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

• Published in: European journal of pharmacology 2017-06, Vol.805, p.14
• Main Authors: Bondarenko, Alexander I, Panasiuk, Olga, Okhai, Iryna, Montecucco, Fabrizio, Brandt, Karim J, Mach, Francois
• Format: Article
• Language: English
• Subjects: Arachidonic Acids - pharmacology; Calcium - metabolism; Cell Membrane - drug effects; Cell Membrane - metabolism; Cholesterol - metabolism; Endocannabinoids - pharmacology; Human Umbilical Vein Endothelial Cells - cytology; Human Umbilical Vein Endothelial Cells - drug effects; Human Umbilical Vein Endothelial Cells - metabolism; Humans; Ion Channel Gating - drug effects; Large-Conductance Calcium-Activated Potassium Channels - metabolism; Polyunsaturated Alkamides - pharmacology; Potassium Channels, Voltage-Gated - metabolism; Receptors, Cannabinoid - metabolism
• ISSN: 1879-0712

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 -dependent K channels expressed in endothelial cell line EA.hy926. Under resting conditions, 10µM anandamide did not significantly influence the resting membrane potential. In a Ca -free solution the cells were depolarized by ~10mV. Further administration of 10µM anandamide hyperpolarized the cells by ~8mV. In voltage-clamp mode, anandamide elicited the outwardly rectifying whole-cell current sensitive to paxilline but insensitive to GDPβS, a G-protein inhibitor. Administration of 70µM Mn , 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 (0.1-30µM) facilitated single BK channel activity in a concentration-dependent manner within a physiological Ca range and a wide range of voltages, mainly by reducing mean closed time. The effect is essentially eliminated following chelation of Ca from the cytosolic face and pre-exposure to cholesterol-reducing agent methyl-β-cyclodextrin. O-1918 (3µM), a cannabidiol analog used as a selective antagonist of endothelial anandamide receptor, reduced BK 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 BK opener. The action does not require cell integrity or integrins and is caused by direct modification of BK channel activity.