Abstract 3634: Endothelial cGMP-dependent Protein Kinase (PKG1) Directly Activates eNOS and Plays a Key Role in Modulation of Vascular Tone
It is known that PKG1 is expressed in various types of vascular cells, however, its physiological impact on regulation of vascular tone has been primarily studied in smooth muscle cells. Here we show for the first time that endothelial PKG1 directly controls eNOS and plays an important role in regulation of nitric oxide production. Overexpression of PKG1α using an adenoviral vector which produced a 3–5 fold increase in PKG1 protein expression and activity, resulted in a concomitant 15% increase in NO2/NO3 in culture medium and a 35% increase in eNOS activity by DAF2 fluorescence, as compared to cells transfected with reporter gene only (Fig.1⇓). A 3–5 fold increase in P-eNOS(S1179, S1177) expression was observed by western blot analysis. Using bovine siRNA, we were able to selectively downregulate mRNA of α and β subunits of PKG1 which resulted in a significant decrease in PKG1 protein expression and activity by 55% and 70%, respectively. In addition, silencing PKG1 gene inhibited both eNOS phosphorylation at S1179 by 50% and calcium dependent NOS activity by 60% (Fig.2⇓). Finally, we mutated PKG1 gene at Asp502 to Ala (AdDsRed-PKG1α-D502A) which created a kinase activity deficient form of cGMP-dependent protein kinase. Interestingly, this was associated with a statistically significant increase in cAMP (P<0.001), suggesting a role of PKA in this process. These findings collectively demonstrate that endothelial PKG1 is a direct regulator of eNOS activity. In addition, our data suggest that both PKG and PKA work in concert in controlling eNOS pools within endothelial cells. This implicates a new insight into our current knowledge on endothelial dysfunction and other vascular diseases.