N-arachidonoyl glycine suppresses Na(+)/Ca(2+) exchanger-mediated Ca(2+) entry into endothelial cells and activates BK(Ca) channels independently of GPCRs

BACKGROUND AND PURPOSE: N-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 APPROACH: The effects of NAGly on endothelial electrical signalling were studied in combination with vascular reactivity. KEY RESULTS: In EA.hy926 cells, the sustained hyperpolarization to histamine was inhibited by the non-selective Na(+)/Ca(2+) 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, [Ca(2+)](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 Ca(2+)-activated K(+) (BK(Ca)) channels in a GDPβS-insensitive, paxilline-sensitive manner and produced a sustained hyperpolarization. In cell-free inside-out patches, NAGly stimulated single BK(Ca) channel activity. CONCLUSION AND IMPLICATIONS: Our data showed that NCX is a Ca(2+) 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 Ca(2+) entry, while in unstimulated cells, it promotes hyperpolarization via receptor-independent activation of BK(Ca) channels.