enkephalin--ala(2)-mephe(4)-gly(5)- and 3-(2-hydroxy-4-(1-1-dimethylheptyl)phenyl)-4-(3-hydroxypropyl)cyclohexanol

Classically, G protein-coupled receptor activation by a ligand has been viewed as producing a defined response such as activation of a G protein, activation or inhibition of adenylyl cyclase, or stimulation of phospholipase C and/or alteration in calcium flux. Newer concepts of ligand-directed signaling recognize that different ligands, ostensibly acting at the same receptors, may induce different downstream effects, complicating the selection of a screening assay. Dynamic mass redistribution (DMR), a label-free technology that uses light to measure ligand-induced changes in the mass of cells proximate to the biosensor, provides an integrated cellular response comprising multiple pathways and cellular events. Using DMR, signals induced by opioid or cannabinoid agonists in cells transfected with these receptors were blocked by pharmacologically appropriate receptor antagonists as well as by pertussis toxin. Differences among compounds in relative potencies at DMR versus ligand-stimulated GTPγS or receptor binding endpoints, suggesting functional selectivity, were observed. Preliminary evidence indicates that inhibitors of intermediate steps in the cell signaling cascade, such as receptor recycling inhibitors, mitogen-activated protein kinase kinase/p38 mitogen-activated protein kinase inhibitors, or cytoskeletal disruptors, altered or attenuated the cannabinoid-induced response. Notable is the finding that mitogen-activated protein kinase kinase 1/2 inhibitors attenuated signaling induced by the cannabinoid type 2 receptor inverse agonist AM630 but not that stimulated by the agonist CP 55,940. Thus, DMR has the potential to not only identify ligands that activate a given G protein-coupled receptor, but also ascertain the signaling pathways engaged by a specific ligand, making DMR a useful tool in the identification of biased ligands, which may ultimately exhibit improved therapeutic profiles.

Topics: Analgesics, Opioid; Animals; Butadienes; Cannabinoids; Carrier Proteins; Chemistry Techniques, Analytical; CHO Cells; Cricetinae; Cyclohexanols; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; GTP-Binding Protein alpha Subunits; Indoles; Male; Morphine; Nitriles; Optical Phenomena; Pertussis Toxin; Protein Serine-Threonine Kinases; Rats; Rats, Wistar; Receptor, Cannabinoid, CB2; Receptors, G-Protein-Coupled; Receptors, Opioid; Signal Transduction

Interactions between mu-opioid receptor (muOR) and cannabinoid CB1 receptor (CB1R) were examined by morphological and electrophysiological methods. In baby hamster kidney (BHK) cells coexpressing muOR fused to the yellow fluorescent protein Venus and CB1R fused to the cyan fluorescent protein Cerulean, both colors were detected on the cell surface; and fluorescence resonance energy transfer (FRET) analysis revealed that muOR and CB1R formed a heterodimer. Coimmunoprecipitation and Western blotting analyses also confirmed the heterodimers of muOR and CB1R. [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin (DAMGO) or CP55,940 elicited K+ currents in Xenopus oocytes expressing muOR or CB1R together with G protein activated-inwardly rectifying K+ channels (GIRKs), respectively. In oocytes coexpressing both receptors, either of which was fused to the chimeric Galpha protein Gqi5 that activates the phospholipase C pathway, both DAMGO and CP55,940 elicited Ca2+-activated Cl(-) currents, indicating that each agonist can induce responses through Gqi5 fused to either its own receptor or the other. Experiments with endogenous Gi/o protein inactivation by pertussis toxin (PTX) supported the functional heterodimerization of muOR/CB1R through PTX-insensitive Gqi5(m) fused to each receptor. Thus, muOR and CB1R form a heterodimer and transmit a signal through a common G protein. Our electrophysiological method could be useful for determination of signals mediated through heterodimerized G protein-coupled receptors.

Topics: Animals; Blotting, Western; Calcium; Cell Line; Cell Membrane; Chloride Channels; Cyclohexanols; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Fluorescence Resonance Energy Transfer; G Protein-Coupled Inwardly-Rectifying Potassium Channels; GTP-Binding Protein alpha Subunits, Gq-G11; Humans; Immunoprecipitation; Membrane Potentials; Patch-Clamp Techniques; Pertussis Toxin; Protein Multimerization; Rats; Receptor, Cannabinoid, CB1; Receptors, Opioid, mu; Recombinant Fusion Proteins; Signal Transduction; Transfection; Type C Phospholipases; Xenopus

In the present study we investigated the signal transduction pathways leading to the activation of extracellular signal-regulated kinase (ERK) by opioid or cannabinoid drugs, when their receptors are coexpressed in the same cell-type. In N18TG2 neuroblastoma cells, the opioid agonist etorphine and the cannabinoid agonist CP-55940 induced the phosphorylation of ERK by a similar mechanism that involved activation of delta-opioid receptors or CB1 cannabinoid receptors coupled to Gi/Go proteins, matrix metalloproteases, vascular endothelial growth factor (VEGF) receptors and MAPK/ERK kinase (MEK). In HEK-293 cells, these two drugs induced the phosphorylation of ERK by separate mechanisms. While CP-55940 activated ERK by transactivation of VEGFRs, similar to its effect in N18TG2 cells, the opioid agonist etorphine activated ERK by a mechanism that did not involve transactivation of a receptor tyrosine kinase. Interestingly, the activation of ERK by etorphine was resistant to the inhibition of MEK, suggesting the possible existence of a novel, undescribed yet mechanism for the activation of ERK by opioids. This mechanism was found to be specific to etorphine, as activation of ERK by the micro-opioid receptor (MOR) agonist DAMGO ([D-Ala(2), N-Me-Phe(4), Gly(5)-ol] enkephalin) was mediated by MEK in these cells, suggesting that etorphine and DAMGO activate distinct, ligand-specific, conformations of MOR. The characterization of cannabinoid- and opioid-induced ERK activation in these two cell-lines enables future studies into possible interactions between these two groups of drugs at the level of MAPK signaling.

Topics: Analgesics; Analgesics, Opioid; Animals; Cell Line; Cell Line, Tumor; Central Nervous System; Cyclohexanols; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enzyme Activation; Etorphine; Extracellular Signal-Regulated MAP Kinases; Humans; MAP Kinase Kinase 1; Mice; Neuroblastoma; Neurons; Rats; Receptor, Cannabinoid, CB1; Receptors, Cannabinoid; Receptors, G-Protein-Coupled; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, mu; Vascular Endothelial Growth Factor Receptor-1

Early cannabinoid consumption may predispose individuals to the misuse of addictive drugs later in life. However, there is a lack of experimental evidence as to whether cannabinoid exposure during adolescence might differently affect opiate reinforcing efficacy and the opioid system in adults of both sexes. Our aim was to examine whether periadolescent chronic exposure to the cannabinoid agonist CP-55,940 could exert sex-dependent effects on morphine reinforcing and the opioid system in adulthood. Morphine reinforcing was studied under a progressive ratio (PR) reinforcement schedule in adult male and female rats that previously acquired morphine self-administration under a fixed ratio 1 (FR1) schedule. Binding levels and functionality of mu-opioid receptors were also evaluated. Periadolescent cannabinoid exposure altered morphine self-administration and the opioid system in adult rats in a sex-dependent manner. CP-55,940-exposed males exhibited higher self-administration rates under a FR1, but not under a PR schedule. In females, CP-55,940 did not modify morphine self-administration under either schedule. Moreover, CP-55,940 also increased mu-opioid receptor levels in the subcallosal streak of pre-treated animals and decreased mu-opioid receptor functionality in the nucleus accumbens shell but again, only in males. Our data indicate that adult male rats exposed to the cannabinoid in adolescence self-administer more morphine than females, but only when the demands required by the schedule of reinforcement are low, which might be related to the decrease in mu-opioid receptor functionality in the NAcc-shell observed in these animals.

Topics: Analgesics; Analgesics, Opioid; Animals; Autoradiography; Behavior, Animal; Brain; Conditioning, Operant; Cyclohexanols; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Female; Guanosine 5'-O-(3-Thiotriphosphate); Male; Morphine; Protein Binding; Rats; Rats, Wistar; Receptors, Opioid, mu; Reinforcement Schedule; Self Administration; Sex Characteristics; Statistics, Nonparametric; Sulfur Isotopes

This study examines the effect of intravenous self-administration (SA) of either heroin or the cannabinoid receptor agonist WIN 55,212-2 on levels and functionality of mu-opioid (MOR) and CB1-cannabinoid receptors (CB1R) in reward-related brain areas, such as the prefrontal cortex (PFC), nucleus accumbens (NAc), caudate putamen (CP), hippocampus (Hippo), amygdala (Amy), hypothalamus (Hypo) and ventral tegmental area (VTA). [3H]DAMGO and [3H]CP-55,940 autoradiography and agonist-stimulated [35S]GTPgammaS binding were performed on brain sections of rats firmly self-administering heroin or WIN 55,212-2. Animals failing to acquire heroin or cannabinoid SA behaviour as well as drug-naïve animals never exposed to experimental apparatus or procedure (home-control group) were used as controls. With respect to control groups, which displayed very similar values, rats SA heroin showed increased MOR binding in the NAc (+174%), CP (+165%), Hippo (+121%), VTA (+175%), an enhanced CB1R density localized in the Amy (+147%) and VTA (+37%), and a widespread increased CB1 receptor functionality in the PFC (+95%), NAc (+313%), CP (+265%), Hippo (+38%), Amy (+221%). In turn, cannabinoid SA differently modulates CB1R binding in the Amy (+47%), Hypo (+94%), Hippo (-23%), VTA (-15%), and increases MOR levels (PFC: +124%; NAc: +68%; CP: +80%; Hippo: +73%; Amy: +99%) and efficiency (Hippo: +518%; Amy: +173%; Hypo: +188%). These findings suggest that voluntary chronic intake of opioids or cannabinoids induces reciprocal but differential regulation of MORs and CB1Rs density and activity in brain structures underlying drug-taking and drug-seeking behaviour, which could represent long-term neuroadaptations contributing to the development of drug addiction and dependence.

Topics: Analgesics, Opioid; Animals; Benzoxazines; Brain; Cannabinoids; Cyclohexanols; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Guanosine 5'-O-(3-Thiotriphosphate); Heroin; Humans; Male; Morpholines; Naphthalenes; Rats; Receptor, Cannabinoid, CB1; Receptors, Opioid, mu; Self Administration

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 mu 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]GTPgammaS binding showed a significant decrease in receptor/G protein coupling in the limbic area. In CP-55,940 exposed rats, mu opioid receptor binding was significantly raised in the lateral thalamus and periaqueductal gray (PAG), with an increase in DAMGO-stimulated [35S]GTPgammaS 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.

Topics: Animals; Area Under Curve; Behavior, Animal; Brain; Brain Chemistry; Cannabinoids; Cyclic AMP; Cyclohexanols; Dose-Response Relationship, Drug; Drug Administration Schedule; Drug Interactions; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Guanosine 5'-O-(3-Thiotriphosphate); Male; Narcotics; Pain Measurement; Radioligand Assay; Rats; Rats, Sprague-Dawley; Receptors, Cannabinoid; Receptors, Opioid; Sulfur Isotopes; Tritium

The nonpsychoactive plant cannabinoid, (-)-cannabidiol, modulates in vivo responses to Delta(9)-tetrahydrocannabinol. We have found that cannabidiol can also interact with cannabinoid CB(1) receptor agonists in the mouse vas deferens, a tissue in which prejunctional cannabinoid CB(1) receptors mediate inhibition of electrically evoked contractions by suppressing noradrenaline and/or ATP release. Cannabidiol (0.316-10 microM) attenuated the ability of (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo-[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone (R-(+)-WIN55212) to inhibit contractions in a concentration-related, surmountable manner with a K(B) value (120.3 nM) well below its reported cannabinoid receptor CB(1)/CB(2) K(i) values. Cannabidiol (10 microM) also antagonized (-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol (CP55940; K(B)=34 nM) and [D-Ala(2), NMePhe(4), Gly-ol]enkephalin (DAMGO; K(B)=5.6 microM) and attenuated contractile responses to noradrenaline, phenylephrine and methoxamine but not to beta, gamma-methyleneadenosine 5'-triphosphate. At 3.16-10 microM, it increased the amplitude of evoked contractions, probably by enhancing contractile neurotransmitter release. We conclude that cannabidiol antagonizes R-(+)-WIN55212 and CP55940 by acting at prejunctional sites that are unlikely to be cannabinoid CB(1) or CB(2) receptors.

Topics: Adenosine Triphosphate; Animals; Benzoxazines; Cannabidiol; Cyclohexanols; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; In Vitro Techniques; Male; Methoxamine; Mice; Morpholines; Muscle Contraction; Naphthalenes; Norepinephrine; Phenylephrine; Receptors, Cannabinoid; Receptors, Drug; Vas Deferens; Vasoconstrictor Agents

Systemic administration of 3-nitropropionic acid (3NPA) in experimental animals produces bilateral striatal lesions similar to those seen in Huntington's disease (HD) caudate and putamen. 3H[-CP55,940 binding to cannabinoid receptors in human basal ganglia nuclei has been shown to be highly susceptible to the earliest pathological changes in the HD brain. In this study, to assess further the suitability of 3NPA-induced striatal lesions as a model for HD neuropathology, we examined the effects of striatal lesions induced by the systemic administration of 3NPA on the binding of 3H[-CP55,940 to pre- and postsynaptic cannabinoid receptors in striatum, globus pallidus, entopeduncular nucleus and substantia nigra pars reticulata and also the effect of 3NPA-induced striatal lesions on the binding of 3H[-DAMGO to mu-opioid receptors in striatal striosomes. Systemic administration of 3NPA induced bilateral and symmetrical lesions in dorsolateral striatum. Within the lesion core, 3H[-CP55,940 and 3H[-DAMGO binding density was reduced to background levels. Beyond the immediate borders of the central core of the 3NPA-induced lesion, striatal binding density was not significantly different from that measured in unlesioned rats. 3H[-CP55,940 binding in globus pallidus, entopeduncular nucleus and substantia nigra in 3NPA-lesioned rats was significantly reduced compared to controls, and the individual decreases were similar for each site. However, these reductions were statistically marginal. These data suggest that, while producing striatal lesions which bear some similarity to those seen in HD, the consequences of 3NPA for striatopallidal and striatonigral efferent projections do not reflect the reported neurodegenerative changes seen in the HD brain.

Topics: Animals; Autoradiography; Basal Ganglia; Cannabinoids; Caudate Nucleus; Corpus Striatum; Cyclohexanols; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Humans; Male; Neurotoxins; Nitro Compounds; Propionates; Putamen; Rats; Receptors, Cannabinoid; Receptors, Drug; Receptors, Opioid, mu; Tritium

In vitro receptor binding and quantitative autoradiography were used to assess the pre- and postsynaptic distribution of cannabinoid receptors in the cervical dorsal horn of the rat spinal cord. An extensive unilateral dorsal rhizotomy was performed across seven or eight successive spinal segments from C3 to T1 or T2. The densities of cannabinoid and mu opioid receptors in the central (C6) spinal segment were assessed 2, 4, 8, and 16 days post rhizotomy and compared with those of untreated rats. Rhizotomy induced approximately a 50% ipsilateral loss in the [3H]CP55,940 binding to spinal cannabinoid receptors that was maximal at 8 days post-rhizotomy. By comparison, the binding of [3H][d-Ala2-MePhe4, Gly-ol5]enkephalin (DAMGO) to mu receptors was depleted approximately 60% in near-adjacent sections. By contrast, changes in [3H]CP55,940 binding contralateral to the deafferentation were largely absent at all post-lesion delays. These data suggest that under conditions in which a spinal segment is completely deafferented, approximately 50% of cannabinoid receptors in the cervical (C6) dorsal horn reside presynaptically on central terminals of primary afferents. The present data provide anatomical evidence for presynaptic as well as postsynaptic localization of cannabinoid receptors in the spinal dorsal horn.

Topics: Analgesics; Analgesics, Opioid; Animals; Arachidonic Acids; Autoradiography; Calcium Channel Blockers; Cyclohexanols; Endocannabinoids; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Functional Laterality; Male; Neurons, Afferent; Polyunsaturated Alkamides; Presynaptic Terminals; Rats; Rats, Sprague-Dawley; Receptors, Cannabinoid; Receptors, Drug; Receptors, Opioid, mu; Rhizotomy; Spinal Cord; Tritium

In vitro receptor binding and quantitative autoradiography were used to determine whether cannabinoid receptors in rat lumbar spinal cord are localized to the central terminals of nociceptive primary afferents. Rats were treated as neonates with capsaicin to destroy sensory C-fibers. The densities of cannabinoid and mu opioid receptors in the spinal cord of the adult rats were compared with age-matched vehicle controls. Neonatal capsaicin produced a moderate but reliable suppression (16%) of [3H]CP55,940 binding to cannabinoid receptors. By contrast, the binding of [3H][D-Ala2-MePhe4,Gly-ol5]enkephalin (DAMGO) to mu receptors was depleted by approximately 60% in near adjacent sections. These data suggest that only a subpopulation of cannabinoid receptors is situated on the central terminals of primary afferent C-fibers. The present data provide anatomical evidence for a dissociation between cannabinoid and mu opioid modulation of sensory transmission at the level of the primary afferent inputs to the spinal cord.

Topics: Analgesics, Opioid; Animals; Animals, Newborn; Autoradiography; Capsaicin; Cyclohexanols; Down-Regulation; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Female; Immunosuppressive Agents; Nerve Fibers; Nociceptors; Pregnancy; Rats; Rats, Sprague-Dawley; Receptors, Cannabinoid; Receptors, Drug; Receptors, Opioid, mu; Spinal Cord; Tritium