Studies on the mechanisms and kinetics of apoptosis induced by microinjection of cytochrome c in rat kidney tubule epithelial cells (NRK-52E)

Recent reports substantiating the role of cytochrome c in the induction of apoptosis led us to examine the kinetics and mechanisms involved in this pr ocess as an extension of our ongoing studies of cell injury and cell death. Microinjection of cytochrome c into NRK-52E kidney cells produced rapid ap optosis, which usually began within 30 minutes and reached a maximum of 60- 70% by 3 hours. The changes that occurred included four phases: an initial shrinkage phase, an active phase, a spherical phase, and a necrotic phase. For morphological purposes, the progressive changes were followed by phase- contrast and fluorescence microscopy, transmission and scanning electron mi croscopy, and time-lapse video microscopy, Cells first showed shrinkage, th en displayed multiple pseudopods, which rapidly extended and retracted, giv ing the cells a bosselated appearance. During this active phase there was c hromatin condensation, mitochondria were swollen but retained membrane pote ntial, and the endoplasmic reticulum was dilated. Within 2-4 hours, active- phase cells became spherical and smooth-surfaced but were still alive, the nuclei showed chromatin clumping, the mitochondria underwent high-amplitude swelling but retained membrane potential, the endoplasmic reticulum was hi ghly dilated, acid many large apical vacuoles were present. Elevation of [C a2+](i) was seen at the late spherical phase, shortly before cell death. Pr etreatment with the caspase 3 inhibitor (Ac-DEVD-CHO) prevented apoptosis, whereas overexpression of Bcl-2 did not. Depletion of cellular ATP by cyani de inhibition of energy metabolism prevented cytochrome c from inducing the active and later phases of apoptosis, The results clearly indicate that cy tochrome c-induced apoptosis is a dynamic and energy-requiring process that has a distinct active and spherical phase before cell death.