Calcium-dependent and calcium-independent activation of the endothelial NO synthase.
Largely assumed to be a Ca2(+)-/calmodulin-dependent enzyme, the endothelial constitutive nitric oxide (NO) synthase (NOS III) can be activated by agonists as a consequence of an increase in the intracellular concentration of free Ca2+ ([Ca2+]i). This increase in [Ca2+]i is elicited by an increase in inositol 1,4,5-trisphosphate which is the consequence of tyrosine phosphorylation and activation of phospholipase C-gamma1 as well as protein tyrosine phosphatases. Following the mobilization of intracellular Ca2+, the depleted Ca2+ stores signal to cation channels in the plasma membrane by a pathway which appears to involve activation of both tyrosine and serine/threonine kinases since this portion of the Ca2+ response is attenuated by both tyrosine kinase inhibitors and serine phosphatase inhibitors. In response to fluid shear stress the continuous production of NO by native and cultured endothelial cells is associated with only a transient and minimal increase in [Ca2+]i. In the absence of extracellular Ca2+ and in the presence of the calmodulin antagonist, shear stress stimulates a continuous production of NO which is sensitive to the nonspecific kinase inhibitor staurosporine and the tyrosine kinase inhibitor erbstatin A. A pharmacologically identical activation of NOS III can be induced by protein phosphatase inhibitors suggesting that the tyrosine phosphorylation of NOS III or an associated regulatory protein is crucial for its Ca2(+)-independent activation. Thus in a departure from widely held beliefs, we propose that the endothelial cells are able to respond to mechanical and humoral stimuli activating NOS III by at least two separate pathways.
Fleming
I
I
Zentrum der Physiologie, Klinikum der J.W. Goethe-Universität, Frankfurt/Main, Deutschland. Fleming@em.uni-frankfurt.de
Bauersachs
J
J
Busse
R
R
D002118:Calcium / Q000502:physiology*; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology*; D004789:Enzyme Activation; D006801:Humans; D009569:Nitric Oxide / Q000096:biosynthesis; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D015398:Signal Transduction; D013314:Stress, Mechanical
D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D002118:Calcium