Endothelial nitric oxide synthase phosphorylation in treadmill‐running mice: role of vascular signalling kinases

Abstract

The intracellular signalling kinases Akt/protein kinase B (Akt), protein kinase A (PKA) and adenosine monophosphate-activated protein kinase (AMPK) are phosphorylated in response to increased mechanical force or perfusion rate in cultured endothelial cells or isolated blood vessels. All three kinases phosphorylate endothelial nitric oxide synthase (eNOS) on serine (S) 1177, while Akt and PKA additionally phosphorylate eNOS on S617 and S635 respectively. Although these kinases might contribute to subsequent activation of eNOS during dynamic exercise, the specific mediators of exercise-induced eNOS phosphorylation and activation in vivo are unknown. We determined the impact of 50 min of treadmill running on the phosphorylation of Akt, AMPK, cyclic adenosine monophosphate response element binding protein (CREB -a target of PKA) and eNOS (S 1177, 635 and 617 and threonine (T) 495) in the presence or absence of pharmacological inhibition of PI3 kinase (PI3K) and Akt signalling using wortmannin. Compared to arteries from sedentary mice, eNOS enzyme activity was greater in vessels from treadmill-running animals and was associated with increased phosphorylation of Akt (S473), CREB (S133), AMPK (T172), and eNOS at S1177 and S617 but not at S635 or T495. These data suggest that Akt signalling is a major mediator of eNOS activation. To confirm this, treadmill-running was performed in the presence of vehicle (DMSO) or PI3K inhibition. Compared to results from sedentary mice, vascular Akt phosphorylation and eNOS phosphorylation at S617 during treadmill-running were prevented by wortmannin but not vehicle treatment, whereas exercise-related increases in AMPK and CREB phosphorylation were similar between groups. Arterial eNOS phosphorylation at S1177 increased during exercise after wortmannin treatment relative to values obtained from sedentary animals, but the elevation was blunted by ∼50% compared to results from vehicle-treated mice. These findings indicate that Akt and AMPK contribute importantly to vascular eNOS S1177 phosphorylation during treadmill-running, and that AMPK is sufficient to activate p-eNOS S1177 in the presence of PI3K inhibition. Frictional forces along the surface of the endothelium exerted by flowing blood are potent stimuli for endothelial nitric oxide (NO) production (Jo et al. 1997). These forces are proportional to the product of blood viscosity and blood velocity at the endothelial wall and collectively have been termed 'endothelial shear stress' . Endothelial surfaces possess mechanoreceptors that detect shear stress and data concerning the pathways responsible for phosphorylation and activation of endothelial NO synthase (eNOS) are emerging (

Exercise training improves endothelial function via adiponectin-dependent and independent pathways in type 2 diabetic mice

“…Previous studies showed inconsistent effects of exercise on eNOS expression in the vasculature. For example, several groups reported that both low and moderate exercise increased eNOS protein expression, whereas others indicated that exercise did not change the expression of eNOS in the vasculature (12,32,37,55). In addition, Bitar et al (5) showed that diabetes increased eNOS protein, whereas our previous study showed that diabetes status was not associated with a change in eNOS protein expression within the aorta (54).…”

Delayed preconditioning prevents ischemia/reperfusion-induced endothelial injury in rats: role of ROS and eNOS

“…27,28,[39][40][41][42] eNOSderived NO has been known to have a pivotal role in the modulation of vascular tone and function. 6,7 Taken together, our results allow us to propose that, ROS/RNS excess in peripheral resistance vessels during I/R injury may blunt PI3K/Akt/eNOS pathway, thus triggering endothelial dysfunction. A schematic representation of the proposed mechanism is shown in Figure 10.…”

“…It should be noted that NO is the primary regulator of vascular function, because it induces vasodilatation, inhibits platelet adhesion and aggregation, and reduces leukocyte-endothelial cell interaction. [5][6][7] This could explain the critical role of NO in IPC-induced endothelial protection.…”

“…[1][2][3][4] Moreover, the arterial endothelial nitric oxide synthase (eNOS) protein expression is severely reduced after I/R injury. 3 Given the well-known role of nitric oxide (NO) not only as a vasodilator but also as an inhibitor of neutrophil adhesion and platelet aggregation, [5][6][7] the amelioration of the endothelial dysfunction associated with I/R through the maintenance of an adequate level of eNOS could be important. Reactive oxygen/nitrogen species (ROS/RNS) generation on reperfusion is also generally accepted as a contributing factor to I/R-induced vascular injury.…”

The Physiological Responses to 5000 m Open Water Swimming Exercise in Children

“…It has been reported that both acute and regular aerobic exercise in swimmers and sedentary group increase blood NOx levels. 23,47,48 The best-established stimulus of NO production is shear stress, which increases during increased blood flow and can increase NOS activity. Furthermore, it has been reported that all isoforms of NOS can be regulated by transcription with hypoxia.…”