OPIOIDOPIOID RECEPTORSRECEPTORS defined or “classical” types of opioid receptor µ,dk and . Alistair Corbett, Sandy McKnight and Graeme Genes encoding for these receptors have been cloned.5, Henderson 6,7,8 More recently, cDNA encoding an “orphan” receptor Dr Alistair Corbett is Lecturer in the School of was identified which has a high degree of homology to Biological and Biomedical Sciences, Glasgow the “classical” opioid receptors; on structural grounds Caledonian University, Cowcaddens Road, this receptor is an opioid receptor and has been named Glasgow G4 0BA, UK. ORL (opioid receptor-like).9 As would be predicted from 1 Dr Sandy McKnight is Associate Director, Parke- their known abilities to couple through pertussis toxin- Davis Neuroscience Research Centre, sensitive G-proteins, all of the cloned opioid receptors Cambridge University Forvie Site, Robinson possess the same general structure of an extracellular Way, Cambridge CB2 2QB, UK. N-terminal region, seven transmembrane domains and Professor Graeme Henderson is Professor of intracellular C-terminal tail structure. There is Pharmacology and Head of Department, pharmacological evidence for subtypes of each Department of Pharmacology, School of Medical receptor and other types of novel, less well- Sciences, University of Bristol, University Walk, characterised opioid receptors,eliz , , , , have also been Bristol BS8 1TD, UK. postulated. Thes -receptor, however, is no longer regarded as an opioid receptor. Introduction Receptor Subtypes Preparations of the opium poppy papaver somniferum m-Receptor subtypes have been used for many hundreds of years to relieve The MOR-1 gene, encoding for one form of them - pain. In 1803, Sertürner isolated a crystalline sample of receptor, shows approximately 50-70% homology to the main constituent alkaloid, morphine, which was later shown to be almost entirely responsible for the the genes encoding for thedk -(DOR-1), -(KOR-1) and orphan (ORL ) receptors. Two splice variants of the analgesic activity of crude opium. The rigid structural 1 and stereochemical requirements essential for the MOR-1 gene have been cloned, differing only in the analgesic actions of morphine and related opioids led to presence or absence of 8 amino acids in the C-terminal the theory that they produce their effects by interacting tail. The splice variants exhibit differences in their rate of onset and recovery from agonist-induced with a specific receptor.1 The concept that there is more than one type of opioid receptor arose to explain the internalization but their pharmacology does not appear dual actions of the synthetic opioid nalorphine, which to differ in ligand binding assays.10 Furthermore, in the antagonises the analgesic effect of morphine in man but MOR-1 knockout mouse, morphine does not induce also acts as an analgesic in its own right. Martin (1967) antinociception demonstrating that at least in this concluded that the analgesic action of nalorphine is species morphine’s analgesia is not mediated through mediated by a receptor, later called thek -opioid dk- or -receptors.11 Similarly morphine did not exhibit positive reinforcing properties or an ability to induce receptor, that is different from the morphine receptor.2 physical dependence in the absence of the MOR-1 Evidence for multiple receptors,mk , and s , came from gene. the demonstration of different profiles of pharmacological activity in the chronic spinal dog with mmand : The mm / subdivision was proposed by the prototype agonists morphine, ketazocine and N- 12 12 Pasternak and colleagues to explain their observations, allylnormetazocine (SKF 10047).3 The existence of the d-receptor was subsequently proposed to explain the made in radioligand binding studies, that [3 H]-labelled-m , profile of activityin vitro of the enkephalins (the first -dk and - ligands displayed biphasic binding endogenous opioid peptides), and on the basis of the characteristics.12 Each radioligand appeared to bind to the same very high affinity site (m ) as well as to the relative potency of the non-selective opioid antagonist 1 naloxone to reverse endogenous opioid peptide appropriate high affinity site (md , or k ) depending on the inhibition of the nerve-evoked contractions of the radioligand used. Naloxazone and naloxonazine were mouse vas deferens.4 Its existence was further reported to abolish the binding of each radioligand to confirmed by radioligand binding studies using rat brain them -site. Furthermore, inin vivo studies it was 1 homogenates. observed that naloxazone selectively blocked morphine-induced antinociception but did not block It is now clear from work carried out in many morphine-induced respiratory depression or the laboratories over the last 20 years that there are 3 well- induction of morphine dependence.13,14 Subsequent work Tocris Cookson Ltd., UK Tocris Cookson Inc., USA Tel: + 44 (0)117 982 6551 Tel: (800) 421-3701 Fax: + 44 (0)117 982 6552 www.tocris.com Fax: (800) 483-1993 e-mail: [email protected] [email protected] e-mail: [email protected] Is there another, novel form of them -opioid naltriben or naltrindole 5¢¢ -isothiocyanate (5 -NTII).18,19,20 receptor? Furthermore, while mice develop tolerance to the Several related observations suggest the existence of a antinociceptive effects of repeated injections of either novel form ofm -receptor at which analogues of DPDPE or deltorphin II, this tolerance appears to be morphine with substitutions at the 6 position (e.g. homologous in that there is no cross tolerance between these ligands.21 In vivo, dd - and -receptor-induced morphine-6b -glucuronide, heroin and 6-acetyl 12 morphine) are agonists, but with which morphine itself antinociception can be differentially antagonised by does not interact.15 In antinociception tests on mice it has blockers of different types of potassium channels.22 been reported that morphine does not exhibit cross tolerance with morphine-6b -glucuronide, heroin or 6- The best evidence fromin vitro experiments to support thedd and subdivision of d -receptors comes from acetyl morphine. Furthermore, in mice of the CXBX 12 strain morphine is a poor antinociceptive agent inhibition of adenylyl cyclase activity in membranes whereas morphine-6b -glucuronide, heroin and 6-acetyl from rat brain24,25 and from thed -receptor-mediated morphine are all potently antinociceptive. The 6- elevations of intracellular Ca2+ in the ND8-47 cell line26 substituted morphine analogues do not appear to be where BNTX selectively antagonised DPDPE, and acting throughdk - or -receptors because the naltriben selectively antagonised deltorphin II. antinociception they induce is not blocked by selective Surprisingly, little selectivity was seen in radioligand dk-- or receptor antagonists, whereas 3- displacement studies.24 The converse has been methoxynaltrexone has been reported to antagonise observed in studies on neuronal cell lines. Two distinct morphine-6b -glucuronide- and heroin-induced d-receptor binding sites were observed in radioligand antinociception without affecting that induced by binding experiments on SK-N-BE cells.27 Studies on NG108-15 cells28 or the human neuroblastoma cell line, morphine, [D-Pen25 , D-Pen ]enkephalin (DPDPE ,d - SH-SY5Y,29,30 have failed to find any functional evidence selective) or U50488 (k -selective).16 ford -receptor subtypes. Recently it has been reported that heroin and morphine- 6-glucuronide, but not morphine, still produce The pharmacological properties of the cloned DOR-1 antinociception in MOR-1 knockout mice in which the receptor are somewhere between those predicted for either thedd or subtypes. DPDPE and deltorphin II are disruption in the MOR-1 gene was engineered in exon- 12 both potent displacers of [3 H]-diprenorphine binding to 1.17 The same authors observed that in other MOR-1 knockout mice in which exon-2, not exon-1, had been mouse and human recombinant receptors, which is not consistent with either thedd or classifications.31 In disrupted, all three agonists were ineffective as 12 antinociceptive agents. They conclude that the contrast, [3 H]-diprenorphine binding to the mouse antinociceptive actions of heroin and morphine-6- recombinant receptor is more potently displaced by glucuronide in the exon-1 MOR-1 mutant mice are naltriben than BNTX, suggesting that the cloned receptor is of thed subtype. It will be of importance to mediated through a receptor produced from an 2 alternative transcript of the MOR-1 gene differing from determine in the DOR-1 knockout mouse if analgesia can still be induced by eitherdd - or -receptor selective the MOR-1 gene product, them -opioid receptor, in the 12 exon-1 region. To substantiate this conclusion they agonists. report that in RT-PCR experiments using primers spanning exons 2 and 3, a MOR-1 gene product was ddand : The dd and subdivision of d -receptors was cx ncx cx nx c still detected in MOR-1 knockout mice. based on the hypothesis that one type ofdd -receptor ( ) cx was complexed withmk -receptors (and perhaps also - d-Receptor subtypes receptors) whereas the other type ofdd -receptor ( ) was ncx The DOR-1 gene is the onlyd -receptor gene cloned to not associated with an opioid receptor complex.32 It was date. However, two, overlapping subdivisions ofd - originally observed that sub-antinociceptive doses of agonists at thed receptor (e.g. low doses of DPDPE), receptor have been proposed (dd / and dd / ) on the cx 12 cxncx basis ofin vivo and in vitro pharmacological potentiatedm -receptor-mediated analgesia, an effect experiments. which could be antagonised by 5¢ -NTII. On the other hand, at higher doses, DPDPE then acted as an agonist ddand : The subdivision of the d -receptor into dd and at thed -receptor and itself induced analgesia which 12 12 ncx subtypes was proposed primarily on the basis of in vivo was reversed by DALCE. Data obtained from pharmacological studies (Table 1). In rodents in vivo, subsequent radioligand binding studies have been the supraspinal antinociceptive activity of DPDPE can interpreted as demonstrating the existence of further be selectively antagonised by 7-benzylidenenaltrexone subtypes of theddd receptor i.e.