Activation of initiator and effector caspases, mitochondrial changes involving a reduction in its membrane potential and release of cytochrome c (cyt c) into the cytosol, are characteristic features of apoptosis. These changes are associated with cell acidification in some models of apoptosis. The hierarchical relationship between these events has, however, not been deciphered. We have shown that somatostatin (SST), acting via the Src homology 2 bearing tyrosine phosphatase SHP-1, exerts cytotoxic action in MCF-7 cells, and triggers cell acidification and apoptosis. We investigated the temporal sequence of apoptotic events linking caspase activation, acidification, and mitochondrial dysfunction in this system and report here that (i) SHP-1-mediated caspase-8 activation is required for SST-induced decrease in pH i . (ii) Effector caspases are induced only when there is concomitant acidification. (iii) Decrease in pH i is necessary to induce reduction in mitochondrial membrane potential, cyt c release and caspase-9 activation and (iv) depletion of ATP ablates SST-induced cyt c release and caspase-9 activation, but not its ability to induce effector caspases and apoptosis. These data reveal that SHP-1-/caspase-8-mediated acidification occurs at a site other than the mitochondrion and that SST-induced apoptosis is not dependent on disruption of mitochondrial function and caspase-9 activation.Apoptosis is a physiological process of cell death indispensable for the maintenance of multicellular organisms. This process drives the cell into self-destruction via a common execution pathway. The cellular machinery utilized for this process creates distinct apoptotic features of cell shrinkage, cytoplasmic and nuclear condensation, membrane blebbing, chromatin compaction, and fragmentation of chromosomal DNA into 180-base pair multimers. A central event in the process of apoptosis is the activation of cysteine aspartate proteases (caspases) (1). Active caspases consist of dimeric complexes of ϳ20-and 10-kDa fragments derived from the procaspases that exist as inactive zymogens by internal proteolytic cleavage at cysteineaspartate sites (2). Mammalian caspases can be divided into initiator (e.g. caspases 2, 8, 9, 10) and effector (caspases 3, 4, 5, 6, 7, 11, 12, and 13) enzymes. A feature of apoptosis that impinges on caspases is altered mitochondrial function characterized by a reduction in the electrochemical gradient across the mitochondrial membrane (⌬ m ) 1 and release of mitochondrial cyt c into the cytoplasm (3-15). Cyt c is necessary for caspase-9 activation (16, 17). Caspase-9 can function as an initiator caspase when mitochondrial dysfunction is the primary event in apoptosis, whereas it serves to amplify the apoptotic signaling of other initiator caspases under conditions in which disruption of mitochondria is a late event (16 -19).In some models of apoptosis activation of caspases is associated with intracellular acidification (20 -23). The question of whether intracellular acidification is necessary for inducing ...