AJP - Heart and Circulatory Physiology, Vol 270, Issue 3 837-H848, Copyright © 1996 by American Physiological Society

ARTICLES

Agonist- and CPA-induced elevation of cytoplasmic free Ca2+ in intact valvular endothelium from rabbits

L. Li and C. van Breemen
Department of Pharmacology and Therapeutics, University of British Columbia, Faculty of Medicine, Vancouver, Canada.

Intracellular Ca2+ signals of intact endothelium from rabbit aortic or pulmonic valves loaded with fura 2 were studied using imaging fluorescence microscopy. Agonists such as ATP or carbachol and inhibitors of endoplasmic reticulum (ER) Ca(2+)-ATPase such as cyclopiazonic acid (CPA), thapsigargin, and 2', 5'-di(tert-butyl)-1, 4,-benzohydroquinone (BHQ) induced an increase in cytoplasmic free Ca2+ concentration ([Ca2+]i) that was maintained above the prestimulated levels as long as extracellular Ca2+ was present, indicating that the maintained [Ca2+]i increase is dependent on the entry of extracellular Ca2+. The voltage-gated channel was not found to contribute to [Ca2+]i increase. SK&F-96365, a receptor-operated cation channel (ROC) blocker, and 2-nitro-4-carboxyphenyl-N, N-diphenylcarbamate (NCDC), a postulated phospholipase C inhibitor, were shown to effectively block ROC, since they greatly reduced the maintained [Ca2+]i increase caused by ATP, but not that caused by CPA, which was blocked by Ni2+. To further investigate the Ca2+ influx involved in this process, divalent cation entry was measured as Mn2+ quenching of fura 2 fluorescence at 360-nm excitation wavelength. At rest, fluorescence quenching at 360 nm by Mn2+ was observed, which was inhibited by Ni2+ but not by NCDC, indicating Mn2+ entry through the leak pathway. The quenching was enhanced following ATP stimulation, and this enhancement was abolished by pretreatment with NCDC. In contrast, the rate of Mn2+ quenching was unaffected by the application of CPA. These results demonstrate that ATP stimulates divalent cation influx that is not related to the Ca2+ content of ER. Abolition of Ca2+ uptake into ER was postulated to increase the effectiveness of the Ca2+ leak in raising [Ca2+]i.

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