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.


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/cm2, 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.
Department of Bioengineering, University of California San Diego, La Jolla, Calif, USA.
N
N
Dusserre
N
N
L'Heureux
K S
KS
Bell
H Y
HY
Stevens
J
J
Yeh
L A
LA
Otte
L
L
Loufrani
J A
JA
Frangos
D000818:Animals; D001011:Aorta / Q000166:cytology / Q000201:enzymology / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism; D042783:Endothelial Cells / Q000201:enzymology; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology*; D004789:Enzyme Activation; D047468:Immunoprecipitation / Q000379:methods; D007366:Intercostal Muscles / Q000098:blood supply / Q000201:enzymology; D051379:Mice; D008810:Mice, Inbred C57BL; D008815:Mice, Inbred Strains; D018345:Mice, Knockout; D019001:Nitric Oxide Synthase / Q000276:immunology / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D013314:Stress, Mechanical; D014471:Umbilical Veins / Q000166:cytology
D019408:Platelet Endothelial Cell Adhesion Molecule-1; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; C496315:Nos3 protein, mouse; D006152:Cyclic GMP