{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,23]],"date-time":"2026-10-23T06:47:32Z","timestamp":1792738052433,"version":"4.5.0"},"reference-count":64,"publisher":"Wiley","issue":"2","license":[{"start":{"date-parts":[[2010,1,26]],"date-time":"2010-01-26T00:00:00Z","timestamp":1264464000000},"content-version":"vor","delay-in-days":1792,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Microcirculation"],"published-print":{"date-parts":[[2005,3]]},"abstract":"<jats:title>ABSTRACT<\/jats:title><jats:p><jats:bold>Objective:<\/jats:bold> Hyperpolarization modulates Ca<jats:sup>2+<\/jats:sup> influx during agonist stimulation in many endothelial cells, but the effects of hyperpolarization on Ca<jats:sup>2+<\/jats:sup> influx in freshly isolated arteriolar endothelial cells are unknown. Therefore, the purpose of the present study was to characterize agonist\u2010induced Ca<jats:sup>2+<\/jats:sup> transients in freshly isolated arteriolar endothelial cells and to test the hypothesis that membrane hyperpolarization augments agonist\u2010induced Ca<jats:sup>2+<\/jats:sup> influx into these cells.<\/jats:p><jats:p><jats:bold>Methods:<\/jats:bold> Arterioles were removed from hamster cremaster muscles and arteriolar endothelial cells were enzymatically isolated. Endothelial cells were loaded with Fura 2\u2010AM and the Fura 2 ratio measured photometrically as an index of intracellular Ca<jats:sup>2+<\/jats:sup>. The cells were then stimulated with the muscarinic, cholinergic agonist, methacholine, and the resulting Ca<jats:sup>2+<\/jats:sup> transients were measured.<\/jats:p><jats:p><jats:bold>Results:<\/jats:bold> Methacholine (1 \u03bc M) increased the endothelial cell Fura 2 ratio from a baseline of 0.81 \u00b1 0.02 to an initial peak of 1.17 \u00b1 0.05 (<jats:italic>n<\/jats:italic> = 17) followed by a sustained plateau of 1.12 \u00b1 0.07. The plateau phase of the Ca<jats:sup>2+<\/jats:sup> transient was inhibited by removal of extracellular Ca<jats:sup>2+<\/jats:sup> (<jats:italic>n<\/jats:italic> = 12, <jats:italic>p<\/jats:italic> &lt; .05), or the nonselective cation channel blockers Gd<jats:sup>3+<\/jats:sup> (30 \u03bc M; <jats:italic>n<\/jats:italic> = 7, <jats:italic>p<\/jats:italic> &lt; .05) or La<jats:sup>3+<\/jats:sup> (50 \u03bc M; <jats:italic>n<\/jats:italic> = 7, <jats:italic>p<\/jats:italic> &lt; .05) without significant effect on the baseline or peak (<jats:italic>p<\/jats:italic> &gt; .05). The initial peak of methacholine\u2010induced Ca<jats:sup>2+<\/jats:sup> transients was inhibited by the IP<jats:sub>3<\/jats:sub>\u2010receptor antagonist xestospongin D (10 \u03bc M, <jats:italic>n<\/jats:italic> = 5, <jats:italic>p<\/jats:italic> &lt; .05). The methacholine\u2010induced Ca<jats:sup>2+<\/jats:sup> transients were accompanied by endothelial cell hyperpolarization of approximately 14\u201318 mV, as assessed by experiments using the potentiometric dye, di\u20108\u2010ANEPPS as well as by patch\u2010clamp experiments. However, inhibition of hyperpolarization by blockade of Ca<jats:sup>2+<\/jats:sup>\u2010activated K<jats:sup>+<\/jats:sup> channels with charybdotoxin (100 nM) and apamin (100 nM) (<jats:italic>n<\/jats:italic> = 5), or exposure of endothelial cells to 80 or 145 mM KCl (both <jats:italic>n<\/jats:italic> = 7) had no effect on the plateau phase of methacholine\u2010induced Ca<jats:sup>2+<\/jats:sup> transients (<jats:italic>p<\/jats:italic> &gt; .05).<\/jats:p><jats:p><jats:bold>Conclusions:<\/jats:bold> Freshly isolated arteriolar endothelial cells display agonist\u2010induced Ca<jats:sup>2+<\/jats:sup> transients. For the muscarinic agonist, methacholine, these Ca<jats:sup>2+<\/jats:sup> transients result from release of Ca<jats:sup>2+<\/jats:sup> from intracellular stores through IP<jats:sub>3<\/jats:sub> receptors, followed by sustained influx of extracellular Ca<jats:sup>2+<\/jats:sup>. While these changes in intracellular Ca<jats:sup>2+<\/jats:sup> are associated with endothelial cell hyperpolarization, the methacholine\u2010induced, sustained increase in intracellular Ca<jats:sup>2+<\/jats:sup> appears to be independent from this change in membrane potential. These data suggest that arteriolar endothelial cells may possess a novel Ca<jats:sup>2+<\/jats:sup> influx pathway, or that the relationship between intracellular Ca<jats:sup>2+<\/jats:sup> and Ca<jats:sup>2+<\/jats:sup> influx is more complex than that observed in other endothelial cells.<\/jats:p>","DOI":"10.1080\/10739680590904973","type":"journal-article","created":{"date-parts":[[2007,7,7]],"date-time":"2007-07-07T20:45:45Z","timestamp":1183841145000},"page":"169-182","source":"Crossref","is-referenced-by-count":39,"title":["Membrane Hyperpolarization Is Not Required for Sustained Muscarinic Agonist\u2010Induced Increases in Intracellular Ca<sup>2+<\/sup> in Arteriolar Endothelial Cells"],"prefix":"10.1111","volume":"12","author":[{"given":"KENNETH D.","family":"COHEN","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"WILLIAM 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