Intracellular pH-dependent peroxynitrite-evoked synergistic death of glucose-deprived astrocytes

Previously, we reported that glucose-deprived astrocytes were highly vulnerable to peroxynitrite (ONOO^-). Here we demonstrate that the increased vulnerability caused by glucose deprivation and ONOO^- depends on intracellular pH. The ONOO^- releasing reagent 3-morpholinosydnonimine (SIN-1) markedly induced the release of lactate dehydrogenase (LDH, the marker of cytotoxicity) in glucose-deprived astrocytes. Morphological studies and caspase activity assay showed that astrocytes treated together with glucose deprivation and ONOO^- died mostly in a necrotic mode. Alkalinization of pH from 7.4 to 7.8 increased LDH release, whereas acidification from pH 7.4 to 7.0 decreased it. However, intracellular pH (pH_i), not extracellular pH (pH_e), appeared to play a critical role in the synergistic death. Thus, without a change in pH_e (7.4) cytosolic acidification by a weak acid salt, sodium acetate, and a Na⁺/H⁺ antiporter inhibitor, amiloride, reduced LDH release. In contrast, a weak base, NH₄Cl, and a Na⁺/H ⁺ antiporter stimulator, monensin, increased pH_i and greatly enhanced LDH release. The augmented death was found to be due, in part, to the preceding decrease in the level of reduced glutathione, the ONOO ^- scavenger, and collapse of the mitochondrial transmembrane potential at alkaline pH.