Mitochondrial Na+/Ca2+-Exchanger Blocker CGP37157 Protects against Chromaffin Cell Death Elicited by Veratridine
- Santos M. Nicolau,
- Antonio M. G. de Diego,
- Lorena Cortés,
- Javier Egea,
- José C. González,
- Marta Mosquera,
- Manuela G. López,
- Jesús Miguel Hernández-Guijo and
- Antonio G. García
- Instituto Teófilo Hernando (S.M.N., A.M.G.D., L.C., J.E., J.C.G., M.M., M.G.L., J.M.H.-G., A.G.G.), Servicio de Farmacología Clínica, Hospital Universitario de la Princesa (A.G.G.), and Departamento de Farmacología y Terapéutica, Facultad de Medicina, Universidad Autónoma de Madrid (S.M.N., A.M.G.D., L.C., J.E., J.C.G., M.M., M.G.L., J.M.H.-G., A.G.G.), Madrid, Spain
- Address correspondence to:
Dr. Antonio G. García, Instituto Teófilo Hernando, Facultad de Medicina, Universidad Autónoma de Madrid, C/Arzobispo Morcillo, 4, 28029-Madrid, Spain. E-mail: agg{at}uam.es
Abstract
Mitochondrial calcium (Ca2+) dyshomeostasis constitutes a critical step in the metabolic crossroads leading to cell death. Therefore, we have studied here whether 7-chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one (CGP37157; CGP), a blocker of the mitochondrial Na+/Ca2+-exchanger (mNCX), protects against veratridine-elicited chromaffin cell death, a model suitable to study cell death associated with Ca2+ overload. Veratridine produced a concentration-dependent cell death, measured as lactate dehydrogenase released into the medium after a 24-h incubation period. CGP rescued cells from veratridine-elicited death in a concentration-dependent manner; its EC50 was approximately 10 μM, and 20 to 30 μM caused near 100% cytoprotection. If preincubated for 30 min and washed out for 3 min before adding veratridine, CGP still afforded significant cytoprotection. At 30 μM, CGP blocked the veratridine-elicited free radical production, mitochondrial depolarization, and cytochrome c release. At this concentration, CGP also inhibited the Na+ and Ca2+ currents by 50 to 60% and the veratridine-elicited oscillations of cytosolic Ca2+. This drastic cytoprotective effect of CGP could be explained in part through its regulatory actions on the mNCX.
Footnotes
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This work was supported by the “Consolider Program Ingenio-2010,” Ministerio de Ciencia e Innovación, Spain [Grant SAF2006-03589] (to A.G.G.); Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación, Spain [Grant RETICS-RD06/0026] (to A.G.G. and M.G.L.); Comunidad Autónoma de Madrid, Spain [Grant S-SAL-0275-2006] (to A.G.G.); Mútua Madrileña, Madrid, Spain (to A.G.G. and M.G.L.); Agencia Lain Entralgo, Madrid, Spain [Grant NDG07/9] (to A.G.G.); FIS-Instituto de Salud Carlos III [Grant PI080227] (to J.M.H.-G.); Ministerio de Ciencia e Innovación, Spain [Grant SAF2006-0854] (to M.G.L.); a fellowship from Faculdade de Medicina-Universidade Agostinho Neto, government of Angola, to S.M.N.; and a fellowship from Ministério de Ciencia e Innovación, Spain (to J.C.G.).
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.109.154765.
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ABBREVIATIONS: mNCX, mitochondrial Na+/Ca2+-exchanger; DMSO, dimethyl sulfoxide; FPL64176, FPL, 2,5-dimethyl-4-[2-(phenylmethyl)benzoyl]-1H-pyrrole-3-carboxylic acid methyl ester; 30 K+/FPL, 30 mM K+/0.3 μM FPL; MTT formazan, 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan, thiazolyl blue formazan; CGP37157, 7-chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one; TTX, tetrodotoxin citrate, octahydro-12-(hydroxymethyl)-2-imino-5,9:7,10a-dimethan o-10aH-[1,3] dioxocino[6,5-d]pyrimidine-4,7,10,11,12-pentol; DMEM, Dulbecco's modified Eagle′s medium; JC-1, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide; Olig, oligomycin; Rot, rotenone; CM-H2DCFDA, 5-(and 6-) chloromethyl-2′,7′-dichlorodihydro-fluorescein-diacetate acetyl ester; Ver, veratridine; LDH, lactate dehydrogenase; ROS, reactive oxygen species; HRP, horseradish peroxidase.
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- Received April 6, 2009.
- Accepted June 8, 2009.
- The American Society for Pharmacology and Experimental Therapeutics
