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Influence of Intra- and Extracellular Acidification on Free Radical Formation and Mitochondria Membrane Potential in Rat Brain Synaptosomes

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Abstract

Brain ischemia is accompanied by lowering of intra- and 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.

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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’-1-ethanesulfonic 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

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Acknowledgments

This work was supported by Committee for Aid and Education in Neurochemistry-International Society for Neurochemistry (CAEN-ISN). Foundation body had no involvement in study design, in the collection, analysis, and interpretation of data, in the writing of the report, and in the decision to submit the article for publication.

We thank Mrs. Claire Roulston for improvement of English.

Declaration of competing interests

Authors declare no competing financial, personal, or other interests.

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Authors and Affiliations

  1. Laboratory of Biophysics and Engineering of Cell, Institute of Biophysics and Cell Engineering, Akademicheskaya St., 27, Minsk, 220072, Belarus

    Tatyana G. Pekun, Valeriya V. Lemeshchenko, Tamara I. Lyskova, Tatyana V. Waseem & Sergei V. Fedorovich

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  1. Tatyana G. Pekun
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  2. Valeriya V. Lemeshchenko
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  3. Tamara I. Lyskova
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  4. Tatyana V. Waseem
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  5. Sergei V. Fedorovich
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Correspondence to Sergei V. Fedorovich.

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Pekun, T.G., Lemeshchenko, V.V., Lyskova, T.I. et al. Influence of Intra- and Extracellular Acidification on Free Radical Formation and Mitochondria Membrane Potential in Rat Brain Synaptosomes. J Mol Neurosci 49, 211–222 (2013). https://doi.org/10.1007/s12031-012-9913-3

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  • DOI: https://doi.org/10.1007/s12031-012-9913-3

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