Dynamic changes in the distribution of the calcium-activated neutral protease in human red blood cells following cellular insult and altered Ca 2+ homeostasis

Abstract

Mechanistic studies were conducted to examine the relationship between oxidative membrane protein damage, altered Ca 2+ homeostasis, and changes in the levels of plasma membrane-bound Ca 2+-activated neutral protease, μCANP. Alterations in the levels of plasma membrane-bound μCANP in erythrocytes and hemolysate following cumene hydroperoxide (CHP) insult were monitored using SDS-PAGE and immunoblot analyses. Free radical scavengers, antioxidant and EGTA effects on membrane-bound μCANP levels in CHP-treated cells and hemolysate were also examined. CHP (2 m m) addition to red cells caused a significant decrease/loss in intensity of numerous protein bands in the SDS-PAGE pattern, to include bands 1,2,2.1,4.1,4.2, and an ∼60-kDa protein. N-acetylcysteine (20 m m), dithiothreitol (50 m m), and dimethylthiourea (50 m m) diminished CHP-mediated membrane protein damage; in contrast, dimethylfuran (50 m m) exacerbated CHP-mediated membrane protein damage. Dimethylsulfoxide (50 m m) was without significant effect. The free radical scavengers and antioxidants differentially affected membrane-bound μCANP levels largely in parallel with their ability to modulate membrane protein damage. Immunoblot analysis of 1 m m CHP-treated red cells revealed a time-dependent loss of membrane-bound μCANP, with a complete loss of μCANP monitored at 8 hr. Treatment of erythrocytes with CHP also resulted in concentration-dependent alterations in the level of membrane-bound μCANP: at 0.5 or 1.0 m m CHP a decreased level of membrane-bound μCANP was detected relative to control, whereas an increase in the level of bound enzyme was monitored from 2 to 4 m m CHP. CHP addition to hemolysate produced a decrease in membrane-bound μCANP levels comparable to that observed with erythrocytes; addition of the Ca 2+ chelator EGTA or Calpain Inhibitor I ( N-acetyl-leucyl-leucyl-leucyl-nor-leucinal) to hemolysate effectively inhibited this decrease. In contrast, treatment of erythrocytes with Ca 2+ in the presence of the Ca 2+ ionophore A23187 resulted in changes in the SDS-PAGE protein bands and membrane-bound μCANP levels that were comparable to those produced by CHP. Inclusion of EGTA in this system prevented μCANP binding. These data provide evidence for membrane damage and concomitant dynamic alterations in membrane-bound μCANP levels in the red cell or hemolysate following oxidative insult, and show that this process can be modulated by free radical scavengers and antioxidant, simulated by treating cells with Ca 2+ in the presence of ionophore, and inhibited by EGTA or Calpain Inhibitor I.