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

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 μM, 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 96365 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.