ROLE OF CALCIUM AND CALMODULIN IN FLOW-INDUCED NITRIC-OXIDE PRODUCTION IN ENDOTHELIAL-CELLS

These experiments demonstrate that exposure of cultured endothelial ce lls (EC) to well-defined laminar fluid flow results in an elevated rat e of NO production. NO production was monitored by release of NOx (NO2 - + NO32-) and by cellular guanosine 3',5'-cyclic monophosphate (cGMP) concentration. NO synthase (NOS) inhibitor blocked the flow-mediated stimulation of both NOx and cGMP, indicating that both measurements re flect NO production. Exposure to laminar flow increased NO release in a biphasic manner, with an initial rapid production consequent to the onset of flow followed by a less rapid, sustained production. A simila r rapid increase in NO production resulted from an increase in flow ab ove a preexisting level. The rapid initial production of NO was not de pendent on shear stress within a physiological range (6-25 dyn/cm(2)) but may be dependent on the rate of change in shear stress. The sustai ned release of NO was dependent on physiological levels of shear stres s. The calcium (Ca2+) or calmodulin (CaM) dependence of the initial an d sustained production of NO was compared with bradykinin (BK)-mediate d NO production. Both BK and the initial production were inhibited by Ca2+ and CaM antagonists. In contrast, the sustained shear stress-medi ated NO production was not affected, despite the continued functional presence of the antagonists. Dexamethasone had no effect on either the initial or the sustained shear stress-mediated NO production. An indu cible NOS does not, therefore, explain the apparent Ca2+/CaM independe nce of the sustained shear stress-mediated NO production. In summary, these data reveal that initial laminar fluid flow-stimulated NO produc tion is very similar to BK-mediated production: rapid and Ca2+/CaM dep endent. Continued exposure stimulates NO production via shear stress i n a physiologically Ca2+/CaM-independent (activated by resting levels) manner.