Abstract
Disturbances of endoplasmic reticulum (ER) Ca2+ homeostasis or protein processing can lead to ER stress-induced cell death. Increasing evidence suggests that oxidative stress (OS) plays an important role in a variety of cell death mechanisms. To investigate the role of OS in ER stress, we measured OS in response to three ER stress agents: econazole (Ec), which stimulates ER Ca2+ release and blocks Ca2+ influx; thapsigargin (Tg), a sarco(endo)plasmic reticulum Ca2+ ATPase inhibitor that releases ER Ca2+ and stimulates Ca2+ influx; and tunicamycin (Tu), a glycosylation inhibitor that causes protein accumulation in the ER. Ec, but not Tg or Tu, caused a rapid increase in OS. Reactive oxygen species (ROS) generation was observed within mitochondria immediately after exposure to Ec. Furthermore, Ec hyperpolarized the mitochondrial membrane and inhibited adenine nucleotide transport in cell-free mitochondria, suggesting a mitochondrial target. Antimycin A, an inhibitor of complex III in electron transport, reversed mitochondrial hyperpolarization, OS generation, ER Ca2+ depletion, and cell death by Ec, suggesting complex III dependence for these effects. Antioxidants butylated hydroxytoluene and N-Acetyl-l-cysteine prevented ER Ca2+ depletion and cell death by Ec. However, inhibition of Ca2+ influx by Ec was unaffected by either antimycin A or the antioxidants, suggesting that this target is distinct from the mitochondrial target of Ec. Atractyloside, an adenine nucleotide transport inhibitor, generated ROS and stimulated ER Ca2+ release, but it did not block Ca2+ influx, deplete the ER or induce cell death. Taken together, these results demonstrate that combined mitochondrial ROS generation and Ca2+ influx blockade by Ec is required for cell death.
Footnotes
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This work was supported by grants from the National Cancer Institute of Canada (to S.A.B.).
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ABBREVIATIONS: ER, endoplasmic reticulum; SERCA, sarco(endo)plasmic reticulum Ca2+ ATPase; Tg, thapsigargin; Ec, econazole; Atra, atractyloside; ROS, reactive oxygen species; OS, oxidative stress; Tu, tunicamycin; FBS, fetal bovine serum; AA, antimycin A; BHT, butylated hydroxytoluene; Nac, N-Acetyl-l-cysteine; PI, propidium iodide; CM-H2DCFDA, 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate; AM, acetoxymethyl ester; MMP, mitochondrial membrane potential; JC-1, 5,5′6,6′-tetraethylbenzimidazolcarbocyanine iodide; FCCP, carbonyl cyanide p-(trifluomethoxy) phenylhydrazone; ANT, adenine nucleotide transporter; PBS, phosphate-buffered saline; PMA, phorbol 12-myristate 13-acetate; DCF, 2′,7′-dichlorofluorescein.
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↵1 Current affiliation: Department of Hematology, Tongji Hospital, Tongji Medical College, and Huazhong University of Science and Technology, Wuhan China, 430030.
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↵2 Current affiliation: Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201.
- Received March 9, 2006.
- Accepted July 18, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
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