Abstract 13174: Calcium Mediated Changes in Phosphorylation Co-Regulate Argininosuccinate Synthase and Endothelial Nitric Oxide Synthase Localization in Vascular Endothelial Cells
The dynamic endothelial environment that maintains homeostasis of nitric oxide (NO) production, thus vascular function, requires an integrated response by argininosuccinate synthase (AS) and nitric oxide synthase (eNOS) to physiological cues. A well-studied facet of NO regulation involves the phosphorylation of eNOS, where phosphorylation at serine-1177 promotes activity. In addition, increase in intracellular calcium mediates the translocation of eNOS from the plasma membrane to the cytosol where it actively produces NO. Our laboratory has previously shown that AS co-localizes with eNOS to plasmalemmal caveolae which suggests that the proximal relationship of the two enzymes is required for NO production. Although regulation of AS, which catalyzes the rate-limiting step of the citrulline/NO cycle, has mainly been described as transcriptional, it has recently been demonstrated that AS is also regulated by phosphorylation. Here we show by membrane enrichment that total AS was found in both the membrane fraction and the cytosolic fraction, while AS phosphorylated at serine-328 (pS328 AS) was found only in the cytosolic fraction. Furthermore, immunofluorescent confocal microscopy of bovine aortic endothelial cells (BAECs) showed cytosolic co-localization of pS328 AS and eNOS. These data led to the hypothesis that pS328 regulates AS localization from the plasma membrane to the cytosol in order to provide substrate to cytosolic eNOS. Endothelial stimulants that lead to eNOS translocation such as calcium ionophore A23187 were used to treat BAECs followed by western blot analysis to assess AS and eNOS phosphorylation. BAEC treatment with calcium A23187 resulted in an increase of phosphorylation of AS at serine-328 (52.41% ± 12.15) and eNOS at serine-1177 (41.02% ± 15.16) relative to non-treated cells (n=3, p<0.05). In contrast, insulin, which promotes activation of plasma membrane localized eNOS independent of calcium, led to a decrease in AS serine-328 phosphorylation (-31.46% ± 0.89), while eNOS phosphorylation at S1177 increased (46.5% ± 6.05) relative to non-treated cells (n=2, p<0.05). These data suggest that serine-328 phosphorylation promotes AS co-localization with cytosolic eNOS in response to calcium mediated physiological cues.
- © 2011 by American Heart Association, Inc.