P346Ionophore A23187 facilitates cyclosporine A inhibition of cardiac mitochondrial swelling induced by calcium, tert-butyl hydroperoxide, and phenylarsine oxide

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Abstract

Purpose

The effects of cyclosporine A (CsA) were studied on the cardiac mitochondrial swelling induced either by calcium alone or supplemented with prooxidant tert-butyl hydroperoxide (t-BuOOH), or the thiol cross-linker phenylarsine oxide (PhAsO), in the presence or absence of ionophore A23187, in order to investigate the mechanisms involved in the mitochondrial permeability transition pore (mPTP) inhibition.

Methods

Mitochondria were isolated from the myocardium of rats by standard differential centrifugation. Mitochondria (0.5 mg protein/ml) were suspended in incubation medium with or without 2 μM A23187 incubated for 5 min before swelling induction. Swelling was induced by Ca2+ alone (500 μM), or by the combination of Ca2+ (50 μM) + t-BuOOH (100 μM) or PhAsO (2 μM). In each experiment, the potential inhibitory effect of CsA was tested at different concentrations (from 0.01 μM to 10 μM), and compared with the control conditions (DMSO), incubated for 5 min before swelling induction. Swelling was monitored with a spectrophotometer by measuring OD at 540nm for 10 min. It was calculated at 10 min and expressed in % of maximal OD after calcium addition.

Results

The mitochondrial swelling induced by calcium alone was slightly inhibited by 1 μM CsA (-15% vs control) in the absence of A23187. In the presence of A23187, the swelling was strongly inhibited by CsA in a dose-response relationship (-0%, -11%, -70%, and -84% vs control, respectively with 0.01, 0.1, 1, and 10 μM of CsA). A23187 also potentiated the inhibitory effect of CsA on mitochondrial swelling induced by t-BuOOH (-48%, and -79% vs control without A23187, respectively with 0.1, and 10 μM of CsA; compared to -84%, and -100% vs control in the presence of A23187). Finally, the mitochondrial swelling induced by PhAsO was inhibited by CsA in the presence of A23187 (-29%, and -89% vs control, respectively with 0.1, and 1 μM of CsA), while this inhibition was completely abolished without A23187 (-0% vs control with 0.1, 1, and 10 μM of CsA).

Conclusions

Our results demonstrated that ionophore A23187 facilitates CsA inhibition of the cardiac mitochondrial swelling induced by calcium complemented or not by t-BuOOH or PhAsO. We hypothesized that A23187 in these experiments potentiated the inhibitory effect of CsA, potentially by either providing phosphate that has been shown essential for inhibition of the mPTP by CsA (E. Basso, J Biol Chem, 2008) or by leading to matrix acidification which is also known to be inhibitory as A23187 has been described to allow proton uptake and calcium release from the mitochondria (D.R. Pfeiffer, Biochemistry, 1976).

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