Mitochondrial Na /Ca -Exchanger Blocker CGP37157 Protects against Chromaffin Cell Death Elicited by Veratridine

Mitochondrial calcium (Ca ) 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 /Ca -exchanger (mNCX), protects against veratridine-elicited chromaffin cell death, a model suitable to study cell death associated with Ca 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 M, and 20 to 30 M 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 M, CGP blocked the veratridine-elicited free radical production, mitochondrial depolarization, and cytochrome c release. At this concentration, CGP also inhibited the Na and Ca currents by 50 to 60% and the veratridine-elicited oscillations of cytosolic Ca . This drastic cytoprotective effect of CGP could be explained in part through its regulatory actions on the mNCX. In general, it is accepted that a dysregulation of the mechanism that fine tunes the transient or more sustained levels of the cytosolic Ca concentrations ([Ca ]c), leads to excitotoxic neuronal death (Schanne et al., 1979) and to neurodegeneration (Mattson, 2007). However, Ca may behave as both a cell survival supporter and a cell death inducer. For instance, cell depolarization and subsequent Ca entry into the cytosol helps to sustain the survival of cerebellar granule cells (Gallo et al., 1987) and bovine chromaffin cells (Orozco et al., 2006). However, chronic elevation of [Ca ]c by ionophores induces apoptosis (Martikainen et al., 1991). The opposite is also true, i.e., Ca antagonists that reduce [Ca ]c also cause neuronal death (Koh and Cotman, 1992) and chromaffin cell death (Novalbos et al., 1999). These apparent contradictory findings may be explained in the frame of the hypothesis suggesting that the [Ca ]c changes occurring during cell activation must move within a critical set point; beyond this point a cytoprotective signal might turn into a cytotoxic one (Koike et al., 1989). In this context, the suggestion of Nicholls (1985) and White and Reynolds (1995) that Ca accumulation into mitochondria could play a neuroproThis 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.). Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.109.154765. ABBREVIATIONS: mNCX, mitochondrial Na /Ca -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 M 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. 0022-3565/09/3303-844–854$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 330, No. 3 Copyright © 2009 by The American Society for Pharmacology and Experimental Therapeutics 154765/3502870 JPET 330:844–854, 2009 Printed in U.S.A. 844 at A PE T Jornals on M ay 4, 2017 jpet.asjournals.org D ow nladed from tective role by removing Ca from the cytoplasm fits well in the hypothesis. Conversely, by slowing down the rate of Ca exit into the cytosol through the mNCX, the brisk [Ca ]c changes could be mitigated and protect cells from a cytotoxic insult. In this study we have analyzed such hypothesis by using the mNCX blocker CGP as a pharmacological tool to slow down Ca efflux from Ca -loaded mitochondria into the cytosol (Montero et al., 2000). We have used bovine adrenal medulla chromaffin cells, a paraneuronal cell type that as neurons, possess Na and Ca channels as well as K channels. Furthermore, as stated above, mitochondrial Ca fluxes including the use of CGP have been thoroughly studied in these cells (Garcia et al., 2006). However, to explore the mNCX function we needed a model of cell death elicited by Na and Ca overload; hence, we resorted to veratridine that induces cell death by causing Na and Ca overload (Maroto et al., 1994, 1996; Novalbos et al., 1999), and augments superoxide production (Jordán et al., 2000). We have found that CGP affords drastic protection against chromaffin cell death elicited by veratridine. Such protection is linked to the preservation of mitochondrial function in veratridinetreated cells. Materials and Methods Reagents. Amphotericin B, cadmium, dimethyl sulfoxide (DMSO), FPL64176, 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan, thiazolyl blue formazan (MTT formazan), oligomycin, rotenone, veratridine, the salts to make the saline solutions, and the lactate dehydrogenase (LDH) kit were obtained from Sigma (Madrid, Spain). CGP37157 (CGP) and tetrodotoxin citrate (TTX) were purchased from Tocris (Biogen Científica, Spain). Dulbecco s modified Eagle s medium (DMEM), fetal calf serum, and penicillin/streptomycin were purchased from Invitrogen (Madrid, Spain). Fluo-4AM, JC-1, and 5(and 6-) chloromethyl-2 ,7 -dichlorodihydro-fluorescein-diacetate acetyl ester (CM-H2DCFDA), pluronic acid were purchased from Invitrogen Molecular Probes (Madrid, Spain). Cytochrome c Elisa Kit was purchased from Millipore (Madrid, Spain). Preparation of Cells. Adrenal glands were obtained from the city slaughterhouse under the supervision of the local veterinary service. Bovine adrenal medullary chromaffin cells were isolated as described previously (Livett, 1984), with some modifications (Moro et al., 1990). We used the Percoll gradients for the cell isolation procedure; thus, we had in our cultures a mixture of adrenergic (60–70%) and noradrenergic cells (30–40%). Cells were suspended in DMEM supplemented with 5% fetal bovine serum, 50 IU/ml penicillin, and 50 g/ml streptomycin. Cells were preplated for 30 min and proliferation inhibitors (10 M cytosine arabinoside, 10 M fluorodeoxyuridine, and 10 M leucine methyl ester) were added to the medium to prevent excessive growth of fibroblasts that would mask the chromaffin cell death measurements. The total cell number was determined as described previously. For cell death studies, cells were plated at a density of 5 10 cells/well on 24-well dishes. Cultures were maintained in an incubator at 37°C in a water-saturated atmosphere with 5% CO2. Cell treatments were performed in DMEM free of serum, because serum interferes with LDH measurements. Measurement of Cell Death through LDH Activity. Samples of incubation media were collected at the end of the 24-h period of veratridine, CGP, and veratridine/CGP exposure to estimate extracellular LDH, an indication of cell death (Koh and Choi, 1987). LDH activity was measured in the cells (5 10 per well) after treatment with 10% Triton X-100 (intracellular LDH). LDH activity was measured spectrophotometrically at 490 to 620 nm with use of a microplate reader (iEMS reader MF; Thermo Fisher Scientific, Waltham, MA). Total LDH activity was defined as the sum of intracellular plus extracellular LDH activity. Released LDH was defined as the percentage of extracellular compared with total LDH activity (Cano-