Effect of Nitric Oxide Exposure on the Red Cell Rheology

In Relation to Oxidative Crosslinking of Membrane Proteins

Abstract

Human and rat (Sprague-Dawley, male; Body weight, 320+10 g) erythrocytes were nitrosylated anaerobically and the rheological alterations induced by exposure to air were examined. Further, acute exposure experiment on rats to 25-250 ppm NO was carried out by using an exposure chamber for single animal.
(1) The suspension viscosity of human erythrocytes (measured by a cone-plate viscometer) increased in 10-20% in low shear rates compared with the control after 4 hr exposure to air. The deformability of erythrocytes (measured by a high shear rheoscope) was not altered significantly, but the rouleaux formation of erythrocytes in autologous plasma (measured by a low shear rheoscope combined with TV-image analyzer) was suppressed. The phenomena were explained by the echinocytic transformation of nitrosylated erythrocytes after exposing to air. The biochemical analyses of erythrocyte membrane revealed (a) no changes of the composition of major fatty acids of phospholipids but (b) oxidative crosslinking of proteins by disulfide bridge formation (detected by 1% sodium dodecyl sulfate-4% polyacrylamide gel electrophoresis).
(2) The similar experiments for rat erythrocytes showed the remarkable and rapid oxidative crosslinking among membrane proteins (mainly, spectrin) and denatured globin. The crosslinking was restored completely by a reducing agent, mercaptoethanol.
(3) In in vitro experiment in rats to 25 250 ppm NO for 1 hr, 0.08-3% nitrosylhemoglobin and 0.6-20 % methemoglobin were formed dependent on exposed NO concentration. The viscosity of rat blood was not influenced by NO exposure within experimental error, but some echinocytes (with poor deformability) were detected. The oxidative crosslinking of membrane proteins as observed in in vitro experiments was not detected, perhaps due to the analytical limitation, some repairing mechanisms in erythrocytes, the selective removal of damaged erythrocytes in the reticuloendotherial system, and so forth.