Isometric contraction induces the Ca2+-independent activation of the endothelial nitric oxide synthase

Shear stress and tyrosine phosphatase inhibitors have been shown to activat e the endothelial NO synthase (eNOS) in a Ca2+/calmodulin-independent manne r. We report here that isometric contraction of rabbit aorta activates eNOS by a pharmacologically identical pathway. Endothelium-intact aortic rings were precontracted under isometric conditions up to 60% of the maximal phen ylephrine-induced tone. The NO synthase inhibitor N(G)nitro-L-arginine (L-N A) and the soluble guanylyl cyclase inhibitor NS 2028 induced an additional contraction, the amplitude of which depended on the level of precontractio n. The maximal production of NO by isometrically contracted aortic rings (a s estimated by the increase in cGMP in detector smooth muscle cells in a su perfusion bioassay) was observed during the initial phase of isometric cont raction and was greater than that detected following the application of ace tylcholine. The supplementary L-NA-induced increase in vascular tone was in hibited by the nonselective kinase inhibitor staurosporine and the tyrosine kinase inhibitors erbstatin A and herbimycin A. Another tyrosine kinase in hibitor, genistein, the calmodulin antagonist calmidazolium, and the select ive protein kinase C inhibitor, Ro 31-8220, had no effect, Coincident with the enhanced NO formation during isometric contraction was an increase in t he tyrosine phosphorylation of endothelial proteins, which also correlated with the level of precontraction, Thus, isometric contraction activates eNO S via a Ca2+-independent, tyrosine kinase inhibitor-sensitive pathway and, like shear stress, seems to be an independent determinant of mechanically i nduced NO formation.