Lead and Calcium Produce Rod Photoreceptor Cell Apoptosis by Opening the Mitochondrial Permeability Transition Pore

doi:10.1074/jbc.275.16.12175

Lead and Calcium Produce Rod Photoreceptor Cell Apoptosis by Opening the Mitochondrial Permeability Transition Pore*

From the ^‡Department of Biology and Biochemistry, and the ^¶College of Optometry, University of Houston, Houston, Texas 77204-6052

Abstract

Calcium overload is suggested to play a fundamental role in the process of rod apoptosis in chemical-induced and inherited retinal degenerations. However, this hypothesis has not been tested directly. We developed an in vitro model utilizing isolated rat retinas to determine the mechanisms underlying Ca²⁺- and/or Pb²⁺-induced retinal degeneration. Confocal microscopy, histological, and biochemical studies established that the elevated [Ca²⁺] and/or [Pb²⁺] were localized to photoreceptors and produced rod-selective apoptosis. Ca²⁺ and/or Pb²⁺ induced mitochondrial depolarization, swelling, and cytochrome c release. Subsequently caspase-9 and caspase-3 were sequentially activated. Caspase-7 and caspase-8 were not activated. The effects of Ca²⁺ and Pb²⁺ were additive and blocked completely by the mitochondrial permeability transition pore (PTP) inhibitor cyclosporin A, whereas the calcineurin inhibitor FK506 had no effect. The caspase inhibitors carbobenzoxy-Leu-Glu-His-Asp-CH₂F and carbobenzoxy-Asp-Glu-Val-Asp-CH₂F, but not carbobenzoxy-Ile-Glu-Thr-Asp-CH₂F, differentially blocked post-mitochondrial events. The levels of reduced and oxidized glutathione and pyridine nucleotides in rods were unchanged. Our results demonstrate that rod mitochondria are the target site for Ca²⁺ and Pb²⁺. Moreover, they suggest that Ca²⁺ and Pb²⁺ bind to the internal metal (Me²⁺) binding site of the PTP and subsequently open the PTP, which initiates the cytochrome c-caspase cascade of apoptosis in rods.

Footnotes

↵* This work was partially funded by National Institutes of Health Grants ES03183 and EY07024, a Fight-for-Sight Foundation Student Fellowship, a University of Houston Program for Enhanced External Research Grant, and a College of Optometry Vision Research Starter Grant.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.
↵§ Contributed equally to this work.
↵‖ To whom reprint requests should be addressed: College of Optometry, University of Houston, 4901 Calhoun Blvd., Houston, TX 77204-6052. Tel.: (713) 743-1964; Fax: (713) 743-2053; E-mail: dafox@uh.edu.
↵2 L. He, A. T. Poblenz, C. J. Medrano, and D. A. Fox, unpublished data.
↵3 L. He, A. T. Poblenz, C. J. Medrano, and D. A. Fox, unpublished observations.
Abbreviations:

PTP

permeability transition pore

CsA

cyclosporin A

ROS

reactive oxygen species

DEVD-fmk

carbobenzoxy-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluromethylketone

LEHD-fmk

carbobenzoxy-Leu-Glu(OMe)-His-Asp(OMe)-fluromethylketone

IETD-fmk

carbobenzoxy-Ile-Glu(OMe)-Thr-Asp(OMe)-fluromethylketone

YVAD-cmk

N-acetyl-Tyr-Val-Ala-Asp-chloromethylketone

DEVD-pNA

N-acetyl-Asp-Glu-Val-Asp-p-nitroanilide

PARP

poly(ADP-ribose) polymerase

DMQD-pNA

N-acetyl-Asp-Met-Gln-Asp-p-nitroanilide

LEHD-pNA

N-acetyl-Leu-Glu-His-Asp-p-nitroanilide

PKC

protein kinase C

JC-1

5,5′,6,6′-tetrachloro-1,1′,3,3′,-tetraethylbenzimidazolycarbocyanine iodide

DMQD-CHO

N-acetyl-Asp-Met-Gln-Asp-aldehyde

IBMX

3-isobutyl-1-methylxanthine

HMW

high molecular weight

EtBr

ethidium bromide

kbp

kilobase pair

AO

acridine orange

CHAPS

3-[(3-cholamidopropyl) dimethylammonil]-1-propane-sulfonate)

DEVD-CHO

N-acetyl-Asp-Glu-Val-Asp-aldehyde

ΔΨ_m

mitochondrial membrane potential
- Received November 2, 1999.
- Revision received January 4, 2000.
The American Society for Biochemistry and Molecular Biology, Inc.