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The aim of this work was to characterize the effect of bongkrekic acid (BKA), atractyloside (ATR) and carboxyatractyloside (CAT) on single channel properties of chloride channels from mitochondria. Mitochondrial membranes isolated from a rat heart muscle were incorporated into a bilayer lipid membrane (BLM) and single chloride channel currents were measured in 250/50 mM KCl cis/trans solutions. BKA (1-100 microM), ATR and CAT (5-100 microM) inhibited the chloride channels in dose-dependent manner. The inhibitory effect of the BKA, ATR and CAT was pronounced from the trans side of a BLM and it increased with time and at negative voltages (trans-cis). These compounds did not influence the single channel amplitude, but decreased open dwell time of channels. The inhibitory effect of BKA, ATR and CAT on the mitochondrial chloride channel may help to explain some of their cellular and/or subcellular effects.

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We studied the effects of the chloride channel blockers, 5-nitro-2-(phenylpropylamino)-benzoate (NPPB), dihydro-4,4' diisothiocyanostilbene-2,2'-disulphonic acid (DIDS), and phloretin on H2O2-induced primary culture cardiomyocyte apoptosis and activity of intracellular chloride channels obtained from rat heart mitochondrial and lysosomal vesicles. The chloride channel blockers (100 micromol/l) inhibited the H2O2-induced cardiomyocytes apoptosis. We characterized the effect of the blockers on single channel properties of the chloride channels derived from the mitochondrial and lysosomal vesicles incorporated into a bilayer lipid membrane. The single chloride channel currents were measured in 250:50 mmol/l KCl cis/trans solutions. NPPB, DIDS, and phloretin inhibited the chloride channels by decreasing the channel open probability in a concentration-dependent manner with EC50 values of 42, 7, and 20 micromol/l, respectively. NPPB and phloretin inhibited the channel's conductance and open dwell time, indicating that they could affect the chloride selective filter, pore permeability, and gating mechanism of the chloride channels. DIDS and NPPB inhibited the channels from the other side than bongkrekic acid and carboxyatractyloside. The results may contribute to understand a possible involvement of intracellular chloride channels in apoptosis and cardioprotection.

Hypoxic brain cell injury is a complex process that results from a series of intracellular events. In this work, we tested whether severe hypoxia for 6 h can affect gene expression and protein levels of intracellular calcium channels, ryanodine receptors, and inositol 1,4,5-trisphosphate receptors in mouse cerebellum. In addition, we tested the effect of hypoxia on cerebellar granular cells of rats. We have found that gene expression of types 1 and 2 IP(3) receptors is significantly increased after the exposure of mice to hypoxic stimulus for 6 h and also in rat cerebellar granular cells. Increased gene expression of IP(3) receptors was reflected in increased protein levels of these channels as well. In this process, reactive oxygen species are most probably involved, as antioxidant quercetin abolished hypoxia-induced increase in both types 1 and 2 IP3 receptor. Ryanodine receptors of types 1 and 2 and sarco(endo)plasmic reticulum Ca(2+)-ATPase were not affected by hypoxia on the level of messenger RNA. To test physiological consequences, we measured levels of intracellular calcium. We observed significantly elevated calcium level in hypoxic compared to normoxic cells. Deeper understanding of mechanisms, through which hypoxia regulates intracellular calcium, could point towards the development of new therapeutic approaches to reduce or suppress the pathological effects of cellular hypoxia, such as those seen in stroke or ischemia.