Darren P Casey, Kenichi Ueda, Lauren Wegman-Points, Gary L Pierce

We determined if local increases in brachial artery shear during repetitive muscle contractions induce changes in protein expression of endothelial nitric oxide synthase (eNOS) and/or phosphorylated eNOS at serine 1177 (P-eNOS(ser1177)), the primary activation site on eNOS, in endothelial cells (ECs) of humans. Seven young males (25±1 yr) performed 20 separate bouts (3 min each) of rhythmic forearm exercise at 20% of max over a 2-hr period. Each bout of exercise was separated by 3 min of rest. An additional six male subjects (24±1 yr) served as time controls (no exercise). ECs were freshly isolated from the brachial artery using sterile J-wires through an arterial catheter at baseline and again following the 2 hr exercise or time control period. Expression of eNOS or P-eNOS(ser1177) in ECs was determined via immunofluorescence. Brachial artery mean shear rate was elevated compared to baseline and the time control group throughout the 2-hr exercise protocol (P<0.001). P-eNOS(ser1177) expression was increased 57% in ECs in the exercise group (0.06±0.01 vs. 0.10±0.02 a.u., P=0.02) but not in the time control group (0.08±0.01 vs. 0.07±0.01 a.u., P=0.72). In contrast, total eNOS expression did not change in either the exercise (0.13±0.04 vs. 0.12±0.03 a.u.) or time control (0.12±0.03 vs. 0.11±0.03 a.u.) group (P>0.05 for both). Our novel results suggest that elevations in brachial artery shear increase eNOS(ser1177) phosphorylation in the absence of changes in total eNOS in ECs of young healthy males, suggesting that this model is sufficient to alter post-translational modification of eNOS activity in vivo in humans.