Nitric oxide production in human endothelial cells stimulated by histamine requires Ca2+ influx

The causal relationships between cytosolic free-Ca2+ concentration ([Ca2+](i)) increases and production of nitric oxide (NO) have been investigated mostly with indirect methods and remain unclear. Here we demonstrate, by direct real-time measurements of[NO] with a porphyrinic microsensor, that Ca2+ entry, but not an increase in [Ca2+](i), is required for triggering of NO production in human endothelial cells. Histamine, ranging from 0.1 to 100 mu M, increased both NO production and [Ca2+](i) when given in a single dose. However, histamine caused increased NO release but induced progressively smaller [Ca2+](i) changes when cumulatively added. In the absence of a transmembrane Ca2+ gradient, no significant NO release was detectable, despite the marked Ca2+ peak induced by histamine. Inhibition of Ca2+ entry by SK&F 95365 abolished histamine-elicited NO production but only reduced the transient [Ca2+](i) rise. The suppression of the sustained [Ca2+](i) response under these two conditions suggests that NO release was closely associated with Ca2+ entry from the extracellular space. In addition, membrane depolarization, achieved by increasing the extracellular K+ concentration from 5 to 130 mM, reduced both the amplitude of histamine-induced sustained [Ca2+](i) elevation and NO production. These results lead us to propose that the availability of numerous Ca2+ ions around the internal side of the plasma membrane would promote the association between nitric oxide synthase and calmodulin, thereby activating the enzyme.