Molecular mechanisms involved in the asymmetric interaction between cannabinoid and opioid systems

The aim of this work was to study the mechanism of cross-modulation between cannabinoid and opioid systems for analgesia during acute and chronic exposure. Acute coadministration of ineffectual subanalgesic doses of the synthetic cannabinoid CP-55,940 (0.2 mg/kg i.p.) and morphine (2.5 mg/kg i.p.) resulted in significant antinociception. In chronic studies, a low dose of CP-55,940 (0.2 mg/kg, i.p.) that per se did not induce analgesia in naive animals produced a significant degree of antinociception in rats made tolerant to morphine, whereas in rats made tolerant to CP-55,940, morphine challenge did not produce any analgesic response. To identify the mechanism of these asymmetric interactions during chronic treatment, we investigated the functional activity of cannabinoid and μ opioid receptors and their effects on the cyclic AMP (cAMP) cascade. Autoradiographic-binding studies indicated a slight but significant reduction in cannabinoid receptor levels in the hippocampus and cerebellum of morphine-tolerant rats, whereas CP-55,940-stimulated [³⁵S]GTPγS binding showed a significant decrease in receptor/G protein coupling in the limbic area. In CP-55,940 exposed rats, μ opioid receptor binding was significantly raised in the lateral thalamus and periaqueductal gray (PAG), with an increase in DAMGO-stimulated [³⁵S]GTPγS binding in the nucleus accumbens. Finally, we tested the cAMP system's responsiveness to the cannabinoid and opioid in the striatum and dorsal mesencephalon. In vivo chronic morphine did not affect CP-55,940's ability to inhibit forskolin-stimulated cAMP production in vitro and actually induced sensitization in striatal membranes. In contrast, in vivo chronic CP-55,940 desensitized DAMGO's efficacy in inhibiting forskolin-stimulated cAMP production in vitro. The alterations to the cAMP system seem to mirror the behavioral responses, indicating that the two systems may interact at the postreceptor level. This might open up new therapeutic opportunities for relief of chronic pain through cannabinoid–opioid coadministration.     

Peripheral interactions between cannabinoid and opioid systems contribute to the antinociceptive effect of crotalphine

Background and Purpose

Crotalphine is an antinociceptive peptide that, despite its opioid-like activity, does not induce some of the characteristic side effects of opioids, and its amino acid sequence has no homology to any known opioid peptide. Here, we evaluated the involvement of the peripheral cannabinoid system in the crotalphine effect and its interaction with the opioid system.

Experimental Approach

Hyperalgesia was evaluated using the rat paw pressure test. Involvement of the cannabinoid system was determined using a selective cannabinoid receptor antagonist. Cannabinoid and opioid receptor activation were evaluated in paw slices by immunofluorescence assays using conformation state-sensitive antibodies. The release of endogenous opioid peptides from skin tissue was measured using a commercial enzyme immunoassay (EIA).

Key Results

Both p.o. (0.008–1.0 μg·kg⁻¹) and intraplantar (0.0006 μg per paw) administration of crotalphine induced antinociception in PGE₂-induced hyperalgesia. Antinociception by p.o. crotalphine (1 μg·kg⁻¹) was blocked by AM630 (50 μg per paw), a CB₂ receptor antagonist, and by antiserum anti-dynorphin A (1 μg per paw). Immunoassay studies confirmed that crotalphine increased the activation of both κ-opioid (51.7%) and CB₂ (28.5%) receptors in paw tissue. The local release of dynorphin A from paw skin was confirmed by in vitro EIA and blocked by AM630.

Conclusions and Implications

Crotalphine-induced antinociception involves peripheral CB₂ cannabinoid receptors and local release of dynorphin A, which is dependent on CB₂ receptor activation. These results enhance our understanding of the mechanisms involved in the peripheral effect of crotalphine, as well as the interaction between the opioid and cannabinoid systems.     

mu opioid and CB1 cannabinoid receptor interactions: reciprocal inhibition of receptor signaling and neuritogenesis

Several studies have described functional interactions between opioid and cannabinoid receptors; the underlying mechanism(s) have not been well explored. One possible mechanism is direct receptor-receptor interactions, as has been demonstrated for a number of G-protein-coupled receptors. In order to investigate interactions between opioid and cannabinoid receptors, we epitope tagged mu, delta and kappa opioid receptors with Renilla luciferase and CB1 cannabinoid or CCR5 chemokine receptors with yellow fluorescent protein and examined the extent of substrate hydrolysis induced bioluminescence resonance energy transfer (BRET) signal. We find that coexpression of opioid receptors with cannabinoid receptors, but not with chemokine receptors, leads to a significant increase in the level of BRET signal, suggesting that the opioid-cannabinoid interactions are receptor specific. In order to examine the implications of these interactions to signaling, we used GTPgammaS binding and mitogen-activated protein kinase (MAPK) phosphorylation assays and examined the effect of receptor activation on signaling. We find that the mu receptor-mediated signaling is attenuated by the CB1 receptor agonist; this effect is reciprocal and is seen in heterologous cells and endogenous tissue expressing both receptors. In order to explore the physiological consequences of this interaction, we examined the effect of receptor activation on the extent of Src and STAT3 phosphorylation and neuritogenesis in Neuro-2A cells. We find that the simultaneous activation of mu opioid and CB1 cannabinoid receptors leads to a significant attenuation of the response seen upon activation of individual receptors, implicating a role for receptor-receptor interactions in modulating neuritogenesis.     

Molecular basis of opioid dependence: role of signal regulation by G-proteins

1. Morphine and opiate narcotics are potent analgesics that have a high propensity to induce tolerance and physical dependence following their repeated administration. 2. The molecular basis of opiate dependence has not been completely elucidated, although the participation of opioid receptors is a prerequisite. Cellular dependence on opioids is believed to result from the chronic stimulation of opioid-regulated signalling networks. 3. As G-protein-coupled receptors, the opioid receptors must rely on heterotrimeric G-proteins for signal transduction. Recent advances in our understanding of G-protein signalling have unveiled novel signalling molecules and mechanisms, some of which may be intricately involved in the manifestation of opiate dependence. 4. In the present review, we will attempt to trace chronic opioid signals along elaborate G-protein-regulated pathways.     

Molecular basis for cellular effects of naturally occurring polyamines.

The naturally occurring polyamines, putrescine, spermidine and spermine, and the analogue cadaverine, induce a dose-dependent histamine release from rat peritoneal mast cells. Spermine was the most active among these polycationic metabolites, followed by spermidine and putrescine. The histamine release was inhibited by a 2 h pretreatment of the cells with pertussis toxin (100 ng/ml), demonstrating the involvement of a pertussis toxin-sensitive GTP-binding regulatory protein during the exocytotic process. Experiments performed with purified Go/Gi proteins reconstituted into phospholipid vesicles showed a direct stimulation of GTPase activity by the polyamines. This direct stimulation of G proteins and the consequent activation of the coupled effectors may represent a new mechanism of action for natural polyamines controlling receptor-dependent processes.     

Synergistic efects of opioid and cannabinoid antagonists on food intake

RATIONALE: Central cannabinoid systems have been implicated in appetite regulation through the hyperphagic effects of exogenous and endogenous cannabinoids. These effects may involve activation of reward systems and be mediated in part by opioidergic processes. OBJECTIVE: Cannabinoid-opioid interactions in feeding were examined by testing the combined effects on food intake of sub-anorectic doses of selective antagonists for CB1 and opioid receptors. METHODS: Male rats (n = 8) received subcutaneous injections of naloxone (0, 0.1, 0.5, 1.0 mg/kg) and SR141716 (0, 0.1, 0.5, 1.0 mg/kg) before l-h, nocturnal food (chow) intake tests. RESULTS: Neither naloxone nor SR141716 reliably affected feeding when administered alone. By contrast, combined administration of the two antagonists significantly suppressed chow intake at each dose combination. Joint administration of the highest doses of each antagonist suppressed intake by 73%, a significantly greater effect than produced by either naloxone (32%) or SR141716 alone (17%). CONCLUSION: The data reveal a synergistic interaction between the effects of naloxone and SR141716 on feeding, provide further evidence of important functional relationships between endogenous cannabinoid and opioid systems, and strengthen the postulated role for endocannabinoids in reward processes contributing to the normal control of appetite.     

Molecular biology of cannabinoid receptors

To date, two cannabinoid receptors have been isolated by molecular cloning. The CB1 and CB2 cannabinoid receptors are members of the G protein-coupled receptor family. There is also evidence for additional cannabinoid receptor subtypes. The CB1 and CB2 receptors recognize endogenous and exogenous cannabinoid compounds, which fall into five structurally diverse classes. Mutagenesis and molecular modeling studies have identified several key amino acid residues involved in the selective recognition of these ligands. Numerous residues involved in receptor activation have been elucidated. Regions of the CB1 receptor mediating desensitization and internalization have also been discovered. The known genetic structures of the CB1 and CB2 receptors indicate polymorphisms and multiple exons that maybe involved in tissue and species-specific regulation of these genes. The cannabinoid receptors are regulated during chronic agonist exposure, and gene expression is altered in disease states. There is a complex molecular architecture of the cannabinoid receptors that allows a single receptor to recognize multiple classes of compounds and produce an array of distinct downstream effects.     

Differential effect of opioid and cannabinoid receptor blockade on heroin-seeking reinstatement and cannabinoid substitution in heroin-abstinent rats

BACKGROUND AND PURPOSE

Opioids and cannabinoids interact in drug addiction and relapse. We investigated the effect of the opioid receptor antagonist naloxone and/or the cannabinoid CB₁ receptor antagonist rimonabant on cannabinoid-induced reinstatement of heroin seeking and on cannabinoid substitution in heroin-abstinent rats.

EXPERIMENTAL APPROACH

Rats were trained to self-administer heroin (30 µg·kg⁻¹ per infusion) under a fixed-ratio 1 reinforcement schedule. After extinction of self-administration (SA) behaviour, we confirmed the effect of naloxone (0.1–1 mg·kg⁻¹) and rimonabant (0.3–3 mg·kg⁻¹) on the reinstatement of heroin seeking induced by priming with the CB₁ receptor agonist WIN55,212-2 (WIN, 0.15–0.3 mg·kg⁻¹). Then, in a parallel set of heroin-trained rats, we evaluated whether WIN (12.5 µg·kg⁻¹ per infusion) SA substituted for heroin SA after different periods of extinction. In groups of rats in which substitution occurred, we studied the effect of both antagonists on cannabinoid intake.

KEY RESULTS

Cannabinoid-induced reinstatement of heroin seeking was significantly attenuated by naloxone (1 mg·kg⁻¹) and rimonabant (3 mg·kg⁻¹) and fully blocked by co-administration of sub-threshold doses of the two antagonists. Moreover, contrary to immediate (1 day) or delayed (90 days) drug substitution, rats readily self-administered WIN when access was given after 7, 14 or 21 days of extinction from heroin, and showed a response rate that was positively correlated with the extinction period. In these animals, cannabinoid intake was increased by naloxone (1 mg·kg⁻¹) and decreased by rimonabant (3 mg·kg⁻¹).

CONCLUSIONS AND IMPLICATIONS

Our findings extend previous research on the crosstalk between cannabinoid and opioid receptors in relapse mechanisms, which suggests a differential role in heroin-seeking reinstatement and cannabinoid substitution in heroin-abstinent rats.

LINKED ARTICLES

This article is part of a themed issue on Cannabinoids in Biology and Medicine. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2011.163.issue-7     

Molecular pharmacology of opioid receptors

We cloned kappa and mu opioid receptor cDNAs. Using these cDNAs, first, we examined the molecular mechanism for the subtype selectivity of opioid ligands, especially a mu-selective ligand DAMGO. Binding experiments using various chimera and mutated receptors revealed that DAMGO discriminates between mu and delta receptors by recognizing the difference in only one amino acid residue, that is, N(127) in mu and K(108) in delta, at the first extracellular loop, and that it distinguishes between mu and kappa receptors by the difference in four amino acid residues at the third extracellular loop. Second, we established the cell lines expressing the cloned mu, delta, or kappa receptor and elucidated the pharmacological properties, that is, binding affinity and agonistic activity of several opioid agonists. Third, distribution of the mRNAs for mu, delta, and kappa receptors in the brain, spinal cord, and DRG was examined by in situ hybridization histochemistry (ISHH). Double ISHH demonstrated that most of the substance P-producing DRG neurons express the micro receptor. Recently, we are interested in the emotional aspect of pain and its regulation by opioids. Behavioral and microdialysis studies showed that sustained pain evoked by the intraplanter injection of formalin induced conditioned place aversion through the increment of glutamate release followed by the activation of NMDA receptors in the basolateral nucleus of amygdala (BLA). Intra-BLA injection of morphine suppressed the place aversion by inhibiting the glutamate release.     

Effect of opioid and cannabinoid receptor antagonism on orphanin FQ-induced hyperphagia in rats

Feeding induced in rats by cerebroventricular (i.c.v.) injection of orphanin FQ was potently and dose-dependently reversed by peripheral injection of either the opioid antagonist naloxone or the cannabinoid CB(1) receptor antagonist SR 141716[N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophelyl)-4-methyl-3-pyrazole-carboxamine]. The combination of these two agents inhibited food intake in a manner suggestive of additivity or supra-additivity.     

Synergistic and additive interactions of the cannabinoid agonist CP55,940 with mu opioid receptor and alpha2-adrenoceptor agonists in acute pain models in mice

1. Cannabinoid receptor agonists elicit analgesic effects in acute and chronic pain states via spinal and supraspinal pathways. We investigated whether the combination of a cannabinoid agonist with other classes of antinociceptive drugs exerted supra-additive (synergistic) or additive effects in acute pain models in mice. 2. The interactions between the cannabinoid agonist CP55,940, alpha2-adrenoceptor agonist dexmedetomidine and mu-opioid receptor agonist morphine were evaluated by isobolographic analysis of antinociception in hot plate (55 degrees C) and tail flick assays in conscious male Swiss mice. Drug interactions were examined by administering fixed-ratio combinations of agonists (s.c.) in 1:1, 3:1 and 1:3 ratios of their respective ED50 fractions. 3. CP55,940, dexmedetomidine and morphine all caused dose-dependent antinociception. In the hot plate and tail flick assays, ED50 values (mg kg(-1)) were CP55,940 1.13 and 0.51, dexmedetomidine 0.066 and 0.023, and morphine 29.4 and 11.3, respectively. Synergistic interactions existed between CP55,940 and dexmedetomidine in the hot plate assay, and CP55,940 and morphine in both assays. Additive interactions were found for CP55,940 and dexmedetomidine in the tail flick assay, and dexmedetomidine and morphine in both assays. 4. Thus, an alpha2-adrenoceptor agonist or mu opioid receptor agonist when combined with a cannabinoid receptor agonist showed significant synergy in antinociception in the hot plate test. However, for the tail flick nociceptive response to heat, only cannabinoid and mu opioid receptor antinociceptive synergy was demonstrated. If these results translate to humans, then prudent selection of dose and receptor-specific agonists may allow an improved therapeutic separation from unwanted side effects.     

阿片耐受的分子药理机制

在使用阿片类药物进行疼痛治疗的过程中,机体会对药物产生生理耐受现象,即随着用药时间的延长,各种药理作用(包括止痛作用)的剂量-效力曲线发生右移,从而导致要维持相同的药效,必须不断加大用药剂量.过去数十年的研究表明,其分子机制极为复杂. 1阿片受体以及阿片类激动剂作用于受体后的效应 阿片受体按照其与不同高度选择性配体的特异结合力以及它们激动后产生的不同生理效应划分为μδ、κ 3种,每种又分为数个亚型.其中μ受体对吗啡和其它临床使用的阿片镇痛药的亲和力明显高于δ和κ受体.     

Molecular mechanisms of cannabinoid protection from neuronal excitotoxicity

Cannabinoids protect neurons from excitotoxic injury. We investigated the mechanisms involved by studying N-methyl-D-aspartate (NMDA) toxicity in cultured murine cerebrocortical neurons in vitro and mouse cerebral cortex in vivo. The cannabinoid agonist R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)-methyl]pyrrolo[1,2,3-de]-1,4-benzoxazin-yl]-(1-naphthalenyl)-methanone mesylate [R(+)-Win 55212] reduced neuronal death in murine cortical cultures treated with 20 microM NMDA, and its protective effect was attenuated by the CB1 cannabinoid receptor (CB1R) antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-cichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride (SR141716A). Cultures from CB1R-knockout mice were more sensitive to NMDA toxicity than were cultures from wild-type mice. The in vitro protective effect of R(+)-Win 55212 was reduced by pertussis toxin, consistent with signaling through CB1R-coupled G-proteins. The nitric-oxide synthase (NOS) inhibitors 7-nitroindazole (7-NI) and N-omega-nitro-L-arginine methyl ester also reduced NMDA toxicity. In addition, CB1R and neuronal NOS were coexpressed in cultured cortical neurons, suggesting that cannabinoids might reduce NMDA toxicity by interfering with the generation of NO. NOS activity in cerebral cortex was higher in CB1R-knockouts than in wildtype mice, and 7-NI reduced NMDA lesion size. R(+)-Win 55212 inhibited NO production after NMDA treatment of wild-type cortical neuron cultures, measured with 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate, and this effect was reversed by SR141716A. In contrast, R(+)-Win 55212 failed to inhibit NO production in cultures from CB1R knockouts. Dibutyryl-cAMP blocked the protective effect of R(+)-Win 55212, and this was reversed by the protein kinase A (PKA) inhibitor N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide (H89). Cannabinoids seem to protect neurons against NMDA toxicity at least in part by activation of CB1R and downstream inhibition of PKA signaling and NO generation.     

Molecular recognition of opioid receptor ligands

The cloning of the opioid receptors and subsequent use of recombinant DNA technology have led to many new insights into ligand binding. Instead of focusing on the structural features that lead to increased affinity and selectivity, researchers are now able to focus on why these features are important. Site-directed mutagenesis and chimeric data have often been at the forefront in answering these questions. Herein, we survey pharmacophores of several opioid ligands in an effort to understand the structural requirements for ligand binding and selectivity. Models are presented and compared to illustrate key sites of recognition for both opiate and nonopiate ligands. The results indicate that different ligand classes may recognize different sites within the receptor, suggesting that multiple epitopes may exist for ligand binding and selectivity.     

Molecular pharmacology of opioid receptor mechanisms

The molecular basis of opioid receptor mechanisms was studied in reconstitution experiments using purified or membrane-bound opioid receptors and purified GTP-binding proteins (G-proteins). mu-Opioid receptor exclusively purified from rat brains was reconstituted with G-proteins in lipid vesicles. The mu-agonist stimulated the G-protein activity in both G1 or Go-reconstituted vesicles. The stoichiometry revealed that one molecule of mu-receptor is functionally coupled to plural numbers of Gi or Go molecules and that mu-receptor exists in at least two different subtypes, mu i and mu o, separately coupled to Gi and Go, respectively. In addition, when the mu-receptor was phosphorylated by cAMP-dependent protein kinase, the mu-agonist-stimulation of G-protein activity disappeared, while the guanine nucleotide-sensitivity of agonist binding was unchanged. These findings suggest that there are independent domains in the receptor which are related to functional coupling to G-protein and to the agonist-binding modulation by G-protein. kappa-Opioid receptor agonist inhibited the G-protein activity in guinea pig cerebellar membranes. Further experiments revealed that the kappa-opioid receptor is functionally coupled to an inhibition of phospholipase C activity via an inhibition of Gi-activity. Such a receptor-mediated inhibition of G-protein activity may be the first demonstration of a signal transduction mechanism. The delta-opioid receptor agonist showed no effect on G-protein activity in guinea pig striatal and rat cortical membranes, while it stimulated it in NG108-15 cells. In all these membranes, the delta-agonist binding was markedly reduced by GTP gamma S in the presence of MgCl2. These findings suggest that delta-receptors in the brain might be coupled to G-protein without signal transduction.     

Molecular basis of chronopharmaceutics

Many pathophysiological circumstances vary during 24 h periods. Many physiologic processes undergo biological rhythms, including the sleep-wake rhythm and metabolism. Disruptive effect in the 24 h variations can manifest as the emergence or exacerbation of pathological conditions. So, chronotherapeutics is gaining increasing interest in experimental biology, medicine, pharmacy, and drug delivery. This science and the plethora of information should be used intelligently for optimizing the effectiveness and safety of the drug, relying on the timing of drug intake. These chronopharmacological findings are affected by not only the pharmacodynamics but also pharmacokinetics of drugs. The mammalian circadian pacemaker is located in the suprachiasmatic nucleus. The molecular mechanisms are associated with Clock genes that control the circadian rhythms in physiology, pathology, and behavior. Clock controls several diseases such as metabolic syndrome, cancer, and so on. CLOCK mutation influences the expression of both rhythmic and nonrhythmic genes in wild-type tissues. These genotypic changes lead to phenotypic changes, affecting the drug pharmacokinetic and pharmacodynamic parameters. This review is intended to elaborate system regulating biological rhythms and the applicability in pharmaceutics from viewpoints of the intraindividual and interindividual variabilities of Clock genes.     

Membrane pathways for drug/ion channel interactions: Molecular basis for pharmacokinetic properties

This review focuses on the interactions of certain classes of drugs that are known to bind to membrane-bound ion channel/receptors. The concepts described here have broad applications to various ion channels as well as membrane bound receptors that are not ion channels such as the superfamily of G protein coupled receptors. Since data spanning from the molecular to the clinical field currently exists for drugs that bind to the voltage gated calcium channel, this review will highlight this rather narrow scope. It is to be appreciated that the concepts, as described, may have applicability to other membrane-bound receptors. The focus will be on the calcium channel antagonist drugs of the 1,4 dihydropyridine type that bind to the L type calcium channel. Clearly an evolution in their molecular design has been brought to a point where these molecules are not only amphiphilic but increasingly lipophilic. Simply stated, this means that these drugs can readily transport across cell membranes accessing both hydrophilic and hydrophobic environments, although they have also become more soluble in the membrane bilayer. From an equilibrium point of view these molecules prefer to reside in the lipid bilayer hydrocarbon core; from a kinetic point of view they spend more time, on average, solvated within membranes than outside membranes, but their rates of entry into and exit from membranes do not appear to be related solely to their intramembranal equilibrium concentration. This biophysical understanding appears not only to define the molecular pathways for drug binding to the calcium channel receptor, but also to explain differences in the overall clinical pharmacokinetics observed for different drugs in this class. The potency of calcium antagonists is primarily related to their binding affinity to the calcium channel with a variable degree of dependency on the solvation of the drug in the membrane. The pharmacokinetic profile of calcium antagonists, although influenced to some degree by interactions with the target receptor, appears to be largely dictated by their interactions with the cell membranes at the molecular level. These interactions are distinctly different for each calcium antagonist but they can be classified into a few subgroups to explain drug onset and duration of action. © 1994 Wiley-Liss, Inc.     

Molecular neuropharmacology of nociceptive transmission and opioid receptors

This review summarizes our studies using pharmacological, neurochemical and molecular biological methods on the nociception in the CNS and opioid receptors (OPRs). We designed an in vitro fluorometric on-line monitoring system including an immobilized glutamate dehydrogenase column, and for the first time actually demonstrated that capsaicin induced the release of glutamate from rat dorsal horn slices containing the terminal area of primary afferents, in concentration-dependent, extracellular Ca(2+)-dependent and tetrodotoxin-resistant manners. Further, such a release was shown to be inhibited through mu- and delta-opioid receptors and alpha 2-adrenoceptors. On the other hand, we found that intracerebroventricular injections of interleukin (IL)-1 beta in rats produced biphasic effects on the mechanical nociception in rats (hyperalgesia in lower concentrations but analgesia in higher ones) and that similar injections of cytokine-induced neutrophil chemoattractant-1 (CINC-1) facilitated mechanical nociception in rats. The above described facts suggest that glutamate and some sorts of cytokines (IL-1 beta and CINC-1) contribute to nociception at least from the primary afferents to the spinal dorsal horn neurons and in higher brain, respectively. We have cloned rat kappa- and mu-opioid receptors. Using cloned cDNA for OPRs, we demonstrated (1) the distribution of mRNAs for OPRs in the rat central nervous system, (2) coexistence of each type of mRNA for mu-, delta- and kappa-OPRs and pre-protachykinin A mRNA in the dorsal root ganglion neurons, (3) an increased expression of mu- and kappa-OPR mRNAs in the I-II layers of rat lumbar dorsal horn with an adjuvant arthritis in the hind limb, (4) the inhibitions of N- and Q-types of Ca2+ channels by mu- and kappa-OPR agonists and (5) cross-desensitization of the inhibition through a common intracellular phosphorylation-independent mechanism, (6) pharmacological characterization of "antagonist analgesics" as partial agonists at every type of OPRs, and (7) the key-structure(s) of OPRs for discriminative binding of DAMGO to mu-OPR.