Mitochondrial Na⁺/Ca²⁺-Exchanger Blocker CGP37157 Protects against Chromaffin Cell Death Elicited by Veratridine

Abstract

Mitochondrial calcium (Ca 2ϩ ) 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 . 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 2ϩ concentrations ([Ca 2ϩ ] c ), leads to excitotoxic neuronal death (Schanne et al., 1979) and to neurodegeneration (Mattson, 2007). However, Ca 2ϩ may behave as both a cell survival supporter and a cell death inducer. For instance, cell depolarization and subsequent Ca 2ϩ 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 2ϩ ] c by ionophores induces apoptosis (Martikainen et al., 1991). The opposite is also true, i.e., Ca 2ϩ antagonists that reduce [Ca 2ϩ ] 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 2ϩ ] 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 2ϩ accumulation into mitochondria could play a neuropro- Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.109.154765.ABBREVIATIONS: mNCX, mitochondrial Na ϩ /Ca 2ϩ -exchanger; DMSO, dimethyl sulfoxide; FPL64176, FPL, 2,5-dimethyl-4-[2-(phenylmethyl)benzoyl]-1H-pyrrole-3-carboxylic acid methyl ester; 30 K ϩ /FPL, 30 mM K ϩ /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

Benzothiazepine CGP37157 and Its Isosteric 2′-Methyl Analogue Provide Neuroprotection and Block Cell Calcium Entry

“…overload cell death models, 27 as it was unable to rescue chromaffin cells subjected to a toxic stimulus related to the mitochondrial disruption-derived oxidative stress, for example, blockade of the mitochondrial respiratory chain by combining 10 μM oligomycin A and 30 μM rotenone. Rotenone and oligomycin A (O/R) block complexes I and V, respectively, of the mitochondrial electron transport chain, thereby causing free radical generation and blockade of ATP synthesis.…”

“…In fact, the mNCX blocker CGP37157 protected bovine chromaffin cells against cytotoxicity induced by veratridine with an EC 50 of 10 μM. 27 We selected 30 μM veratridine as toxic stimulus based on our previous study where this concentration afforded a reliable neuronal damage. 27 Veratridine augmented the release of LDH with respect to nontreated cells by 31%.…”

“…27 We selected 30 μM veratridine as toxic stimulus based on our previous study where this concentration afforded a reliable neuronal damage. 27 Veratridine augmented the release of LDH with respect to nontreated cells by 31%. This release was reduced by increasing concentrations of ITH12505, though it was only statistically significant at 30 μM.…”

“…42 CGP37157 not only failed against the O/R exposure, but in fact augmented cell-damaging effects of O/R in chromaffin cells. 27 Herein, SH-SY5Y cells were incubated with CGP37157 or ITH12505 before the addition of O/R, and coincubated with compounds plus O/R for an additional 24 h period. Cell viability at the end of this period was evaluated by the MTT method.…”

“…In this regard, we recently discovered that CALHM1 generates transient elevations of both [Ca 2+ ] c and mitochondrial Ca 2+ concentrations ([Ca 2+ ] m ); however, its P86L mutated form also causes cell Ca 2+ entry, but generates a plateau of [Ca 2+ ] m that was prolonged in time. 26 Furthermore, we have described the neuroprotective properties of the mitochondrial Na + /Ca 2+ exchanger (mNCX) blocker CGP37157 to rescue cultured cells 27 and hippocampal slices 28 from the toxic stimulus exerted by veratridine, a model of cell death by Na + and Ca 2+ overload. Indeed, the scientific community devoted to the research on neurodegenerative diseases has not paid attention to the mNCX until recently, due to its emerging role in the mitochondrial/cytosolic [Ca 2+ ] trafficking 29 and its implication in the development of neurological diseases.…”