Abstract 4885: Folic Acid Modulates eNOS Phosphorylation Status/Activity via Phosphoinositide 3-Kinase-Dependent and Independent Pathways
While evidence suggests that folic acid can improve endothelial function through the regulation of endothelial nitric oxide synthase (eNOS), the exact mechanism(s) involved in this process remain elusive. Given that the phosphorylation status of eNOS can play a significant role in modifying its activity, the present study investigated the effect of folic acid on the phosphorylation of eNOS at important regulatory sites. Cultured porcine aortic endothelial cells (PAEC) were exposed to folic acid (0 –50μM for 24 hours) in the absence or presence of phosphoinositide 3-kinase (PI3K) inhibition (wortmannin 100nM or LY294002 10μM for 30 minutes before folic acid). The phosphorylation status of eNOS as well as total eNOS protein levels were then determined by western blotting. In other experiments similarly treated PAEC were subsequently exposed to calcimycin (10μM for 30 minutes) before being lysed. The cGMP content of the cell lysates was then measured by ELISA to provide an index of eNOS activity. Exposure of PAEC to folic acid promoted significant (p<0.05) concentration-dependent dephosphorylation of eNOS at Ser116 and Thr495 (negative regulatory sites) and increased phosphorylation at Ser635 and Ser1177 (positive regulatory sites). In the presence of PI3K inhibition the observed folic acid-induced changes in phosphorylation at Ser116, Ser635 and Ser1177, but not Thr495 (a regulatory site well known to be independent of PI3K control), were prevented. Importantly, the expression of total eNOS protein was not affected by any of the treatments outlined above. The folic acid-induced change in eNOS phosphorylation status was accompanied by increased eNOS activity (as measured by calcimycin-induced cGMP production). Again this increase in eNOS activity was prevented by the presence of PI3K inhibition. In conclusion, these data would suggest that exposure of PAEC to folic acid modulates eNOS phosphorylation status/activity via both PI3K-dependent and independent pathways. Such changes may underlie the beneficial effect of folic acid on endothelial function described by many previous studies.