Membrane hyperpolarization is not required. for sustained muscarinic agonist-induced increases in intracellular Ca2+ in arteriolar endothelial cells

Objective: Hyperpolarization modulates Ca2+ influx during agonist stimulation in many endothelial cells, but the effects of hyperpolarization on Ca2+ influx in freshly isolated arteriolar endothelial cells are unknown. Therefore, the purpose of the present study was to characterize agonist-induced Ca2+ transients in freshly isolated arteriolar endothelial cells and to test the hypothesis that membrane hyperpolarizaiton augments agonist-induced Ca2+ influx into these cells. Methods: Arterioles were removed from hamster cremaster muscles and arteriolar endothelial cells were enzymatically isolated. Endothelial cells were loaded with Fura 2-AM and the Fura 2 ratio measured photometrically as an index of intracellular Ca2+. The cells were then stimulated with the muscarinic, cholinergic agonist, methacholine, and the resulting Ca2+ transients were measured. Results: Methacholine (1 mu M) increased the endothelial cell Fura 2 ratio from a baseline of 0.81 +/- 0.02 to an initial peak of 1.17 +/- 0.05 (n = 17) followed by a sustained plateau of 1.12 +/- 0.07. The plateau phase of the Ca2+ transient was inhibited by removal of extracellular Ca2+ (n = 12, p < .05), or the nonselective cation channel blockers Gd3+ (30 mu M; n = 7, p < .05) or La3+ (50 mu M; n = 7, p < .05) without significant effect on the baseline or peak (p > .05). The initial peak of methacholine-induced Ca2+ transients was inhibited by the IP3-receptor antagonist xestospongin D (10 mu M, n = 5, p < .05). The methacholine-induced Ca2+ transients were accompanied by endothelial cell hyperpolarization of approximately 14-18 mV, as assessed by experiments using the potentiometric dye, di-8-ANEPPS as well as by patch-clamp experiments. However, inhibition of hyperpolarization by blockade of Ca2+-activated K+ channels with charybdotoxin (100 nM) and apamin (100 nM) (n = 5), or exposure of endothelial cells to 80 or 145 mM KCl (both n = 7) had no effect on the plateau phase of methacholine-induced Ca2+ transients (p > .05). Conclusions: Freshly isolated arteriolar endothelial cells display agonist-induced Ca2+ transients. for the muscarinic agonist, methacholine, these Ca2+ transients result from release of Ca2+ from intracellular stores through IP3 receptors, followed by sustained influx of extracellular Ca2+. While these changes in intracellular Ca2+ are associated with endothelial cell hyperpolarization, the methacholine-induced, sustained increase in intracellular Ca2+ appears to be independent from this change in membrane potential. These data suggest that arteriolar endothelial cells may possess a novel Ca2+ influx pathway, or that the relationship between intracellular Ca2+ and Ca2+ influx is more complex than that observed in other endothelial cells.