The mitochondrial redistribution of ENOS is regulated by AKT1 and dimer status.

Previously, we have shown that endothelial nitric-oxide synthase (eNOS) dimer levels directly correlate with the interaction of eNOS with hsp90 (heat shock protein 90). Further, the disruption of eNOS dimerization correlates with its redistribution to the mitochondria. However, the causal link between these events has yet to be investigated and was the focus of this study. Our data demonstrates that simvastatin, which decreases the mitochondrial redistribution of eNOS, increased eNOS-hsp90 interactions and enhanced eNOS dimerization in cultured pulmonary arterial endothelial cells (PAEC) from a lamb model of pulmonary hypertension (PH). Our data also show that the dimerization of a monomeric fraction of human recombinant eNOS was stimulated in the presence of hsp90 and ATP. The over-expression of a dominant negative mutant of hsp90 (DNHsp90) decreased eNOS dimer levels and enhanced its mitochondrial redistribution. We also found that the peroxynitrite donor3-morpholinosydnonimine (SIN-1) increased the mitochondrial redistribution of eNOS in PAEC and this was again associated with decreased eNOS dimer levels. Our data also show in COS-7 cells, the SIN-1 mediated mitochondrial redistribution of wildtype eNOS (WT-eNOS) is significantly higher than a dimer stable eNOS mutant protein (C94R/C99R-eNOS). Conversely, the mitochondrial redistribution of a monomeric eNOS mutant protein (C96A-eNOS) was enhanced. Finally, we linked the SIN-1-mediated mitochondrial redistribution of eNOS to the Akt1-mediated phosphorylation of eNOS at Serine(S)(617) and showed that the accessibility of this residue to phosphorylation is regulated by dimerization status. Thus, our data reveal a novel mechanism of pulmonary endothelial dysfunction mediated by mitochondrial redistribution of eNOS, regulated by dimerization status and the phosphorylation of S(617).