Opiate analgesia: evidence for mediation by a subpopulation of opiate receptors.

Naloxazone, a hydrazone derivative of the opiate antagonist naloxone, has a high affinity for opiate receptor binding sites. Naloxazone injections reduce opiate receptor binding to extensively washed mouse brain membranes for more than 24 hours, suggesting that the effect is irreversible. High-affinity binding sites are abolished by this treatment, whereas low-affinity sites are unaffected. Naloxazone treatment blocks the analgesic effects of morphine for at least 24 hours but does not prevent death from high doses of morphine. Thus analgesic but nonlethal opiate effects may be mediated by the high-affinity subpopulation of opiate receptors.

High and low doses of cocaine intake are differentially regulated by dopamine D2 receptors in the ventral tegmental area and the nucleus accumbens.

Dopamine D2 receptors (D2Rs) in the ventral tegmental area (VTA) and the nucleus accumbens (NAc) are associated with vulnerability to addiction; however, whether D2Rs in these two brain regions play differential roles in regulation of drug intake is unknown. Here, we compared the effect of decreased mRNA level of Drd2 in each region on cocaine self-administration in a dose-response function. Drd2 mRNA levels in rat VTA or NAc were knocked down by bilateral microinjection of lentivirus coding shRNAs against rat Drd2 or scrambled shRNA. Drd2 knockdown was persistent and stable between 20 and 90 days after lentiviral infection. Animals were trained to self-administer cocaine 20 days after Drd2 shRNA treatment. Compared to scrambled shRNA treated rats, Drd2 knockdown in the VTA increased cocaine self-administration at all tested doses (0.02-0.56 mg/kg/infusion) producing an upward shift (both the ascending and descending limb) in the dose-response curve of cocaine self-administration. In contrast, intra-NAc knockdown increased cocaine self-administration only on the ascending limb of the dose-response curve (0.02-0.07 mg/kg/infusion). These data suggest that D2Rs in the VTA, not in the NAc, regulate high-dose cocaine intake. The present study not only demonstrates that low levels of D2Rs in either region increase low doses of cocaine intake, but also reveals for the first time their dissociable roles in limiting high doses of cocaine self-administration.

Opioid receptors: pinning down the opiate targets.

Recent studies of knockout mice conclusively show that the mu opioid receptor mediates the analgesic effects of morphine; they further suggest that the mu opioid receptor also has a role in mediating the effects of other opioid agonists, and provide insights into the non-analgesic effects of mu opioids.

Sigma 2 receptors as potential biomarkers of proliferation in breast cancer.

sigma 1 and sigma 2 receptors have been shown to exist in a number of rodent and human tumor cell lines. Although their expression is heterogeneous and their function is unknown, sigma receptors have been proposed as potential targets for diagnostic tumor-imaging agents. In this study, the density of sigma 2 receptors in proliferative (P) and quiescent (Q) cells of the mouse mammary adenocarcinoma, line 66, was examined. Scatchard analyses of sigma 2 receptors were performed on membrane preparations of 66 P cells from 3-day cultures and 66 Q cells from 7-, 10-, and 12-day cultures. The Scatchard studies revealed that 66 P cells had approximately 10 times more sigma 2 receptors/cell than the 66 Q cells from 10-day cultures. Although > 97% of the cells were quiescent after 7 days in culture, the maximum differential in the sigma 2 expression between 66 P and 66 Q cells was not attained until these cells had been in culture for 10 days. These data suggest that ligands labeled with positron-emitting or single photon-emitting radionuclides, which selectively bind sigma 2 receptors, have the potential to noninvasively assess the proliferative status of human breast tumors.

Calcium-binding proteins in electroplax and skeletal muscle. Comparison of the parvalbumin and phosphodiesterase activator protein of Electrophorus electricus.

A soluble calcium-binding protein has been isolated from the red skeletal muscle of the electric eel (Electrophorus electricus). The purification procedure involved ammonium sulfate precipitation, gel filtration on Sephadex G-75 in the presence of 45Ca2+, and chromatography on QAE-Sephadex. This procedure resulted in the isolation of a protein which was homogeneous upon polyacrylamide gel electrophoresis. The calcium-binding protein was found to be a typical parvalbumin by the following criteria: (1) molecular weight of 11 000; (2) pI of 4.7; (3) 1.9 mol Ca2+ bound per mol protein; Kd of approx. 10(-7) M; (4) no detectable phosphorus; (5) amino acid composition included nine residues of phenylalanine, single arginine, and no tyrosine or tryptophan; (6) ximax at 259 nm; (7) 260 nm:280 nm absorbance ratio of 4.78. Only one parvalbumin could be detected in muscle. Immunoprecipitation assay revealed that the parvalbumin was a major soluble component of skeletal muscle (0.10 mg/mg soluble protein), but could not be detected in liver, kidney, brain, spleen, heart or electroplax. Comparison of the parvalbumin with a calcium-binding protein previously isolated from electroplax revealed that the two proteins were different as judged by a variety of chemical criteria. These results suggest that during embryological development of electroplax the parvalbumin is lost and that it is not required for the function of electric tissue.

Isolation and purification of calcium-binding protein from electroplax of Electrophorus electricus.

An acidic calcium-binding phosphoprotein has been isolated from a cholinergic tissue, electroplax from Electrophorus electricus. The purification procedures included (NH4)2SO4 fractionation, boiling treatment, ECTEOLA-cellulose chromatography, and gel filtration on Sephadex G-100. Experiments were performed to compare this protein and a calcium-binding protein isolated from mammalian brain, adrenal medulla, and testis. These experiments showed that the two proteins were identical in terms of molecular weight (14 000), calcium-binding dissociation constant (kd=2.1-10(-5) M), electrophoretic mobility at pH 8.7 in 15% polyacrylamide gels, and phosphorus content (1 mol phosphorus per mol protein). In addition, the two proteins had similar amino acid compositions and peptide maps. Although the electroplax protein was not present in eel skeletal muscle, preliminary experiments indicated that small amounts of the protein were present in other eel tissues, namely brain, liver and spleen. These results suggest an identity between the electroplax and mammalian calcium-binding proteins and extend the findind of comparatively large amounts of the protein from mammalian nervous tissue to a cholinergic nervous tissue, electroplax. The close similarity of the proteins suggests a conservation of structure during evolution which may be required to fulfill a role in neuronal function.

Regional differences in mu and kappa opioid receptor G-protein activation in brain in male and female prairie voles.

Prairie voles are unusual mammals in that, like humans, they are capable of forming socially monogamous pair bonds, display biparental care, and engage in alloparental behaviors. Both mu and kappa opioid receptors are involved in behaviors that either establish and maintain, or result from pair bond formation in these animals. Mu and kappa opioid receptors both utilize inhibitory G-proteins in signal transduction mechanisms, however the efficacy by which these receptor subtypes stimulate G-protein signaling across the prairie vole neuraxis is not known. Utilizing [(35)S]GTPγS autoradiography, we characterized the efficacy of G-protein stimulation in coronal sections throughout male and female prairie vole brains by [D-Ala2,NMe-Phe4,Gly-ol5]-enkephalin (DAMGO) and U50,488H, selective mu and kappa opioid agonists, respectively. DAMGO stimulation was highest in the forebrain, similar to that found with other rodent species. U-50,488H produced greater stimulation in prairie voles than is typically seen in mice and rats, particularly in select forebrain areas. DAMGO produced higher stimulation in the core versus the shell of the nucleus accumbens (NAc) in females, while the distribution of U-50,488H stimulation was the opposite. There were no gender differences for U50,488H stimulation of G-protein activity across the regions examined, while DAMGO stimulation was greater in sections from females compared to those from males for NAc core, entopeduncular nucleus, and hippocampus. These data suggest that the kappa opioid system may be more sensitive to manipulation in prairie voles compared to mice and rats, and that female prairie voles may be more sensitive to mu agonists in select brain regions than males.

Anatomical distribution of mu, delta, and kappa opioid- and nociceptin/orphanin FQ-stimulated [35S]guanylyl-5'-O-(gamma-thio)-triphosphate binding in guinea pig brain.

An in vitro autoradiographic technique has recently been developed to visualize receptor-activated G-proteins by using agonist-stimulated [35S]guanylyl-5'-O-(gamma-thio)-triphosphate ([35S]GTPgammaS) binding in the presence of excess guanosine 5'-diphosphate. This technique was used to localize opioid-activated G-proteins in guinea pig brain, a species that contains the three major types of opioid receptors. This study used selective mu, delta, and kappa opioid agonists as well as nociceptin or orphanin FQ (N/OFQ) peptide, an endogenous ligand for an orphan opioid receptor-like (ORL1) receptor, to stimulate [35S]GTPgammaS binding in guinea pig brain sections. Opioid receptor specificity was confirmed by blocking agonist-stimulated [35S] GTPgammaS binding with the appropriate antagonists. In general, the distribution of agonist-stimulated [35S]GTPgammaS binding correlated with previous reports of receptor binding autoradiography, although quantitative differences suggest regional variations in receptor coupling efficiency. Mu, delta, and kappa opioid-stimulated [35S]GTPgammaS binding was found in the caudate-putamen, nucleus accumbens, amygdala, and hypothalamus. Mu-stimulated [35S]GTPgammaS binding predominated in the hypothalamus, amygdala, and brainstem, whereas kappa-stimulated [35S]GTPgammaS binding was particularly high in the substantia nigra and cortex and was moderate in the cerebellum. N/OFQ-stimulated [35S] GTPgammaS binding was highest in the cortex, hippocampus, and hypothalamus and exhibited a unique anatomical distribution compared with opioid-stimulated [35S]GTPgammaS binding. The present study extends previous reports on opioid and ORL1 receptor localization by anatomically demonstrating functional activity produced by mu, delta, and kappa opioid and ORL1 receptor activation of G-proteins.

Functional and anatomical localization of mu opioid receptors in the striatum, amygdala, and extended amygdala of the nonhuman primate.

The subregional distribution of mu opioid receptors and corresponding G-protein activation were examined in the striatum, amygdala, and extended amygdala of cynomolgus monkeys. The topography of mu binding sites was defined using autoradiography with [(3)H]DAMGO, a selective mu ligand. In adjacent sections, the distribution of receptor-activated G proteins was identified with DAMGO-stimulated guanylyl 5'(gamma-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding. Within the striatum, the distribution of [(3)H]DAMGO binding sites was characterized by a distinct dorsal-ventral gradient with a higher concentration of binding sites at more rostral levels of the striatum. [(3)H]DAMGO binding was further distinguished by the presence of patch-like aggregations within the caudate, as well as smaller areas of very dense receptor binding sites, previously identified in human striatum as neurochemically unique domains of the accumbens and putamen (NUDAPs). The amygdala contained the highest concentration of [(3)H]DAMGO binding sites measured in this study, with the densest levels of binding noted within the basal, accessory basal, paralaminar, and medial nuclei. In the striatum and amygdala, the distribution of DAMGO-stimulated G-protein activation largely corresponded with the distribution of mu binding sites. The central and medial nuclei of the amygdala, however, were notable exceptions. Whereas the concentration of [(3)H]DAMGO binding sites in the central nucleus of the amygdala was very low, the concentration of DAMGO-stimulated G-protein activation in this nucleus, as measured with [(35)S]GTPgammaS binding, was relatively high compared to other portions of the amygdala containing much higher concentrations of [(3)H]DAMGO binding sites. The converse was true in the medial nucleus, where high concentrations of binding sites were associated with lower levels of DAMGO-stimulated G-protein activation. Finally, [(3)H]DAMGO and [(35)S]GTPgammaS binding within the amygdala, particularly the medial nucleus, formed a continuum with the substantia innominata and bed nucleus of the stria terminalis, supporting the concept of the extended amygdala in primates.

Cannabinoid receptors: G-protein-mediated signal transduction mechanisms.

The recent discovery and cloning of cannabinoid receptors has provided a major breakthrough in the understanding of the biochemical mechanisms of action of delta 9-tetrahydrocannibinol (delta 9-THC). Cannabinoid receptors are coupled to G-proteins and inhibit adenylyl cyclase in a variety of systems. In the brain, cannabinoid-inhibited adenylyl cyclase and the receptors are particularly prevalent in the cerebellum, where they are localized to cerebellar granule cells (Fig. 1). In these cells, cannabinoid receptors are co-localized with other Gi/o-linked receptors such as gamma-aminobutyric acid (GABAB) receptors, where they share common effector systems (adenylyl cyclase catalytic units) but not common G-proteins. This sharing of effectors leads to the phenomenon of receptor convergence, in which agonists of different receptor types can produce the same biological response in certain cells. In cultured hippocampal neurons, cannabinoids also act through G-proteins to increase potassium conductance. In these cells, the predominant electrophysiological response at relatively low (microM) concentrations of cannabinoids is mediated through a voltage-sensitive potassium A current (IA) (Fig. 1). The action of cannabinoid receptors in this system is to shift the voltage sensitivity of IA channels to higher voltage ranges, thus increasing K+ conductance at lower membrane potentials and decreasing the probability of multiple action potentials. When combined with data from other groups showing a cannabinoid receptor-mediated decrease in calcium conductance, along with the unique localization of cannabinoid receptors in the brain, it is clear that these receptor-effector combinations are well situated to mediate many of the well-known neurobiological effects of delta 9-THC.

Agonist-stimulated [35S]GTPgammaS binding in brain modulation by endogenous adenosine.

Coupling of receptors to G-proteins can be assessed by the ability of specific agonists to stimulate [35S]GTPgammaS binding in both brain membranes and sections in the presence of excess GDP. In some brain regions, however, high basal activity makes it difficult to detect agonist-stimulated [35S]GTPgammaS binding. The present study suggests a modification of the assay to reduce basal [35S]GTPgammaS binding and thus increase the signal:noise ratio. Adenosine A1 receptors belong to the class of G-protein-coupled receptors that activate Gi/Go proteins in brain. In the present study, the A1 agonist R(-)N6-(2-phenylisopropyl)adenosine (R-PIA) stimulated [35S]GTPgammaS binding in brain regions known to contain A1 receptors, including cerebellum, hippocampus and dentate gyrus, medial geniculate body, superior colliculus, certain thalamic nuclei, cerebral cortex, piriform cortex, caudate-putamen, and nucleus accumbens. Treatment of sections and membranes with adenosine deaminase (ADase), which is typically used in adenosine assays to eliminate endogenous adenosine, reduced basal [35S]GTPgammaS binding. In addition, for cannabinoid and mu-opioid agonists, the percent stimulation of [35S]GTPgammaS binding was approximately doubled when ADase was included in the assay. These results suggest that endogenous adenosine contributes significantly to basal [35S]GTPgammaS binding in certain brain regions, and that this activity may be reduced by the addition of ADase, thus improving the signal:noise ratio of agonist-stimulated [35S]GTPgammaS binding.

Prolonged dopamine and serotonin transporter inhibition after exposure to tropanes.

Cocaine and tropane analogs are known to interact with biogenic monoamine transporters by inhibiting amine uptake. Previous in vivo studies have demonstrated that some of these tropanes produce a longer lasting behavioral effect compared with cocaine. We have previously examined several tropane analogs and found a difference in their relative affinities for dopamine (DA) and serotonin (5-HT) transporters. The purpose of this study was to determine the recovery time of transporter function in vitro and in vivo comparing cocaine with the tropane analogs WF-11 (PTT, selective for DA transporters), WF-31 (selective for 5-HT transporters) and WF-23 (highly potent at both DA and 5-HT transporters). In vitro, using primary rat brain cultures of either midbrain or raphe regions, the recovery of the ability to transport either [3H]dopamine or [3H]serotonin, respectively was evaluated at 0, 3, 24, 48, 120 and 240 h after a 1 h exposure to cocaine and tropane analogs. The tropanes exhibited clearance half-lives ranging from 12 to 69 h, while cocaine, on the other hand, exhibited a clearance half-life of approximately 6 h. In studies utilizing [125I]RTI-55 binding, intraperitoneal injections of cocaine and WF-23 into the rat resulted in striatal clearance half-lives ex vivo that were almost identical to those obtained in vitro. These data suggest that the tropanes bind to and reduce transporter function for prolonged periods of time (up to 10-fold longer than cocaine) and those compounds with the highest affinity may produce a pseudo-irreversible inhibition of transporter function.

Synthesis of 3 beta-aryl-8-azabicyclo[3.2.1]octanes with high binding affinities and selectivities for the serotonin transporter site.

A novel entry to tropane analogs of cocaine was developed based on the reaction of rhodium-stabilized vinylcarbenoids with pyrroles. These analogs were tested in binding to dopamine, serotonin (5-HT), and norepinephrine transporters in membranes from rat striatum and frontal cortex. In all the analogs, the aryl group at the 3 position was directly bound to the tropane ring and an ethyl ketone moiety was present at the 2 position. By appropriate modification of the aryl and nitrogen substituents, highly potent and 5-HT selective tropanes were prepared. The most potent and selective compound was 3 beta-[4-(1-methylethenyl)phenyl]-2 beta-propanoyl-8-azabicyclo[3.2.1]octane (13b) which had a Ki of 0.1 nM at 5-HT transporters and was 150 times more potent at 5-HT vs dopamine transporters and almost 1000 times more potent at 5-HT vs norepinephrine transporters.

Synthesis of 2 beta-acyl-3 beta-aryl-8-azabicyclo[3.2.1]octanes and their binding affinities at dopamine and serotonin transport sites in rat striatum and frontal cortex.

A novel entry to tropane analogs of cocaine was developed on the basis of the reaction of rhodium-stabilized vinylcarbenoids with pyrroles. These analogs were tested in binding to dopamine and serotonin (5-HT) transporters in membranes from rat striatum and frontal cortex. In all the analogs, the aryl group at the 3-position was directly bound to the tropane ring (as in WIN-35,428), and methyl or ethyl ketone moieties were present at the 2-position instead of the typical ester group. The series of analogs containing a 2-naphthyl group at the 3-position were most potent, with Ki values < 1 nM in binding to both dopamine and 5-HT transporters. Although the unsubstituted 2-naphthyl analog was nonselective at dopamine and 5-HT transport sites, other compounds were selective for either site. In general, compounds with relatively small substituents on the aromatic moiety (such as p-methyl or p-fluoro) were relatively selective for the dopamine transporters, while a p-isopropylphenyl derivative was selective for the 5-HT transport sites. This latter compound represents the first N-methyltropane derivative specific for 5-HT transporters. Resolution of two of the most significant analogs was achieved by HPLC on a chiral stationary phase; the active enantiomer of a 2-naphthyl analog exhibited Ki values of < 0.1 nM at both dopamine and 5-HT transporter sites.

Synthesis and quantitative structure-activity relationships of N-(1-benzylpiperidin-4-yl)phenylacetamides and related analogues as potent and selective sigma1 receptor ligands.

A series of N-(1-benzylpiperidin-4-yl)phenylacetamide derivatives was synthesized and evaluated for affinity at sigma1 and sigma2 receptors. Most of these compounds showed a high affinity for sigma1 receptors and a low to moderate affinity for sigma2 receptors. The unsubstituted compound N-(1-benzylpiperidin-4-yl)phenylacetamide, 1, displayed a high affinity and selectivity for sigma1 receptors (Ki values of 3.90 nM for sigma1 receptors and 240 nM for sigma2 receptors). The influence of substitutions on the phenylacetamide aromatic ring on binding at both the sigma1 and sigma2 receptor has been examined through Hansch-type quantitative structure-activity relationship (QSAR) studies. In general, all 3-substituted compounds, except for the OH group, had a higher affinity for both sigma1 and sigma2 receptors when compared with the corresponding 2- and 4-substituted analogues. The selectivity for sigma1 receptors displayed a trend of 3 > 2 approximately 4 for Cl, Br, F, NO2, and OMe substituted analogues. Halogen substitution on the aromatic ring generally increased the affinity for sigma2 receptors while maintaining a similar affinity for sigma1 receptors. Substitution with electron-donating groups, such as OH, OMe, or NH2, resulted in weak or negligible affinity for sigma2 receptors and a moderate affinity for sigma1 receptors. The 2-fluoro-substituted analogue, 11, exhibited the highest selectivity for sigma1 receptors among all compounds tested, with a Ki value of 3.56 nM for sigma1 receptors and 667 nM for sigma2 receptors. Compounds 1, 5, 9, 11, and 20 had no affinity for dopamine D2 (IC50 > 10 000 nM) and D3 (IC50 > 10 000 nM) receptors. The nanomolar binding affinity and high selectivity for sigma1 receptors suggest that these compounds may be developed as potential radiotracers for positron emission tomography or single photon emission computerized tomography imaging studies.

Synthesis of 2beta-acyl-3beta-(substituted naphthyl)-8-azabicyclo[3.2.1]octanes and their binding affinities at dopamine and serotonin transport sites.

A series of 3beta-naphthyltropane derivatives were synthesized and found to show high affinity at both the dopamine and serotonin transporter sites, leading to some of the most potent inhibitors known based on the tropane structure. Comparative molecular field analysis (CoMFA) models were developed for both dopamine and serotonin transporter binding data. These models provide insights into those factors that influence binding at the two transporters.

Mu and kappa1 opioid-stimulated [35S]guanylyl-5'-O-(gamma-thio)-triphosphate binding in cynomolgus monkey brain.

Agonist-stimulated [35S]GTPgammaS binding allows the visualization of receptor-activated G-proteins, thus revealing the anatomical localization of functional receptor activity. In the present study, agonist-stimulated [35S]GTPgammaS binding was used to demonstrate mu and kappa1 opioid-stimulated [35S]GTPgammaS binding in tissue sections and membranes from cynomolgus monkey brain using DAMGO and U50,488H, respectively. Concentrations of agonists required to produce maximal stimulation of [35S]GTPgammaS binding were determined in membranes from the frontal poles of the brain. Receptor specificity was verified in both membranes and sections by inhibiting agonist-stimulated [35S]GTPgammaS binding with the appropriate antagonist. Mu opioid-stimulated [35S]GTPgammaS binding was high in areas including the amygdala, ventral striatum, caudate, putamen, medial thalamus and hypothalamus. Dense mu-stimulated [35S]GTPgammaS binding was also found in brainstem nuclei including the interpeduncular nucleus, parabrachial nucleus and nucleus of the solitary tract. Kappa1 opioid-stimulated [35S]GTPgammaS binding was high in limbic and association cortex, ventral striatum, caudate, putamen, globus pallidus, claustrum, amygdala, hypothalamus and substantia nigra. These results demonstrate the applicability of [35S]GTPgammaS autoradiography to examine receptor-activated G-proteins in the primate brain and reveal functional mu and kappa1 opioid receptor activity that may contribute to the reported central nervous system effects of opiates.

Effects of sodium on agonist efficacy for G-protein activation in mu-opioid receptor-transfected CHO cells and rat thalamus.

1. Sodium ions inhibit spontaneous G(i)/G(o)-coupled receptor activity and promote agonist-induced responses in vitro. The effects of sodium on the relative efficacy of opioid agonists for G-protein activation was measured by guanosine-5'-O-(gamma-(35)S)-triphosphate ([(35)S]-GTPgammaS) binding in membranes from two mu-opioid receptor-containing systems: CHO cells stably transfected with mouse mureceptors (mMOR-CHO cells) and rat thalamus. 2. NaCl inhibited basal [(35)S]-GTPgammaS binding in both systems, and this effect was partially mimicked by KCl. In mMOR-CHO membranes, net [(35)S]-GTPgammaS binding stimulated by partial but not full agonists was inhibited by NaCl with a potency that was inversely proportional to agonist efficacy. Monovalent cations were required for agonist-stimulated [(35)S]-GTPgammaS binding in this system, and increasing NaCl concentrations magnified relative efficacy differences among agonists. 3. In thalamic membranes, which contain a lower receptor:G-protein ratio than mMOR-CHO cells, similar monovalent cation effects were observed, with two exceptions: (1) [(35)S]-GTPgammaS binding stimulated by both full and partial agonists was inhibited by NaCl; and (2) monovalent cations were not required to observe agonist-stimulated [(35)S]-GTPgammaS binding. 4. Basal [(35)S]-GTPgammaS binding stimulated by the absence of monovalent cations resembled that of agonist-stimulated binding and was blocked by pretreatment of mMOR-CHO cells with pertussis toxin. 5. These results indicate that sodium inhibits spontaneous and agonist-occupied mu receptor-mediated G-protein activation in a manner inversely proportional to the efficacy of the agonist, and that spontaneous mu receptor activity and the relative efficacy of partial agonists acting at these receptors are both increased by increases in the stoichiometric ratio of receptors:G-proteins.

Relationship between kappa 1 opioid receptor binding and inhibition of adenylyl cyclase in guinea pig brain membranes.

Previously, we showed that kappa-selective ligands inhibit adenylyl cyclase in guinea pig cerebellar membranes. The present studies explore the relationship between kappa 1 binding sites (as determined with [3H]U-69,593 binding) and kappa 1-inhibition of adenylyl cyclase (using U-50,488H) in guinea pig brain membranes. Various kappa opioids displaced [3H]U-69,593 binding at a single site with subnanomolar affinities. These agonists were several hundred-fold weaker in inhibiting adenylyl cyclase, but for most agonists the rank order of adenylyl cyclase inhibition paralleled the displacement of kappa 1 binding. The correlation of IC50 values for both adenylyl cyclase and binding was significant except for alpha-neo endorphin, which was relatively weak at inhibiting adenylyl cyclase despite a Ki value of 0.08 nM versus kappa 1 binding. Comparison between kappa 1 binding and kappa 1-inhibited adenylyl cyclase across eleven guinea pig brain regions revealed that kappa 1-inhibited adenylyl cyclase was highest in the cerebellum, absent in thalamus and superior colliculus, and moderate in other regions. In most regions, kappa 1 binding correlated with the efficacy of kappa 1-inhibited adenylyl cyclase. However, the hippocampus had high levels of kappa 1-inhibited adenylyl cyclase despite low levels of kappa 1 binding, while cortex exhibited a high density of kappa 1 sites but a relatively low level of kappa 1-inhibited adenylyl cyclase. Reaction of cerebellar kappa receptors with beta-chlornaltrexamine (beta-CNA) blocked both kappa 1 binding and kappa 1-inhibited adenylyl cyclase. The effect of beta-CNA on kappa 1-inhibited adenylyl cyclase was to inhibit efficacy with little decrease in agonist potency, thus suggesting no significant level of kappa receptor reserve for this effector system.

Role of cyclic AMP in the actions of cannabinoid receptors.

Cannabinoids, including delta 9-tetrahydrocannabinol (THC), bind to receptors that couple to Gi/o-proteins and inhibit adenylyl cyclase. However, like other G-protein-coupled receptors, cannabinoid receptors are also coupled to other effector systems. This review examines the characteristics of the cannabinoid-G-protein-adenylyl cyclase system, and explores the role of cyclic AMP in mediating effects of these drugs. Several conclusions emerge from this research. First, the principal actions of cannabinoids are mediated through G-protein-coupled receptors, and the intracellular signaling mechanism that initiates cellular response of cannabinoids is activation of G-proteins. Second, cannabinoid-inhibited adenylyl cyclase is only one of several different effectors coupled to these receptors, and different effectors may be used for different types of responses. Third, cannabinoid inhibition of adenylyl cyclase plays an important role in several aspects of cannabinoid function, including modulating conductance at a voltage-dependent K+ channel ("A" current) in the hippocampus, thus providing an effective rationale for behavioral effects of cannabinoids mediated in this region. Other functions of this system may include production of long-term changes in gene expression by inhibition of cyclic AMP response elements on strategic genes, and inhibition of anandamide synthesis, thus mediating some of the long-term effects of cannabinoids on neuronal function.