Oxidant-induced inhibition of the plasma membrane Ca2+-ATPase in pancreatic acinar cells: role of the mitochondria

Abstract

Impairment of the normal spatiotemporal pattern of intracellular Ca(2+) ([Ca(2+)](i)) signaling, and in particular, the transition to an irreversible "Ca(2+) overload" response, has been implicated in various pathophysiological states. In some diseases, including pancreatitis, oxidative stress has been suggested to mediate this Ca(2+) overload and the associated cell injury. We have previously demonstrated that oxidative stress with hydrogen peroxide (H(2)O(2)) evokes a Ca(2+) overload response and inhibition of plasma membrane Ca(2+)-ATPase (PMCA) in rat pancreatic acinar cells (Bruce JI and Elliott AC. Am J Physiol Cell Physiol 293: C938-C950, 2007). The aim of the present study was to further examine this oxidant-impaired inhibition of the PMCA, focusing on the role of the mitochondria. Using a [Ca(2+)](i) clearance assay in which mitochondrial Ca(2+) uptake was blocked with Ru-360, H(2)O(2) (50 microM-1 mM) markedly inhibited the PMCA activity. This H(2)O(2)-induced inhibition of the PMCA correlated with mitochondrial depolarization (assessed using tetramethylrhodamine methylester fluorescence) but could occur without significant ATP depletion (assessed using Magnesium Green fluorescence). The H(2)O(2)-induced PMCA inhibition was sensitive to the mitochondrial permeability transition pore (mPTP) inhibitors, cyclosporin-A and bongkrekic acid. These data suggest that oxidant-induced opening of the mPTP and mitochondrial depolarization may lead to an inhibition of the PMCA that is independent of mitochondrial Ca(2+) handling and ATP depletion, and we speculate that this may involve the release of a mitochondrial factor. Such a phenomenon may be responsible for the Ca(2+) overload response, and for the transition between apoptotic and necrotic cell death thought to be important in many disease states.

Fig. 1.

Validation of plasma membrane Ca²⁺-ATPase (PMCA) activity assay. A and B: representative traces showing the intracellular Ca²⁺ ([Ca²⁺]_i) clearance assay (see results) in untreated fura-2-loaded pancreatic acinar cells (A) and cells treated with 10 μM Ru-360 for 30 min, to inhibit mitochondrial Ca²⁺ uptake (B). Cells were treated with 30 μM cyclopiazonic acid (CPA), as indicated by the arrow, in the absence of external Ca²⁺ (0 Ca²⁺) to deplete endoplasmic reticulum Ca²⁺. CPA was present in all solutions throughout the remainder of each experiment. [Ca²⁺]_i clearance was initiated by the addition of 20 mM external Ca²⁺ (20 Ca²⁺), followed by the subsequent removal of external Ca²⁺ (0 Ca²⁺). Arrows represent the steady-state [Ca²⁺]_i (see results) and the standardized [Ca²⁺]_i (300 nM) from which the linear rate of [Ca²⁺]_i clearance was measured (E). The inset in A represents the mean steady-state [Ca²⁺]_i (SS [Ca²⁺]_i) plotted against the mean linear clearance rate measured from the steady-state [Ca²⁺]_i value (SS rate, gray triangles) or standardized 300 nM value (Std rate, black circles). There was a clear correlation between SS rate and SS [Ca²⁺]_i (slope = 1.1 min⁻¹, which was significantly different from zero; P = 0.01), but there was no such correlation between Std rate and SS [Ca²⁺]_i (slope = 0.16 min⁻¹, which was not significantly different from zero; P = 0.16). C and D: inhibition of PMCA activity with 1 mM La³⁺ in control (C) and Ru-360-treated cells (D). EGTA (1 mM) was used to remove the La³⁺ and restore PMCA activity. E: mean standardized linear [Ca²⁺]_i clearance rate in untreated control cells (shown in A), compared with Ru-360-treated cells with or without La³⁺ (*statistical significance as assessed using an unpaired t-test).

Fig. 2.

H₂O₂ (50–1,000 μM) inhibited PMCA activity under conditions in which mitochondrial Ca²⁺ uptake was inhibited. Pancreatic acinar cells were pretreated with 10 μM Ru-360 for 30 min, to inhibit mitochondrial Ca²⁺ uptake, immediately before the start of the [Ca²⁺]_i clearance assay in all experiments. H₂O₂ was added 2–5 min before the addition of 20 mM external Ca²⁺. Representative traces show control cells (A) and the effects of 50 μM (B), 100 μM (C), 500 μM (D), and 1 mM (E) H₂O₂. Gray arrows indicate the point at which CPA was added, which was then present throughout the experiment. Black arrows in D and E show rapid loss of dye due to cell lysis. F: mean data showing the standardized linear [Ca²⁺]_i clearance rate following treatment with varying concentrations of H₂O₂ (*P < 0.001, as assessed using an unpaired t-test).

Fig. 3.

Antimycin-A and CCCP, but not oligomycin, inhibited PMCA activity. As in Figs. 1 and 2, cells were pretreated with Ru-360 (10 μM for 30 min) to inhibit mitochondrial Ca²⁺ uptake in all experiments. Representative traces show control cells (A) and the effects of 4 μM CCCP (Bi and Bii), 0.5 μM antimycin-A (C), and 10 μM oligomycin (D). Arrows indicate the point at which CPA was added, which was then present throughout the experiment. In 60% of cells, CCCP evoked an increase in [Ca²⁺]_i when added in the absence of external Ca²⁺ (Bi) but had no effect in the remaining 40% of cells (Bii). E: mean data showing the standardized linear [Ca²⁺]_i clearance rate following treatment with each mitochondrial inhibitor (*P < 0.05, as assessed using an unpaired t-test). Con, control; Anti, antimycin; Oligo, oligomycin.

Fig. 4.

Effects of H₂O₂, CCCP, antimycin, and oligomycin on ATP depletion (ATP-dep). Pancreatic acinar cells were loaded with 4 μM Magnesium Green (MgGreen) for 30 min at room temperature to indirectly measure cytosolic ATP concentration. Representative traces show the relative MgGreen fluorescence (F/F₀; arbitrary units) in response to the “ATP depletion” cocktail with (gray trace) or without (black trace) preincubation with 10 μM BAPTA (A), 50 μM H₂O₂ (B), 500 μM H₂O₂ (C), 4 μM CCCP (D), 0.5 μM antimycin (E), and 10 μM oligomycin (F). The ATP depletion cocktail consisted of 100 μM carbachol (CCh), 10 μM oligomycin, and 2 mM iodoacetate and was used as a positive control to induce maximum ATP depletion. Cells that exhibited a rapid increase followed by a rapid decrease below the baseline fluorescence (see inset in A) were excluded from analysis because these likely represented cells undergoing cell lysis. Vertical bar represents 0.3 F/F₀ and horizontal bar represents 5 min. G: mean data were quantified and normalized by expressing the change in F/F₀ as a percentage of the ATP depletion cocktail response (*P < 0.05, as assessed using a one-sample t-test).

Fig. 5.

Effects of mitochondrial inhibitors and H₂O₂ on the mitochondrial membrane potential (ΔΨm). Representative traces show the relative tetramethylrhodamine methylester (TMRM) fluorescence (F/F₀; arbitrary units) and corresponding TMRM fluorescent images of time-matched control (A) and cells treated with 0.5 μM antimycin-A (B), 10 μM oligomycin (C), 50 μM H₂O₂ (D), 500 μM H₂O₂ (E), and 500 μM H₂O₂ in the presence of 5 μM cyclosporin (F). A decrease in relative TMRM fluorescence represents mitochondrial depolarization. Inset images in A–C were taken at the corresponding points on the trace as indicated by the arrows. Image A from each experiment indicates a punctate TMRM fluorescence distribution consistent with mitochondrial staining, from which fluorescence changes were measured. In each experiment, 4 μM CCCP was used as a positive control to evoke maximum mitochondrial depolarization. G: mean data were quantified and normalized by expressing the change in F/F₀ as a percentage of the CCCP response (*P < 0.05, as assessed using a Mann-Whitney test). CS-A, cyclosporin-A.

Fig. 6.

Inhibition of the mitochondrial permeability transition pore partially protects the H₂O₂ induced inhibition of the PMCA. Representative traces show a paired experimental design whereby two repetitive [Ca²⁺]_i clearance phases (R₁ and R₂) were invoked by successive application of 20 mM (20 Ca²⁺) and zero external Ca²⁺ (0 Ca²⁺). A: a time-matched control. B: effect of 500 μM H₂O₂ applied during the second clearance phase (R₂). C: combined effect of 500 μM H₂O₂ and 5 μM cyclosporin-A (applied at the same time as CPA and throughout the experiment). D: effect of 500 μM H₂O₂ following preincubation with 50 μM bongkrekic acid for 30 min. E: mean data were quantified and normalized by expressing the linear rate (standardized at 300 nM Ca²⁺) during the second clearance phase as a percentage of the linear rate during the first clearance phase (R₂/R₁ × 100%). *P < 0.05, statistical significance as assessed using a paired one-sample t-test; †P < 0.05, statistical significance as assessed using a Mann-Whitney test.