Abstract

The ability of the therapeutic agents phenelzine (PZ) and hydralazine (HD) to stimulate the rate of protein degradation and H2O2 production in human red blood cells (RBC) was characterized. PZ- and HD-stimulated proteolysis, as monitored either by a fluorescence assay or by high-pressure liquid chromatography, occurred in a dose-, time- and hematocrit-dependent manner. The more potent PZ (0.5 mM), in RBC suspension or hemolysate, stimulated the rate of leucine release by 131 and 63%, respectively, whereas HD (1.0 mM) in RBC suspension or hemolysate produced increases of 133 and 66% in the rate of leucine release, respectively. PZ (0.75 mM) addition to red cells resulted in a rapid stimulation of H2O2 generation during the first hour of incubation, whereas HD (0.75 mM) addition to red cells produced a gradual increase in the rate of H2O2 production over 5 h of incubation. Substantial inhibition of PZ- and HD-stimulated proteolysis in RBC was observed with N-acetylcysteine, N-ethylmaleimide and the inhibitors of methemoglobin reduction, NADP and 2'AMP. In contrast, antioxidants dithiothreitol, dimethylthiourea, dimethyl sulfoxide and dimethylfuran had little effect on the rates of PZ- and HD-stimulated protein degradation. Western blot analysis demonstrated little change in the membrane-bound levels of the calcium-activated neutral protease calpain after incubation with PZ or HD. However, PZ- and HD-stimulated amino acid release was inhibited (approximately 30-50%) by the calcium chelator EGTA, suggesting a potential role for calcium-activated neutral protease and divalent metal cations in PZ- and HD-stimulated proteolysis.