Mitochondrial Nitric-oxide Synthase Stimulation Causes Cytochromec Release from Isolated Mitochondria

doi:10.1074/jbc.274.44.31185

Mitochondrial Nitric-oxide Synthase Stimulation Causes Cytochromec Release from Isolated Mitochondria

EVIDENCE FOR INTRAMITOCHONDRIAL PEROXYNITRITE FORMATION*

From the Institute of Biochemistry, Swiss Federal Institute of Technology (ETH), CH-8092 Zurich, Switzerland

Abstract

Nitric oxide (NO) is synthesized by members of the NO synthase (NOS) family. Recently the existence of a mitochondrial NOS (mtNOS), its Ca²⁺ dependence, and its relevance for mitochondrial bioenergetics was reported (Ghafourifar, P., and Richter, C. (1997) FEBS Lett. 418, 291–296; Giulivi, C., Poderoso, J. J., and Boveris, A. (1998) J. Biol. Chem. 273, 11038–11043). Here we report on the possible involvement of mtNOS in apoptosis. We show that uptake of Ca²⁺ by mitochondria triggers mtNOS activity and causes the release of cytochrome c from isolated mitochondria in a Bcl-2-sensitive manner. mtNOS-induced cytochrome c release was paralleled by increased lipid peroxidation. The release of cytochrome c as well as increase in lipid peroxidation were prevented by NOS inhibitors, a superoxide dismutase mimic, and a peroxynitrite scavenger. We show that mtNOS-induced cytochromec release is not mediated via the mitochondrial permeability transition pore because the release was aggravated by cyclosporin A and abolished by blockade of mitochondrial calcium uptake by ruthenium red. We conclude that, upon Ca²⁺-induced mtNOS activation, peroxynitrite is formed within mitochondria, which causes the release of cytochrome c from isolated mitochondria, and we propose a mechanism by which elevated Ca²⁺ levels induce apoptosis.

Footnotes

↵* This work was financially supported by the Kantonale Zürcherische Krebsliga (Zurich, Switzerland) (to P. G.) and by Leica and Avina-Foundation (Switzerland) (to S.D.K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵‡ To whom correspondence should be addressed. Tel.: +41 1 632 31 36; Fax: +41 1 632 11 21; E-mail: pedramgf@bc.biol.ethz.ch.
Abbreviations:

NO

nitric oxide

NOS

nitric-oxide synthase

mtNOS

mitochondrial nitric-oxide synthase

ΔΨ

mitochondrial transmembrane potential

ONOO⁻

peroxynitrite

SOD

superoxide dismutase

LPO

lipid peroxidation

l-NMMA

N ^ω-monomethyl-l-arginine

1400W

N-(3-(aminomethyl)benzyl) acetamidine

MnTBAP

manganese (III) tetrakis (4-benzoic acid) porphyrin

BH₄

tetrahydrobiopterin

CSA

cyclosporin A

RR

ruthenium red
- Received June 2, 1999.
The American Society for Biochemistry and Molecular Biology, Inc.