Interaction between eNOS translocation and phosphorylation in flow –induced NO production

Abstract

Flow‐induced eNOS activation may involve the interaction of several signaling processes, such as eNOS phosphorylation, binding to Ca2+‐calmodulin, dissociation from caveolin (cav‐1) and/or translocation from membrane. We studied NO production (chemiluminescence), eNOS subcellular distribution and phosphorylation in intact resistance vessels of the isolated rat mesenteric bed submitted to step changes of flow rate. Flow increase (2 to 10 mL/min) induced a biphasic rise in NO production: a transient phase that peaked at 15‐sec and lasted 3–5‐ min, 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, which was paralleled by a 50% reduction in the eNOS‐cav‐1 association. In addition, the transient NO production, eNOS translocation, and eNOS dissociation from cav‐1 depended on extracellular Ca2+. In control or Ca2+‐free conditions, flow rise increased eNOS phosphorylation at ser‐1177 in an Akt‐dependent manner. Blockade of the PI3K‐Akt pathway abolished the sustained, but not the transient phase, of flow‐induced NO release. We conclude that flow‐induced NO production involves rapid Ca2+‐dependent eNOS translocation from membrane, while PI3K‐Akt‐ dependent phosphorylation accounts for the sustained eNOS activation by prevailing flow.Fondecyt 1090757 & Anillos‐Act71