Cytotoxic action of lindane in neocortical GABAergic neurons is primarily mediated by interaction with flunitrazepam-sensitive GABAA receptors

Abstract

The cytotoxic action of the gamma-isomer of hexachlorocyclohexane (y-HCH; lindane) was studied in cultured mouse neocortical neurons by measurements of the reduction in mitochondrial function using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) test. The cells were exposed to 30-300 microM lindane in the culture medium for different periods of time and lindane cytotoxicity was found to be time- and concentration-dependent. Lindane cytotoxicity could be ameliorated by addition of gamma aminobutyric acid (GABA) in a concentration-dependent manner but this effect of GABA was not blocked by bicuculline or picrotoxinin (PTX). Lindane induced cytotoxicity was also reduced by the GABA(A) receptor agonists muscimol and THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol). This effect was enhanced by the simultaneous presence of flunitrazepam but only at the highest lindane concentrations studied (200 and 300 microM). Flunitrazepam by itself had no effect on lindane-induced cytotoxicity. The protective effect of GABA plus flunitrazepam was blocked by the benzodiazepine receptor antagonist flumazenil and by the GABA(A) antagonist bicuculline, suggesting the involvement of central benzodiazepine receptors allosterically coupled to the GABA recognition site at the GABA(A) receptor. When 100 microM PTX was used to suppress the protective effect of GABA and flunitrazepam, a significant effect of PTX was observed only at 300 microM lindane. The GABA(B) receptor agonist, baclophen, only marginally reduced the cytotoxic effect induced by the highest lindane concentrations. It is concluded that the cytotoxic action of lindane in neocortical neurons in culture is mediated primarily through an interaction with allosterically coupled GABA-benzodiazepine recognition sites at the GABA(A) receptor.

Cytotoxic action of lindane in cerebellar granule neurons is mediated by interaction with inducible GABAB receptors

“…Although in agreement with a previous report (Rosa et al, 1996), the finding that in the granule cells grown in the absence of THIP, lindane cytotoxicity could not be ameliorated by GABA and benzodiazepines, was at variance with the observation in other types of neurons that agonists and allosteric modulators of GABA A receptors protect against lindane cytotoxicity (Pomés et al, 1994b;Vale et al, 1998). This was even more unexpected in light of the fact that the cerebellar granule cells are known to abundantly express benzodiazepine-sensitive high-affinity GABA A receptors (Squires et al, 1990;Belhage et al, 1991;Vale et al, 1997), a prerequisite in neocortical neurons for the protective action of GABA and benzodiazepines in lindane cytotoxicity (see Vale et al, 1998). It should, however, be noted that in contrast to neocortical neurons where exposure to lindane does not lead to aberrations in intracellular Ca ϩϩ homeostasis (Pomés et al, 1994b), exposure of cerebellar granule cells to lindane has profound effects on intracellular Ca ϩϩ resulting from increased influx as well as release from intracellular stores (Rosa et al, 1996(Rosa et al, , 1997.…”

“…The ␥-HCH isomer (lindane) is a widely used pesticide which produces hyperexcitability and convulsions in humans and other mammals (Tusell et al, 1987;Brown et al, 1995), these effects being primarily mediated by its interaction with the GABA A receptor (Cole and Casida, 1986;Suñol et al, 1989). In keeping with this, a companion paper (Vale et al, 1998) as well as a previous report (Pomés et al, 1994b) showed that lindane cytotoxicity in cultured neocortical neurons can be ameliorated by agents that allosterically modify the GABA A receptor complex.…”

“…To achieve this purpose, primary cultures of cerebellar granule cells were grown in the absence or presence of THIP, leading to differential expression of components of this neurotransmission (see above). Subsequently, cells were exposed to lindane and mitochondrial function of the cells was monitored using the MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5diphenyl-tetrazolium bromide) test as detailed by Vale et al (1998).…”

Intravenous flumazenil versus oxazepam tapering in the treatment of benzodiazepine withdrawal: a randomized, placebo‐controlled study

“…In fact, GABA A and BZD receptors are coupled allosterically, and occupation of either receptor site increases the affinity of the other. 37 Chronic exposure of primary neuronal cultures to BZD agonists reduces these allosteric interactions. FLU has been found to reverse uncoupling and facilitate coupling processes of the BZD site with the GABA site on the GABA macromolecular complex.…”

Profile for Amyloid-β and Tau Expression in Primary Cortical Cultures from 3xTg-AD Mice

“…Primary cortical neurons were prepared from embryonic day 15-18 (E15-18) NonTg and homozygous 3xTg-AD mice as previously described (Vale et al 1998(Vale et al , 1999Garcia et al 2006). Briefly, cerebral cortices were removed and dissociated by mild trypsinization at 37°C, followed by trituration in a DNAse solution (0.005% w/v) containing a soybean trypsin inhibitor (0.05% w/v).…”