δ-Opioid receptor agonists produce antinociception and [35S]GTPγS binding in μ receptor knockout mice

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Abstract

We examined the effects of [d-Pen2,d-Pen5]enkephalin (DPDPE), [d-Ala2,Glu4]deltorphin (DELT), and (+)-4-[(αR)-α((2S,5R)-4-Allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide (SNC80) on [35S]GTPγS binding in brain membranes prepared from μ-opioid receptor knockout (−/−) mice. The potency and maximal response (Emax) of these agonists were unchanged compared to control mice. In contrast, while the potency of [d-Pen2,pCl-Phe4,d-Pen5]enkephalin (pCl-DPDPE) was not significantly different, the Emax was reduced as compared to controls. In the tail-flick test, intracerebroventricular (i.c.v.) or intrathecal (i.th.) DELT produced antinociceptive effects in −/− mice with potency that did not differ significantly from controls. In contrast, the antinociceptive potency of i.c.v. and i.th. DPDPE was displaced to the right by 4- and 9-fold in −/− compared to control mice, respectively. Reduced DPDPE antinociceptive potency in −/− mice, taken together with reduced DPDPE- and pCl-DPDPE- stimulated G protein activity in membranes prepared from −/− mice, demonstrate that these agonists require μ-opioid receptors for full activity. However, because DELT mediated G protein activation and antinociception were both comparable between −/− and wild type mice, we conclude that the μ-opioid receptor is not a critical component of δ-opioid receptor function.

Introduction

Many studies indicate that δ-opioid receptors mediate antinociception. δ-opioid receptor-selective compounds have been shown to produce antinociception that can be blocked by δ-selective antagonists Heyman et al., 1987, Porreca et al., 1987, Shah et al., 1994 but not by μ-selective antagonists (Heyman et al., 1987). Prolonged agonist exposure produces antinociceptive tolerance to δ-opioids without significant cross-tolerance to μ-opioid receptor-selective agonists (Mattia et al., 1991). These data support a direct role for δ-opioid receptors in mediating antinociception. Furthermore, treatment of animals with antisense oligodeoxynucleotides targeted at the cloned δ-opioid receptor selectively attenuated the antinociception produced by δ-opioid, but not μ- or κ-opioid agonists, suggesting that δ receptor selective agonists such as [d-Ala2, Glu4]deltorphin act predominantly through δ receptors Tseng et al., 1994, Bilsky et al., 1996, Standifer et al., 1997.

Germ line mutation of opioid receptor genes have produced transgenic mice which lack specific opioid receptors; these models have been used to evaluate the role of opioid receptors in physiological and pathological function. It was recently reported that mice with null mutation of the cloned μ-opioid receptor (MOR) (−/− mice) did not exhibit antinociceptive response to two putative δ-opioid receptor selective agonists, Tyr-d-Ser(O-t-butyl)-Gly-Phe-Leu-Thr-(O-t-butyl) (BUBU) Matthes et al., 1996, Matthes et al., 1998 and [d-Pen2, d-Pen5]enkephalin (DPDPE) (Sora et al., 1997a). These observations led to the postulation that antinociceptive efficacy of δ-opioid receptor selective agonists requires the presence of μ-opioid receptors in the central nervous system. On the other hand, it may also be plausibly argued that the ligands used in this case are not highly selective for δ receptors as previously thought. The present study aims to test the hypothesis that in the −/− mice the δ-opioid receptors are functionally fully active and mediate antinociception. We have determined the activation of G proteins by δ-opioid selective agonists in both brain and spinal cord membranes prepared from control or −/− mice, and the antinociceptive actions of δ-selective agonists in both control and −/− mice. Our data indicate that the δ-opioid receptor remains functionally coupled to G proteins and produces antinociceptive actions in −/− mice. Thus, expression of μ-opioid receptors is not required for δ-opioid receptor functions.

Section snippets

Generation of μ-opioid receptor knockout mice

The generation of μ-opioid receptor knockout mice in a C57/129Sv genetic background has been previously described (Sora et al., 1997b). Briefly, a 16.5-kb DNA fragment encoding part of the 5′ flanking region, the first exon and part of the first intron of the μ-opioid receptor was isolated from a mouse λ library. A deletion mutation of the fragment was produced in which the translation initiation site was omitted and this fragment was subcloned into the pPGKneo expression vector. Embryonic stem

Results

We examined G-protein activation by the μ-opioid receptor-selective agonist DAMGO, and the opioid agonists DPDPE, pCl-DPDPE, SNC80 and DELT in whole brain membranes prepared from control and −/− mice. These latter drugs have previously been shown to be highly δ-opioid receptor-selective Kramer et al., 1993, Knapp et al., 1996. Activation was determined as drug-induced binding of [35S]GTPγS to membranes (Fig. 1). In membranes prepared from control mouse brains, mean maximal responses of 118,

Discussion

Recent reports using μ-opioid receptor knockout mice have suggested that δ-opioid receptor-mediated antinociception is dependent upon the μ receptor Matthes et al., 1996, Matthes et al., 1998, Sora et al., 1997a. Such findings were unexpected in light of an extensive literature supporting antinociception mediated by the δ-opioid receptor. We thus compared δ-opioid receptor-mediated antinociception and G protein activation in control and −/− mice using several δ-selective agonists. The

Acknowledgements

This research was supported in part by USPHS Grants from the NIDA (DA06284) and the Arizona Disease Control Research Commission.

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