Selective Ligands and Cellular Effectors of a G Protein- Coupled Endothelial Cannabinoid Receptor L ´ ASZL ´ O OFFERT ´ ALER, FONG-MING MO, S ´ ANDOR B ´ ATKAI, JIE LIU, MALCOLM BEGG, RAJ K. RAZDAN, BILLY R. MARTIN, RICHARD D. BUKOSKI, and GEORGE KUNOS Laboratory of Physiologic Studies, National Institute on Alcoholism and Alcohol Abuse, National Institutes of Health, Bethesda, Maryland (L.O., S.B., J.L., M.B., G.K.); Organix, Inc., Woburn, Massachusetts (R.K.R.); Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, Virginia (B.R.M.); and Cardiovascular Disease Research Program, North Carolina Central University, Durham, North Carolina (R.D.B.) Received October 10, 2002; accepted December 3, 2002 This article is available online at http://molpharm.aspetjournals.org ABSTRACT 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  -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 M, but it does cause concentration-dependent (1–30 M) inhibition of the vasorelaxant effects of abn-cbd and anan- damide. In anesthetized mice, O-1918 dose-dependently inhib- its the hypotensive effect of abn-cbd but not the hypotensive effect of the CB 1 receptor agonist (-)-11-OH- 9 -tetrahydro- cannabinol 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 phosphatidylino- sitol 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. Endocannabinoids are recently identified lipid mediators that act as natural ligands for cannabinoid receptors and elicit biological effects similar to those of plant-derived can- nabinoids (Mechoulam et al., 1998). In addition to their well- known neurobehavioral effects, cannabinoids influence a number of physiological functions, including cardiovascular variables (Hillard, 2000; Kunos et al., 2000; Randall et al., 2002). It has long been known that  9 -tetrahydrocannabinol (THC), the main psychoactive ingredient of the marijuana plant, can cause long-lasting hypotension in rodents (Vollmer et al., 1974). The endocannabinoid anandamide also causes hypotension in anesthetized rats and mice, which is suscep- tible to inhibition by CB 1 receptor antagonists (Varga et al., 1995; Lake et al., 1997) and is absent in mice devoid of CB 1 receptors (Jarai et al., 1999; Ledent et al., 1999), clearly implicating CB 1 receptors. Additional findings ruled out a central mechanism for this effect (Varga et al., 1996). Can- nabinoids inhibit norepinephrine release via presynaptic CB 1 receptors on postganglionic sympathetic nerves (Ishac et al., 1996), which probably accounts for their bradycardic effect (Kunos et al., 2000; Wagner et al., 2001). However, cannabi- noids can decrease blood pressure to levels lower than that achieved by elimination of sympathetic tone (Lake et al., 1997), which points to a direct vasodilator mechanism. In- deed, anandamide and its metabolically stable analog (R)- methanandamide cause vasodilation in the coronary (Wag- ner et al., 2001; Ford et al., 2002) and cerebral vasculatures (Ellis et al., 1995; Gebremedhin et al., 1999; Wagner et al., 2001), as tested in anesthetized animals or in isolated or- gans. Surprisingly, anandamide-induced mesenteric vasodila- tion, although moderately sensitive to inhibition by SR141716A, could be dissociated from CB 1 receptors by its presence in mice deficient in CB 1 or in both CB 1 and CB 2 receptors (Jarai et al., 1999). Furthermore, THC or potent synthetic CB 1 receptor agonists do not cause mesenteric va- sodilation (Wagner et al., 1999). Anandamide binds to va- nilloid VR 1 receptors with micromolar affinity, and this in- L.O. and F.-M.M. contributed equally to this work. ABBREVIATIONS: THC,  9 -tetrahydrocannabinol; abn-cbd, (-)-4-(3–3,4-trans-p-menthadien-[1,8]-yl)-olivetol (abnormal cannabidiol); HUVEC, human umbilical vein endothelial cells; L-NAME, N  -nitro-L-arginine methyl ester; CGRP, calcitonin gene-related peptide; PI3, phosphatidylinositol 3; MAP, mitogen-activated protein; NO, nitric oxide; SR141716A, N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyra- zole-3-carboxamide; BK Ca , large-conductance calcium-activated potassium channel. 0026-895X/03/6303-699 –705 MOLECULAR PHARMACOLOGY Vol. 63, No. 3 U.S. Government work not protected by U.S. copyright 2209/1047483 Mol Pharmacol 63:699–705, 2003 Printed in U.S.A. 699 at ASPET Journals on September 5, 2016 molpharm.aspetjournals.org Downloaded from