ACETYLCHOLINE-SENSITIVE INTRACELLULAR CA2-CELLS AND EVIDENCE FOR RYANODINE RECEPTORS( STORE IN FRESH ENDOTHELIAL)

In a freshly isolated endothelial cell preparation from rabbit aorta, the regulation of the acetylcholine (ACh)-sensitive intracellular Ca2 store and the effects of the Ca2+ induced Ca2+ release agonists ryano dine and caffeine were studied using fura 2 imaging fluorescence micro scopy. ACh (10 mu mol/L) caused a transient release of Ca2+ from an in tracellular store, presumably via an inositol tris-phosphate-sensitive mechanism. This ACh response could be repeated in the presence of ext racellular Ca2+ but was obtained only once in Ca2+-free bathing soluti on, which shows that a depleted intracellular Ca2+ store can be rapidl y refilled from the extracellular space. Refilling can be prevented by the endoplasmic reticulum Ca2+-ATPase inhibitor cyclopiazonic acid (1 0 mu mol/L), implying that Ca2+ enters the cytoplasm before accumulati on in the endoplasmic reticulum. Ionomycin (10 mu mol/L) caused a larg e Ca2+ release even after the ACh-releasable store had been emptied, i ndicating the existence of other ACh-insensitive stores, perhaps inclu ding the mitochondria. In one third of the cells studied, ACh induced oscillations in [Ca2+](i) that were dependent on extracellular Ca2+. A lso investigated were the effects of caffeine and ryanodine. In this c ell preparation neither caffeine nor ryanodine induced a Ca2+ transien t but instead slowly increased [Ca2+](i). It was observed that both ca ffeine and ryanodine were able to slowly deplete the ACh-sensitive sto re. These results indicate the presence of functional ryanodine recept ors in native endothelial cells and demonstrate overlap between the ca ffeine and agonist-sensitive Ca2+ stores. We also found that caffeine was able to directly inhibit the process of ACh-induced Ca2+ release. It is hypothesized that endothelial endoplasmic reticulum contains bot h inositol tris-phosphate receptors and ryanodine receptors but that t he former class are more densely distributed.