Abstract 10574: Folic Acid Activates eNOS Through CaMKII-Mediated Phosphorylation and Increased Membrane Targeting
Folic acid improves endothelial function via modulation of endothelial nitric oxide synthase (eNOS) phosphorylation status/activity and regulatory protein interactions. While Ca2+/calmodulin (CaM) interaction, CaM-dependent kinase II (CaMKII)-dependent phosphorylation and subcellular translocation also play a pivotal role in eNOS activation the effects of folic acid thereon are unknown. In the present study cultured primary porcine aortic endothelial cells (PAEC) were exposed to folic acid (0-50 µM, 24 hours) before lysate eNOS, CaM, CaMKII, caveolin-1 (Cav-1) and actin levels were measured via western blot. Co-immunoprecipitation was used to extract eNOS-associated proteins before western blotting for CaM. Separation of membrane and cytosolic fractions or light Cav-1-enriched membranes from heavy membranes was achieved by ultracentrifugation or discontinuous sucrose gradient ultracentrifugation respectively before eNOS, Cav-1 and actin detection by western blot. PAEC were exposed to CaMKII inhibitors (autocamtide-2-related inhibitory peptide 80 nM or CaMKIINtide 1 µM) in the absence or presence of folic acid (50 µM) and eNOS Ser635 and Ser1177 phosphorylation status, total eNOS and actin levels determined via western blot. Activity of eNOS was measured via cGMP assay. Exposure to folic acid did not alter total eNOS, CaM, CaMKII, Cav-1 or actin levels. The association of eNOS with CaM also remained unaltered. A significant (p<0.05) concentration-dependent increase in the proportion of membrane localised eNOS was observed following folic acid treatment. However, there was no redistribution of eNOS between light Cav-1 enriched membranes and heavy membranes. CaMKII inhibition prevented folic acid-induced changes in eNOS Ser635 and Ser1177 phosphorylation as well as eNOS activation. In conclusion, folic acid activates eNOS via CaMKII-mediated phosphorylation in the absence of increased eNOS/CaM association. Activation is accompanied by increased translocation of eNOS to cellular membranes. This effect on eNOS activity may underlie the folic acid-mediated improvement in endothelial function described by many previous studies.
- © 2011 by American Heart Association, Inc.