Dynamic changes in the distribution of the calcium-activated neutral protease in human red blood cells following cellular insult and altered Ca2+ homeostasis.

Mechanistic studies were conducted to examine the relationship between oxidative membrane protein damage, altered Ca2+ homeostasis, and changes in the levels of plasma membrane-bound Ca(2+)-activated neutral protease, microCANP. Alterations in the levels of plasma membrane-bound microCANP 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 microCANP levels in CHP-treated cells and hemolysate were also examined. CHP (2 mM) 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 approximately 60-kDa protein. N-acetylcysteine (20 mM), dithiothreitol (50 mM), and dimethylthiourea (50 mM) diminished CHP-mediated membrane protein damage; in contrast, dimethylfuran (50 mM) exacerbated CHP-mediated membrane protein damage. Dimethylsulfoxide (50 mM) was without significant effect. The free radical scavengers and antioxidants differentially affected membrane-bound microCANP levels largely in parallel with their ability to modulate membrane protein damage. Immunoblot analysis of 1 mM CHP-treated red cells revealed a time-dependent loss of membrane-bound microCANP, with a complete loss of microCANP monitored at 8 hr. Treatment of erythrocytes with CHP also resulted in concentration-dependent alterations in the level of membrane-bound microCANP: at 0.5 or 1.0 mM CHP a decreased level of membrane-bound microCANP was detected relative to control, whereas an increase in the level of bound enzyme was monitored from 2 to 4 mM CHP. CHP addition to hemolysate produced a decrease in membrane-bound microCANP levels comparable to that observed with erythrocytes; addition of the Ca2+ 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 Ca2+ in the presence of the Ca2+ ionophore A23187 resulted in change in the SDS-PAGE protein bands and membrane-bound microCANP levels that were comparable to those produced by CHP. Inclusion of EGTA in this system prevented microCANP binding. These data provide evidence for membrane damage and concomitant dynamic alterations in membrane-bound microCANP 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 Ca2+ in the presence of ionophore, and inhibited by EGTA or Calpain Inhibitor I.