Inhibition by activated neutrophils of the Ca2+ pump ATPase of intact red blood cells.
Human neutrophils, activated by phorbol myristate acetate in the presence of intact red blood cells (RBCs), caused inhibition of the Ca2+ pump ATPase of the RBCs and fragmentation of the enzyme as well as other membrane proteins. Inhibition of the Ca2+ pump ATPase of intact RBCs was directly related to the neutrophil concentration and the time of incubation. Ca2+ pump ATPase activity was partially protected by the addition of exogenous glutathione-glutathione peroxidase, but not by superoxide dismutase. The addition of sodium azide, a potent inhibitor of endogenous RBC catalase, enhanced inhibition of the Ca2+ pump ATPase of intact RBCs. Examination by SDS-polyacrylamide gel electrophoresis of membrane proteins isolated from RBCs preincubated with activated neutrophils showed gross changes in banding patterns as compared to controls. Thus, a significant amount of methemoglobin appeared to be associated with the membrane proteins, and, in general, protein bands appeared to be more diffuse and less defined than proteins in control lanes. In addition, there was an increase in the low molecular weight protein bands. Using a monoclonal antibody to the Ca2+ pump ATPase, it was shown that the 140 kDa band representing the Ca2+ pump ATPase decreased, with concomitant appearance of two low molecular weight bands running at 8.2 and 6.8 kDa in the membrane proteins from RBCs preincubated with activated neutrophils. The data are interpreted to suggest that inhibition of the Ca2+ pump ATPase in intact RBCs under these conditions occurred as a result of: neutrophil-derived superoxide, dismutation of superoxide, to H2O2, diffusion of H2O2 into RBCs, a Fenton type reaction between oxyhemoglobin, and H2O2 producing hydroxyl radical and/or a ferryl radical capable of promoting protein fragmentation of RBC membrane proteins, including the plasma membrane Ca2+ pump ATPase.
Rohn
T T
TT
Department of Pharmacology, University of Washington, Seattle 98195, USA.
Hinds
T R
TR
Vincenzi
F F
FF
D015153:Blotting, Western; D000252:Calcium-Transporting ATPases; D002478:Cells, Cultured / Q000187:drug effects; D004912:Erythrocytes / Q000502:physiology*; D005609:Free Radicals; D006801:Humans; D009504:Neutrophils / Q000502:physiology*; D017382:Reactive Oxygen Species; D013997:Time Factors
D005609:Free Radicals; D017382:Reactive Oxygen Species; D000252:Calcium-Transporting ATPases