Cannabinoid and lipid-mediated vasorelaxation in retinal microvasculature.

The endocannabinoid system plays a role in regulation of vasoactivity in the peripheral vasculature; however, little is known about its role in regulation of the CNS microvasculature. This study investigated the pharmacology of cannabinoids and cannabimimetic lipids in the retinal microvasculature, a CNS vascular bed that is autoregulated. Vessel diameter (edge detector) and calcium transients (fura-2) were recorded from segments of retinal microvasculature isolated from adult, male Fischer 344 rats. Results showed that abnormal cannabidiol (Abn-CBD), an agonist at the putative endothelial cannabinoid receptor, CBe, inhibited endothelin 1 (ET-1) induced vasoconstriction in retinal arterioles. These actions of Abn-CBD were independent of CB1/CB2 receptors and were not mediated by agonists for GPR55 or affected by nitric oxide synthase (NOS) inhibition. However, the vasorelaxant effects of Abn-CBD were abolished when the endothelium was removed and were inhibited by the small Ca(2+)-sensitive K channel (SKCa) blocker, apamin. The effects of the endogenous endocannabinoid metabolite, N-arachidonyl glycine (NAGly), a putative agonist for GPR18, were virtually identical to those of Abn-CBD. GPR18 mRNA and protein were present in the retina, and immunohistochemistry demonstrated that GPR18 was localized to the endothelium of retinal vessels. These findings demonstrate that Abn-CBD and NAGly inhibit ET-1 induced vasoconstriction in retinal arterioles by an endothelium-dependent signaling mechanism that involves SKCa channels. The endothelial localization of GPR18 suggests that GPR18 could contribute to cannabinoid and lipid-mediated retinal vasoactivity.

Indirect sympatholytic actions at ß-adrenoceptors account for the ocular hypotensive actions of cannabinoid receptor agonists.

Intraocular pressure (IOP) is the primary risk factor for glaucoma, a blinding eye disease. Cannabinoid agonists have long been known to decrease IOP, suggesting they may be useful in glaucoma treatment. However, the specific mechanism by which cannabinoids generate this ocular hypotensive effect remains unknown. The current evidence suggests the cannabinoids reduce IOP through actions at cannabinoid 1 (CB(1)) receptors within the eye, and adrenergic receptors (ARs) may also contribute to this action of cannabinoids. Considering this, the present study aimed to elucidate the mechanism behind the ocular hypotensive properties of cannabinoids through the use of mice genetically lacking either cannabinoid receptors or ßARs. Cannabinoid agonists, ßAR antagonists, and ßAR agonists decreased IOP in wild-type mice and CB(2)(-/-) mice. In contrast, none of these compounds were found to reduce IOP in ßAR(-/-) or CB(1)(-/-) mice. Desensitization of the ßARs and depletion of catecholamines in wild-type mice also eliminated the ability of the cannabinoid agonist (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate (WIN 55,212-2) to reduce IOP, strongly implicating a role for both ßARs and catecholamines in the ocular hypotensive properties of cannabinoids. Finally, CB(1) receptors were shown to colocalize with tyrosine hydroxylase, a marker for adrenergic neurons. Taken together, these findings suggest that ßARs are required for the ocular hypotensive properties of cannabinoids, and cannabinoids reduce IOP by acting as indirect sympatholytics and inhibiting norepinephrine release within the eye.

CP47,497-C8 and JWH073, commonly found in 'Spice' herbal blends, are potent and efficacious CB(1) cannabinoid receptor agonists.

'Spice' is an herbal blend that has been reported to produce cannabis-like effects when smoked and is marketed as an alternative to marijuana. Synthetic additives have been identified in numerous 'Spice' preparations from different sources. Common among many of the preparations were the compounds JWH018 and a dimethyloctyl variant of CP47,497 (CP47,497-C8) and, more recently JWH073. The synaptic effects of each of these compounds were uncharacterized. We previously reported that JWH018 is a potent and efficacious CB(1) cannabinoid receptor agonist. In this study we have examined the abilities of CP47,497-C8 and JWH073 to inhibit neurotransmission in cultured autaptic hippocampal neurons. Each inhibited EPSCs with an efficacy and potency similar to JWH018. We also analyzed these compounds' effects on promoting internalization of CB(1) receptors in HEK293 cells stably expressing CB(1) receptors. Similar to our neurotransmission data, CP47,497-C8 internalized CB(1) in a fashion indistinguishable from JWH018. However, JWH073 was less potent and produced slower internalization than JWH018 and CP47,497-C8. It appears that 'Spice' contains a number of cannabinoid receptor agonists that activate CB(1) receptors to inhibit synaptic transmission with similar potencies and efficacies. It is highly probable that the cannabis-like effects of 'Spice' are due to the presence of these and analogous synthetic additives acting on CB(1) receptors.

JWH018, a common constituent of 'Spice' herbal blends, is a potent and efficacious cannabinoid CB receptor agonist.

BACKGROUND AND PURPOSE: 'Spice' is an herbal blend primarily marketed in Europe as a mild hallucinogen with prominent cannabis-like effects and as a legal alternative to cannabis. However, a recent report identified a number of synthetic additives in samples of 'Spice'. One of these, the indole derivative JWH018, is a ligand for the cannabinoid receptor 1 (CB(1)) cannabinoid receptor and inhibits cAMP production in CB(1) receptor-expressing CHO cells. Other effects of JWH018 on CB(1) receptor-mediated signalling are not known, particularly in neurons. Here we have evaluated the signalling pathways activated by JWH018 at CB(1) receptors. EXPERIMENTAL APPROACH: We investigated the effects of JWH018 on neurotransmission in cultured autaptic hippocampal neurons. We further analysed its activation of ERK1/2 mitogen activated protein kinase (MAPK) and internalization of CB(1) receptors in HEK293 cells stably expressing this receptor. KEY RESULTS: In cultured autaptic hippocampal neurons, JWH018 potently inhibited excitatory postsynaptic currents (IC(50)= 14.9 nM) in a concentration- and CB(1) receptor-dependent manner. Furthermore, it increased ERK1/2 MAPK phosphorylation (EC(50)= 4.4 nM). We also found that JWH018 potently induced rapid and robust CB(1) receptor internalization (EC(50)= 2.8 nM; t(1/2)= 17.3 min). CONCLUSIONS AND IMPLICATIONS: JWH018, a prominent component of several herbal preparations marketed for their psychoactivity, is a potent and effective CB(1) receptor agonist that activates multiple CB(1) receptor signalling pathways. Thus, it is likely that the subjective effects of 'Spice' are due to activation of cannabinoid CB(1) receptors by JWH018, added to this herbal preparation.

Incensole acetate, an incense component, elicits psychoactivity by activating TRPV3 channels in the brain.

Burning of Boswellia resin as incense has been part of religious and cultural ceremonies for millennia and is believed to contribute to the spiritual exaltation associated with such events. Transient receptor potential vanilloid (TRPV) 3 is an ion channel implicated in the perception of warmth in the skin. TRPV3 mRNA has also been found in neurons throughout the brain; however, the role of TRPV3 channels there remains unknown. Here we show that incensole acetate (IA), a Boswellia resin constituent, is a potent TRPV3 agonist that causes anxiolytic-like and antidepressive-like behavioral effects in wild-type (WT) mice with concomitant changes in c-Fos activation in the brain. These behavioral effects were not noted in TRPV3(-/-) mice, suggesting that they are mediated via TRPV3 channels. IA activated TRPV3 channels stably expressed in HEK293 cells and in keratinocytes from TRPV3(+/+) mice. It had no effect on keratinocytes from TRPV3(-/-) mice and showed modest or no effect on TRPV1, TRPV2, and TRPV4, as well as on 24 other receptors, ion channels, and transport proteins. Our results imply that TRPV3 channels in the brain may play a role in emotional regulation. Furthermore, the biochemical and pharmacological effects of IA may provide a biological basis for deeply rooted cultural and religious traditions.