Background and PurposeN-arachidonoyl glycine (NAGly) is a lipoamino acid with vasorelaxant properties. We aimed to explore the mechanisms of NAGly's action on unstimulated and agonist-stimulated endothelial cells.Experimental ApproachThe effects of NAGly on endothelial electrical signalling were studied in combination with vascular reactivity.Key ResultsIn EA.hy926 cells, the sustained hyperpolarization to histamine was inhibited by the non-selective Na+/Ca2+ exchanger (NCX) inhibitor bepridil and by an inhibitor of reversed mode NCX, KB-R7943. In cells dialysed with Cs+-based Na+-containing solution, the outwardly rectifying current with typical characteristics of NCX was augmented following histamine exposure, further increased upon external Na+ withdrawal and inhibited by bepridil. NAGly (0.3–30 μM) suppressed NCX currents in a URB597- and guanosine 5′-O-(2-thiodiphosphate) (GDPβS)-insensitive manner, [Ca2+]i elevation evoked by Na+ removal and the hyperpolarization to histamine. In rat aorta, NAGly opposed the endothelial hyperpolarization and relaxation response to ACh. In unstimulated EA.hy926 cells, NAGly potentiated the whole-cell current attributable to large-conductance Ca2+-activated K+ (BKCa) channels in a GDPβS-insensitive, paxilline-sensitive manner and produced a sustained hyperpolarization. In cell-free inside-out patches, NAGly stimulated single BKCa channel activity.Conclusion and ImplicationsOur data showed that NCX is a Ca2+ entry pathway in endothelial cells and that NAGly is a potent G-protein-independent modulator of endothelial electrical signalling and has a dual effect on endothelial electrical responses. In agonist pre-stimulated cells, NAGly opposes hyperpolarization and relaxation via inhibition of NCX-mediated Ca2+ entry, while in unstimulated cells, it promotes hyperpolarization via receptor-independent activation of BKCa channels.
Endothelium-dependent component of cannabinoid-induced vasodilation has been postulated to require G-protein-coupled non-CB/CB endothelial cannabinoid (eCB) receptor. GPR18 was proposed as a candidate for eCBR. To address the hypothesis that the effects attributed to eCBR are mediated by G-protein-coupled receptor (GPCR)-independent targets, we studied the electrical responses in endothelial cells, focusing on BK channels. In patches excised from endothelial-derived EA.hy926 cells, N-arachidonoyl glycine (NAGly) and abnormal cannabidiol (abn-cbd), prototypical agonists for eCB receptor, stimulate single BK activity in a concentration- and Ca-dependent manner. The postulated eCB receptor inhibitors rimonabant and AM251 were found to inhibit basal and stimulated by NAGly- and abn-cbd BK activity in cell-free patches. In isolated mice aortas, abn-cbd and NAGly produced endothelial cell hyperpolarization that was sensitive to paxilline, a selective BK inhibitor, but not to GPR18 antibody, and mimicked by NS1619, a direct BK opener. In excised patches from mice aortic endothelium, single channel activity with characteristics similar to BK was established by the addition of abn-cbd and NAGly. We conclude that the two cannabinoids abn-cbd and NAGly initiate a GPR18-independent activation of BK channels in mice aortic endothelial cells that might contribute to vasodilation to cannabinoids.
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.
Lysophosphatidylinositol (LPI) and lysophosphatidylcholine (LPC) are lipid signaling molecules that induce endothelium-dependent vasodilation. In addition, LPC suppresses acetylcholine (Ach)-induced responses. We aimed to determine the influence of LPC and LPI on hyperpolarizing responses in vitro and in situ endothelial cells (EC) and identify the underlying mechanisms. Using patch-clamp method, we show that LPI and LPC inhibit EC hyperpolarization to histamine and suppress Na/Ca exchanged (NCX) currents in a concentration-dependent manner. The inhibition is non-mode-specific and unaffected by intracellular GDPβS infusion and tempol, a superoxide dismutase mimetic. In excised mouse aorta, LPI strongly inhibits the sustained and the peak endothelial hyperpolarization induced by Ach, but not by SKA-31, an opener of Ca-dependent K channels of intermediate and small conductance. The hyperpolarizing responses to consecutive histamine applications are strongly reduced by NCX inhibition. In a Ca-re-addition protocol, bepridil, a NCX inhibitor, and KB-R7943, a blocker of reversed NCX, inhibit the hyperpolarizing responses to Ca-re-addition following Ca stores depletion. These finding indicate that LPC and LPI inhibit endothelial hyperpolarization to Ach and histamine independently of G-protein coupled receptors and superoxide anions. Reversed NCX is critical for ER Ca refilling in EC. The inhibition of NCX by LPI and LPC underlies diminished endothelium-dependent responses and endothelial dysfunction accompanied by increased levels of these lipids in the blood.
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