Abstract
One of the pathological site effects in excitotoxic activation is Zn2+ overload to postsynaptic neurons. Such an effect is considered to be equivalent to the glutamate component of excitotoxicity. Excessive uptake of Zn2+ by active voltage-dependent transport systems in these neurons may lead to significant neurotoxicity. The aim of this study was to investigate whether and which antagonists of the voltage gated calcium channels (VGCC) might modify this Zn2+-induced neurotoxicity in neuronal cells. Our data demonstrates that depolarized SN56 neuronal cells may take up large amounts of Zn2+ and store these in cytoplasmic and mitochondrial sub-fractions. The mitochondrial Zn2+ excess suppressed pyruvate uptake and oxidation. Such suppression was caused by inhibition of pyruvate dehydrogenase complex, aconitase and NADP-isocitrate dehydrogenase activities, resulting in the yielding of acetyl-CoA and ATP shortages. Moreover, incoming Zn2+ increased both oxidized glutathione and malondialdehyde levels, known parameters of oxidative stress. In depolarized SN56 cells, nifedipine treatment (L-type VGCC antagonist) reduced Zn2+ uptake and oxidative stress. The treatment applied prevented the activities of PDHC, aconitase and NADP-IDH enzymes, and also yielded the maintenance of acetyl-CoA and ATP levels. Apart from suppression of oxidative stress, N- and P/Q-type VGCCs presented a similar, but weaker protective influence. In conclusion, our data shows that in the course of excitotoxity, impairment to calcium homeostasis is tightly linked with an excessive neuronal Zn2+ uptake. Hence, the VGCCs types L, N and P/Q share responsibility for neuronal Zn2+ overload followed by significant energy-dependent neurotoxicity. Moreover, Zn2+ affects the target tricarboxylic acid cycle enzymes, yields acetyl-CoA and energy deficits as well.
Highlights
Zn2+ is an essential trace metal playing a regulatory role in diverse cell functions including gene expression, neurotransmision or being a co-factor of over 300 metalloproteins [1,2,3]
Our double staining with DAPI and specific antibody against L-voltage gated calcium channels (VGCC), P/Q-VGCC as well as N-VGCC revealed that all viable SN56 cells were positive for studied channels (Fig 1A–1C)
The basal Zn2+ content in SN56 cell was equal 0.6 nmol/mg protein (0.08 mmol/L of cell water), which is comparable with 0.1–0.2 mmol/L cation levels reported for the whole brain or primary neurons (Tables 1 and 2)[43,44,45,46]
Summary
Zn2+ is an essential trace metal playing a regulatory role in diverse cell functions including gene expression, neurotransmision or being a co-factor of over 300 metalloproteins [1,2,3]. It has been proven that Zn2+ inter- and intracellular redistributions in postsynaptic neurons of gluzinergic synapses are essential for proper learning and memory storage processes, taking place in hippocampus [9]. The other data demonstrate that prolonged depolarization of presynaptic gluzinergic terminals may cause an excessive postsynaptic Zn2+ uptake, which may trigger the onset of neurodegeneration [6,7, 10,11,12,13,14,15,16,17,18]. Several pathological conditions may cause prolonged depolarization of glutaminergic neurons, which exert excitotoxic effects on postsynaptic neurons through excessive co-release of glutamate and Zn2+ [4,5, 11, 24]. Our past studies revealed that the excessive accumulation of Zn2+ by SN56 septal cholinergic neuronal cells decreased their acetyl-CoA level and suppressed its utilization in pathways of N-
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