Proteasome inhibition increases eNOS phosphorylation in cultured vascular endothelial cells exposed to shear stress

It is known that temporal gradients in shear stress lead to a transient increase in endothelial NO synthase (eNOS) phosphorylation at S1177/activation. Proteasome inhibition is known to reduce eNOS activation in statically‐cultured endothelial cells. The proteasome role in shear stress‐induced eNOS activation hasn’t been clarified. When cultured human umbilical vein endothelial cells (HUVECs) were exposed to steady laminar shear stress (10 dynes/cm 2 ), eNOS phosphorylation at S1177 and Akt phosphorylation at S473 occurred at 15 min, peaked at 30 min and declined by 60 min. Some cells were incubated with wortmannin (500 nM), MG132 (10 μM) or a combination for 30 min prior to 30 min shear exposure. eNOS and Akt phosphorylation were reduced by wortmannin, but were enhanced by MG132, whereas wortmannin+MG132 reversed the MG132‐induced phosphorylation increases. Preincubation with MG132 increased Akt protein levels without changing eNOS, PDK1 or HSP‐90 protein levels compared to sheared controls. Hence, short‐term proteasome inhibition transiently enhances the shear‐induced increase in eNOS activation and the PI3K/Akt pathway is responsible for this effect. When HUVECs were flow‐conditioned under normoxic conditions (10 dynes/cm 2 , 20% O 2 , 16 h) followed by ischemia (0.5 dynes/cm 2 , <1% O 2 , 2 h)/reperfusion (10 dynes/cm2, 20% O 2 , 2 h) and MG132 was added during ischemia, MG132 enhanced the reperfusion‐induced increase in eNOS phosphorylation. Proteasome inhibition may improve endothelial function and diminish the reperfusion‐associated injury by enhancing eNOS activity. Funding by NIH RO1 HL67027 (B.R.A.).