CA2-INDEPENDENT ACTIVATION OF THE ENDOTHELIAL NITRIC-OXIDE SYNTHASE IN RESPONSE TO TYROSINE PHOSPHATASE INHIBITORS AND FLUID SHEAR-STRESS()

Fluid shear stress enhances NO formation via a Ca2+-independent tyrosi ne kinase inhibitor-sensitive pathway, In the present study, we invest igated the effects of the protein tyrosine phosphatase inhibitor pheny larsine oxide and of fluid shear stress on endothelial NO production a s well as on the membrane association and phosphorylation of the NO sy nthase (NOS) III, Phenylarsine oxide (IO mu mol/L) induced an immediat e and maintained NO-mediated relaxation of isolated rabbit carotid art eries, which was insensitive to the removal of extracellular Ca2+ and the calmodulin antagonist calmidazolium. This phenylarsine oxide-induc ed vasodilatation was unaffected by genistein but abrogated by the tyr osine kinase inhibitor erbstatin A, Incubation of native or cultured e ndothelial cells with phenylarsine oxide resulted in a time-dependent tyrosine phosphorylation of mainly Triton X-100-insoluble (cytoskeleta l) proteins, along with a parallel change in the detergent solubility of NOS III, such that the enzyme was recovered in the cytoskeletal fra ction. A similar, though slightly delayed, phenomenon was also observe d after the application of fluid shear stress but not in response to a ny receptor-dependent agonist. Although Ca2+-independent NO formation was sensitive to erbstatin A, phenylarsine oxide treatment was associa ted with the tyrosine dephosphorylation of NOS III rather than its hyp erphosphorylation. Proteins that also underwent redistribution in resp onse to the tyrosine phosphatase inhibitor included paxillin, phosphol ipase C-gamma(1), mitogen-activated protein kinase, and the tyrosine k inases Src and Fyn. We envisage that, fluid shear stress and tyrosine phosphatase inhibitors may alter the conformation and/or protein coupl ing of NOS III, facilitating its interaction with specific phospholipi ds, proteins, and/or protein kinases that enhance/maintain its Ca2+-in dependent activation.