μ Opioid receptor-mediated G-protein activation by heroin metabolites: evidence for greater efficacy of 6-monoacetylmorphine compared with morphine ¹

Abstract

The efficacy of heroin metabolites for the stimulation of μ opioid receptor-mediated G-protein activation was investigated using agonist-stimulated [³⁵S]guanosine-5′-O-(γ-thio)-triphosphate binding. In rat thalamic membranes, heroin and its primary metabolite, 6-monoacetylmorphine (6-MAM), were more efficacious than morphine or morphine-6-βd-glucuronide. This increased efficacy was not due to increased action of heroin and 6-MAM at δ receptors, as determined by competitive antagonism by naloxone, lack of antagonism by naltrindole, and competitive partial antagonism with morphine. In agreement with this interpretation, the same relative efficacy profile of heroin and its metabolites was observed at the cloned human μ opioid receptor expressed in C6 glioma cells. Moreover, these efficacy differences were GDP-dependent in a manner consistent with accepted mechanisms of receptor-mediated G-protein activation. The activity of heroin was attributed to in vitro deacetylation to 6-MAM, as confirmed by HPLC analysis. These results indicate that the heroin metabolite 6-MAM possesses higher efficacy than other heroin metabolites at μ opioid receptors, which may contribute to the higher efficacy of heroin compared with morphine in certain behavioral paradigms in vivo.

Introduction

3,6-Diacetylmorphine (heroin) and morphine are both effective analgesics, but also produce undesirable effects such as respiratory depression, tolerance, and dependence. Morphine is widely used clinically, but heroin is an illicit opiate that accounts for a major portion of non-medical opiate use in the United States. Although heroin generally is more potent than morphine when administered in vivo, previous studies have found no advantages of heroin over morphine in the treatment of chronic or post-surgical pain [1]. However, a recent study of the agonist actions of opioids in a rodent neuropathic pain model has demonstrated that heroin is both a more potent and efficacious anti-allodynic agent than morphine [2]. Thus, although the increased agonist potency of heroin compared with morphine may be due to pharmacokinetic differences between these two drugs 3, 4, it is also possible that a greater intrinsic efficacy of heroin resulting in greater receptor reserve may contribute to its increased potency in vivo. Because heroin is metabolized rapidly to 6-MAM, and then to morphine 3, 5, it is likely that any greater intrinsic efficacy of heroin over morphine is due to the primary metabolite, 6-MAM. Moreover, since most of the in vivo effects of morphine are due primarily to its actions at μ-type opioid receptors 6, 7, 8, 9 and since most opiate alkaloids bind to μ receptors with high affinity [10], any increased efficacy of heroin versus morphine may be due to a greater efficacy of 6-MAM as a μ receptor agonist. Alternatively, heroin could exert actions at different opioid receptor subtypes, as suggested by recent studies that demonstrated that heroin analgesia could be mediated by μ receptor splice variants 11, 12.

Agonist efficacy at μ opioid receptors can be measured in vitro using agonist-stimulated [³⁵S]GTPγS binding 13, 14, 15, which is a direct measure of receptor-mediated G-protein activation 16, 17, 18. Thus, under conditions where there is no receptor reserve for G-protein activation, maximal stimulation of [³⁵S]GTPγS binding by the agonist-occupied receptor directly correlates with intrinsic efficacy [19]. In the present study, [³⁵S]GTPγS binding was used to examine the efficacy of heroin and its metabolites, 6-MAM, morphine, and M-6-G, compared with opioids that are known to be full agonists for G-protein activation by μ opioid receptors in brain, DAMGO and methadone 15, 19, 20. These experiments were conducted in membranes from rat thalamus, which contains predominantly μ opioid receptors 21, 22, and from transfected C6 glioma cells expressing a pure population of human μ opioid receptors.