Direct activation of Ca(2+) 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 Ca(2+)-dependent K(+) channels expressed in endothelial cell line EA.hy926. Under resting conditions, 10 microM anandamide did not significantly influence the resting membrane potential. In a Ca(2+)-free solution the cells were depolarized by ~10 mV. Further administration of 10 microM anandamide hyperpolarized the cells by ~8 mV. In voltage-clamp mode, anandamide elicited the outwardly rectifying whole-cell current sensitive to paxilline but insensitive to GDPbetaS, a G-protein inhibitor. Administration of 70 microM Mn(2+), 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 microM) facilitated single BKCa channel activity in a concentration-dependent manner within a physiological Ca(2+) range and a wide range of voltages, mainly by reducing mean closed time. The effect is essentially eliminated following chelation of Ca(2+) from the cytosolic face and pre-exposure to cholesterol-reducing agent methyl-beta-cyclodextrin. O-1918 (3microM), 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.