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Mitochondrial Free Radical Production Induced by Glucose Deprivation in Cerebellar Granule Neurons

N. K. Isaev1,2*, E. V. Stelmashook2, U. Dirnagl3, E. Yu. Plotnikov1, E. A. Kuvshinova1, and D. B. Zorov1

1Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119992 Moscow, Russia; fax: (495) 939-3181; E-mail: isaev@genebee.msu.su

2Brain Research Department, Institute of Neurology, Russian Academy of Medical Sciences, Pereulok Obukha 5, 105064 Moscow, Russia

3Department of Neurology, Charite Hospital, Humboldt University, Germany; E-mail: ulrich.dirnagl@charite.de

* To whom correspondence should be addressed.

Received May 22, 2007; Revision received July 5, 2007
Using a fluorescent probe for superoxide, hydroethidine, we have demonstrated that glucose deprivation (GD) activates production of reactive oxygen species (ROS) in cultured cerebellar granule neurons. ROS production was insensitive to the blockade of ionotropic glutamate channels by MK-801 (10 µM) and NBQX (10 µM). Inhibitors of mitochondrial electron transport, i.e. rotenone (complex I), antimycin A (complex III), or sodium azide (complex IV), an inhibitor of mitochondrial ATP synthase--oligomycin, an uncoupler of oxidative phosphorylation--CCCP, a chelator of intracellular Ca2+--BAPTA, an inhibitor of electrogenic mitochondrial Ca2+ transport--ruthenium red, as well as pyruvate significantly decreased neuronal ROS production induced by GD. GD was accompanied by a progressive decrease in the mitochondrial membrane potential and an increase in free cytosolic calcium ions, [Ca2+]i. Pyruvate, BAPTA, and ruthenium red lowered the GD-induced calcium overload, while pyruvate and ruthenium red also prevented mitochondrial membrane potential changes induced by GD. We conclude that GD-induced ROS production in neurons is related to potential-dependent mitochondrial Ca2+ overload. GD-induced mitochondrial Ca2+ overload in neurons in combination with depletion of energy substrates may result in the decrease of the membrane potential in these organelles.
KEY WORDS: hypoglycemia, reactive oxygen species, free radicals, mitochondria, calcium, cerebellar granule neuron, cell culture

DOI: 10.1134/S0006297908020053