Abstract 3375: Endogenous And Exogenous S100A1 Modulates Nitric Oxide Synthase Phosphorylation In Endothelial Cells And Cardiomyocytes
S100A1, a Ca 2+ -binding protein, is expressed in myocardium and endothelial cells (EC) and regulates cardiac muscle contractility. S100A1 knockout mice (KO) are hypertensive with impaired endothelial-dependent vasodilatation. We hypothesized that endothelial dysfunction found in S100A1 KO mice can be attributed to altered regulation of eNOS content or phosphorylation. EC were isolated from pulmonary vasculature of wild-type (WT) and S100A1 KO. Rat neonatal cardiomyocytes (CMC) were co-transfected with plasmids encoding eNOS and the receptor for advanced glycation end products (RAGE) or dominant negative mutant (ΔRAGE). eNOS/S100A1 interaction was tested by co-immunoprecipitation or confocal immunofluorescence. Phosphorylation of eNOS at Ser1177 and Thr495, positive and negative regulatory residues, respectively, was analysed in response to exogenous S100A1 (1μM). Western blotting and immunofluorescence demonstrated endogenous S100A1 in EC and CMC from WT not KO. EC from KO displayed significantly lower basal eNOS protein (4.02+0.19 fold decrease, n=3) and mRNA expression (5–10-fold decrease). S100A1and eNOS were co-immunoprecipitated and co-localized in perinuclear regions of ECs and CMC. In unstimulated ECs from KO, eNOS was strongly phosphorylated at Thr495 but not at Ser1177. Exogenously administered S100A1 resulted in a 7.5–11 fold increase in Ser 1177 phosphorylation in WT but only a 2.2-fold increase in KO. In cardiac myocytes from WT, S100A1 similarly increases Ser 1177 and decreases Thr 495 phosphorylation of transfected eNOS. Exogenous S100A1 effects were mediated by RAGE (inhibited by ΔRAGE) and Akt-dependent. Demonstrating selectivity, the 3.5-fold increase in Erk1/2 phosphorylation was not RAGE-dependent. In EC and CMC, cell lineages expressing endogenous S100A1, S100A1 binds and co-localizes with eNOS. Abrogation of S100A1 in EC reduces eNOS availability and regulates eNOS phosphorylation consistent with decreased activity. Exogenous S100A1 phosphorylates residues that confer increased eNOS activity in a RAGE and Akt-dependent manner. Thus, S100A1 represents a novel endogenous and exogenous regulator of eNOS activity that is amenable to therapeutic intervention.