Modulation of red blood cell nitric oxide synthase phosphorylation in the quiescent and exercising human forearm

Abstract

In vitro investigations demonstrate that human erythrocytes synthesise nitric oxide via a functional isoform of endothelial nitric oxide synthase (RBC-NOS). We tested the hypothesis that phosphorylation of RBC-NOS at serine residue 1177 (RBC-NOS1177) would be amplified in blood draining active skeletal muscle. Further, given hypoxaemia modulates local blood flow and thus shear stress, and nitric oxide availability, we performed duplicate experiments under normoxia and hypoxia. Nine healthy volunteers performed rhythmic handgrip exercise at 60% of individualised maximal workload for 3.5 min while breathing room air (normoxia) and after being titrated to an arterial oxygen saturation 80% (hypoxaemia). We measured brachial artery blood flow and vascular conductance by high resolution Duplex Ultrasound and photoplethysmography, respectively. Blood viscosity was measured to facilitate calculation of accurate shear stresses. Erythrocytes were assessed for levels of phosphorylated RBC-NOS1177 and cellular deformability from blood collected at rest and exercise. Forearm exercise increased blood flow, vascular conductance, and vascular shear stress, which coincided with a 2.7 ± 0.6-fold increase in RBC-NOS1177 phosphorylation (p < 0.0001) and increased cellular deformability (p < 0.0001) under normoxia. When compared to normoxia, hypoxaemia elevated vascular conductance and shear stress (p < 0.05) at rest, while cellular deformability (p < 0.01) and RBC-NOS1177 phosphorylation (p < 0.01) increased. Hypoxaemic exercise elicited further increases in vascular conductance, shear stress, and cell deformability (p < 0.0001), although a subject-specific response in RBC-NOS1177 phosphorylation was observed. Our data yields novel insights into the manner that haemodynamic force and oxygen tension modulates RBC-NOS in vivo.