ABSTRACT
Title
Role of “diazepam binding inhibitor” in oxygen-glucose deprivation and reperfusion-induced damage
Authors
A. Contartese
Doctorate School in Pharmacology and Toxicology
Dept. Neuroscience – University of Siena, Italy
Doctorate School in Pharmacology and Toxicology
Dept. Neuroscience – University of Siena, Italy
Abstract
During brain ischemia, tissues subjected to oxygen/glucose deprivation (OGD) undergo within seconds to disruption of the energy supply, which leads to massive cell depolarisation. This results in a quick rise of extracellular glutamate caused by reversed operation of neuronal glutamate transporters, formation of free radicals, mitochondrial damage and fragmentation of cellular DNA (Saito et al., 2005). GABA and GABA agonist compounds as well, represent physio-pharmacological means to inhibit cellular activity within the brain, possibly blunting ischemic injury (Ricci et al., 2007, 2009). Furthermore protein expression changes so greatly at the early time point of reperfusion that it provides important molecular information about the initiation of ischemia–reperfusion injury cascade (Prajapati et al., 2010).
A proteomic profiling using two-dimensional gel electrophoresis was performed by comparing samples of rat brain slices subjected to control conditions or OGD and reperfusion (OGD/R). Results indicated that 16 spots out of the1550 were differently expressed and among these, the diazepam binding inhibitor (DBI) was found to be completely absent in the OGD samples. Consequently, the role exerted by DBI in OGD/R related damage was investigated in rat brain slices and in human neuroblastoma cells lines (IMR-32). Its neuroprotective/damaging effect was assessed by measuring the release of glutamate and lactate dehydrogenase (LDH) during reperfusion and by determining final tissue water gain, taken as an index of cell swelling (brain slices) or by measuring cell vitality, cell apoptosis or necrosis (IMR-32).
In brain slices, the results showed that in control conditions, DBI (0.001–10 µM) caused a significant increase in glutamate release at the highest concentration used while it did not modify LDH and tissue edema. On the contrary, when the peptide was added to artificial cerebrospinal fluid used during reperfusion, a significant LDH release was observed at DBI concentrations of 0.001–0.1 µM, while higher concentrations were ineffective. Moreover DBIsignificantly increased glutamate release as well as tissue water gain although according to a "bell-shaped", hormetic-like, concentration-response curve, with an efficacy window of 0.1-1 µM.The receptors involved in DBI-mediated effects were further investigated by using selective antagonists for central and mitochondrial DBI receptors (Flumazenil e PK-11195, respectively). Flumazenil antagonised in a concentration dependent fashion DBI-induced LDH and glutamate release and tissue edema while PK-11195 had no effects, suggesting that benzodiazepine central receptors are involved in DBI effects observed in brain slcies.
In IMR-32 cells, DBI (0.01 and 0.1µM ) induced a significant cell necrosis when added to colture medium for 24 or 48 hours as revealed by Annexine V/Propidium Iodide cytofluorimetric assay. Moreover, DBI (0.1 µM) enhanced OGD and reperfusion-induced IMR-32 cell death in atime-dependentfashion.
In conclusion, these results open new fields of study designed to better characterize DBI mechanism of action with the objective to delineate a therapeutic strategy useful to prevent and/or reduce ischemia and reperfusion-induced tissue damage.
Prajapati KD et al.(2010) Brain Res 1327:118
Ricci L et al. (2007) Eur J Pharmacol 561:80
Ricci L et al. (2009) Eur J Pharmacol 621:26
Saito A et al. (2005) Mol. Neurobiol 31: 105
A proteomic profiling using two-dimensional gel electrophoresis was performed by comparing samples of rat brain slices subjected to control conditions or OGD and reperfusion (OGD/R). Results indicated that 16 spots out of the1550 were differently expressed and among these, the diazepam binding inhibitor (DBI) was found to be completely absent in the OGD samples. Consequently, the role exerted by DBI in OGD/R related damage was investigated in rat brain slices and in human neuroblastoma cells lines (IMR-32). Its neuroprotective/damaging effect was assessed by measuring the release of glutamate and lactate dehydrogenase (LDH) during reperfusion and by determining final tissue water gain, taken as an index of cell swelling (brain slices) or by measuring cell vitality, cell apoptosis or necrosis (IMR-32).
In brain slices, the results showed that in control conditions, DBI (0.001–10 µM) caused a significant increase in glutamate release at the highest concentration used while it did not modify LDH and tissue edema. On the contrary, when the peptide was added to artificial cerebrospinal fluid used during reperfusion, a significant LDH release was observed at DBI concentrations of 0.001–0.1 µM, while higher concentrations were ineffective. Moreover DBIsignificantly increased glutamate release as well as tissue water gain although according to a "bell-shaped", hormetic-like, concentration-response curve, with an efficacy window of 0.1-1 µM.The receptors involved in DBI-mediated effects were further investigated by using selective antagonists for central and mitochondrial DBI receptors (Flumazenil e PK-11195, respectively). Flumazenil antagonised in a concentration dependent fashion DBI-induced LDH and glutamate release and tissue edema while PK-11195 had no effects, suggesting that benzodiazepine central receptors are involved in DBI effects observed in brain slcies.
In IMR-32 cells, DBI (0.01 and 0.1µM ) induced a significant cell necrosis when added to colture medium for 24 or 48 hours as revealed by Annexine V/Propidium Iodide cytofluorimetric assay. Moreover, DBI (0.1 µM) enhanced OGD and reperfusion-induced IMR-32 cell death in atime-dependentfashion.
In conclusion, these results open new fields of study designed to better characterize DBI mechanism of action with the objective to delineate a therapeutic strategy useful to prevent and/or reduce ischemia and reperfusion-induced tissue damage.
Prajapati KD et al.(2010) Brain Res 1327:118
Ricci L et al. (2007) Eur J Pharmacol 561:80
Ricci L et al. (2009) Eur J Pharmacol 621:26
Saito A et al. (2005) Mol. Neurobiol 31: 105