o-methyl-serine-dodecylamide-hydrochloride and benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone

Apoptosis is often associated with acidification of the cytosol and since loss of lysosomal proton gradient and release of lysosomal content are early events during apoptosis, we investigated if the lysosomal compartment could contribute to cytosolic acidification. After exposure of U937 cells to tumor necrosis factor-alpha, three populations; healthy, pre-apoptotic, and apoptotic cells, were identified by flow cytometry. These populations were investigated regarding intra-cellular pH and apoptosis-associated events. There was a drop in cytosolic pH from 7.2 +/- 0.1 in healthy cells to 6.8 +/- 0.1 in pre-apoptotic, caspase-negative cells. In apoptotic, caspase-positive cells, the pH was further decreased to 5.7 +/- 0.04. The cytosolic acidification was not affected by addition of specific inhibitors towards caspases or the mitochondrial F(0)F(1)-ATPase. In parallel to the cytosolic acidification, a rise in lysosomal pH from 4.3 +/- 0.3, in the healthy population, to 4.8 +/- 0.3 and 5.5 +/- 0.3 in the pre-apoptotic- and apoptotic populations, respectively, was detected. In addition, lysosomal membrane permeability increased as detected as release of cathepsin D from lysosomes to the cytosol in pre-apoptotic and apoptotic cells. We, thus, suggest that lysosomal proton release is the cause of the cytosolic acidification of U937 cells exposed to TNF-alpha.

Topics: Amides; Amino Acid Chloromethyl Ketones; Apoptosis; Caspase 3; Caspase 8; Caspase Inhibitors; Caspases; Cathepsin D; Cell Nucleus; Cell Size; Cytosol; Detergents; Enzyme Inhibitors; Humans; Hydrogen-Ion Concentration; Intracellular Membranes; Lysosomes; Mitochondrial Proton-Translocating ATPases; Oligomycins; Permeability; Phosphatidylserines; Protein Transport; Protons; Serine; Tumor Necrosis Factor-alpha; U937 Cells