CALCIUM-DEPENDENT AND CALCIUM-INDEPENDENT ACTIVATION OF THE ENDOTHELIAL NO SYNTHASE

Largely assumed to be a Ca2+-/calmodulin-dependent enzyme, the endothe lial constitutive nitric oxide (NO) synthase (NOS III) can be activate d by agonists as a consequence of an increase in the intracellular con centration of free Ca2+ ([Ca2+](i)). This increase in [Ca2+](i) is eli cited by an increase in inositol 1,4,5-trisphosphate which is the cons equence of tyrosine phosphorylation and activation of phospholipase C- gamma(1) as well as protein tyrosine phosphatases. Following the mobil ization of intracellular Ca2+, the depleted Ca2+ stores signal to cati on channels in the plasma membrane by a pathway which appears to invol ve activation of both tyrosine and serine/threonine kinases since this portion of the Ca2+ response is attenuated by both tyrosine kinase in hibitors and serine phosphatase inhibitors. In response to fluid shear stress the continuous production of NO by native and cultured endothe lial cells is associated with only a transient and minimal increase in [Ca2+](i). In the absence of extracellular Ca2+ and in the presence o f the calmodulin antagonist, shear stress stimulates a continuous prod uction of NO which is sensitive to the nonspecific kinase inhibitor st aurosporine and the tyrosine kinase inhibitor erbstatin A. A pharmacol ogically identical activation of NOS III can be induced by protein pho sphatase inhibitors suggesting that the tyrosine phosphorylation of NO S III or an associated regulatory protein is crucial for its Ca2+-inde pendent activation. Thus in a departure from widely held beliefs, we p ropose that the endothelial cells are able to respond to mechanical an d humoral stimuli activating NOS III by at least two separate pathways .