Endothelial Nitric Oxide Synthase Interaction with Actin Contributes to Mild Hyperoxia‐induced Endothelial Wound Repair.

Abstract

We previously reported that endothelial nitric oxide synthase (eNOS) association with actin increases eNOS activity in pulmonary artery endothelial cells (PAEC). In the present study, PAEC were exposed to 40% O2 for 1-24 h after which eNOS activity and endothelial monolayer wound repair was measured, and eNOS/actin association was evaluated by using confocal microscopy and immunoprecipitation. We found that exposure of PAEC to 40% O2 for 24 h resulted in increases in endothelial monolayer wound repair and eNOS activity and caused partial translocation of eNOS to plasma membrane where it co-localizes with cortical F-actin. We did not detect eNOS co-localization with stress fibers in normoxic or hyperoxic PAEC. Immunoprecipitation study revealed that mild hyperoxia increased association of eNOS with actin in both Triton X-100-soluble and Triton X-100-insoluble fractions. Knockdown of actin in PAEC by actin siRNA reduced the actin level and eNOS/actin association, and thus attenuated hyperoxia-induced increase in eNOS activity and in endothelial monolayer wound repair. These results indicate that mild hyperoxia increases eNOS activity and eNOS/actin association and that increased eNOS activity due to eNOS/actin association may contribute to mild hyperoxia-induced wound repair of PAEC. Supported by Grants from: Florida DOH, FAMRI, AHAFL, ALAF, NIH, and VA