Proceedings of The Physiological Society

University of Oxford (2011) Proc Physiol Soc 23, C24

Oral Communications

Exercise increases endothelial nitric oxide synthase phosphorylation in the microvasculature and sarcolemma of human skeletal muscle

M. Cocks1, S. Shepherd1, A. M. Ranasinghe2, T. A. Barker2, C. S. Shaw1, A. J. Wagenmakers1

1. Sport and Exercise Sciences, University of Birmingham, Birmingham, United Kingdom. 2. Clinical and Experimental Medicine, University of Birmingham, Birmingham, United Kingdom.


Nitric oxide (NO) plays a multifaceted role in the physiological responses and adaptive processes during and following exercise within the skeletal muscle and its microvasculature. Endothelial nitric oxide synthase (eNOS) is one of the three NOS isoforms responsible for synthesis of NO. eNOS is expressed in both vascular endothelial cells and skeletal muscle and therefore changes in its activity may be responsible for the increase in NO production during exercise. The aim of the current study was to assess eNOS serine1177 phosphorylation in response to acute exercise, using quantitative immunofluorescence microscopy in both the skeletal muscle sarcolemma and microvascular endothelium in humans. Eight young sedentary males (age 21 ± 2 years, BMI 23 ± 1 kg/m2, VO2peak 42 ± 8 ml.kg.min-1) completed one hour of cycling at approximately 65% VO2peak. Muscle biopsies (1% lidocaine as local anaesthesia) from the vastus lateralis were taken prior to, and immediately after exercise. eNOS serine1177 phosphorylation was assessed in the skeletal muscle microvascular endothelium and sarcolemma independently, using quantitative immunofluorescence microscopy. eNOS serine1177 phosphorylation is displayed as fold change from rest ± standard error. eNOS serine1177 staining was present in the microvessels in both resting and post exercise images, and colocalised with the endothelial marker UEA-I-FITC. eNOS serine1177 staining was also present at the sarcolemma in both resting and post exercise images, and colocalised with the membrane marker wheat germ agglutinin-350. Exercise increased endothelial eNOS serine1177 phosphorylation by 25% (1.25 ± 0.08 fold change from resting, P < 0.05). Exercise also increased sarcolemmal eNOS serine1177 phosphorylation by 14% (1.14 ± 0.05 fold change from rest, P < 0.05). We have shown that eNOS serine1177 phosphorylation is increased following exercise in both the sarcolemma and microvascular endothelium in skeletal muscle. These results suggest that eNOS is at least partially responsible for NO production in both the microvasculature and sarcolemma during exercise. Quantitative immunofluorescence microscopy allowed for the independent analysis of eNOS phosphorylation in the microvasculature and sarcolemma of skeletal muscle. This novel approach may improve our understanding of the mechanisms and location of increased NO production within the skeletal muscle in response to exercise.

Where applicable, experiments conform with Society ethical requirements