PECAM-1 interacts with nitric oxide synthase in human endothelial cells - Implication for flow-induced nitric oxide synthase activation

Objective - We have previously shown that fluid shear stress (FSS) triggers endothelial nitric oxide synthase ( eNOS) activity in endothelial cells and that the mechanotransduction mechanisms responsible for activation discriminate between rapid changes in FSS and FSS per se. We hypothesized that the particular sublocalization of eNOS at the cell - cell junction would render it responsive to activation by FSS temporal gradients. Methods and Results - In human umbilical vein endothelial cells (HUVECs), immunofluorescence revealed strong eNOS membrane staining at the cell - cell junction colocalizing with platelet/endothelial cell adhesion molecule-1 (PECAM-1). In PECAM-1 -/- mouse aorta, eNOS junctional localization seen in the wild type was absent. Similarly, junctional staining was lost in wild-type aorta near intercostal artery branches. eNOS/PECAM-1 association in HUVECs was confirmed by coimmunoprecipitation. When HUVECs were subjected to a 0.5s impulse of 12 dynes/cm(2), a transient disruption of the eNOS/PECAM-1 complex was observed, accompanied by an increase in eNOS activity ( cGMP production). Ramped flow did not trigger complex dissociation or an increase in cGMP production. In a cell-free system, a direct inhibition of eNOS activity by PECAM-1 is shown. Conclusions - These results suggest that eNOS is complexed with PECAM-1 at the cell - cell junction and is likely involved in the modulation of eNOS activity by FSS temporal gradients but not by FSS itself.