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The perivascular sensory nerve (PvN) Ca(2+)-sensing receptor (CaR) is implicated in Ca(2+)-induced relaxation of isolated, phenylephrine (PE)-contracted mesenteric arteries, which involves the vascular endogenous cannabinoid system. We determined the effect of inhibition of diacylglycerol (DAG) lipase (DAGL), phospholipase A(2) (PLA(2)), and cytochrome P-450 (CYP) on Ca(2+)-induced relaxation of PE-contracted rat mesenteric arteries. Our findings indicate that Ca(2+)-induced vasorelaxation is not dependent on the endothelium. The DAGL inhibitor RHC 802675 (1 microM) and the CYP and PLA(2) inhibitors quinacrine (5 microM) (EC(50): RHC 802675 2.8 +/- 0.4 mM vs. control 1.4 +/- 0.3 mM; quinacrine 4.8 +/- 0.4 mM vs. control 2.0 +/- 0.3 mM; n = 5) and arachidonyltrifluoromethyl ketone (AACOCF(3), 1 microM) reduced Ca(2+)-induced relaxation of mesenteric arteries. Synthetic 2-arachidonoylglycerol (2-AG) and glycerated epoxyeicosatrienoic acids (GEETs) induced concentration-dependent relaxation of isolated arteries. 2-AG relaxations were blocked by iberiotoxin (IBTX) (EC(50): control 0.96 +/- 0.14 nM, IBTX 1.3 +/- 0.5 microM) and miconazole (48 +/- 3%), and 11,12-GEET responses were blocked by IBTX (EC(50): control 55 +/- 9 nM, IBTX 690 +/- 96 nM) and SR-141716A. The data suggest that activation of the CaR in the PvN network by Ca(2+) leads to synthesis and/or release of metabolites of the CYP epoxygenase pathway and metabolism of DAG to 2-AG and subsequently to GEETs. The findings indicate a role for 2-AG and its metabolites in Ca(2+)-induced relaxation of resistance arteries; therefore this receptor may be a potential target for the development of new vasodilator compounds for antihypertensive therapy.

The cannabinoid analog abnormal cannabidiol [abn-cbd; (-)-4-(3-3,4-trans-p-menthadien-[1,8]-yl)-olivetol] does not bind to CB(1) or CB(2) receptors, yet it acts as a full agonist in relaxing rat isolated mesenteric artery segments. Vasorelaxation by abn-cbd is endothelium-dependent, pertussis toxin-sensitive, and is inhibited by the BK(Ca) channel inhibitor charybdotoxin, but not by the nitric-oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester or by the vanilloid VR1 receptor antagonist capsazepine. The cannabidiol analog O-1918 does not bind to CB(1) or CB(2) receptors and does not cause vasorelaxation at concentrations up to 30 microM, but it does cause concentration-dependent (1-30 microM) inhibition of the vasorelaxant effects of abn-cbd and anandamide. In anesthetized mice, O-1918 dose-dependently inhibits the hypotensive effect of abn-cbd but not the hypotensive effect of the CB(1) receptor agonist (-)-11-OH-Delta(9)-tetrahydrocannabinol dimethylheptyl. In human umbilical vein endothelial cells, abn-cbd induces phosphorylation of p42/44 mitogen-activated protein kinase and protein kinase B/Akt, which is inhibited by O-1918, by pertussis toxin or by phosphatidylinositol 3 (PI3) kinase inhibitors. These findings indicate that abn-cbd is a selective agonist and that O-1918 is a selective, silent antagonist of an endothelial "anandamide receptor", which is distinct from CB(1) or CB(2) receptors and is coupled through G(i)/G(o) to the PI3 kinase/Akt signaling pathway.

Background: Type II diabetes in humans is associated with pathology of both the cardiovascular and peripheral sensory nervous systems. Because abnormal vasodilator responses have been reported in animals of type II diabetes and perivascular sensory nerves are a source of vasodilator substances, we tested the hypothesis that sensory nerve-dependent relaxation is abnormal in arteries of the Zucker diabetic fatty (ZDF) rat model of type II diabetes., Methods: The ZDF rats and genetic controls were studied at 26 weeks of age. Tail-cuff systolic blood pressure (BP) was measured, serum was obtained for chemical determinations, and mesenteric branch arteries were isolated for wire myograph analysis and confocal-based measurement of calcitonin gene-related peptide (CGRP) positive nerve density., Results: No differences in BP were detected. Serum glucose, triglycerides, and cholesterol were significantly elevated in ZDF. Sensory nerve-dependent vasodilation was assessed by measuring relaxation of phenylephrine preconstricted arterial segments to cumulative addition of divalent calcium ion (Ca2+) or capsaicin. Neither Ca(2+)-nor capsaicin-induced relaxation were different in ZDF versus control (maximal ZDF response to Ca2+ = 64% +/- 2% v 59% +/- 4%; ED50 for Ca2+ = 3.7 +/- 0.5 mmol/L v 3.2 +/- 0.5 mmol/L; n = 5, P = not significant [NS]; maximal ZDF response to capsaicin = 68% +/- 9% v 74% +/- 4%; ZDF ED50 = 3.8 +/- 0.5 nmol/L v 9.8 +/- 7 nmol/L; n = 5, P = NS). In contrast, the maximal relaxation response to acetylcholine was impaired in ZDF (maximal ZDF response = 83% +/- 5% v 94% +/- 2%, n = 4, P = .039; ED50 for acetylcholine = 8.1 +/- 2.9 nmol/L for ZDF v 33.5 +/- 18.2; n = 4 per group, P = .086). The CGRP positive nerve density was not different between groups., Conclusions: Blood pressure, perivascular sensory nerve CGRP content, and dilator function is normal in the ZDF model of type II diabetes, whereas endothelium-dependent relaxation is impaired.

Mesenteric branch arteries isolated from cannabinoid type 1 receptor knockout (CB(1)(-/-)) mice, their wild-type littermates (CB(1)(+/+) mice), and C57BL/J wild-type mice were studied to test the hypothesis that murine arteries undergo high sensitivity Ca(2+)-induced relaxation that is CB(1) receptor dependent. Confocal microscope analysis of mesenteric branch arteries from wild-type mice showed the presence of Ca(2+) receptor-positive periadventitial nerves. Arterial segments of C57 control mice mounted on wire myographs contracted in response to 5 micromol/L norepinephrine and responded to the cumulative addition of extracellular Ca(2+) with a concentration-dependent relaxation that reached a maximum of 72.0 +/- 6.3% of the prerelaxation tone and had an EC(50) for Ca(2+) of 2.90 +/- 0.54 mmol/L. The relaxation was antagonized by precontraction in buffer containing 100 mmol/L K(+) and by pretreatment with 10 mmol/L tetraethylammonium. Arteries from CB(1)(-/-) and CB(1)(+/+) mice also relaxed in response to extracellular Ca(2+) with no differences being detected between the knockout and their littermate controls. SR141716A, a selective CB(1) antagonist, caused concentration-dependent inhibition of Ca(2+)-induced relaxation in both the knockout and wild-type strains (60% inhibition at 1 micromol/L). O-1918, a cannabidiol analog, had a similar blocking effect in arteries of both wild-type and CB(1)(-/-) mice at 10 micromol/L. In contrast, 1 micromol/L SR144538, a cannabinoid type 2 receptor antagonist, or 50 micromol/L 18alpha-glycyrrhetinic acid, a gap junction blocker, were without effect. SR141716A (1 to 30 micromol/L) was also assessed for nonspecific actions on whole-cell K(+) currents in isolated vascular smooth muscle cells. SR141716A inhibited macroscopic K(+) currents at concentrations higher than those required to inhibit Ca(2+)-induced relaxation, and appeared to have little effect on currents through large conductance Ca(2+)-activated K(+) channels. These data indicate that arteries of the mouse relax in response to cumulative addition of extracellular Ca(2+) in a hyperpolarization-dependent manner and rule out a role for CB(1) or CB(2) receptors in this effect. The possible role of a nonclassical cannabinoid receptor is discussed.