A Role for N-Arachidonylethanolamine (Anandamide) as the Mediator of Sensory Nerve-Dependent Ca-Induced Relaxation

We tested the hypothesis that an endogenous cannabinoid (CB) receptor agonist, such as N-arachidonylethanolamine (anandamide), is the transmitter that mediates perivascular sensory nerve-dependent Ca-induced relaxation. Rat mesenteric branch arteries were studied using wire myography; relaxation was determined after inducing contraction with norepinephrine. Cumulative addition of Ca caused dosedependent relaxation (ED50 5 2.2 6 0.09 mM). The relaxation was inhibited by 10 mM TEA and 100 nM iberiotoxin, a blocker of large conductance Ca-activated K channels, but not by 5 mM glibenclamide, 1 mM 4-aminopyridine, or 30 nM apamin. Ca-induced relaxation was also blocked by the selective CB receptor antagonist SR141716A and was enhanced by pretreatment with 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (pefabloc; 30 mM), an inhibitor of anandamide metabolism. Anandamide also caused dose-dependent relaxation (ED50 5 .72 6 0.3 mM). The relaxation was not inhibited by endothelial denudation, 10 mM indomethacin, or 1 mM miconazole, but was blocked by 3 mM SR141716A, 10 mM TEA, precontraction with 100 mM K, and 100 nM iberiotoxin, and was enhanced by treatment with 30 mM pefabloc. Mesenteric branch arteries were 200-fold more sensitive to the relaxing action of anandamide than arachidonic acid (ED50 5 160 6 7 mM). These data show that: 1) Ca and anandamide cause hyperpolarization-mediated relaxation of mesenteric branch arteries, which is dependent on an iberiotoxin-sensitive Caactivated K channel, 2) relaxation induced by both Ca and anandamide is inhibited by CB receptor blockade, and 3) relaxation induced by anandamide is not dependent on its breakdown to arachidonic acid and subsequent metabolism. These findings support the hypothesis that anandamide, or a similar cannabinoid receptor agonist, mediates nerve-dependent Ca-induced relaxation in the rat. We recently demonstrated that perivascular sensory nerve endings express a membrane-spanning receptor for extracellular Ca (CaR; Bukoski et al., 1997; Mupanomunda et al., 1998) that is homologous to the CaR expressed in the parathyroid (Brown et al., 1993), kidney (Riccardi et al., 1995), and brain (Ruat et al., 1995). We also showed that stimulation of isolated arteries with physiologic concentrations of extracellular Ca causes vasodilation that is unaffected by endothelial denudation, but is sensory nerve-dependent and associated with the release of a vasodilator from the periadventitial surface of the artery (Bukoski et al., 1997; Mupanomunda et al., 1998). The relaxation is completely blocked by precontraction of the arterial segments with depolarizing concentrations of K, and during nonselective blockade of K channels with TEA (Bian et al., 1995; Bukoski et al., 1997). The relaxation is not, however, affected by pharmacologic blockade of receptors for known sensory peptide neurotransmitters, i.e., calcitonin gene-related peptide, Substance P, and neurokinin A. These observations, and more recent data obtained using in situ microdialysis-based measurements of interstitial Ca in the duodenal submucosa (Mupanomunda and Bukoski, 1998), have led us to propose that the periadventitial nerve CaR is activated by dynamic changes in interstitial Ca that occur during transcellular Ca movement, and that activation of the receptor is coupled to the release of a nonpeptide-hyperpolarizing dilator (Bukoski et