Acidosis and 5-(N-ethyl-N-isopropyl)amiloride (EIPA) Attenuate Zinc/Kainate Toxicity in Cultured Cerebellar Granule Neurons

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E. V. Stelmashook^1*, S. V. Novikova¹, G. A. Amelkina², E. G. Ivashkin¹, E. E. Genrikhs¹, L. G. Khaspekov¹, and N. K. Isaev^1,2*

¹Research Center of Neurology, Russian Academy of Medical Sciences, 125367 Moscow, Russia; E-mail: estelmash@mail.ru

²Lomonosov Moscow State University, Belozersky Institute of Physico-Chemical Biology, 119991 Moscow, Russia; E-mail: isaev@genebee.msu.ru

^* To whom correspondence should be addressed.

Received December 10, 2014

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Cultured cerebellar granule neurons (CGNs) are resistant to the toxic effect of ZnCl₂ (0.005 mM, 3 h) and slightly sensitive to the effect of kainate (0.1 mM, 3 h). Simultaneous treatment of CGNs with kainate and ZnCl₂ caused intensive neuronal death, which was attenuated by external acidosis (pH 6.5) or 5-(N-ethyl-N-isopropyl)amiloride (EIPA, Na⁺/H⁺ exchange blocker, 0.03 mM). Intracellular zinc and calcium ion concentrations ([Zn²⁺]_i and [Ca²⁺]_i) were increased under the toxic action of kainate + ZnCl₂, this effect being significantly decreased on external acidosis and increased in case of EIPA addition. Neuronal Zn²⁺ imaging demonstrated that EIPA increases the cytosolic concentration of free Zn²⁺ on incubation in Zn²⁺-containing solution. These data imply that acidosis reduces ZnCl₂/kainate toxic effects by decreasing Zn²⁺ entry into neurons, and EIPA prevents zinc stores from being overloaded with zinc.

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KEY WORDS: zinc, calcium, kainate, cerebellar granule neurons, acidosis, EIPA

DOI: 10.1134/S000629791508012X

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