Evidence for a contribution of store-operated Ca2+ channels to NO-mediated endothelium-dependent relaxation of guinea-pig aorta in response to a Ca2+ ionophore, A23187

Abstract

A23187 (6S-[6α,8β,9β,11α]-5-(methylamino)-2-[[3,9,11-trimethyl-8-[1-methyl-2-oxo-2-(1H-pyrrol-2-yl)ethyl]-1,7-dioxaspiro[5.5]undec-2-yl]methyl]-4-benzoxazolecarboxylic acid, calcimycin), an antibiotic Ca²⁺ ionophore, produces an endothelium-dependent vascular relaxation. In the present study, pharmacological features were functionally characterized of endothelium-dependent relaxant response of guinea-pig aorta to A23187, especially focusing on the possible Ca²⁺ source and Ca²⁺ mobilization mechanisms in endothelial cells responsible for the vasorelaxant response to the Ca²⁺ ionophore. A23187-induced endothelium-dependent relaxation was suppressed profoundly by N ^G-nitro-L-arginine (L-NNA; 3×10^–4 M) or calmidazolium (3×10^–5 M), suggesting that nitric oxide (NO) produced by the enhanced activation of Ca²⁺/calmodulin-dependent endothelial NO synthase (eNOS) is largely responsible for the relaxant response of this artery to A23187. In the Ca²⁺-free solution without EGTA, NO-mediated endothelium-dependent relaxation induced by A23187 was almost abolished, which suggests that Ca²⁺ entry from extracellular space into endothelial cells plays the key role in the A23187-induced functional vasorelaxation. On the other hand, SK&F96365 (1-[β-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole; 5×10^–5 M) and Ni²⁺ (3×10^–4 M), both of which inhibit capacitative Ca²⁺ influx through store-operated Ca²⁺ channels (SOCCs), attenuated significantly NO-mediated endothelium-dependent relaxation by A23187. Furthermore, A23187-induced endothelium-dependent relaxation was suppressed more strongly than endothelium-independent relaxation induced by SIN-1 (3-morpholino-sydnonimine), an NO donor, when aortic preparation was preconstricted with high KCl instead of agonistic stimulation (prostaglandin F_{2 α}). These findings suggest that NO-mediated endothelium-dependent relaxant response of guinea-pig aorta to A23187 is preceded by the increase in endothelial cytosolic free Ca²⁺ concentration ([Ca²⁺]_cyt) due to the enhanced Ca²⁺ influx from extracellular space. In the enhanced Ca²⁺ entry leading to the stimulation of eNOS and NO-mediated functional relaxant response of guinea-pig aorta to A23187, activation of SOCCs but not the Ca²⁺ entry through plasma membrane Ca²⁺-specific routes made by A23187 seems to play the predominant role. It is most likely that A23187 acts primarily at the Ca²⁺ store sites in endothelial cells, which subsequently depletes stored Ca²⁺ to activate SOCCs via unidentified mechanisms.