guanosine-5--o-(3-thiotriphosphate) and Morphine-Dependence

It has been demonstrated that opioid agonists that preferentially act at μ-opioid receptors to activate G protein signaling over βarrestin2 recruitment produce antinociception with less respiratory suppression. However, most of the adverse effects associated with opioid therapeutics are realized after extended dosing. Therefore, we tested the onset of tolerance and dependence, and assessed for neurochemical changes associated with prolonged treatment with the biased agonist SR-17018. When chronically administered to mice, SR-17018 does not lead to hot plate antinociceptive tolerance, receptor desensitization in periaqueductal gray, nor a super-sensitization of adenylyl cyclase in the striatum, which are hallmarks of opioid neuronal adaptations that are seen with morphine. Interestingly, substitution with SR-17018 in morphine-tolerant mice restores morphine potency and efficacy, whereas the onset of opioid withdrawal is prevented. This is in contrast to buprenorphine, which can suppress withdrawal, but produces and maintains morphine antinociceptive tolerance. Biased agonists of this nature may therefore be useful for the treatment of opioid dependence while restoring opioid antinociceptive sensitivity.

Topics: Analgesics, Opioid; Animals; Dose-Response Relationship, Drug; Drug Tolerance; Female; Guanosine 5'-O-(3-Thiotriphosphate); Infusion Pumps, Implantable; Male; Mice; Mice, Inbred C57BL; Morphine; Morphine Dependence; Oxycodone; Pain Measurement; Receptors, Opioid, mu; Substance Withdrawal Syndrome

Extensive 3' alternative splicing of the mu opioid receptor gene OPRM1 creates multiple C-terminal splice variants. However, their behavioral relevance remains unknown. The present study generated 3 mutant mouse models with truncated C termini in 2 different mouse strains, C57BL/6J (B6) and 129/SvEv (129). One mouse truncated all C termini downstream of Oprm1 exon 3 (mE3M mice), while the other two selectively truncated C-terminal tails encoded by either exon 4 (mE4M mice) or exon 7 (mE7M mice). Studies of these mice revealed divergent roles for the C termini in morphine-induced behaviors, highlighting the importance of C-terminal variants in complex morphine actions. In mE7M-B6 mice, the exon 7-associated truncation diminished morphine tolerance and reward without altering physical dependence, whereas the exon 4-associated truncation in mE4M-B6 mice facilitated morphine tolerance and reduced morphine dependence without affecting morphine reward. mE7M-B6 mutant mice lost morphine-induced receptor desensitization in the brain stem and hypothalamus, consistent with exon 7 involvement in morphine tolerance. In cell-based studies, exon 7-associated variants shifted the bias of several mu opioids toward β-arrestin 2 over G protein activation compared with the exon 4-associated variant, suggesting an interaction of exon 7-associated C-terminal tails with β-arrestin 2 in morphine-induced desensitization and tolerance. Together, the differential effects of C-terminal truncation illustrate the pharmacological importance of OPRM1 3' alternative splicing.

Topics: Alternative Splicing; Analgesics, Opioid; Animals; Brain; Codon, Nonsense; Dose-Response Relationship, Drug; Drug Tolerance; Exons; Gastrointestinal Transit; Guanosine 5'-O-(3-Thiotriphosphate); Locomotion; Male; Mice, 129 Strain; Mice, Inbred C57BL; Morphine; Morphine Dependence; Protein Binding; Protein Isoforms; Receptors, Opioid, mu

Yokukansan (YKS) is a traditional Japanese medicine consisting of seven medicinal herbs that is used for the treatment of neurosis, insomnia, and the behavioral/psychological symptoms of dementia. This study examined the effects of YKS on morphine tolerance and physical dependence in mice. Daily oral administration of YKS (0.5 or 1.0 g/kg) for 3 weeks significantly attenuated morphine tolerance and naloxone-precipitated morphine withdrawal signs (jumps and body weight loss) without affecting the analgesic effect of morphine. The inhibitory effect of YKS on withdrawal jumps in morphine-dependent mice was blocked by a single pretreatment with an α(2)-adrenoceptor antagonist, yohimbine, but not by an α(1)-adrenoceptor antagonist, prazosin. A similar inhibitory effect on withdrawal jumps was observed by repeated administration of yohimbine. The membrane expression of α(2A)-adrenoceptors in the pons/medulla was decreased in morphine withdrawn animals; this reduction was prevented by repeated administration of YKS or yohimbine. Competitive radioligand and [(35)S]guanosine-5'-O-(3-thiotriphosphate) binding assays revealed that YKS and its constituent herbs, Glycyrrhiza (GR) and Uncaria hook (UH), had specific binding affinity for and antagonist activity against the α(2A)-adrenoceptor. Certain chemical constituents, including GR -derived glycyrrhizin and its metabolite, 18β-glycyrrhetinic acid, and UH-derived geissoschizine methyl ether (GME), shared such activities. Repeated administration of GR, UH, glycyrrhizin or GME significantly inhibited morphine withdrawal signs. These results suggest that YKS and its active constituents inhibit morphine tolerance and physical dependence, and that the latter is due at least in part to the prevention of the decreased membrane expression of the α(2A)-adrenoceptor in the brainstem by its prolonged blockade.

Topics: Adrenergic Agents; Adrenergic alpha-Antagonists; Adrenergic beta-Antagonists; Analysis of Variance; Animals; Behavior, Addictive; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Tolerance; Drugs, Chinese Herbal; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Isotopes; Male; Mice; Morphine Dependence; Norepinephrine Plasma Membrane Transport Proteins; Pain Threshold; Propranolol; Protein Binding; Radioligand Assay; Receptors, Adrenergic, alpha-2; Time Factors; Tropanes

As important pharmacological probes, highly selective opioid receptor antagonists are essential in opioid receptor structural characterization and opioid agonist functional studies. At present, a nonpeptidyl, highly selective, and reversible mu opioid receptor antagonist is still not available. Among a series of novel naltrexamine derivatives that have been designed and synthesized following molecular modeling studies, two compounds, NAP and NAQ, were identified as leads based on the results of in vitro and in vivo pharmacological assays. Both of them displayed high binding affinity and selectivity to the mu opioid receptor. Further pharmacokinetic and functional characterization revealed that NAP seems to be a peripheral nervous system agent while NAQ seems to be a central one. Such characteristics provide two distinguished potential application routes for these two agents and their derivatives. These results also supported our hypothesis that they may serve as leads to develop more potent and selective antagonists for the mu opioid receptor.

Topics: Animals; Caco-2 Cells; CHO Cells; Cricetinae; Cricetulus; Drug Implants; Guanosine 5'-O-(3-Thiotriphosphate); Heterocyclic Compounds; Humans; Male; Membranes; Mice; Morphine; Morphine Dependence; Naltrexone; Narcotic Antagonists; Narcotics; Rats; Receptors, Opioid, mu; Substance Withdrawal Syndrome; Thalamus

The present study was undertaken to investigate pharmacological actions induced by morphine and oxycodone under a neuropathic pain-like state. In the mu-opioid receptor (MOR) binding study and G-protein activation, we confirmed that both morphine and oxycodone showed MOR agonistic activities. Mice with sciatic nerve ligation exhibited the marked neuropathic pain-like behavior. Under these conditions, antinociception induced by subcutaneously (s.c.) injected morphine was significantly decreased by sciatic nerve ligation, whereas s.c. injection of oxycodone produced a profound antinociception in sciatic nerve-ligated mice. There were no significant differences in spinal or supraspinal antinociception of morphine and oxycodone between sham operation and nerve ligation. Moreover, either morphine- or oxycodone-induced increase in guanosine-5'-o-(3-thio) triphosphate ([(35)S]GTPgammaS) binding in the spinal cord, periaqueductal gray matter and thalamus in sciatic nerve-ligated mice was similar to that in sham-operated mice. Antinociception induced by s.c., intrathecal, or intracerebroventricular injection of the morphine metabolite morphine-6-glucuronide (M-6-G) was significantly decreased by sciatic nerve ligation. Furthermore, the increase in the G-protein activation induced by M-6-G was eliminated in sciatic nerve ligation. In addition, either morphine- or oxycodone-induced rewarding effect was dramatically suppressed under a neuropathic pain-like state. The increased [(35)S]GTPgammaS binding by morphine or oxycodone was significantly lower in the lower midbrain of mice with sciatic nerve ligation compared with that in control mice. These findings provide further evidence that oxycodone shows a profound antinociceptive effect under a neuropathic pain-like state with less of a rewarding effect. Furthermore, the reduction in G-protein activation induced by M-6-G may, at least in part, contribute to the suppression of the antinociceptive effect produced by morphine under a neuropathic pain-like state.

Topics: Animals; Central Nervous System; Conditioning, Operant; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Administration Routes; Drug Interactions; Freund's Adjuvant; Guanosine 5'-O-(3-Thiotriphosphate); Inflammation; Male; Mice; Mice, Inbred ICR; Morphine; Morphine Dependence; Narcotic Antagonists; Narcotics; Oxycodone; Pain Measurement; Protein Binding; Sciatica; Sulfur Isotopes

Acute morphine administration produces analgesia and reward, but prolonged use may lead to analgesic tolerance in patients chronically treated for pain and to compulsive intake in opioid addicts. Moreover, long-term exposure may induce physical dependence, manifested as somatic withdrawal symptoms in the absence of the drug. We set up three behavioral paradigms to model these adaptations in mice, using distinct regimens of repeated morphine injections to induce either analgesic tolerance, locomotor sensitization or physical dependence. Interestingly, mice tolerant to analgesia were not sensitized to hyperlocomotion, whereas sensitized mice displayed some analgesic tolerance. We then examined candidate molecular modifications that could underlie the development of each behavioral adaptation. First, analgesic tolerance was not accompanied by mu opioid receptor desensitization in the periaqueductal gray. Second, cdk5 and p35 protein levels were unchanged in caudate-putamen, nucleus accumbens and prefrontal cortex of mice displaying locomotor sensitization. Finally, naloxone-precipitated morphine withdrawal did not enhance basal or forskolin-stimulated adenylate cyclase activity in nucleus accumbens, prefrontal cortex, amygdala, bed nucleus of stria terminalis or periaqueductal gray. Therefore, the expression of behavioral adaptations to chronic morphine treatment was not associated with the regulation of micro opioid receptor, cdk5 or adenylate cyclase activity in relevant brain areas. Although we cannot exclude that these modifications were not detected under our experimental conditions, another hypothesis is that alternative molecular mechanisms, yet to be discovered, underlie analgesic tolerance, locomotor sensitization and physical dependence induced by chronic morphine administration.

Topics: Adenylyl Cyclases; Analgesics; Animals; Behavior, Animal; Brain; Cyclin-Dependent Kinase 5; Drug Administration Schedule; Drug Tolerance; Female; Guanosine 5'-O-(3-Thiotriphosphate); Locomotion; Male; Mice; Mice, Inbred C57BL; Morphine; Morphine Dependence; Naloxone; Narcotic Antagonists; Narcotics; Protein Binding; Receptors, Opioid, mu; Time Factors

We have recently demonstrated that the combination of methadone and morphine enhances the ability of morphine to induce mu-opioid peptide (MOP) receptor endocytosis. As a result, rats receiving both drugs show reduced morphine tolerance and dependence. In the present study, we identify the biochemical basis for the protective effect of the drug combination. In rats treated with morphine alone, the inhibitory effect of DAMGO on forskolin-stimulated adenylyl cyclase activity was significantly reduced in a brain-region-selective manner. Importantly, these reductions were prevented in animals receiving the drug combination. We found that these changes were not due to alterations in MOP receptor density, or MOP receptor-G protein coupling, as no significant change in these parameters was observed. Together these data demonstrate that neither changes in receptor number nor function are required for morphine tolerance and dependence. Rather, brain-region-selective changes in adenylyl cyclase signal transduction are critical, and both these biochemical changes and the behavioral effects are prevented by facilitating endocytosis of the MOP receptor.

Topics: Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Analgesics, Opioid; Animals; Brain; Dose-Response Relationship, Drug; Drug Interactions; Drug Tolerance; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Male; Methadone; Morphine; Morphine Dependence; Radioligand Assay; Rats; Rats, Sprague-Dawley; Receptors, Opioid, mu

Naloxone administration produces a robust conditioned place aversion (CPA) in opiate-naive rodents by blocking the action of enkephalins at mu opioid receptors. This aversive response is potentiated by prior exposure to morphine. In vitro studies indicate that morphine treatment may promote constitutive activity of mu opioid receptors. We hypothesized that such enhanced constitutive activity in vivo may underlie the increased aversive property of naloxone by uncovering the inverse agonist property of this drug. The CPA produced by naloxone was compared with that produced by the neutral antagonists 6-alpha- and 6-beta-naloxol in mice with and without prior morphine exposure. While all three drugs produced CPA, only naloxone CPA was enhanced by morphine given 20 h prior to each naloxone injection. Furthermore, only naloxone produced withdrawal jumping when given 20 h after morphine, even though 6-alpha-naloxol was able to produce jumping when given 4 h after morphine. These data suggest that morphine may enhance naloxone CPA by increasing levels of constitutively active mu receptors and further support the role of such constitutive activity in mediating naloxone-precipitated physical withdrawal. Such long-term changes in constitutive activity of the mu receptor induced by exogenous opiate exposure may thus be an important factor in hedonic homeostatic dysregulation proposed to underlie the addictive process.

Topics: Animals; Avoidance Learning; Conditioning, Operant; Cyclic AMP; Dose-Response Relationship, Drug; Guanosine 5'-O-(3-Thiotriphosphate); Habituation, Psychophysiologic; Male; Mice; Mice, Inbred C57BL; Morphine Dependence; Naloxone; Narcotic Antagonists; Receptors, Opioid, mu; Substance Withdrawal Syndrome

Repeated intake of opioids is associated with dose escalation and alterations in signal transduction at the G-protein-coupled receptor level. The current study utilized two experiments to identify factors in rats that influence consumption rates such as daily intake of self-administered morphine and receptor desensitization. In Experiment 1, rats self-administered either 0.30, 1.00, or 3.00 mg/kg/infusion morphine sulfate (morphine) during 7 daily 4-h sessions. For Experiment 2, rats were assigned to groups that self-administered either 1) self-regulated escalating doses of morphine, 2) a fixed dose of morphine, or 3) saline during 18-h sessions for 7 days to determine if dose control would increase consumption without significantly decreasing response rate. We then assessed morphine-stimulated [(35)S]GTPgammaS binding in the amygdala and thalamus from these three groups in Experiment 2. Results from Experiment 1 demonstrated that 0.30 mg/kg/morphine did not support stable self-administration. For Experiment 2, the self-escalation group self-administered more morphine than the fixed-dose group, yet maintained similar response rates. Additionally, self-escalation rats demonstrated decreased morphine-stimulated [(35)S]GTPgammaS binding in membranes prepared from amygdalar and thalamic nuclei compared to the fixed-dose and control groups. Our results suggest that session length inversely affects consumption rates for fixed doses of morphine. Self-regulated dosing of morphine is also associated with rapid escalation of daily consumption and no significant alterations in consumption rates. These results suggest subject-regulated dosing is a useful approach for modeling dose escalation associated with opioid dependence.

Topics: Amygdala; Animals; Dose-Response Relationship, Drug; Drug Tolerance; Guanosine 5'-O-(3-Thiotriphosphate); Male; Morphine; Morphine Dependence; Narcotics; Rats; Self Administration; Thalamus; Time Factors

Pseudoginsenoside-F11 (PF11), an ocotillol type saponin isolated from Panax quinquefolium L., has been shown to antagonize the behavioral actions of morphine. Biochemical experiments revealed that PF11 could inhibit diprenorphine (DIP) binding with an IC50 of approximately 6.1 microM and reduced the binding potency of morphine in Chinese hamster ovary (CHO)-mu cells. Furthermore, PF11 significantly attenuated morphine-stimulated [35S]GTPgammaS binding in a dose dependent manner, and strongly decreased the efficacy of morphine to inhibit intracellular cAMP production. In addition, PF11 pretreatment could also significantly inhibit naloxone induced cAMP overshoot in the morphine-pretreated cells. However, PF11 per se had no effect on either [35S]GTPgammaS binding or intracellular cAMP accumulation. These data suggested that PF11 antagonized the morphine stimulated opioid receptor signalling directly at the cellular level.

Topics: Adenylyl Cyclases; Analgesics, Opioid; Animals; Binding Sites; Binding, Competitive; Cell Membrane; CHO Cells; Cricetinae; Cyclic AMP; Dose-Response Relationship, Drug; Drug Interactions; Ginsenosides; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Morphine; Morphine Dependence; Narcotic Antagonists; Receptors, Opioid, mu; Saponins; Signal Transduction; Sulfur Radioisotopes

The mu opioid receptor, MOR, displays spontaneous agonist-independent (basal) G protein coupling in vitro. To determine whether basal MOR signaling contributes to narcotic dependence, antagonists were tested for intrinsic effects on basal MOR signaling in vitro and in vivo, before and after morphine pretreatment. Intrinsic effects of MOR ligands were tested by measuring GTPgammaS binding to cell membranes and cAMP levels in intact cells. beta-CNA, C-CAM, BNTX, and nalmefene were identified as inverse agonists (suppressing basal MOR signaling). Naloxone and naltrexone were neutral antagonists (not affecting basal signaling) in untreated cells, whereas inverse agonistic effects became apparent only after morphine pretreatment. In contrast, 6alpha- and 6beta-naltrexol and -naloxol, and 6beta-naltrexamine were neutral antagonists regardless of morphine pretreatment. In an acute and chronic mouse model of morphine-induced dependence, 6beta-naltrexol caused significantly reduced withdrawal jumping compared to naloxone and naltrexone, at doses effective in blocking morphine antinociception. This supports the hypothesis that naloxone-induced withdrawal symptoms result at least in part from suppression of basal signaling activity of MOR in morphine-dependent animals. Neutral antagonists have promise in treatment of narcotic addiction.

Topics: Analgesics, Opioid; Animals; Benzylidene Compounds; Cell Line; Cell Membrane; Cyclic AMP; Dose-Response Relationship, Drug; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Kidney; Magnesium; Male; Mice; Mice, Inbred ICR; Morphine; Morphine Dependence; Naloxone; Naltrexone; Narcotic Antagonists; Nociceptors; Receptors, Opioid, mu; Signal Transduction; Substance Withdrawal Syndrome; Sulfur Radioisotopes; Transfection; Tritium

The mechanisms by which prolonged exposure to morphine leads to tolerance are not fully understood. We investigated the effects of etorphine (ET) on [(35)S]guanosine 5'-(-thio)-triphosphate ([(35)S]GTP-gamma-S) binding in brains of rats made tolerant to morphine via the implantation of morphine (or placebo) pellets. Binding surface analysis was used to characterize the interactions of ET, Gpp(Np)H and GTP-gamma-S with sites labeled by [(35)S]GTP-gamma-S. Data sets were fitted to one- and two-site binding models using the nonlinear least squares curve fitting program MLAB-PC (Civilized Software, Bethesda, MD, USA). Two binding sites were readily resolved. Chronic morphine significantly increased the B(max) and K(d) of the high affinity binding site. ET stimulated [(35)S]GTP-gamma-S binding in placebo membranes via an increase in the B(max) of the high affinity binding site. In contrast, ET stimulated [(35)S]GTP-gamma-S in chronic morphine membranes via a large decrease in the K(d) of the high affinity site. These results suggest that chronic morphine treatment alters the mechanism by which ET stimulates [(35)S]GTP-gamma-S binding to G-proteins. Since proper G-protein/receptor coupling increases [(35)S]GTP-gamma-S binding via an increase in B(max) values, these results suggest that opioid receptors in chronic morphine membranes are not normally coupled to G-proteins. These findings corroborate earlier studies that reported changes in G-protein function in morphine tolerant animals.

Topics: Animals; Binding Sites; Binding, Competitive; Brain; Etorphine; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; In Vitro Techniques; Male; Morphine Dependence; Narcotic Antagonists; Narcotics; Radioligand Assay; Rats; Rats, Sprague-Dawley; Receptors, Opioid

The tolerance and dependence after chronic medication with morphine are thought to be representative models for studying the plasticity, including the remodeling of neuronal networks. To test the hypothesis that changes in neuronal plasticity observed in opioid tolerance or dependence are derived from increased activity of the anti-opioid nociceptin system, the effects of chronic treatments with morphine were examined using nociceptin receptor knock-out (NOR(-/-)) mice and a novel nonpeptidic NOR antagonist, J-113397, which shows a specific and potent NOR antagonist activity in in vitro [(35)S]GTPgammaS binding assay and in vivo peripheral nociception test. The NOR(-/-) mice showed marked resistance to morphine analgesic tolerance without affecting morphine analgesic potency in tail-pinch and tail-flick tests. The NOR(-/-) mice also showed marked attenuation of morphine-induced physical dependence, manifested as naloxone-precipitated withdrawal symptoms after repeated morphine treatments. Similar marked attenuation of morphine tolerance was also observed by single subcutaneous (10 mg/kg) or intrathecal (1 nmol) injection of J-113397, which had been given 60 min before the test in morphine-treated ddY mice. However, the intracerebroventricular injection (up to 3 nmol) did not affect the tolerance. On the other hand, morphine dependence was markedly attenuated by J-113397 that had been subcutaneously given 60 min before naloxone challenge. There was also observed a parallel enhancement of NOR gene expression only in the spinal cord during chronic morphine treatments. Together, these findings suggest that the spinal NOR system develops anti-opioid plasticity observed on morphine tolerance and dependence.

Topics: Animals; Benzimidazoles; Binding, Competitive; Brain; Cell Membrane; Disease Models, Animal; Drug Administration Schedule; Drug Antagonism; Drug Tolerance; Guanosine 5'-O-(3-Thiotriphosphate); Male; Mice; Mice, Knockout; Morphine; Morphine Dependence; Naloxone; Narcotic Antagonists; Neuronal Plasticity; Nociceptin; Nociceptin Receptor; Opioid Peptides; Pain Measurement; Piperidines; Receptors, Opioid; Spinal Cord; Substance Withdrawal Syndrome

Morphine is a powerful pain reliever, but also a potent inducer of tolerance and dependence. The development of opiate tolerance occurs on continued use of the drug such that the amount of drug required to elicit pain relief must be increased to compensate for diminished responsiveness. In many systems, decreased responsiveness to agonists has been correlated with the desensitization of G-protein-coupled receptors. In vitro evidence indicates that this process involves phosphorylation of G-protein-coupled receptors and subsequent binding of regulatory proteins called beta-arrestins. Using a knockout mouse lacking beta-arrestin-2 (beta arr2-/-), we have assessed the contribution of desensitization of the mu-opioid receptor to the development of morphine antinociceptive tolerance and the subsequent onset of physical dependence. Here we show that in mice lacking beta-arrestin-2, desensitization of the mu-opioid receptor does not occur after chronic morphine treatment, and that these animals fail to develop antinociceptive tolerance. However, the deletion of beta-arrestin-2 does not prevent the chronic morphine-induced up-regulation of adenylyl cyclase activity, a cellular marker of dependence, and the mutant mice still become physically dependent on the drug.

Topics: Adenylyl Cyclases; Analgesics, Opioid; Animals; Arrestins; beta-Arrestin 2; beta-Arrestins; Brain Stem; Drug Implants; Drug Tolerance; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Membranes; Mice; Mice, Inbred C57BL; Mice, Knockout; Morphine; Morphine Dependence; Mutation; Receptors, Opioid, mu

The present study was designed to test the possible existence of changes in brain cannabinoid receptors in morphine-dependent mice. To this end, we compared cannabinoid receptor binding and WIN 55,212-2-stimulated [35S]guanylyl-5'-O-(gamma-thio)-triphosphate ([35S]GTP gamma S) binding in several brain regions of mice chronically exposed to morphine or saline. The existence of opiate dependence in morphine-injected mice was assessed by analyzing the well-known jumping behavior induced by the blockade of opioid receptors with naloxone, whereas these animals were unresponsive to the blockade of cannabinoid receptors with SR141716. The different structures analyzed exhibited similar cannabinoid receptor binding levels in morphine-dependent and control mice, with the only exception of the globus pallidus, which exhibited a very small, but statistically significant, increase. In addition, the activation of cannabinoid receptors with WIN 55,212-2 increased [35S]GTP gamma S binding in most of the structures examined. The increase was of similar magnitude in morphine-dependent and control mice, except in the substantia nigra, where morphine-dependent mice exhibited lesser [35S]GTP gamma S binding levels in basal conditions, although a significantly higher WIN 55,212-2-stimulated binding. Other structures, such as the central gray substance, where there was a poor agonist-induced stimulation in control mice, exhibited, however, higher levels of WIN 55,212-2-stimulated [35S]GTP gamma S binding in morphine-dependent mice, whereas these animals tended to exhibit a higher [35S]GTP gamma S binding levels in basal conditions, although a lesser and not statistically significant WIN 55,22-2-stimulated binding, in the deep layers of the cerebral cortex. Thus, the data support the potential existence of a specific effect of morphine in the coupling of cannabinoid receptors to GTP-binding proteins, rather than on receptor binding, although this was observed only in the substantia nigra and central gray substance.

Topics: Analgesics; Analysis of Variance; Animals; Autoradiography; Benzoxazines; Brain; Brain Mapping; Cannabinoids; Drug Interactions; Drug Tolerance; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Male; Mice; Morphine; Morphine Dependence; Morpholines; Naphthalenes; Radioligand Assay; Receptors, Cannabinoid; Receptors, Drug; Sulfur Radioisotopes