Enhancement of Pressure-Induced Hemolysis by Aquaporin-1 Inhibitors in Human Erythrocytes

Abstract

Abstract Pressure-induced hemolysis reflects sensitively membrane perturbations in erythrocytes. The functions of transporters and channels are inhibited by sodium p-chloromercuribenzoate (pCMB). So, we tested the effects of pCMB on hemolysis of human erythrocytes at 200 MPa. Pressure-induced hemolysis was enhanced by pCMB, but restored by dithiothreitol. Such effects of pCMB were also observed in N-ethylmaleimide-pretreated erythrocytes. Similar hemolytic properties were observed by aquaporin-1 (AQP1) inhibitors such Hg2+ and Au3+, but not by inhibitors of Na+–K+ pump, Gardos channels, and glucose transporter. In fact, the results of atomic absorption spectrometry showed the binding of mercury to AQP1. From the 1H spin–spin relaxation time of water, we found that pressure-induced hemolysis was enhanced upon inhibition of water transport. Flow cytometric analysis demonstrated the enhancement of hemolysis by suppression of fragmentation under pressure. Detachment of cytoskeletal proteins from the membrane by low ionic strength buffer was unaffected by pCMB. Moreover, diameters of vesicles released from intact and pCMB-treated erythrocytes at 200 MPa were 438 and 584 nm, respectively. In both vesicles, spectrin contents were almost the same. Taken together, these data suggest that pCMB bound to AQP1 inhibits water transport and enhances the hemolysis due to the suppression of fragmentation under pressure, but not affect membrane–cytoskeleton interactions.