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

Jean François Dumas,Laurent Argaud,Cécile Cottet-Rousselle,Guillaume Vial,Cécile Gonzalez,Dominique Detaille,Xavier Leverve,Eric Fontaine

doi:10.1074/jbc.m900926200

Abstract

To study the effect of mitochondrial permeability transition pore (PTP) opening on NAD(P)H localization, intact cells were exposed to the Ca(2+) 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, tetramethylrhodamine methyl ester, 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, whereas 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, whereas 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.

Highlights

Under normal physiological conditions, the mitochondrial inner membrane is impermeable to almost all metabolites and ions
When incubated in the standard media used with isolated organelles, permeability transition pore (PTP) opening leads to mitochondrial swelling because of the presence of matrix proteins that cannot diffuse through the open pore, creating an oncotic pressure gradient
This oncotic pressure gradient can be eliminated by incubating mitochondria in media containing osmotic molecules larger than 1500 Da, a particular condition in which PTP opening occurs in the absence of mitochondrial swelling [9]

Summary

Introduction

The mitochondrial inner membrane is impermeable to almost all metabolites and ions. Observations where calcein decompartmentalization occurred without mitochondrial depolarization indicate that PTP opening may be reversible in intact cells [16]. It is not known whether PTP opening inhibits Complex 1 in intact cells, as it does in isolated mitochondria.

Results

Conclusion