Calcium‐dependent nitric oxide synthesis in endothelial cytosol is mediated by calmodulin

Abstract

We investigated whether calmodulin mediates the stimulating effect of Ca²⁺ on nitric oxide synthase in the cytosol of porcine aortic endothelial cells. Nitric oxide was quantified by activation of a purified soluble guanylate cyclase. The Ca²⁺‐sensitivity of nitric oxide synthase was lost after anion exchange chromatography of the endothelial cytosol and could only be reconstituted by addition of calmodulin or heat‐denatured endothelial cytosol. The Ca²⁺‐dependent activation of nitric oxide synthase in the cytosol was inhibited by the calmodulin‐binding peptides/proteins melittin, mastoparan, and calcineurin (IC₅₀ 450, 350 and 60 nM, respectively), but not by the calmodulin antagonist, calmidazolium. In contrast, Ca²⁺‐calmodulin‐reconstituted nitric oxide synthase was inhibited with similar potency by melittin and calmidazolium. The results suggest that the Ca²⁺‐dependent activation of nitric oxide synthase in endothelial cells is mediated by calmodulin.