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1 Department of Medicine, University of Florida, Gainesville, FL, USA
2 Department of Medicine, University of Florida, Gainesville, FL, USA; Research Service, Malcom Randall Veterans Affairs Medical Center, Gainesville, FL, USA
* To whom correspondence should be addressed. E-mail: ysu{at}ufl.edu.
In the present study, the association of endothelial nitric oxide synthase (eNOS) with the actin cytoskeleton in pulmonary artery endothelial cells (PAEC) was examined. We found that the protein contents of eNOS, actin and caveolin-1 were significantly higher in the caveolar fraction of plasma membranes than in the non-caveolar fraction of plasma membranes in PAEC. Immunoprecipitation of eNOS from lysates of caveolar fractions of plasma membranes in PAEC resulted in the co-precipitation of actin, and immunoprecipitation of actin from lysates of caveolar fractions resulted in the co-precipitation of eNOS. Confocal microscopy of PAEC, in which eNOS was labeled with fluorescein, F-actin was labeled with Texas-red-phalloidin, and G-actin was labeled with deoxyribonuclease I conjugated with Texas-red also demonstrated an association between eNOS and F-actin or G-actin. Incubation of purified eNOS with purified F-actin and G-actin resulted in an increase in eNOS activity. The increase in eNOS activity caused by G-actin was much higher than that caused by F-actin. Incubation of PAEC with swinholide A, an actin filament disruptor, resulted in an increase in eNOS activity, in eNOS protein content, and in association of eNOS with G-actin and resulted in a decrease in the association of eNOS with F-actin. The increase in eNOS activity was higher than that in eNOS protein content in swinholide A-treated cells. In contrast, exposure of PAEC to phalloidin, an actin filament stabilizer, caused decreases in eNOS activity and association of eNOS with G-actin and increases in association of eNOS with F-actin. These results suggest that eNOS is associated with actin in PAEC and that actin and its polymerization state play an important role in the regulation of eNOS activity.
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