Fluctuations in mitochondrial membrane potential in single isolated brain mitochondria: modulation by adenine nucleotides and Ca2+

Abstract

In this study we investigated fluctuations in mitochondrial membrane potential (DeltaPsim) in single isolated brain mitochondria using fluorescence imaging. Mitochondria were attached to coverslips and perfused with K+-based buffer containing 20 microM EDTA, supplemented with malate and glutamate, and rhodamine 123 for DeltaPsim determination. DeltaPsim fluctuations were triggered by mitochondrial Ca2+ uptake since they were inhibited by both ruthenium red, a Ca2+-uniporter blocker, and by high concentrations of EGTA. A very low concentration of Ca2+ (approximately 30 nM) was required to initiate the fluctuations. Both ATP and ADP reversibly inhibited DeltaPsim fluctuations, with maximal effects occurring at 100 microM. The effect of nucleotides could not be explained by the reversed mode of mitochondrial ATP-synthase, since oligomycin was not effective and nonhydrolysable analogs of ATP and ADP did not stop the fluctuations. The effects of adenine nucleotides were abolished by blockade of the adenine nucleotide translocator with carboxyatractyloside, but were insensitive to another inhibitor, bongkrekic acid. ATP-sensitive K+-channels are not involved in the mechanism of DeltaPsim fluctuations, since the inhibitor 5-hydroxydecanoate or the activator diazoxide did not affect dynamics of DeltaPsim. We suggest DeltaPsim fluctuations in brain mitochondria are not spontaneous, but are triggered by Ca2+ and are modulated by adenine nucleotides, possibly from the matrix side of the inner mitochondrial membrane.

FIGURE 1

Phase-contrast (A) and fluorescence (B) images of single isolated brain mitochondria attached to the coverslip. Mitochondria were incubated with HBS containing 200 nM Rh123.

FIGURE 2

Stabilizing effect of adenine nucleotides on ΔΨ_m. (A and B) Representative traces of three individual mitochondria show changes in Rh123 fluorescence in control and during application of 100 μM ATP (panel A) or ADP (panel B). (C) Concentration dependence of adenine nucleotide effects. Each histogram represents the mean (± SE) of four experiments, with ∼150 mitochondria analyzed in each experiment.

FIGURE 3

Adenine nucleotide analogs and inhibition of mitochondrial ATP-synthase have no effect on ΔΨ_m dynamics. (A and B) Representative traces of individual mitochondria exposed to 300 μM AMP-PNP and 100 μM ATP (A); 300 μM AMP-CP and 100 μM ADP (B); (C) application of 100 μM ATP in the presence of 2 μM oligomycin; and (D) Summarized data show the frequency of ΔΨ_m fluctuations at the experimental conditions illustrated in A–C. Each histogram represents the mean (± SE) of four experiments with 100–150 mitochondria analyzed in each experiment.

FIGURE 4

Effect of ANT inhibition on ΔΨ_m dynamics and mitochondrial respiration. (A i and ii) representative traces of ΔΨ_m measured in individual mitochondria exposed to 2 μM CA before and after ATP (100 μM) application. (A iii) an example of ΔΨ_m dynamics in individual mitochondria exposed to 2 μM BA against a background of 100 μM ADP. (A iv) Summarized data shows the frequency of ΔΨ_m fluctuations in mitochondria treated with 100 μM ATP or ADP, 2 μM CA or BA and the combinations of these drugs. Each bar represents the mean value of 5–8 experiments (± SE), 100–150 mitochondria were analyzed in each experiment. (B) The rate of respiration measured by Clark electrode (as described in Methods section) in mitochondria stimulated by ADP. The effect of 2 μM CA and BA is shown on the records from the individual experiments (i and ii) and as mean (± SE) respiration rate calculated from five experiments (iii). The rate of oxygen consumption was normalized to that measured at ADP application.

FIGURE 5

Effect of inhibition of mitochondrial Ca²⁺ uptake on ΔΨ_m fluctuations. (A and B) the changes in Rh123 fluorescence in the individual mitochondria in response to 2 μM RuRed and 150 μM EGTA. (C) Summarized data of the experiments illustrating in A and B; the mean (± SE) frequency of fluctuations was calculated from four experiments, 100 individual mitochondria were analyzed in each experiment.

FIGURE 6

ADP stops Ca²⁺-triggered fluctuations in ΔΨ_m. EGTA (230 μM), Ca²⁺ (15 μM), and ADP (100 μM) were added to normal HBS; the free Ca²⁺ concentrations calculated by using Max-Chelator program (see Methods) were 30.65 nM in EGTA + Ca²⁺ and 30.58 nM in EGTA + Ca²⁺ + ADP.