor mRNA is intensely expressed in the substantia nigra, especially the compact part, and the ventral tegmental area. These areas contain a number of dopaminergic neurons whose axons widely innervate various brain regions, including the caudate putamen, globus pallidus, nucleus accumbens, amygdala, and cerebral cortex. The release of dopamine from striatal and cortical slices is inhibited by x-opioid agonist, but not by CL-or (i-opioid agonists (Werling et al., 1988). These findings suggest that x-opioid receptor is synthesized and transported to the terminals of dopaminergic neurons and that it regulates the release of dopamine. This inhibitory action of x-opioid agonists on the release of dopamine might be involved in their effects on locomotion. The mRNA for x-opioid receptor is moderately expressed in the superior and inferior colliculi. There are also the signals in the PAG and widely spread in the medulla oblongata, which are believed to play a major role in antinociceptive processes. These regions might be the supraspinal sites where x-opioid agonists produce their antinociceptive effects (Millan et al., 1989). Furthermore, the density of x-opioid binding sites in the PAG increases in adjuvant arthritic rats, a model of chronic pain (Millan et al., 1987). There are several species differences in the distribution of x-opioid receptor mRNA among rat, mouse, and guinea pig brains. Intense expression of the mRNA is demonstrated in the dentate gyrus and cerebellum of the guinea pig brain, whereas there is little or no expression in these regions of the rat and mouse brains. The mRNA is intensely expressed in the locus coeruleus of the mouse brain, whereas only a small number of cells express it in this region of the rat brain. Such species differences suggest differences in the roles of the x-opioid receptor in several brain regions across the species. In the rat spinal cord (Maekawa et al., 1994), cells intensely express x-opioid receptor mRNA in laminae I and II, where nociceptive C and A6 fibers of primary afferents principally terminate (Light and Perl, 1977) and dynorphin immunoreactive fibers abundantly innervate (Botticelli et al., 1981). This suggests that the x-opioid receptor plays an important role in the modulation of nociceptive information at postsynaptic sites of primary afferents. The expression of the x-opioid receptor mRNA is moderate to intense throughout laminae III-VIII. Especially, intense signals are concentrated in the medial part of lamina IV. The finding of intense expression of x-opioid receptor mRNA, but slight expression of p-opioid receptor mRNA, in laminae III-IV morphologically supports the reports that x-, but not p-opioid agonists inhibit the nociceptive responses of multireceptive cells in these laminae (Hope et al., 1990). The mRNA is expressed moderately in lamina X, but very slightly in lamina IX. Mansour et al. (1995) have reviewed the distribution of opioid receptor mRNAs and opioid receptor binding sites in the rat CNS. 4.2. Opioid Receptor Expression 4.2.1.

in the Dorsal Root Ganglia

In Situ Hybridization Histochemistry of ,u-, 6-, and x-Opioid Receptors

The analgesic actions of opiates such as morphine and opioid peptides are attributed, at least in part, to their ability to inhibit the release of neurotransmitter(s) from primary afferent terminals

Opioid receptors

Fig. . Coexistence of the mRNA for c- (A), 6- (B), and X- (C) opioid receptors Wiith PI?TA mR? Lin the rat DRG. Autoradiographic silver grains (white dots in this figure) are a cc umul,ated on tl opioid receptor mRNA-positive cells. PPTA mRNA-positive cells are stain ed with .the Idphosphatase-reaction product. The arrows in A and C show double positii Irecells that ~1sboth c- (A) or X- (C) opioid receptor mRNA and PPTA mRNA. Bars = 50 IJ.m. 9

357

358

M. Satoh and M. Minami Table 4. Expression of the mRNA for Each Subtype of Opioid Receptor (OPR) in the PITA mRNA-Positive Cells of the Rat DRG OPR mRNA Background

Low

High

23 cells (2.1%)

87 (8.0%)

971 (89.9%)

S-OPR mRNA (919 PPTA mRNApositive cells)

568 (62.2%)

316 (34.6%)

29 (3.2%)

X-OPR mRNA (1409 PPTA mRNApositive cells)

913 (64.8%)

87 (6.2 %)

409 29.0%)

p-OPR mRNA (1081 PPTA mRNApositive cells)

Double in situ hybridization histochemistry was performed using a mixture of antisense RNA probes labeled with 35S for each subtype of OPR and digoxigenin for PPTA. Eighteen sections of the rat DRG (L4-L6) were examined for each subtype of OPR. The level of OPR mRNA expression was estimated by counting the microautoradiographic silver grains in each cell (O-14 grains/cell, background, 15-49; grains/cell, low expression; over 50 grains/cell, high expression). The expression of PPTA mRNA was estimated by a comparison with the sections hybridized to the digoxigenin-labeled sense RNA probe.

at the spinal dorsal horn. In the dorsal root ganglia (DRG), where the cell bodies of primary afferent neurons are located, the expression of p-, 6-, and x-opioid receptor mRNAs is intense (Maekawa et al. , 1994; Minami et al., 1995). They are expressed on cells morphologically regarded as neurons. The p-opioid receptor mRNA is highly expressed in about 55% of DRG neurons, which is more than the number of 6- (20%) and X- (18%) opioid receptor mRNA-positive neurons. 4.2.2.

Coexistence of Opioid Receptors with Substance P

Substance P (SP), which is a neuropeptide present in primary afferent neurons, plays a role in the transmission of nociceptive information from the periphery to the spinal cord (Otsuka and Konishi, 1977, Kuraishi et al., 1985, 1989), and its release from the spinal dorsal horn is regulated by opioids (Aimone and Yaksh, 1989; Hirota et al., 1985; Jesse11 and Iversen, 1977; Lembeck and Donnerer, 1985). Figure 4 shows the coexistence of p-, a-, and x-opioid receptor mRNAs with the mRNA for preprotachykinin A (PPTA), a precursor of SP, in the DRG using double in situ hybridization histochemistry with a 35S-labeled antisense RNA probe for each subtype of opioid receptor and digoxigenin-labeled antisense RNA probe for PPTA (Minami et al., 1995). The expression of p- and x-opioid receptor mRNAs is intense in about 90 and 30% of the SP-containing neurons, respectively (Table 4), suggesting that p and, at least in part, x-agonists act directly on the primary afferent terminals of SP-containing neurons to presynaptically modulate the SP release. On the other hand, b-opioid receptor mRNA is weakly or scarcely expressed in most SP-containing neurons, implying that the b-opioid receptor is not involved in the regulation of SP release. However, b-opioid selective agonists, such as DPDPE, suppress the SP release in both in vivo (Aimone and Yaksh, 1989) and in vitro (Mauborgne et al., 1987) environments. A small amount of &opioid receptor on neurons containing SP may efficiently couple to the inhibitory systems to suppress SP release. Alternatively, the b-opioid receptor might be indirectly involved in the regulation of the SP release. The b-opioid receptor mRNA is highly expressed in about 20% of DRG neurons, most of which are PPTA mRNAnegative. It is likely that these b-opioid receptors modulate the release of other neurotransmitters and/or neuromodulators more than SP, the release of which is indirectly regulated. Indeed, Dado et al. (1993) demonstrated the colocalization of b-opioid receptor with calcitonin gene-related peptide, a peptide that enhances the SP release from the primary afferent terminals at the spinal dorsal horn (Oku et al., 1987).

Opioid receptors 5. CONCLUDING

359

REMARKS

To date, five cDNAs have been cloned that belong to the opioid receptor gene family. Among them, three clones have been pharmacologically identified as CL-,6-, and x-opioid receptors. The other two clones have yet to be pharmacologically characterized. Pharmacological studies have further classified p-, 6-, and x-opioid receptors into subtypes, that is, ~1 and ,u2, 61 and 62, and xl and x2, respectively. Whether these subtypes are attributed to different genes or posttranslational processing needs to be clarified. Cloning of’the cDNAs for p-, 6-, and x-opioid receptors has given us powerful molecular tools with which to investigate opioid receptors. Studies with chimeric and mutated receptors will clarify which structures and amino acid residues in each receptor are important to its ligand binding and coupling to second messenger systems. While the physiological and pharmacological significance of opioid receptors is being evaluated by antisense oligodeoxynucleotide strategy, further progress will be attained by molecular genetic means, such as constructing transgenic mice and gene targeting. The cDNA cloning of the p-, 6-, and x-opioid receptors has allowed the cells expressing these receptor mRNAs to be mapped by in situ hybridization. Furthermore, mapping of the receptors themselves are in progress using antibodies against the amino acid sequences deduced from the nucleotide sequences of the cloned cDNAs. Double labeling by in situ hybridization or immunohistochemistry will characterize the cells expressing each subtype of opioid receptor- that is, identify what kinds of neurotransmitters, neuropeptides, receptors, channels, and enzymes they possess. Clarification of the physiological and pharmacological roles of opioid receptors will be useful for the development of novel analgesics, antitussives, or antidiarrheals that are more selective for each condition of a disease and exhibit lower side effects, such as dependence and addiction, than those now clinically available.

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