Abstract 13450: Role of PKC in Metabolic Regulation of Endothelial SK Channels

Abstract

Introduction: Diabetes is associated with increases in metabolites NADH, and PKC. NADH has been recently found to inactivate endothelial SK channels. Overexpressed and activated PKC has been shown to play an important role in diabetes-induced endothelial dysfunction. However, it is undefined if PKC is involved in the NADH dysregulation of endothelial SK channels. Hypothesis: We hypothesized that PKC in a signaling cascade whereby NADH dysregulates endothelial SK channels and coronary relaxation function. Methods: SK channel currents of human coronary artery endothelial cells were measured by whole-cell patch-clamp method in the presence or absence of NADH, and/or PKC activator PMA, or PKC inhibitors or endothelial PKC α /PKC β knock-down by using short interfering RNA. The in-vitro coronary arterioles (n = 5) from the harvested atrial tissue samples of patients undergoing cardiac surgery were perfused with NADH in the presence or absence of PKC β inhibitor LY333531. Vascular reactivity was measured by vessel myography in the presence of vasoconstrictor and vasodilators. Results: NADH (30-300μM, n=7-9) or PKC activator PMA (30-300μM, n=6-12) reduced endothelial SK current density (p<0.05 vs. control (n=15), Fig. A-D), whereas the selective PKC β inhibitor LY333531 (50nM, n=12) significantly reversed the NADH-induced SK channel inhibition (p>0.05 vs. control (n=15), Fig. E). PKC α knock-down failed to affect NADH (n=13) and PMA (n=10) inhibition of endothelial SK currents (Fig. F). In contrast, PKC β knock-down significantly prevented NADH (n=12) and PMA (n=6)-induced SK inhibition (p>0.05, vs. control, Fig. G). Incubation of vessels with NADH significantly reduced relaxation response to the SK activator NS309 and this effect was abolished in the presence of LY333531. Conclusions: The metabolite NADH dysregulation of endothelial SK channels and impairment of endothelium-dependent relaxation function was via the PKC β signaling pathway.