Tolerance-Inducing Dose of 3-Nitropropionic Acid Modulates bcl-2 and bax Balance in the Rat Brain: A Potential Mechanism of Chemical Preconditioning

Abstract

Many studies have reported ischemia protection using various preconditioning techniques, including single dose 3-nitropropionic acid (3-NPA), a mitochondrial toxin. However, the cellular signal transduction cascades resulting in ischemic tolerance and the mechanisms involved in neuronal survival in the tolerant state still remain unclear. The current study investigated the mRNA and protein expression of the antiapoptotic bcl-2 and the proapoptotic bax. two antagonistic members of the bcl-2 gene family, in response to a single dose of 3-NPA, to global cerebral ischemia-reperfusion. and to the combination of both 3-NPA-pretreatment and subsequent global cerebral ischemia-reperfusion. Brain homogenates of adult Wistar rats (n = 25) were analyzed for bcl-2 and bax mRNA expression using a new highly sensitive and quantitative polymerase chain reaction (PCR) technique that allows real-time fluorescence measurements of the PCR product (LightCycler; Roche Diagnostics, Mannheim, Germany). Animals for mRNA analysis received 3-NPA (20 mg/kg, intraperitoneal; "chemical preconditioning") or vehicle (normal saline), and were either observed for 24 plus 3 hours or were subjected to 15 minutes of global cerebral ischemia 24 hours after the pretreatment and observed for 3 hours of reperfusion. Immunohistochemistry was applied to serial brain sections of additional rats (n = 68) to determine amount and localization of the respective Bcl-2 and Bax protein expression in various brain areas. One set of animals was injected with 3-NPA and observed for 3, 12, 24, and 96 hours; a second set was exposed to 15 minutes global cerebral ischemia, 3, 12, and 24 hours reperfusion; and a third set was pretreated with 3-NPA or saline 24 hours before the ischemic brain insult and observed for 96 hours of reperfusion. The authors found single dose 3-NPA treatment to be associated with an elevated bcl-2:bax ratio (increased bcl-2 expression, decreased bax expression), both on the transcriptional (mRNA) and the translational (protein) level. The differential influence of 3-NPA was maintained during early recovery from global cerebral ischemia (3 hours), when 3-NPA pretreated animals showed higher bcl-2 and lower bax mRNA levels compared with rats with saline treatment. Respective changes in protein expression were localized predominately in neurons vulnerable to ischemic damage. Compared with baseline, Bcl-2 protein was significantly higher in surviving neurons at 96 hours after the insult, whereas Bax protein remained unchanged. However, at this late time of postischemic recovery (96 hours), the protein expression pattern of surviving neurons was not different between animals with and without 3-NPA pretreatment. To the authors' knowledge, the current study is the first report on the differential expression of pro- and antiapoptotic genes after a single, nonlethal dose of 3-NPA. The current results suggest alterations in the balance between pro- and antiapoptotic proteins as a potential explanation for the reported protection provided by chemical pr...

Verge: A Novel Vascular Early Response Gene

“…cDNA was synthesized using oligo-dT primers and Superscript II reverse transcriptase (RT) and purified by using silicabased columns (Qiagen, Hilden, Germany). Quantitative PCR was performed using the LightCycler system (Roche) as described previously (Brambrink et al, 2000). In brief, cDNAs were serially threefold diluted to obtain four to five concentration points for quantification.…”

Opening of Mitochondrial ATP-Sensitive Potassium Channels Is a Trigger of 3-Nitropropionic Acid–Induced Tolerance to Transient Focal Cerebral Ischemia in Rats

“…27 Taken together, it is possible to speculate that mitoK ATP opening leads to oxygen radical release, enhanced protein synthesis, and resultant ischemic tolerance. It is not clear at the present time which proteins are involved in development of preconditioning associated with 3-NPA, but such diverse proteins as the adenosine A3 receptor 28 or anti-apoptosis proteins like bcl-2 15 have been suggested. In our model, the chemical preconditioning conferred no protective effect on striatum despite its regional vulnerability to 3-NPA toxicity.…”

“…A single dose of 20 mg/kg 3-NPA is documented to confer significant neuroprotection but not abnormalities in neurological and histopathological outcomes. 3,12,14,15 In group 1 (nϭ32), animals were chemically preconditioned with 3-NPA (Sigma; 20 mg/kg, diluted in sodium chloride 0.9% to a concentration of 1 mg/mL, buffered [pH 7.4] with NaOH, IP) 24 hours (nϭ8), 48 hours (nϭ8), or 72 hours (nϭ16) before MCAO. In the following groups, all drugs were given 72 hours before MCAO.…”

“…13 On the other hand, several studies have shown that a single dose of 20 mg/kg 3-NPA given systemically confers no histopathological abnormalities but provides delayed cerebral ischemic tolerance. 3,12,14,15 Although a previous study reported that 3-NPA has the potential to activate sK ATP in hippocampal cells, 16 the role of mitoK ATP in mediating the delayed ischemic tolerance with 3-NPA has not been documented in intact brain. The purpose of this study was to examine the involvement of mitoK ATP opening in the generation of delayed ischemic tolerance with 3-NPA in brain.…”

Services to Operate and Maintain a Microwave Research Laboratory

“…Bcl-2, a protein tiiat is a key regulator of apoptosis, is increased by two injections of 10 mg/kg separated by 12 h at 24 h after the first injection . Activity has been detected in both proapoptotic and antiapoptotic molecular pathways at 12 and 24 h after a single 3-NP injection and two closely spaced 3-NP injections Brambrink et al, 2000;Duan et al, 2000;Antonawich et al, 2002; and in cultured rat striatal neurons . This activity includes release of cytochrome-c from mitochondria, an increase in Bcl-2, increased levels of one or more activated cysteine-dependent, aspartate-specific proteases, caspase-9, caspase-8, and caspase-3, and increased level of calpain.…”