Influence of Intra- and Extracellular Acidification on Free Radical Formation and Mitochondria Membrane Potential in Rat
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Influence of Intra- and Extracellular Acidification on Free Radical Formation and Mitochondria Membrane Potential in Rat Brain Synaptosomes Tatyana G. Pekun & Valeriya V. Lemeshchenko & Tamara I. Lyskova & Tatyana V. Waseem & Sergei V. Fedorovich
Received: 20 September 2012 / Accepted: 21 October 2012 / Published online: 6 November 2012 # Springer Science+Business Media New York 2012
Abstract Brain ischemia is accompanied by lowering of intraand extracellular pH. Stroke often leads to irreversible damage of synaptic transmission by unknown mechanism. We investigated an influence of lowering of pHi and pHo on free radical formation in synaptosomes. Three models of acidosis were used: (1) pHo 6.0 corresponding to pHi decrease down to 6.04; (2) pHo 7.0 corresponding to the lowering of pHi down to 6.92: (3) 1 mM amiloride corresponding to pHi decrease down to 6.65. We have shown that both types of extracellular acidification, but not intracellular acidification, increase 2′,7′dichlorodihydrofluorescein diacetate fluorescence that reflects free radical formation. These three treatments induce the rise of the dihydroethidium fluorescence that reports synthesis of superoxide anion. However, the impact of amiloride on superoxide anion synthesis was less than that induced by moderate extracellular acidification. Superoxide anion synthesis at pHo 7.0 was almost completely eliminated by mitochondrial uncoupler carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone. Furthermore, using fluorescent dyes JC-1 and rhodamine-123, we confirmed that pHo lowering, but not intracellular acidification, led to depolarization of intrasynaptosomal mitochondria. We have shown that pHo but not pHi lowering led to oxidative stress in neuronal presynaptic endings that might underlie the long-term irreversible changing in synaptic transmission.
Abbreviations ASIC Acid-sensitive ion channel BCECF-AM 2′7′-bis(2-Carboxyethyl)-5(6)carboxyfluorescein acetoxymethyl ester CCCP Carbonyl cyanide 4(trifluoromethoxy)phenylhydrazone DCF 2′,7′-Dichlorodihydrofluorescein DCFDA 2′,7′-Dichlorodihydrofluorescein diacetate DPI Diphenyleniodinium chloride HEPES 4-(2-Hydroxyethyl)piperazine-N’-1ethanesulfonic acid Ie-Ic, R.U. Fluorescence intensity; the control curve was extracted from the experimental curve; relative units Ifl Fluorescence intensity Ifl, R.U. Fluorescence intensity; relative units IP3 1,4,5-Triphosphate JC-1 5,5′,6,6′-Tetrachloro-1,1′3,3′tetraethylbenzimidazolo-carbocyanine iodide MES 4-Morpholineethanesulfonic acid NMDA N-Methyl-D-aspartate ROS Reactive oxygen species SOD Superoxide dismutase Tris Tris(hydroxymethyl)aminomethane
Keywords Synaptosomes . Acidosis . Ischemia . Presynaptic . Superoxide anion . Mitochondria T. G. Pekun : V. V. Lemeshchenko : T. I. Lyskova : T. V. Waseem : S. V. Fedorovich (*) Laboratory of Biophysics and Engineering of Cell, Institute of Biophysics and Cell Engineering, Akademicheskaya St., 27, Minsk 220072, Belarus e-mail: [email protected]
Introduction Brain ischemia is accompanied by lowering of intra- and ex
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