Muscle contraction induced arterial shear stress increases endothelial nitric oxide synthase phosphorylation in humans

Abstract

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 (p-)eNOS at Ser, the primary activation site on eNOS, in endothelial cells (ECs) of humans. Seven young male subjects (25 ± 1 yr) performed 20 separate bouts (3 min each) of rhythmic forearm exercise at 20% of maximum over a 2-h 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 after the 2-h exercise or time control period. Expression of eNOS or p-eNOS Ser in ECs was determined via immunofluorescence. Brachial artery mean shear rate was elevated compared with baseline and the time control group throughout the 2-h exercise protocol ( < 0.001). p-eNOS Ser expression was increased 57% in ECs in the exercise group [0.06 ± 0.01 vs. 0.10 ± 0.02 arbitrary units (au), = 0.02] but not in the time control group (0.08 ± 0.01 vs. 0.07 ± 0.01 au, = 0.72). In contrast, total eNOS expression did not change in either the exercise (0.13 ± 0.04 vs. 0.12 ± 0.03 au) or time control (0.12 ± 0.03 vs. 0.11 ± 0.03 au) group ( > 0.05 for both). Our novel results suggest that elevations in brachial artery shear increase eNOS Ser phosphorylation in the absence of changes in total eNOS in ECs of young healthy male subjects, suggesting that this model is sufficient to alter posttranslational modification of eNOS activity in vivo in humans. Elevations in brachial artery shear in response to forearm exercise increased endothelial nitric oxide synthase Ser phosphorylation in brachial artery endothelial cells of healthy humans. Our present study provides the first evidence in humans that muscle contraction-induced increases in conduit arterial shear lead to in vivo posttranslational modification of endothelial nitric oxide synthase activity in endothelial cells.

Flow does not alter eNOS phosphoryation at Ser1179 or Thr495 in preconstricted mouse mesenteric arteries

“…) and was likely in the study of human brachial artery, as evidenced by the increase in heart rate (Casey et al. ). Thus, it is possible that the increase in phosphorylation could be due to other factors during exercise rather than the shear stress exclusively, but this is challenging to ascertain with an exercise stimulus in vivo.…”

“…; Casey et al. ). Exercise, however, promotes more than just an increase in shear stress and is likely to lead to neuro‐humoral changes.…”

“…; Casey et al. ). eNOS is activated by increased endothelial cell calcium and phosphorylation at one or more sites, although the relative importance of each of these events to shear stress‐induced activation is not clear (Dimmeler et al.…”

“…Many arteries in the body dilate in response to increased blood flow, such as during exercise or after acute ischemia (Markos et al 2013;Green et al 2014Green et al , 2017. Increased flow causes shear stress on the endothelial cells that line the artery, and the resulting vasodilation is due at least in part to activation of the endothelium enzyme, eNOS, which generates nitric oxide and causes smooth muscle relaxation (Gilligan et al 1994;Dimmeler et al 1999;Boo and Jo 2003;Sessa 2004;Goto et al 2007;Balligand et al 2009;Green et al 2014;Casey et al 2017). eNOS is activated by increased endothelial cell calcium and phosphorylation at one or more sites, although the relative importance of each of these events to shear stressinduced activation is not clear (Dimmeler et al 1999;Boo and Jo 2003;Sessa 2004;Balligand et al 2009;Rafikov et al 2011).…”

Thirty minutes of handgrip exercise potentiates flow‐mediated dilatation in response to sustained and transient shear stress stimuli to a similar extent

“…This probably involves greater endothelial nitric oxide synthase activity, and an increase in phosphorylated endothelial nitric oxide synthase content has been observed in brachial artery endothelial cells after a session of handgrip exercise (Casey et al, 2017). This study was not designed to evaluate mechanisms of FMD potentiation.…”