Abstract
After accumulation of a Ca2+ load, the addition of uncoupler to respiring rat liver mitochondria is followed by opening of the permeability transition pore (MTP), a voltage-dependent channel sensitive to cyclosporin A. The channel's voltage threshold is profoundly affected under conditions of oxidative stress, with a shift to more negative values that may cause MTP opening at physiological membrane potentials. In this paper we further clarify the mechanisms by which oxidative agents affect the apparent voltage dependence of the MTP. We show that two sites can be experimentally distinguished. (i) A first site is in apparent oxidation-reduction equilibrium with the pyridine nucleotide (PN) pool (NADH/NAD + NADPH/NADP); PN oxidation is matched by increased MTP open probability under conditions where the glutathione pool is kept in the fully reduced state; this site can be blocked by N-ethylmaleimide but not by monobromobimane, a thiol-selective reagent. (ii) A second site coincides with the oxidation-reduction-sensitive dithiol we have recently identified (Petronilli, V., Costantini, P., Scorrano, L., Colonna, R., Passamonti, S., and Bernardi, P. (1994) J. Biol. Chem. 269, 16638-16642); dithiol cross-linking at this site by arsenite or phenylarsine oxide is matched by increased MTP open probability under conditions where the PN pool is kept in the fully reduced state; at variance from the first, this site can be blocked by both N-ethylmaleimide and monobromobimane and is probably in equilibrium with the glutathione pool. Based on these findings, we reassess the mechanisms by which many oxidative agents affect the MTP and resolve conflicting reports on the relative role of PN and glutathione oxidation in the permeability transition within the framework of MTP (dys)regulation at two separate sites.
Highlights
Mitochondrial function is affected by oxidative stress, as shown by the permeability increase caused by a wide variety of oxidants (Ref. 1 and references therein)
We show that two sites can be experimentally distinguished. (i) A first site is in apparent oxidation-reduction equilibrium with the pyridine nucleotide (PN) pool (NADH/NAD ؉ NADPH/NADP); PN oxidation is matched by increased MTP open probability under conditions where the glutathione pool is kept in the fully reduced state; this site can be blocked by N-ethylmaleimide but not by monobromobimane, a thiol-selective reagent. (ii) A second site coincides with the oxidation-reductionsensitive dithiol we have recently identified (Petronilli, V., Costantini, P., Scorrano, L., Colonna, R., Passamonti, S., and Bernardi, P. (1994) J
Work by Lehninger and co-workers pointed to the role of the oxidation-reduction level of PN in the modulation of mitochondrial Ca2ϩ fluxes, showing that PN oxidation caused Ca2ϩ efflux, which could be reversed by PN reduction [19]
Summary
Mitochondrial function is affected by oxidative stress, as shown by the permeability increase caused by a wide variety of oxidants (Ref. 1 and references therein). Pfeiffer and co-workers [23] were able to independently modulate the NAD/NADH, NADP/NADPH, and GSSG/GSH ratios and concluded that the latter was the relevant factor since permeabilization could be observed after glutathione oxidation under conditions where the PN pool was kept fully reduced This correlation was subsequently challenged by the finding that the permeability transition induced by organic hydroperoxides could be inhibited by butylhydroxytoluene while glutathione remained oxidized [24] and by the observation that oxidation of glutathione with 1,3-bis(2-chloroethyl)-1nitrosourea (which prevents PN oxidation by inhibiting glutathione reductase) was not followed by Ca2ϩ efflux due to a permeability transition [25].
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