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

The major constituent of the cannabis plant, Δ(9)-tetrahydrocannabinol, has stimulatory and depressant effects on cardiovascular functions. There is evidence from an in vivo study on the urethane-anaesthetized rat that part of the stimulatory effects is related to a tyramine-like activity. In the present study, we examined whether Δ(9)-tetrahydrocannabinol induces carrier-mediated noradrenaline release in vitro. The study was extended to another phytocannabinoid, cannabidiol, to the synthetic cannabinoids CP 55,940 and WIN 55,212-2 and to the endocannabinoids anandamide and 2-arachidonoyl glycerol. Tissue pieces of the renal cortex from the mouse and the rat were preincubated with (3)H-noradrenaline and superfused. The effect of the cannabinoids on basal (3)H-noradrenaline release was studied. Tyramine served as a positive control. In the mouse kidney, basal (3)H-noradrenaline release was increased by tyramine 0.1, 1 and 10 μM by 39, 91 and 212 %, respectively, and, in the rat kidney, (3)H-noradrenaline release was increased by tyramine 10 μM by 158 %. All effects were abolished by desipramine 1 μM, an inhibitor of the neuronal noradrenaline transporter. The cannabinoids at 0.1, 1 and 10 μM (CP 55,940 at 0.1, 1 and 3.2 μM) did not affect (3)H-noradrenaline release in the mouse kidney. The highest concentration of the cannabinoids (10 μM and in the case of CP 55,940 3.2 μM) also failed to affect (3)H-noradrenaline release in the rat kidney. In conclusion, the cannabinoids Δ(9)-tetrahydrocannabinol, cannabidiol, CP 55,940, WIN 55,212-2, anandamide and 2-arachidonoyl glycerol do not possess a tyramine-like effect on noradrenaline release.

Topics: Adrenergic Uptake Inhibitors; Animals; Arachidonic Acids; Benzoxazines; Cannabidiol; Cannabinoids; Cyclohexanols; Dose-Response Relationship, Drug; Dronabinol; Endocannabinoids; Glycerides; Kidney Cortex; Male; Mice; Mice, Inbred C57BL; Morpholines; Naphthalenes; Norepinephrine; Norepinephrine Plasma Membrane Transport Proteins; Polyunsaturated Alkamides; Rats; Rats, Sprague-Dawley; Time Factors; Tyramine

The inflammatory response plays an important role in the pathogenesis of many diseases in the central nervous system. Cannabinoids exhibit diverse pharmacological actions including anti-inflammatory activity. In this study, we tried to elucidate possible effects of cannabinoids on lipopolysaccharide (LPS)-induced expression of inflammatory cytokine mRNAs in rat cerebellar granule cells.. Inhibitory effects of cannabinoids on cytokine induction in cerebellar granule cells were determined by RT-PCR method.. In these cells, both mRNA and protein of cannabinoid receptor 1 (CB(1) ), but not CB(2) , were expressed. LPS (1 µg/ml) produced a marked increase in the induction of inflammatory cytokines, including interleukin-1β, interleukin-6 and tumour necrosis factor-α. CP55940, a synthetic cannabinoid analogue, concentration-dependently inhibited inflammatory cytokine expression induced by LPS. On the other hand, the endocannabinoids 2-arachidonoylglycerol and anandamide were not able to inhibit this inflammatory response. Notably, a CB(1) /CB(2) antagonist NESS0327 (3 µm) did not reverse the inhibition of cytokine mRNA expression induced by CP55940. GPR55, a putative novel cannabinoid receptor, mRNA was also expressed in cerebellar granule cells. Although it has been suggested that G(q) associates with GPR55, cannabinoids including CP55940 did not promote phosphoinositide hydrolysis and consequent elevation of intracellular Ca([2+]) concentration. Furthermore, a putative GPR55 antagonist, cannabidiol, also showed a similar inhibitory effect to that of CP55940.. These results suggest that the synthetic cannabinoid CP55940 negatively modulates cytokine mRNA expression in cerebellar granule cells by a CB and GPR55 receptor-independent mechanism.

Topics: Animals; Anti-Inflammatory Agents; Arachidonic Acids; Calcium; Cannabidiol; Cannabinoid Receptor Antagonists; Cannabinoid Receptor Modulators; Cannabinoids; Cerebellum; Cyclohexanols; Cytokines; Dose-Response Relationship, Drug; Endocannabinoids; Glycerides; Inflammation; Lipopolysaccharides; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Receptors, Cannabinoid; Receptors, G-Protein-Coupled; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

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

A nonpsychoactive constituent of the cannabis plant, cannabidiol has been demonstrated to have low affinity for both cannabinoid CB1 and CB2 receptors. We have shown previously that cannabidiol can enhance electrically evoked contractions of the mouse vas deferens, suggestive of inverse agonism. We have also shown that cannabidiol can antagonize cannabinoid receptor agonists in this tissue with a greater potency than we would expect from its poor affinity for cannabinoid receptors. This study aimed to investigate whether these properties of cannabidiol extend to CB1 receptors expressed in mouse brain and to human CB2 receptors that have been transfected into CHO cells.. The [35S]GTPS binding assay was used to determine both the efficacy of cannabidiol and the ability of cannabidiol to antagonize cannabinoid receptor agonists (CP55940 and R-(+)-WIN55212) at the mouse CB1 and the human CB2 receptor.. This paper reports firstly that cannabidiol displays inverse agonism at the human CB2 receptor. Secondly, we demonstrate that cannabidiol is a high potency antagonist of cannabinoid receptor agonists in mouse brain and in membranes from CHO cells transfected with human CB2 receptors.. This study has provided the first evidence that cannabidiol can display CB2 receptor inverse agonism, an action that appears to be responsible for its antagonism of CP55940 at the human CB2 receptor. The ability of cannabidiol to behave as a CB2 receptor inverse agonist may contribute to its documented anti-inflammatory properties.

Topics: Animals; Anti-Inflammatory Agents; Benzoxazines; Brain; Camphanes; Cannabidiol; Cannabinoid Receptor Agonists; Cannabinoid Receptor Antagonists; Cell Membrane; CHO Cells; Cricetinae; Cricetulus; Cyclohexanes; Cyclohexanols; Dose-Response Relationship, Drug; Guanosine 5'-O-(3-Thiotriphosphate); Humans; In Vitro Techniques; Mice; Mice, Inbred C57BL; Mice, Knockout; Morpholines; Naphthalenes; Phenols; Piperidines; Protein Binding; Pyrazoles; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Receptors, Cannabinoid; Rimonabant; Transfection

Cannabidiol (CBD), a prominent psychoinactive component of cannabis with negligible affinity for known cannabinoid receptors, exerts numerous pharmacological actions, including anti-inflammatory and immunosuppressive effects, the underlying mechanisms of which remain unclear. In the current study, we questioned whether CBD modulates activation of mast cells, key players in inflammation. By using the rat basophilic leukemia mast cell line (RBL-2H3), we demonstrate that CBD (3-10 muM) augments beta-hexosaminidase release, a marker of cell activation, from antigen-stimulated and unstimulated cells via a mechanism, which is not mediated by G(i)/G(o) protein-coupled receptors but rather is associated with a robust rise in intracellular calcium ([Ca(2+)](i)) levels sensitive to clotrimazole and nitrendipine (10-30 muM). This action, although mimicked by Delta(9)-tetrahydrocannabinol (THC), is opposite to that inhibitory, exerted by the synthetic cannabinoids WIN 55,212-2 and CP 55,940. Moreover, the vanilloid capsaicin, a full agonist of transient receptor potential channel VR1, did not affect [Ca(2+)](i)levels in the RBL-2H3 cells, thus excluding the involvement of this receptor in the CBD-mediated effects. Together, these results support existence of yet-to-be identified sites of interaction, i.e., receptors and/or ion channels associated with Ca(2+) influx of natural cannabinoids such as CBD and THC, the identification of which has the potential to provide for novel strategies and agents of therapeutic interest.

Topics: Animals; Benzoxazines; beta-N-Acetylhexosaminidases; Calcium; Cannabidiol; Cannabinoids; Capsaicin; Cell Degranulation; Cell Line; Cyclohexanols; Dronabinol; Intracellular Fluid; Mast Cells; Morpholines; Naphthalenes; Rats; TRPV Cation Channels

Previous experiments with the mouse vas deferens have shown that cannabidiol produces surmountable antagonism of cannabinoid CB(1) receptor agonists at concentrations well below those at which it binds to cannabinoid CB(1) receptors and antagonizes alpha(1)-adrenoceptor agonists insurmountably. It also enhances electrically evoked contractions of this tissue. We have now found that subtle changes in the structure of cannabidiol markedly influence its ability to produce each of these effects, suggesting the presence of specific pharmacological targets for this non-psychoactive cannabinoid. Our experiments were performed with cannabidiol, 6"-azidohex-2"-yne-cannabidiol, abnormal-cannabidiol and 2'-monomethoxy- and 2',6'-dimethoxy-cannabidiol. Of these, 6"-azidohex-2"-yne-cannabidiol was as potent as cannabidiol in producing surmountable antagonism of (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo-[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone (R-(+)-WIN55212) in vasa deferentia. However, it produced this antagonism with a potency that matched its cannabinoid CB(1) receptor affinity, suggesting that, unlike cannabidiol, it is a competitive cannabinoid CB(1) receptor antagonist. Moreover, since it did not enhance the amplitude of electrically evoked contractions, it may be a neutral cannabinoid CB(1) receptor antagonist.

Topics: Adenosine Triphosphate; Adrenergic alpha-Agonists; Animals; Binding, Competitive; Brain Chemistry; Cannabidiol; Cyclohexanols; Electric Stimulation; In Vitro Techniques; Male; Membranes; Mice; Phenylephrine; Receptor, Cannabinoid, CB1; Vas Deferens

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

Delta9-tetrahydrocannabinol (delta9-THC), cannabinol and cannabidiol are three important natural cannabinoids from the Marijuana plant (Cannabis sativa). Using [35S]GTP-gamma-S binding on rat cerebellar homogenate as an index of cannabinoid receptor activation we show that: delta9-THC does not induce the maximal effect obtained by classical cannabinoid receptor agonists such as CP55940. Moreover at high concentration delta9-THC exhibits antagonist properties. Cannabinol is a weak agonist on rat cerebellar cannabinoid receptors and cannabidiol behaves as an antagonist acting in the micromolar range.

Topics: Analgesics; Animals; Binding, Competitive; Brain; Cannabidiol; Cell Membrane; Cerebellar Cortex; Cyclohexanols; Dronabinol; Guanosine Triphosphate; Male; Piperidines; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptors, Cannabinoid; Receptors, Drug; Rimonabant

One aspect of cannabinoid structure-activity relationships (SARs) that has not been thoroughly investigated is the aromatic (A) ring. Although halogenation of the side chain enhances potency, our recent observation that iodination of the A ring also enhanced activity was surprising. The purpose of this investigation was to establish the steric and electrostatic requirements at these sites of the cannabinoid molecule via molecular modeling, while determining pharmacological activity. Molecular modeling was performed using the Tripos molecular mechanics force field and the semiempirical quantum mechanical package AM1. The Ki values for novel cannabinoids were determined in a [3H]CP-55,940 binding assay and ED50 values generated from four different evaluations in a mouse model. The present studies underscore the increase in potency produced by a dimethylheptyl (DMH) side chain. Trifluoro substitutions on the pentyl side chain, or bromination of the DMH side chain, had little effect on the pharmacological activity. Any substitution at the C4 position of the aryl ring resulted in a loss of activity, which appears to be due to steric hindrances. Nitro, but not iodo, substitution at the C2 position essentially produces an inactive analog, and the drastic alteration of the electrostatic potential appears to be responsible. The altered pharmacological profile of the 2-iodo analog seems to be related to an alteration in the highest occupied molecular orbital because there is no alteration in the electron density map compared to delta 8-tetrahydrocannibinol.

Topics: Analgesics; Animals; Body Temperature; Cannabidiol; Cannabinoids; Cyclohexanols; Dronabinol; Male; Mice; Mice, Inbred ICR; Models, Molecular; Molecular Conformation; Motor Activity; Receptors, Cannabinoid; Receptors, Drug; Structure-Activity Relationship