Overexpressed p32 localized in the endoplasmic reticulum and mitochondria negatively regulates calcium-dependent endothelial nitric oxide synthase activity

The p32 protein plays a crucial role in the regulation of cytosolic Ca(2+) concentrations ([Ca2+]c) that contributes to the Ca(2+)-dependent signaling cascade. Using an adenovirus and plasmid p32-overexpression system, the aim of the study was to evaluate the role of p32 in the regulation of [Ca(2+)] and its potential associated with Ca(2+)-dependent endothelial nitric oxide synthase (eNOS) activation in endothelial cells. Using electron and confocal microscopic analysis, p32 overexpression was observed to be localized to mitochondria and the endoplasmic reticulum and played an important role in Ca(2+) translocation, resulting in increased [Ca(2+)] in these organelles and reducing cytosolic [Ca(2+)] ([Ca(2+)]c). This decreased [Ca(2+)]c following p32 overexpression attenuated the Ca(2+)-dependent signaling cascade of calcium/calmodulin dependent protein kinase II (CaMKII)/AKT/eNOS phosphorylation. Moreover, in aortic endothelia of wild-type mice intravenously administered adenovirus encoding the p32 gene, increased p32 levels reduced NO production and accelerated reactive oxygen species (ROS) generation. In a vascular tension assay, p32 overexpression decreased acetylcholine (Ach)-induced vasorelaxation and augmented phenylephrine (PE)-dependent vasoconstriction. Notably, decreased levels of arginase II (ArgII) protein using siArgII were associated with downregulation of overexpressed p32 protein, which contributed to CaMKII-dependent eNOS phosphorylation at Ser1177. These results indicated that increased protein levels of p32 caused endothelial dysfunction through attenuation of the Ca(2+)-dependent signaling cascade and that ArgII protein participated in the stability of p32. Therefore, p32 may be a novel target for the treatment of vascular diseases associated with endothelial disorders.