Direct activation of Ca2+ and voltage-gated potassium channels of large conductance by anandamide in endothelial cells does not support the presence of endothelial atypical cannabinoid receptor

Endocannabinoid anandamide induces endothelium-dependent relaxation commonly attributed to stimulation of the G-protein coupled endothelial anandamide receptor. The study addressed the receptor-independent effect of anandamide on large conductance Ca2+-dependent K+ channels expressed in endothelial cell line EA.hy926. Under resting conditions, 10 mu M anandamide did not significantly influence the resting membrane potential. In a Ca2+-free solution the cells were depolarized by similar to 10 mV. Further administration of 10 mu M anandamide hyperpolarized the cells by similar to 8 mV. In voltage-clamp mode, anandamide elicited the outwardly rectifying whole cell current sensitive to paxilline but insensitive to GDP beta S, a G-protein inhibitor. Administration of 70 mu M Mn2+, an agent used to promote integrin clustering, reversibly stimulated whole-cell current, but failed to further facilitate the anandamide-stimulated current. In an inside-out configuration, anandamide (0.1-30 mu M) facilitated single BKca channel activity in a concentration-dependent manner within a physiological Ca2+ range and a wide range of voltages, mainly by reducing mean closed time. The effect is essentially eliminated following chelation of Ca2+ from the cytosolic face and pm-exposure to cholesterol-reducing agent methyl-P-cyclodextrin. 0-1918 (3 mu M), a cannabidiol analog used as a selective antagonist of endothelial anandamide receptor, reduced BKca, channel activity in inside-out patches. These results do not support the existence of endothelial cannabinoid receptor and indicate that anandamide acts as a direct BKca opener. The action does not require cell integrity or integrins and is caused by direct modification of BKca channel activity.