Increase in the toxic effects of Tl+ on isolated rat liver mitochondria in the presence of nonactin

The effects of Tl(+) ions on isolated rat liver mitochondria were studied in the presence of nonactin, a cyclic ionophore. Nonenergized rat liver mitochondria were increasingly swollen at an elevated concentration of Tl(+) in the 160 mOsm medium containing 0-150 mM sucrose and 0-75 mM TlNO(3) or 0-50 mM Tl acetate. On the contrary, mitochondria in experiments with nonactin were contracted in the medium with 5-25 mM Tl(+) and were swollen only in the medium with 50-75 mM TlNO(3) or 50 mM Tl acetate. State 4 respiration along with swelling of succinate-energized mitochondria followed contraction after their deenergization was further enhanced at increasing concentration of Tl acetate in a medium containing nonactin. Regardless of the presence of nonactin, State 3 and 2,4-dinitrophenol (DNP)-stimulated respiration and the monoamine oxidase (MAO) activity were not affected in the medium with 0-25 mM Tl acetate and sucrose. DNP-stimulated respiration decreased and the MAO activity somewhat increased in the medium containing 50 mM Tl acetate and nonactin. Uptake of (86)Rb(+) by energized mitochondria in the presence of valinomycin was considerably decreased when Tl(+) and nonactin were simultaneously present in the medium. An increase of the toxic effect of Tl(+) on rat liver mitochondria in the presence of nonactin is accounted for by disruption of mitochondria due to their more extensive swelling and uncoupling of mitochondria, resulting in the stimulation of State 4 and depletion of their energy store.     

Properties of Ca(2+) transport in mitochondria of Drosophila melanogaster

We have studied the pathways for Ca(2+) transport in mitochondria of the fruit fly Drosophila melanogaster. We demonstrate the presence of ruthenium red (RR)-sensitive Ca(2+) uptake, of RR-insensitive Ca(2+) release, and of Na(+)-stimulated Ca(2+) release in energized mitochondria, which match well characterized Ca(2+) transport pathways of mammalian mitochondria. Following larger matrix Ca(2+) loading Drosophila mitochondria underwent spontaneous RR-insensitive Ca(2+) release, an event that in mammals is due to opening of the permeability transition pore (PTP). Like the PTP of mammals, Drosophila Ca(2+)-induced Ca(2+) release could be triggered by uncoupler, diamide, and N-ethylmaleimide, indicating the existence of regulatory voltage- and redox-sensitive sites and was inhibited by tetracaine. Unlike PTP-mediated Ca(2+) release in mammals, however, it was (i) insensitive to cyclosporin A, ubiquinone 0, and ADP; (ii) inhibited by P(i), as is the PTP of yeast mitochondria; and (iii) not accompanied by matrix swelling and cytochrome c release even in KCl-based medium. We conclude that Drosophila mitochondria possess a selective Ca(2+) release channel with features intermediate between the PTP of yeast and mammals.     

Complex contribution of cyclophilin D to Ca2+-induced permeability transition in brain mitochondria, with relation to the bioenergetic state

Cyclophilin D (cypD)-deficient mice exhibit resistance to focal cerebral ischemia and to necrotic but not apoptotic stimuli. To address this disparity, we investigated isolated brain and in situ neuronal and astrocytic mitochondria from cypD-deficient and wild-type mice. Isolated mitochondria were challenged by high Ca(2+), and the effects of substrates and respiratory chain inhibitors were evaluated on permeability transition pore opening by light scatter. In situ neuronal and astrocytic mitochondria were visualized by mito-DsRed2 targeting and challenged by calcimycin, and the effects of glucose, NaCN, and an uncoupler were evaluated by measuring mitochondrial volume. In isolated mitochondria, Ca(2+) caused a large cypD-dependent change in light scatter in the absence of substrates that was insensitive to Ruthenium red or Ru360. Uniporter inhibitors only partially affected the entry of free Ca(2+) in the matrix. Inhibition of complex III/IV negated the effect of substrates, but inhibition of complex I was protective. Mitochondria within neurons and astrocytes exhibited cypD-independent swelling that was dramatically hastened when NaCN and 2-deoxyglucose were present in a glucose-free medium during calcimycin treatment. In the presence of an uncoupler, cypD-deficient astrocytic mitochondria performed better than wild-type mitochondria, whereas the opposite was observed in neurons. Neuronal mitochondria were examined further during glutamate-induced delayed Ca(2+) deregulation. CypD-knock-out mitochondria exhibited an absence or a delay in the onset of mitochondrial swelling after glutamate application. Apparently, some conditions involving deenergization render cypD an important modulator of PTP in the brain. These findings could explain why absence of cypD protects against necrotic (deenergized mitochondria), but not apoptotic (energized mitochondria) stimuli.     

Mersalyl prevents the Tl+-induced permeability transition pore opening in the inner membrane of Ca2+-loaded rat liver mitochondria

It was earlier shown that the calcium load of rat liver mitochondria in medium containing TlNO₃ and KNO₃ resulted in the Tl⁺-induced mitochondrial permeability transition pore (MPTP) opening in the inner membrane. This opening was accompanied by an increase in swelling and membrane potential dissipation and a decrease in state 3, state 4, and 2,4-dinitrophenol-uncoupled respiration. This respiratory decrease was markedly leveled by mersalyl (MSL), the phosphate symporter (PiC) inhibitor which poorly stimulated the calcium-induced swelling, but further increased the potential dissipation. All of these effects of Ca²⁺ and MSL were visibly reduced in the presence of the MPTP inhibitors (ADP, N-ethylmaleimide, and cyclosporine A). High MSL concentrations attenuated the ability of ADP to inhibit the MPTP. Our data suggest that the PiC can participate in the Tl⁺-induced MPTP opening in the inner membrane of Ca²⁺-loaded rat liver mitochondria.