Nitric oxide (NO), a vascular signaling molecule, is primarily produced by endothelial NO synthase. Recently, a functional endothelial NO synthase (eNOS) was described in red blood cells (RBC). The RBC-eNOS contributes to the...
moreNitric oxide (NO), a vascular signaling molecule, is primarily produced by endothelial NO synthase. Recently, a functional endothelial NO synthase (eNOS) was described in red blood cells (RBC). The RBC-eNOS contributes to the intravascular NO pool and regulates physiological functions. However the regulatory mechanisms and clinical implications of RBC-eNOS are unknown. The present study investigated regulation and functions of RBC-eNOS under mechanical stimulation. This study shows that mechanical stimuli perturb RBC membrane, which triggers a signaling cascade to activate the eNOS. Extracellular NO level, estimated by the 4-Amino-5-Methylamino-2′, 7′-Difluorofluorescein Diacetate probe, was significantly increased under mechanical stimuli. Immunostaining and western blot studies confirmed that the mechanical stimuli phosphorylate the serine 1177 moiety of RBC-eNOS, and activates the enzyme. The NO produced by activation of RBC-eNOS in vortexed RBCs promoted important endothelial functions such as migration and vascular sprouting. We also show that mechanical perturbation facilitates nitrosylation of RBC proteins via eNOS activation. The results of the study confirm that mechanical perturbations sensitize RBC-eNOS to produce NO, which ultimately defines physiological boundaries of RBC structure and functions. Therefore, we propose that mild physical perturbations before, after, or during storage can improve viability of RBCs in blood banks. The work of Kosaka et al. 1 suggested that hemolysate of erythrocyte membrane has endothelial nitric oxide syn-thase (eNOS) catalytic activity 1. The study of Cortese-Krott et al. 2 demonstrated that the red blood cells (RBCs) possess eNOS and produce nitric oxide (NO) 2. The work of Kleinbongard et al. 3 confirmed that RBC carry eNOS enzyme to generate NO production, and that eNOS localizes in the cytoplasm and plasma membrane of the RBC 3. It is evident that shear stress can regulate NO production by RBC 4,5. In addition, the modulation of NO levels due to activation or inhibition of RBC-eNOS can regulate deformability of RBC membranes 6,7. However, the effects of routine physical perturbations on RBCs in motion have not been examined. This is of particular relevance, since RBCs have structural flexibility in order to fit themselves into both aorta and capillary loops as they traverse through the varied and versatile architecture of the vasculature. In the presence of such complex physical perturbations, RBC membranes can be altered transiently or for longer periods due to presence of a " shape memory " 8. Also, localized strain at a given position on the biconcave RBC surface and resilience within the RBC membrane, may result in uneven physical perturbations which may transiently activate NO production 9. RBCs also collide with each other and other cell types during their sojourn through blood vessels 10. Therefore, our proposition is that colliding RBCs are always under " Off and On " mode of NO production in a given laminar flow condition. This occurs because the deformability of the RBC membrane alters as it undergoes transient changes in shape during each collision. In this context, we hypothesized that a tightly controlled mechano-transduction pathway regulates NO production in RBC. Hemo-rheological disturbances in the patho-physiological microenvi-ronment are associated with aggregation and local accumulation of RBC in microvascular lumina, thus entailing disorders of blood flow. Abnormal blood flow and apparently static milieu of aggregated RBC in hematoma could potentially result in loss of a critical NO pool. Therefore, our primary experimental model involved measurement of NO production by static and mechanically perturbed human RBCs in suspension. After standardizing
