Functional implications of Ca2+ mobilizing properties for nitric oxide production in aortic endothelium.

Abstract We have investigated the relationship between Ca 2+ mobilization and the cellular production of nitric oxide (NO) by using fura-2 and diaminofluorescein-2 (DAF-2), an NO-sensitive dye, in bovine aortic endothelial cells (BAEC). High concentrations of ATP (100 μM) or thapsigargin (1 μM) depleted intracellular Ca 2+ store sites with a single Ca 2+ transient, and induced an increase in DAF-2 fluorescence even in Ca 2+ -free solution, thereby indicating that store depletion leads to NO production. The same level of increase in DAF-2 fluorescence was elicited by low concentrations of ATP (1 μM), which induced Ca 2+ oscillations but did not deplete store sites, only in the presence of extracellular Ca 2+ . Furthermore, inhibition of ATP (1 μM)-induced Ca 2+ entry with La 3+ suppressed DAF-2 fluorescence. ATP (0.3 μM), applied in Ca 2+ -free, Mn 2+ -containing solution induced Mn 2+ entry-coupled fura-2 quenching, repeating shortly after each oscillation peak. These results indicate that NO is produced preferentially by entered Ca 2+ , and that Ca 2+ oscillations, which are induced by low levels of stimulation, play a significant role in NO production by strongly modulating Ca 2+ entry.