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

We have investigated the relationship between Ca²⁺ 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 Ca²⁺ store sites with a single Ca²⁺ transient, and induced an increase in DAF-2 fluorescence even in Ca²⁺-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 Ca²⁺ oscillations but did not deplete store sites, only in the presence of extracellular Ca²⁺. Furthermore, inhibition of ATP (1 μM)-induced Ca²⁺ entry with La³⁺ suppressed DAF-2 fluorescence. ATP (0.3 μM), applied in Ca²⁺-free, Mn²⁺-containing solution induced Mn²⁺ entry-coupled fura-2 quenching, repeating shortly after each oscillation peak. These results indicate that NO is produced preferentially by entered Ca²⁺, and that Ca²⁺ oscillations, which are induced by low levels of stimulation, play a significant role in NO production by strongly modulating Ca²⁺ entry.