virodhamine and 3-(2-hydroxy-4-(1-1-dimethylheptyl)phenyl)-4-(3-hydroxypropyl)cyclohexanol

In human bronchial epithelial (16HBE14o(-)) cells, CB(1) and CB(2) cannabinoid receptors are present, and their activation by the endocannabinoid virodhamine and the synthetic non-selective receptor agonist CP55,940 inhibits adenylyl cyclase and cellular interleukin-8 release. Here, we analyzed changes in intracellular calcium ([Ca2+](i)) evoked by Delta(9)-tetrahydrocannabinol (Delta(9)-THC), CP55,940, and virodhamine in 16HBE14o(-) cells. Delta(9)-THC induced [Ca2+](i) increase and a large transient [Ca2+](i) mobilization, the latter probably reflecting store-depletion-driven capacitative Ca2+ entry (CCE). In contrast, CP55,940 induced a rather moderate Ca2+ influx and a sustained [Ca2+](i) mobilization. CP55,940-induced Ca2+ influx was inhibited by Ni2+, indicating CCE, possibly mediated by transient receptor potential channel TRPC1, the mRNA of which is expressed in 16HBE14o(-) cells. CP55,940-induced calcium alterations were mimicked by virodhamine concentrations below 30 microM. Interestingly, higher virodhamine induced an additional Ca2+ entry, insensitive to Ni2+, but sensitive to the TRPV1 antagonist capsazepine, the TRPV1-TRPV4 inhibitor ruthenium red, and the non-CCE (NCCE) inhibitors La3+ and Gd3+. Such pharmacological profile is supported by the presence of TRPV1, TRPV4, and TRPC6 mRNAs as well as TRPV1 and TRPC6 proteins in 16HBE14o(-) cells. Cannabinoid receptor antagonists increased virodhamine-induced Ca2+ entry. Virodhamine also enhanced arachidonic acid release, which was insensitive to cannabinoid receptor antagonism, but sensitive to the phospholipase A(2) inhibitor quinacrine, and to capsazepine. Arachidonic acid induced [Ca2+](i) increase similar to virodhamine. Collectively, these observations suggest that [Ca2+](i) alterations induced by Delta(9)-THC, CP55,940 and by low concentrations of virodhamine involve mobilization and subsequent CCE mechanisms, whereas such responses by high virodhamine concentrations involve NCCE pathways.

Topics: Arachidonic Acid; Arachidonic Acids; Bronchi; Calcium; Cannabinoid Receptor Modulators; Cannabinoids; Cell Line; Cyclohexanols; Dose-Response Relationship, Drug; Dronabinol; Epithelial Cells; Humans; RNA, Messenger; TRPC Cation Channels

The endocannabinoid system functions through two well characterized receptor systems, the CB1 and CB2 receptors. Work by a number of groups in recent years has provided evidence that the system is more complicated and additional receptor types should exist to explain ligand activity in a number of physiological processes.. Cells transfected with the human cDNA for GPR55 were tested for their ability to bind and to mediate GTPgammaS binding by cannabinoid ligands. Using an antibody and peptide blocking approach, the nature of the G-protein coupling was determined and further demonstrated by measuring activity of downstream signalling pathways.. We demonstrate that GPR55 binds to and is activated by the cannabinoid ligand CP55940. In addition endocannabinoids including anandamide and virodhamine activate GTPgammaS binding via GPR55 with nM potencies. Ligands such as cannabidiol and abnormal cannabidiol which exhibit no CB1 or CB2 activity and are believed to function at a novel cannabinoid receptor, also showed activity at GPR55. GPR55 couples to Galpha13 and can mediate activation of rhoA, cdc42 and rac1.. These data suggest that GPR55 is a novel cannabinoid receptor, and its ligand profile with respect to CB1 and CB2 described here will permit delineation of its physiological function(s).

Topics: Amino Acid Sequence; Animals; Arachidonic Acids; Binding Sites; Binding, Competitive; Cannabidiol; Cannabinoids; Cell Line; Cloning, Molecular; Cyclohexanols; Down-Regulation; Endocannabinoids; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Ligands; Mice; Molecular Sequence Data; Organ Specificity; Polymerase Chain Reaction; Polyunsaturated Alkamides; Rats; Receptors, Cannabinoid; Receptors, G-Protein-Coupled; RNA, Messenger; Signal Transduction; Structure-Activity Relationship

We investigated expression of cannabinoid receptors and the effects of the endogenous cannabinoid virodhamine and the synthetic agonist CP55,940 on cAMP accumulation and interleukin-8 (IL-8) release in human bronchial epithelial cells.. Human bronchial epithelial (16HBE14o(-)) cells were used. Total mRNA was isolated and cannabinoid receptor mRNAs were detected by RT-PCR. Expression of CB(1) and CB(2) receptor proteins was detected with Western blotting using receptor-specific antibodies. cAMP accumulation was measured by competitive radioligand binding assay. IL-8 release was measured by ELISA.. CB(1) and CB(2) receptor mRNAs and proteins were found. Both agonists concentration-dependently decreased forskolin-induced cAMP accumulation. This effect was inhibited by the CB(2) receptor antagonist SR144528, and was sensitive to Pertussis toxin (PTX), suggesting the involvement of CB(2) receptors and G(i/o)-proteins. Cell pretreatment with PTX unmasked a stimulatory component, which was blocked by the CB(1) receptor antagonist SR141716A. CB(2) receptor-mediated inhibition of cAMP production by virodhamine and CP55,940 was paralleled by inhibition of tumor necrosis factor-alpha (TNF-alpha) induced IL-8 release. This inhibition was insensitive to SR141716A. In the absence of agonist, SR144528 by itself reduced TNF-alpha induced IL-8 release.. Our results show for the first time that 16HBE14o(-) cells respond to virodhamine and CP55,940. CB(1) and CB(2) receptor subtypes mediated activation and inhibition of adenylyl cyclase, respectively. Stimulation of the dominant CB(2) receptor signalling pathway diminished cAMP accumulation and TNF-alpha-induced IL-8 release. These observations may imply that cannabinoids exert anti-inflammatory properties in airways by modulating cytokine release.

Topics: Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Analgesics; Arachidonic Acids; Blotting, Western; Bronchi; Camphanes; Cannabinoids; Cell Line; Colforsin; Cyclic AMP; Cyclohexanols; Dose-Response Relationship, Drug; Drug Antagonism; Epithelial Cells; Humans; Immunosuppressive Agents; Interleukin-8; Pertussis Toxin; Piperidines; Pyrazoles; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Reverse Transcriptase Polymerase Chain Reaction; Rimonabant; RNA, Messenger; Tumor Necrosis Factor-alpha

As persistent behavioural changes, such as increased anxiety-related behaviours, can be predicted based on the phenomenon of psychostimulant-induced neuronal plasticity, the time course (3-, 5- and 10-day time points) of the effects of both a single and repeated (daily for 7 days) i.p. administrations of cocaine (COC) and methamphetamine (MA) on anxiety-related behavioural symptoms in the elevated plus-maze test were examined in mice. Furthermore, based on the reported interactions between brain dopamine versus cannabinoid (CB) receptors and the contribution of CB receptors to the occurrence of persistent anxiety-related behavioural symptoms, the interactions of the agonist CP 55940 (CP) and the endogenous ligands anandamide (arachidonylethanolamide: AEA), 2-arachidonylglycerol (ARA), N-arachidonyldopamine (NADA), noladin ether (NL), and virodhamine (VA) with the COC- or MA-induced anxiety-related behaviours were also studied. In both an acute experiment using a single COC (30 mg/kg) or MA (4 mg/kg) dose and a chronic experiment using repeated COC (15 mg/kg) or MA (2 mg/kg) doses, anxiety-related behavioural symptoms were observed similarly at 3- and 5-day time points, but disappeared at the 10-day time point. Among the CB ligands, the agonists CP, AEA, ARA, NADA, and NL provided strong protective effects against each parameter at 3- and 5-day time points. Therefore, it was concluded that both COC and MA caused persistent anxiety-related behavioural symptoms following both a single and repeated treatments. Since these anxiogenic effects were attenuated by the endogenous CB agonists, the involvement of brain CB receptors was suspected.

Topics: Analysis of Variance; Animals; Anxiety; Arachidonic Acids; Behavior, Animal; Cannabinoids; Cocaine; Cyclohexanols; Dopamine; Dose-Response Relationship, Drug; Drug Interactions; Endocannabinoids; Glycerides; Injections, Intraperitoneal; Male; Maze Learning; Methamphetamine; Mice; Mice, Inbred ICR; Polyunsaturated Alkamides; Receptor, Cannabinoid, CB1; Time Factors