Coordinated endothelial nitric oxide synthase activation by translocation and phosphorylation determines flow-induced nitric oxide production in resistance vessels.
OBJECTIVE
Endothelial nitric oxide synthase (eNOS) is associated with caveolin-1 (Cav-1) in plasma membrane. We tested the hypothesis that eNOS activation by shear stress in resistance vessels depends on synchronized phosphorylation, dissociation from Cav-1 and translocation of the membrane-bound enzyme to Golgi and cytosol.


METHODS
In isolated, perfused rat arterial mesenteric beds, we evaluated the effect of changes in flow rate (2-10 ml/min) on nitric oxide (NO) production, eNOS phosphorylation at serine 1177, eNOS subcellular distribution and co-immunoprecipitation with Cav-1, in the presence or absence of extracellular Ca(2+).


RESULTS
Increases in flow induced a biphasic rise in NO production: a rapid transient phase (3-5-min) that peaked during the first 15 s, followed by a sustained phase, which lasted until the end of stimulation. Concomitantly, flow caused a rapid translocation of eNOS from the microsomal compartment to the cytosol and Golgi, paralleled by an increase in eNOS phosphorylation and a reduction in eNOS-Cav-1 association. Transient NO production, eNOS translocation and dissociation from Cav-1 depended on extracellular Ca(2+), while sustained NO production was abolished by the PI3K-Akt blocker wortmannin.


CONCLUSIONS
In intact resistance vessels, changes in flow induce NO production by transient Ca(2+)-dependent eNOS translocation from membrane to intracellular compartments and sustained Ca(2+)-independent PI3K-Akt-mediated phosphorylation.
Departamento de Fisiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago, Chile.
Xavier F
XF
Figueroa
Daniel R
DR
González
Mariela
M
Puebla
Juan P
JP
Acevedo
Daniel
D
Rojas-Libano
Walter N
WN
Durán
Mauricio P
MP
Boric
D000818:Animals; D001783:Blood Flow Velocity; D002118:Calcium / Q000378:metabolism; D051242:Caveolin 1 / Q000378:metabolism; D004789:Enzyme Activation; D008297:Male; D040542:Mechanotransduction, Cellular; D008638:Mesenteric Arteries / Q000201:enzymology*; D009569:Nitric Oxide / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D058539:Phosphatidylinositol 3-Kinase / Q000378:metabolism; D010766:Phosphorylation; D021381:Protein Transport; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D012039:Regional Blood Flow; D012694:Serine; D013152:Splanchnic Circulation; D013314:Stress, Mechanical; D013997:Time Factors; D014655:Vascular Resistance
C495537:Cav1 protein, rat; D051242:Caveolin 1; D009569:Nitric Oxide; D012694:Serine; D052250:Nitric Oxide Synthase Type III; C496316:Nos3 protein, rat; D058539:Phosphatidylinositol 3-Kinase; D051057:Proto-Oncogene Proteins c-akt; D002118:Calcium