Effect of Transient and Permanent Permeability Transition Pore Opening on NAD(P)H Localization in Intact Cells

Abstract

In order to study the effect of mitochondrial Permeability Transition Pore (PTP) opening on NAD(P)H localization, intact cells were exposed to the Ca2+ ionophore A23187. PTP opening, mitochondrial membrane potential, mitochondrial volume and NAD(P)H localization were assessed by time-lapse laser confocal microscopy using the calcein-cobalt technique, TMRM, MitoTracker and NAD(P)H autofluorescence respectively. Concomitant with PTP opening, NAD(P)H fluorescence increased outside mitochondria. These events occurred in all cells and were prevented by cyclosporin A. Mitochondrial membrane potential was not systematically collapsed while mitochondrial volume did not change, confirming that A23187 induced transient PTP opening in a subpopulation of cells, and suggesting that mitochondrial swelling did not immediately occur after PTP opening in intact cells. NAD(P)H autofluorescence remained elevated after PTP opening, particularly after membrane potential had been collapsed by an uncoupler. Extraction of nucleotide for NAD(P)H quantification confirmed that PTP opening led to an increase in NAD(P)H content. Because the oxygen consumption rate decreased while the lactate/pyruvate ratio increased after PTP opening in intact cells, we conclude that PTP opening inhibits respiration and dramatically affects the cytosolic redox potential in intact cells.