The Short-Term Opening of Cyclosporin A-Independent Palmitate/Sr2+-Induced Pore Can Underlie Ion Efflux in the Oscillatory Mode of Functioning of Rat Liver Mitochondria

Abstract

Mitochondria are capable of synchronized oscillations in many variables, but the underlying mechanisms are still unclear. In this study, we demonstrated that rat liver mitochondria, when exposed to a pulse of Sr²⁺ ions in the presence of valinomycin (a potassium ionophore) and cyclosporin A (a specific inhibitor of the permeability transition pore complex) under hypotonia, showed prolonged oscillations in K⁺ and Sr²⁺ fluxes, membrane potential, pH, matrix volume, rates of oxygen consumption and H₂O₂ formation. The dynamic changes in the rate of H₂O₂ production were in a reciprocal relationship with the respiration rate and in a direct relationship with the mitochondrial membrane potential and other indicators studied. The pre-incubation of mitochondria with Ca²⁺(Sr²⁺)-dependent phospholipase A₂ inhibitors considerably suppressed the accumulation of free fatty acids, including palmitic and stearic acids, and all spontaneous Sr²⁺-induced cyclic changes. These data suggest that the mechanism of ion efflux from mitochondria is related to the opening of short-living pores, which can be caused by the formation of complexes between Sr²⁺(Ca²⁺) and endogenous long-chain saturated fatty acids (mainly, palmitic acid) that accumulate due to the activation of phospholipase A₂ by the ions. A possible role for transient palmitate/Ca²⁺(Sr²⁺)-induced pores in the maintenance of ion homeostasis and the prevention of calcium overload in mitochondria under pathophysiological conditions is discussed.

Keywords: cyclosporin A; cyclosporin A-independent palmitate/Ca2+-induced permeability transition pore; ion oscillations; lipid pore; mitochondria; mitochondrial permeability transition; palmitic acid; phospholipase A2.

Figure 1

Single pulse addition of Sr²⁺ (shown by the arrow) induces rapid reversible changes in Sr²⁺ fluxes (a), membrane potential (b), respiratory rate (c), and slow changes in K⁺ fluxes (d) in the suspension of rat liver mitochondria. The incubation medium contained 20 mM sucrose, 1 mM KCl, 1 μM TPP⁺, 1 μM CsA, 1 μM rotenone, 5 mM succinic acid, and 12.5 mM Tris (pH 7.3). Addition: 45 nmol SrCl₂/mg of mitochondrial protein. The dotted arrow indicates a change in the membrane potential. The typical traces are presented (n = 7).

Figure 2

Simultaneous recording of Sr²⁺/valinomycin-induced oscillations in Sr²⁺ and K⁺ fluxes and membrane potential of rat liver mitochondria. The medium contained 20 mM sucrose, 1 mM KCl, 1.5 μM TPP⁺, 1 μM CsA, 1 μM rotenone, 5 mM succinic acid, and 12.5 mM Tris (pH 7.3). Additions: 2 ng valinomycin and 45 nmol SrCl₂/mg of protein. The typical traces are presented (n = 5).

Figure 3

Sr²⁺/valinomycin-induced cyclic changes in matrix volume of rat liver mitochondria. The experimental conditions were the same as in Figure 2. Additions: 2 ng valinomycin and 45 nmol SrCl₂/mg of protein. The typical traces are presented (n = 5).

Figure 4

Sr²⁺/valinomycin-induced dynamic changes in the respiration rate (trace 1), H₂O₂ production rate (trace 2), and the membrane potential (trace 3) of rat liver mitochondria: (a) Reciprocal changes in the rates of oxygen consumption and H₂O₂ production by rat liver mitochondria after the addition of Sr²⁺ and valinomycin. The conditions were the same as in Figure 2. The rate of mitochondrial respiration (O₂ slope, (pmol/s·ml)) was measured polarographically using an Oxygraph-2k respirometer and DatLab software (Oroboros Instruments, Innsbruck, Austria). The rate of H₂O₂ production (H₂O₂, (pmol/s·ml)) by the mitochondria was determined by the Amplex Red/peroxidase assay. The typical traces are presented (n = 5); (b) Synchronous changes in the rate of H₂O₂ production and the membrane potential of rat liver mitochondria after the addition of Sr²⁺ ions and valinomycin. The conditions were the same as in Figure 2. The mitochondrial membrane potential was estimated with the use of tetraphenylphosphonium (TPP⁺) and an electrode selective for TPP⁺ as described in the Materials and Methods section. The amount of TPP⁺ accumulated in mitochondria was determined by measuring the difference between its initial concentration and the concentration after the addition of mitochondria. The typical traces are presented (n = 5).

Figure 5

A comparative analysis of the content of the main free fatty acids in rat liver mitochondria before (control) and after the onset of Sr²⁺/valinomycin (Val)-induced ion oscillations in the absence (0.1% DMSO) or presence of 25 μM aristolochic acid (ArA), a phospholipase A₂ (PLA₂) inhibitor. The medium and conditions were the same as in Figure 2. The inhibitor of PLA₂ ArA or 0.1% DMSO was added to a mitochondrial suspension 1 min before the addition of Sr²⁺. Data represent the means ± SEM of at least four independent experiments. * p < 0.05 vs. the control group (without additions); ^# p < 0.05 vs. the experimental group without PLA₂ inhibitor (0.1% DMSO).

Figure 6

Blocking effect of aristolochic acid (25 μM), a phospholipase A₂ inhibitor, on Sr²⁺/valinomycin-induced cyclic changes in the fluxes of Sr²⁺ (a), K⁺ (b), TPP⁺ (c), respiration rate (d), matrix volume (e), and H₂O₂ production rate (f) in rat liver mitochondria. The medium and conditions were the same as in Figure 2. Additions: 2 ng valinomycin and 45 nmol SrCl₂/mg of protein. The typical traces are presented (n = 5).

Figure 7

Ultrastructural localization of the group IV Ca²⁺(Sr²⁺)-dependent cytosolic phospholipase A₂ (cPLA₂) in isolated rat liver mitochondria. Mitochondria were incubated with specific antibodies against cPLA₂ and secondary antibodies labeled with 10 nm colloidal gold nanoparticles. Black granules (shown by the arrows) are the binding sites for the antibodies to the target proteins. The scale bar is 0.25 μm.