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Piezo1 regulates shear-dependent nitric oxide production in human erythrocytes.

Mature circulating red blood cells (RBC) are classically viewed as passive participants in circulatory function, given erythroblasts eject their organelles during maturation. Endogenous production of nitric oxide (NO) and its effects are of particular significance; however, the integration between RBC sensation of the local environment and subsequent activation of mechano-sensitive signaling networks that generate NO remain poorly understood. The present study investigated endogenous NO-production via the RBC-specific nitric oxide synthase-isoform (RBC-NOS), connecting membrane strain with intracellular enzymatic processes. Isolated RBC were obtained from apparently healthy humans. Intracellular NO was compared at rest and following shear (cellular deformation) using semi-quantitative fluorescent imaging. Concurrently, RBC-NOS phosphorylation at its Serine1177 (ser1177) residue was measured. The contribution of cellular deformation to shear-induced NO-production in RBC was determined by rigidifying RBC with the thiol-oxidizing agent diamide; rigid RBC exhibited significantly impaired (up to 80%) capacity to generate NO via RBC-NOS during shear. Standardizing membrane strain of rigid RBC by applying increased shear did not normalize NO-production, or RBC-NOS activation. Calcium-imaging with Fluo-4 revealed that diamide-treated RBC exhibited a 42%-impairment in Piezo1-mediated calcium-movement when compared with untreated RBC. Pharmacological inhibition of Piezo1 with GsMTx4 during shear inhibited RBC-NOS activation in untreated RBC, while Piezo1-activation with Yoda1 in the absence of shear stimulated RBC-NOS activation. Collectively, a novel, mechanically-activated signaling pathway in mature RBC is described. Opening of Piezo1 and subsequent influx of calcium appears to be required for endogenous production of NO in response to mechanical shear, which is accompanied by phosphorylation of RBC-NOS at ser1177.