Abstract 13010: Folic Acid-induced eNOS Activation is Dependent on Both Protein Kinase A Activity and Interactions with Caveolin-1 and Heat Shock Protein-90
Our previous studies demonstrated that folic acid improves endothelial function via phosphoinositide-3-kinase (PI3K)-dependent modulation of endothelial nitric oxide synthase (eNOS) phosphorylation status/activity. While activation of eNOS is also regulated by protein kinase A (PKA) and interactions with caveolin-1 and heat shock protein-90 (HSP-90), the effects of folic acid thereon are unknown. Therefore the present study investigated the role of both PKA in eNOS phosphorylation and protein/protein interactions in eNOS activation following exposure of cultured primary porcine aortic endothelial cells (PAEC) to folic acid (0–50μM) for 24 hours. Following cell lysis co-immunoprecipitation was used to extract eNOS and associated proteins. Total eNOS, caveolin-1, HSP-90 and actin levels were determined via western blot. PAEC PKA activity was measured using an immuno-based assay for cAMP. In separate experiments PAEC were exposed to PKA inhibitors (RpcAMPs 100nM or PKI 10μM) in the absence or presence of folic acid (50μM for 24 hours). Subsequently the phosphorylation status of eNOS at Ser116, Thr495, Ser635 and Ser1177 as well as total eNOS and actin levels were determined via western blot, followed by eNOS activity measurements via cGMP assay. While exposure to folic acid did not significantly alter total eNOS, caveolin-1, HSP-90 or actin levels co-immunoprecipitation revealed a concentration-dependent decrease in eNOS/caveolin-1 and increase in eNOS/HSP-90 interactions. These changes were accompanied by a significant (p<0.05) concentration-dependent increase in PKA activity. Furthermore PKA inhibition prevented both folic acid-induced changes in eNOS phosphorylation at Ser116, Thr495, Ser635 and Ser1177 and eNOS activation. In conclusion, folic acid modulates eNOS activation via both PKA signalling and dissociation from caveolin−1. Moreover, folic acid also promotes increased eNOS/HSP-90 interaction. As such, this interplay may underlie the beneficial effects of folic acid on endothelial function described by many previous studies.
- © 2010 by American Heart Association, Inc.