AGONIST-INDUCED AND CPA-INDUCED ELEVATION OF CYTOPLASMIC FREE CA2+ ININTACT VALVULAR ENDOTHELIUM FROM RABBITS

Intracellular Ca2+ signals of intact endothelium from rabbit aortic or pulmonic valves loaded with fura 2 were studied using imaging fluores cence microscopy. Agonists such as ATP or carbachol and inhibitors of endoplasmic reticulum (ER) Ca2+-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 extrac ellular Ca2+ was present, indicating that the maintained [Ca2+](i) inc rease is dependent on the entry of extracellular Ca2+. The voltage-gat ed channel was not found to contribute to [Ca2+](i) increase. SK&F-963 65, a receptor-operated cation channel (ROC) blocker, and 2-nitro-4-ca rboxyphenyl-N,N-diphenylcarbmate (NCDC), a postulated phospholipase C inhibitor, were shown to effectively block ROC, since they greatly red uced the maintained [Ca2+](i) increase caused by ATP, but not that cau sed by CPA, which was blocked by Ni2+. To further investigate the Ca2 influx involved in this process, divalent cation entry was measured a s Mn2+ quenching of fura 2 fluorescence at 360-nm excitation wavelengt h. At rest, fluorescence quenching at 360 nm by Mn2+ was observed, whi ch was inhibited by Ni2+ but not by NCDC, indicating Mn2+ entry throug h the leak pathway. The quenching was enhanced following ATP stimulati on, and this enhancement was abolished by pretreatment with NCDC. In c ontrast, 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 C a2+ leak in raising [Ca2+](i).