Phosphorylation and activation of the endothelial nitric oxide synthase by fluid shear stress.
Fluid shear stress activates the endothelial nitric oxide (NO) synthase (eNOS) by a mechanism which does not require an increase in the intracellular concentration of free Ca2+ ([Ca2+]i), and is sensitive to several kinase inhibitors. Although phosphorylation of eNOS has been suggested to regulate enzyme activity, the mechanism of eNOS activation is still unclear. Here we demonstrate that fluid shear stress elicits the phosphorylation of eNOS on tyrosine and serine residues. Inhibition of phosphatidylinositol 3-kinase (PI3K), using wortmannin or a dominant negative mutant of its downstream target, Akt (protein kinase B), prevented the maintained serine phosphorylation and activation of eNOS. Enhancing eNOS phosphorylation by inhibiting serine/threonine phosphatases, increased eNOS activity by approximately twofold, as assessed by the accumulation of intracellular cyclic GMP, without increasing the intracellular concentration of free Ca2+. These data suggest that shear stress activates a pathway involving PI3K and the serine/threonine kinase Akt, which phosphorylates eNOS. This phosphorylation directly increases eNOS activity at resting [Ca2+]i, thus rendering the shear stress-induced activation of eNOS apparently Ca2+-independent.
Fisslthaler
B
B
Institut für Kardiovaskuläre Physiologie, Klinikum der J. W. Goethe-Universität, Theodor-Stern-Kai 7, Frankfurt am Main, Germany.
Dimmeler
S
S
Hermann
C
C
Busse
R
R
Fleming
I
I
D000595:Amino Acid Sequence; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D006152:Cyclic GMP / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology; D004789:Enzyme Activation / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000502:physiology; D051057:Proto-Oncogene Proteins c-akt; D012694:Serine / Q000378:metabolism; D013314:Stress, Mechanical; D013552:Swine; D000077191:Wortmannin
D000730:Androstadienes; D004791:Enzyme Inhibitors; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011518:Proto-Oncogene Proteins; D012694:Serine; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; C494918:AKT1 protein, human; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D010749:Phosphoprotein Phosphatases; D006152:Cyclic GMP; D002118:Calcium; D000077191:Wortmannin