Membrane Hyperpolarization Is Not Required for Sustained Muscarinic Agonist-Induced Increases in Intracellular Ca²⁺ in Arteriolar Endothelial Cells

Abstract

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 hyperpolarization 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 μ 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 μ M; n = 7, p < .05) or La³⁺ (50 μ 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 μ 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.

Keywords:

• arteriole
• cremaster muscle
• endothelial cells
• Fura 2
• intracellular Ca²⁺
• membrane potential
• microcirculation
• muscarinic receptors

The authors are grateful for the superb technical assistance of Sue Kovats. Studies were supported by Public Health Service Grant HL 32469 to Dr. Jackson and American Heart Association Postdoctoral Fellowship 0120607Z to Dr. Cohen.