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, 10µM anandamide did not significantly influence the resting membrane potential. In a Ca2+-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 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 (0.1-30µM) facilitated single BKCachannel 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 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 BKCachannel activity in inside-out patches. These results do not support the existence of endothelial cannabinoid receptor and indicate that anandamide acts as a direct BKCaopener. The action does not require cell integrity or integrins and is caused by direct modification of BKCachannel activity.
Circulatory Physiology Department, Bogomoletz Institute of Physiology NAS of Ukraine, Bogomoletz Str, 4, Kiev 01024, Ukraine; Institute of Molecular Biology and Biochemistry, Medical University of Graz, Harrachgasse 21, 8010 Graz, Austria.
Alexander I
AI
Bondarenko
Circulatory Physiology Department, Bogomoletz Institute of Physiology NAS of Ukraine, Bogomoletz Str, 4, Kiev 01024, Ukraine.
Olga
O
Panasiuk
Circulatory Physiology Department, Bogomoletz Institute of Physiology NAS of Ukraine, Bogomoletz Str, 4, Kiev 01024, Ukraine.
Iryna
I
Okhai
First Clinic of Internal Medicine, Department of Internal Medicine, University of Genoa, 6 viale Benedetto XV, 16132 Genoa, Italy; IRCCS AOU San Martino - IST, Genoa, 10 Largo Benzi, 16132 Genoa, Italy; Centre of Excellence for Biomedical Research (CEBR), University of Genoa, 9 viale Benedetto XV, 16132 Genoa, Italy.
Fabrizio
F
Montecucco
Division of Cardiology, Foundation for Medical Researches, Department of Internal Medicine, University of Geneva, Av. de la Roseraie 64, CH-1211 Geneva 4, Switzerland.
Karim J
KJ
Brandt
Division of Cardiology, Foundation for Medical Researches, Department of Internal Medicine, University of Geneva, Av. de la Roseraie 64, CH-1211 Geneva 4, Switzerland.
Francois
F
Mach
D001095:Arachidonic Acids / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism; D063388:Endocannabinoids / Q000494:pharmacology*; D061307:Human Umbilical Vein Endothelial Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects; D051036:Large-Conductance Calcium-Activated Potassium Channels / Q000378:metabolism*; D053284:Polyunsaturated Alkamides / Q000494:pharmacology*; D024642:Potassium Channels, Voltage-Gated / Q000378:metabolism*; D043882:Receptors, Cannabinoid / Q000378:metabolism
D001095:Arachidonic Acids; D063388:Endocannabinoids; D051036:Large-Conductance Calcium-Activated Potassium Channels; D053284:Polyunsaturated Alkamides; D024642:Potassium Channels, Voltage-Gated; D043882:Receptors, Cannabinoid; D002784:Cholesterol; D002118:Calcium; C078814:anandamide
Anandamide; BK(Ca) channels; Endothelial cannabinoid receptor; Integrins