Changes in intramitochondrial and cytosolic pH: early events that modulatecaspase activation during apoptosis

Mitochondria trigger apoptosis by releasing caspase activators, including c ytochrome c (cytC). Here we show, using a pH-sensitive green fluorescent pr otein (GFP), that mitochondria-dependent apoptotic stimuli (such as Bax, st aurosporine and ultraviolet irradiation) induce rapid, Bcl-2-inhibitable mi tochondrial alkalinization and cytosol acidification, followed by cytC rele ase, caspase activation and mitochondrial swelling and depolarization. Thes e events are not induced by mitochondria-independent apoptotic stimuli, suc h as Fas. Activation of cytosolic caspases by cytC in vitro is minimal at n eutral pH, but maximal at acidic pH, indicating that mitochondria-induced a cidification of the cytosol may be important for caspase activation; this f inding is supported by results obtained from cells using protonophores. Cyt osol acidification and cytC release are suppressed by oligomycin, a FoF1-AT Pase/H+-pump inhibitor, but not by caspase inhibitors. Ectopic expression o f Bax in wild-type, but not FoF1/H+-pump-deficient, yeast cells similarly r esults in mitochondrial matrix alkalinization, cytosol acidification and ce ll death. These findings indicate that mitochondria-mediated alteration of intracellular pH may be an early event that regulates caspase activation in the mitochondrial pathway for apoptosis.