The work has been carried out following Council of Europe Convention on Bioethics (1997) approved by the Ethics Commission on Animal Experiments of A.A. Bogomoletz Institute of Physiology, NAS of Ukraine (protocol N1/23 of 11.01.2023). Adult Wistar-Kyoto female rats with 180–200 g mean body weight were used. Each experiment was repeated 4–6 times, two animals were used in each experimental setting. The rats were euthanized by intraperitoneal injection of 200 mg/kg sodium pentobarbital (40 mg/mL). After the complete lack of reflexes was ascertained, the head was removed by scissors. The brain was washed by cold 0.9% KCl solution (4 °C), minced, and homogenized in 1:5 volume of the isolation medium: 250 mM sucrose, 1 mM EDTA, 20 mM Tris-HCl buffer, 4 °C (pH 7.2). BSA was added at 1 mg/mL. Mitochondria were isolated by centrifugation at 1000 g for 7 min (4 °C). After the pellet was discarded, the supernatant was centrifuged again at 12,000 g for 15 min (4 °C). The final pellet was resuspended in a small volume of isolation medium without EDTA and stored on ice. The protein content was determined by the Lowry method.

Oxygen consumption was studied polarographically in 1 cm³ closed thermostated cell at 26 °C with the platinum electrode at constant stirring in standard incubation medium: 120 mM KCl, 0.5 mM EDTA, 5 mM sodium glutamate, 1 mM KH₂PO₄, 5 mM Tris-HCl buffer (pH 7.4), oligomycin (1 µg/mg protein). Polarograph LP7 (Czech Republic) equipped with a platinum electrode and the chart recorder was used. Depending on the conditions, MgCl₂ (1 mM), ATP (0.3 mM), glibenclamide (5 µM), and 5-HD (100 µM), were added. Diazoxide was added to the standard incubation medium at the concentrations required. In the presence of Mg²⁺ EDTA was replaced by EGTA. Mitochondria were added at 1.5–2.0 mg/mL protein.

Potassium transport was studied based on the light scattering of mitochondrial suspension. Light scattering is known to decrease because of mitochondrial swelling due to water transport, which accompanies the transport of potassium [13]. Initial rates of potassium transport (V₀) were found by monitoring light scattering on a spectrofluorimeter Hitachi F4000 (Japan) at 520 nm excitation/emission wavelengths in 1 cm³ cell starting from the addition of mitochondria at 0.3 mg/mL to standard incubation medium: 150 mM KCl, 0.5 mM EDTA, 5 mM sodium glutamate, 1 mM KH₂PO₄, 5 mM Tris-HCl buffer (pH 7.4), oligomycin (1 µg/mg protein). Depending on the conditions, MgCl₂ (1 mM), ATP (1 mM), glibenclamide (5 µM), and 5-HD (100 µM), were added. Diazoxide was added to the standard incubation medium at the concentrations required. In the presence of Mg²⁺ EDTA was replaced by EGTA.

The mPTP opening was assessed as cyclosporine A-sensitive swelling of mitochondria by monitoring light scattering of mitochondrial suspensions at 520 nm excitation/emission wavelengths in 1 cm³ cell in the incubation medium: 150 mM KCl, 5 mM sodium succinate (glutamate), 1 mM KH₂PO₄, 5 mM Tris-HCl buffer (pH 7.4). CaCl₂ was added at 20 µM, cyclosporine A was added at 1 µM, diazoxide was added at 0.5 µM, and glibenclamide and 5-HD were added at 5 and 100 µM respectively. The registration of the mPTP activity was monitored for 3 min starting from the addition of mitochondria at 0.3 mg/mL. The decrease of light scattering of mitochondrial suspensions under our experimental conditions was caused both by mPTP opening and potassium uptake from the K⁺-based incubation medium. So, mPTP activity was assessed as a cyclosporine-sensitive difference in swelling amplitude found over the time of registration.

Calcium transport was monitored with Ca²⁺ binding probe chlortetracycline (CTC), which indicated the changes in Ca²⁺ concentration in the matrix [32]. The increase of CTC fluorescence upon Ca²⁺ binding in the matrix was monitored at 392/536 excitation/emission wavelengths, bandpass 5 nm, on a spectrofluorimeter Hitachi F4000 in the medium used for the study of the mPTP opening. Mitochondria were added at 1.0 mg/mL; CTC was added at 10 µM, CaCl₂ was added at 20 µM, cyclosporine A was added at 1 µM. Additionally to the light scattering assay, CTC was used to assess the mPTP opening. The mPTP activity was estimated as a cyclosporine-sensitive difference in Ca²⁺ uptake after 3 min registration and expressed in relative units. Using a sucrose-based medium, we ascertained that neither DZ nor the mKATP blockers glibenclamide and 5-HD affected CTC fluorescence per se.

A widely used probe 2^′,7^′-dichlorofluorescein diacetate (DCFH₂DA) was applied for monitoring ROS formation. This compound readily penetrates mitochondrial membranes following the concentration gradient and deacetylates in the matrix forming membrane-impermeable non-fluorescent derivative H₂DCF (2^′,7^′-dihydrodichlorofluorescein), which is oxidized by mitochondrial ROS resulting in highly fluorescent end product 2^′,7^′-dichlorofluorescein, dichlorofluorescein (DCF) [33]. Mitochondria in stock suspension (20 mg/mL) were preloaded with 200 µM of DCFH₂DA for 30 min at 37 °C in the dark, then washed off the excess probe and stored on ice (4 °C). After adding mitochondria at 1 mg/mL to the incubation medium chosen for the study of the mPTP opening, the increase in DCF fluorescence reflecting ROS formation was monitored on a spectrofluorimeter Hitachi F4000 over 5 min time intervals, with monitoring of mPTP opening in parallel experiments. Quasi-linear segments of the time courses of DCF fluorescence were chosen to estimate the rate of ROS production. As we have shown earlier [34], this coincided with keeping stable rates of the state 4 respiration under steady-state conditions. The rate of the increase in DCF fluorescence was assumed to reflect the rate of ROS formation in mitochondria.

Membrane potential ($\mathrm{\Delta }$$\mathrm{\Psi }$_m) was assessed with a membrane potential probe safranin [35] added at 10 µM to standard incubation medium: 150 mM KCl, 0.5 mM EDTA, 5 mM sodium glutamate, 1 mM KH₂PO₄, 5 mM Tris-HCl buffer (pH 7.4), oligomycin (1 µg/mg protein). Fluorescence was measured at 495/586 excitation/emission wavelengths with Hitachi F4000 spectrofluorometer. Complete membrane depolarization was verified by adding 10^-6 M carbonyl cyanide-m-chlorophenylhydrazone (CCCP). The difference between the safranin fluorescence in de-energized and fully energized mitochondria was taken for $\mathrm{\Delta }$$\mathrm{\Psi }$_m.

All reagents were from Sigma-Aldrich (St. Louis, MO, USA). Deionized water was used in all experiments for solutions preparations.

The data were expressed as mean $\pm$ S.E. of 4–6 independent experiments. One-way ANOVA statistics followed by post-hoc Tuckey’s test were applied to find significant differences between groups. p 0.05 was taken as the level of significance. Statictics was calculated based on the online software: https://www.statskingdom.com.

Light scattering (LS) is one of the most direct approaches to assess potassium transport in isolated mitochondria, because of the concomitant water transport, which uptake accompanies K⁺ uptake via K⁺ channels, and the efflux occurs together with K⁺ release via K⁺/H⁺ exchanger [13]. So, we studied the effect of DZ and pharmacological mKATP channel modulators on the light scattering of mitochondrial suspensions.

In Fig. 1A–C representative traces of K⁺ uptake in the presence of different concentrations of DZ and the modulators of the mKATP channels, MgATP, and glibenclamide are shown. As we observed earlier in liver mitochondria, the mKATP channel of brain mitochondria was highly sensitive to sub-micromolar concentrations of DZ (Fig. 1A). An activation of rat brain mitochondria KATP channel by DZ occurred with K_a ~150 nM and reached a plateau at ~0.5 µM (Fig. 1D). ATP-sensitive K⁺ transport was routinely blocked by ATP in the presence of Mg²⁺ (Fig. 1B,E), but individually neither Mg²⁺, nor ATP at 1 mM concentrations affected the sensitivity of mKATP channel to DZ (Fig. 1E, 1–3).

Fig. 1.

Typical records of the light scattering (LS) of mitochondrial suspensions in the presence of the mKATP channels modulators. (A–C) Representative traces of light scattering in the presence of the mKATP channel modulators. (D) Initial rates of swelling in the presence of the mKATP channels modulators. (E) Normalized rates of DZ-stimulated swelling of brain mitochondria. (F) ATP-sensitive component of K⁺ transport found from the blocking by the MgATP, glibenclamide (Glb), and 5-HD. M $\pm$ m, n = 6; # p 0.05 as compared to control; * p 0.05 as compared to Mg²⁺; & p 0.05 as compared to Mg²⁺, DZ; ^ p 0.05 as compared to MgATP. mKATP, mitochondrial KATP chann; DZ, diazoxide; 5-HD, 5-hydroxy decanoate; F, a.u., F, arbitrary units.

In agreement with the common knowledge [21, 22, 23], KATP channels’ activity was routinely restored by the high micromolar concentrations of DZ (30 µM) in the presence of 1 mM MgATP (Fig. 1C,E). To confirm the activation of the mKATP channel, we used KATP channel opener pinacidil at the same concentrations. The effects of pinacidil were similar to the impact of DZ (Fig. 1A,D). The mKATP channel activated by the high micromolar concentrations of DZ in the presence of a MgATP was blocked by glibenclamide (5 µM) and selective mKATP channels blocker 5-HD (100 µM). Similar effects were observed with pinacidil (Fig. 1A,E). Besides the above experiments, which ascertained the activity of the mKATP channel in mitochondrial preparations, we observed the native mKATP channel’s high sensitivity to DZ and its activation by this drug at sub-micromolar concentrations in the absence of a MgATP (Fig. 1A,D). Similar to the activation by DZ, the mKATP channel blockage by glibenclamide and 5-HD did not require a MgATP either (Fig. 1B,E).

To prove the identity of an ATP-sensitive K⁺ transport observed in our experiments with the mKATP channel activity, we used a MgATP for additional blockage of the ATP-sensitive K⁺ transport. As shown in the experiments, combined blockage of native ATP-sensitive K⁺ transport by glibenclamide and ATP in the presence of Mg²⁺ gave the same results as individual blockage of K⁺ transport either by glibenclamide or MgATP (Fig. 1B,E). Close results were obtained by blocking of an ATP-sensitive K⁺ transport activated by sub-micromolar concentrations of DZ by glibenclamide in the absence of a MgATP (Fig. 1C,E). These data indicated a lack of any K⁺ transport activity sensitive to individual mKATP channel blockers (glibenclamide (Glb), 5-hydroxy decanoate (5-HD)), and not sensitive to an MgATP. Worth notion of the equality of the components of native K⁺ transport sensitive to glibenclamide, 5-HD, and MgATP, K⁺ transport activated by DZ in the absence of an MgATP, and K⁺ transport activated by the high concentrations of DZ in the presence of an MgATP (Fig. 1F).

To confirm the results obtained by the light scattering assay, the effects of DZ on the mKATP channel activity were assessed by measuring oxygen consumption in mitochondria. This approach has the advantage of the assessment of the mKATP channels activity ‘in situ’ allowing for the consecutive additions of the mKATP channels modulators to the same suspension of respiring mitochondria in which a constant rate of potassium transport is kept by the K⁺ cycle [13]. In Fig. 2 typical polarographic records of mitochondrial respiration are shown. DZ increased the state 4 oxygen consumption and the rate of K⁺ uptake in a concentration-dependent way (Fig. 2A), which resembled liver mitochondria (Fig. 3A).

Fig. 2.

Representative polarographic records of glutamate-driven respiration of brain mitochondria in the presence of the modulators of the mKATP channel. (A) The effect of DZ on the rate of respiration. (B,C) The effects of Mg²⁺, ATP, and pharmacological KATP channel blockers, Glb and 5-HD, on oxygen consumption under various conditions. The rates of respiration are indicated on the curves in ng-at. O$\cdot$min^-1$\cdot$mg^-1; additions indicated by the arrows.

Fig. 3.

The effects of DZ and the mKATP channel blockers on the oxygen consumption in brain mitochondria respiring on glutamate. (A) Normalized dependence of the respiration rates on DZ concentration in brain and liver mitochondria; on the ordinate axis: normalized oxygen consumption rate. (B) The rates of respiration in the presence of the mKATP channel modulators. (C) The effect of DZ on the MgATP-, Glb-, and 5-HD-sensitive parts of state 4 respiration. M $\pm$ m, n = 6. # p 0.05 as compared to control (0 DZ); * p 0.05 as compared to Mg²⁺; & p 0.05 as compared to MgATP; ^ p 0.05 as compared to MgATP, 30 µM DZ (B). * p 0.05 as compared to MgATP-sensitive part of respiration in the absence of Glb and 5-HD (C).

The sensitivity of K⁺ transport to DZ in native mitochondria without a MgATP well agreed with our earlier results on liver mitochondria (Fig. 3A) and the results obtained by light scattering measurements (Fig. 1). To prove an activation of the mKATP channel by DZ in the absence of a MgATP, we blocked both native and activated channels by a MgATP, glibenclamide, and 5-HD and compared a MgATP-sensitive difference in the rate of native and DZ-stimulated respiration. As we observed, in the absence of a MgATP, DZ increased a MgATP-sensitive part of respiration at low sub-micromolar concentrations, and full activation of oxygen consumption was reached at $\le$0.5 µM DZ (Fig. 3A,B). Similar to light scattering data, a MgATP-sensitive part of respiration equaled to glibenclamide- and 5-HD-sensitive ones in native mitochondria, and showed a stimulation of an ATP-sensitive K⁺ transport by DZ in the absence of a MgATP (Fig. 3C). The rate of respiration stimulated by sub-micromolar concentrations of DZ in the absence of a MgATP equaled the one stimulated by the high micromolar concentrations of DZ in the presence of a MgATP (Fig. 3C).

To prove the identity of the ATP-sensitive K⁺ transport activated by DZ under different conditions, we blocked K⁺ transport by a MgATP, glibenclamide, and 5-HD (Fig. 2B,C; Fig. 3B). Similar to light scattering, oxygen consumption too was blocked by glibenclamide and 5-HD in the absence of a MgATP (Fig. 2B,C). In the presence of Mg²⁺, in native mitochondria, glibenclamide suppressed part of respiration, which was not blocked further by the addition of ATP (Figs. 2B,C). The addition of ATP in the presence of Mg²⁺ further suppressed respiration because of the blockage of the mKATP channel, and this effect was not increased by the following addition of glibenclamide (Fig. 2B,C). Similar results were obtained with 5-HD. The results of the experiments summarized in Fig. 3B indicated an identity of the parts of respiration blocked by MgATP, glibenclamide, and 5-HD in native mitochondria, which allowed us to ascribe these effects to the same mKATP channel activity. With the same approach, we have shown an activation of an ATP-sensitive K⁺ transport by sub-micromolar concentrations of DZ in the absence of an MgATP. As before, a glibenclamide-sensitive part of the respiration equaled a MgATP-sensitive one, and a MgATP-sensitive part of respiration related to the mKATP channel activity equaled the rates of respiration blocked by glibenclamide and 5-HD (Fig. 3C).

An estimation of the rate of ATP-sensitive K⁺ transport from polarographic data, based on K⁺:O stoichiometry 10:1 gave ~40 $\pm$ 10 nmol K⁺$\cdot$min^-1$\cdot$mg^-1 in native mitochondria, ~95 $\pm$ 12 nmol K⁺$\cdot$min^-1$\cdot$mg^-1 after the activation by DZ in the absence of a MgATP, and ~105 $\pm$ 10.5 nmol K⁺$\cdot$min^-1$\cdot$mg^-1 after the routine blocking of the mKATP channel by a MgATP and the reactivation by the high micromolar concentrations of DZ. In all cases, an estimate of the rate of the ATP-sensitive part of the respiration related to the mKATP channel activity was independent of the way of the mKATP channel blockage by MgATP, Glb, or 5-HD.

The increase in CTC fluorescence reflects the elevation of Ca²⁺ concentration in the mitochondrial matrix [32]. As shown in the experiments, Ca²⁺ accumulation observed with CTC well responded to standard assays of Ca²⁺ transport: Ca²⁺ accumulation was followed by Ca²⁺ efflux after the protonophore CCCP addition at 10^-6 M. Using a sucrose-based medium, we ascertained that neither DZ nor the mKATP channel blockers glibenclamide and 5-HD affected CTC fluorescence per se.

Ca²⁺ uptake was elevated by DZ and blocked by glibenclamide and 5-HD without a MgATP (Fig. 4). Assuming that Ca²⁺ uptake followed first-order kinetics, the rate constants (k) found from semi-logarithmic plots were (3.2 $\pm$ 0.2)$\cdot$10^-2/s in control and increased to (4.1 $\pm$ 0.2)$\cdot$10^-2/s (p 0.05) in the presence of DZ. The mKATP channel blocker glibenclamide reduced the rate constant of Ca²⁺ uptake to the control level.

Fig. 4.

The effects of DZ and the mKATP channel blockers on Ca²⁺ uptake and the membrane potential in rat brain mitochondria. (A) Typical records of Ca²⁺ uptake and CCCP-induced Ca²⁺ efflux. (B–D) The effects of DZ, Glb, and 5-HD on Ca²⁺ uptake. (E) The effects of DZ, 5HD, and Glb on the membrane potential of mitochondria. M $\pm$ m; n = 4; * p 0.05 as compared to control; # p 0.05 as compared to DZ. CCCP, carbonyl cyanide-mchlorophenylhydrazone.

Earlier we have shown that the mKATP channels’ activity contributed to the increase in matrix calcium accumulation in native brain mitochondria [29]. As we observed in this study, the mKATP channel activation by DZ still promoted Ca²⁺ uptake, independent of the respiratory substrate (Fig. 4B,C). The mKATP channels blockers, glibenclamide, and 5-HD similarly reduced Ca²⁺ uptake in mitochondria without a MgATP (Fig. 4B,C), contrary to their hyperpolarizing effect in brain mitochondria (Fig. 4E), which proved that the stimulation effect of DZ on Ca²⁺ uptake resulted from the mKATP channels’ activation.

Ca²⁺ uptake in mitochondria via the Ca²⁺ uniporter is known to be strongly dependent on $\mathrm{\Delta }$$\mathrm{\Psi }$_m. The electrochemical potassium transport occurring at the cost of $\mathrm{\Delta }$µ_H+ should depolarize mitochondria, and the blocking of the native mKATP channel, as we showed earlier, produced a hyperpolarizing effect [29, 30]. The blocking of the mKATP channel activated by DZ similarly increased $\mathrm{\Delta }$$\mathrm{\Psi }$_m (Fig. 4E). However, despite the hyperpolarizing effect of the mKATP channel blockers caused by the full blocking of ATP-sensitive K⁺ transport, glibenclamide and 5-HD similarly reduced Ca²⁺ uptake (Fig. 4B–D). Both activation of Ca²⁺ transport by DZ, and the reversal of this effect by glibenclamide and 5-HD occurred without a MgATP (Fig. 4B,C). This confirmed the elevation of Ca²⁺ uptake produced by the mKATP channel opener and ascertained that the stimulation effect of DZ on Ca²⁺ uptake resulted from the activation of ATP-sensitive K⁺ transport in brain mitochondria.

Complex effects of the mKATP channel modulators on ROS production were found, dependent on the respiratory substrate (Fig. 5). So, in mitochondria respiring on glutamate, DZ reduced ROS production (Fig. 5A), which was in line with our earlier data on liver mitochondria [31] and could be explained by the respiratory uncoupling and slight mitochondrial depolarization. Glibenclamide and 5-HD reversed this effect and increased ROS production (Fig. 5A), which correlated with the repolarization of mitochondria resulting from the blockage of the mKATP channel (Fig. 4E).

Fig. 5.

The effects of DZ and the mKATP channel blockers on ROS production in mitochondria. (A–C) Typical time courses of dichlorofluorescein (DCF) fluorescence in mitochondria respiring on glutamate (A) and succinate (B,C). (D) The rate of ROS formation, F/min. M $\pm$ m, n = 6; * p 0.05 as compared to succinate (1); # p 0.05 as compared to succinate+DZ (2); & p 0.05 as compared to succinate+rotenone (5); + p 0.05 as compared to succinate, rotenone, DZ (6); @ p 0.05 as compared to glutamate (9); ^ p 0.05 as compared to glutamate+DZ (10).

However, with succinate as a respiratory substrate, opposing effects were observed: DZ reliably increased ROS production (Fig. 5B, 1, 2). Diazoxide-stimulated increase in ROS production was suppressed by rotenone (Fig. 5B, 3), which could indicate the involvement of the reverse electron transport in the stimulation effect of DZ on ROS formation. In this case, too, activation of ROS formation by DZ was reversed by glibenclamide and 5-HD (Fig. 5B, 4,5), which indicated the involvement of the mKATP channels’ activity in this effect.

As we have found, after the blocking of reverse electron transport (RET) by rotenone, the mKATP channels opening by DZ reduced ROS production (Fig. 5C, 3), indicating the inhibitory action of DZ on ROS formation by forward electron transport. Similarly to what was observed with glutamate, in this case, blocking the mKATP channels by glibenclamide and 5-HD increased ROS production (Fig. 5C, 3). The results of the study of the impact of the mKATP channel modulation on ROS production under different conditions are summarized in Fig. 5D.

The mPTP opening was assessed by the light scattering as the cyclosporine-sensitive swelling of mitochondria. Representative traces reflecting the effects of DZ on the changes in mitochondrial matrix volume sensitive to cyclosporine A (CsA) under different conditions are shown in Fig. 6A–C. The data on the CsA-sensitive amplitude of mitochondrial swelling are summarized in Fig. 6F.

Fig. 6.

The effects of DZ on the mPTP opening in rat brain mitochondria. (A–C) Typical records of the light scattering in mitochondria respiring on glutamate (A), succinate (B,C); with the addition of rotenone (C). (D,E) Typical records of Ca²⁺ transport in mitochondria respiring on succinate in the absence (dotted lines) and presence of CsA (solid lines) at the additions of DZ, rotenone, and Glb; (F) CsA-sensitive swelling amplitude by the light scattering in the presence of DZ. (D,E) The number on the curves corresponds to: 1— succinate, CsA; 2— succinate, CsA, DZ, CsA; 3— succinate; 4— succinate, DZ; 5— succinate, rotenone; 6— succinate, Glb, CsA; 7— succinate, Glb. (F) M $\pm$ m, n = 4; * p 0.05 compared to glutamate; ** p 0.05 compared to succinate; # p 0.05 compared to succinate+Rot. CsA, cyclosporine A; mPTP, mitochondrial permeability transition pore.

As we observed, the effect of DZ on the mPTP opening strongly depended on the respiratory substrate used. Independent of the stimulation of Ca²⁺ uptake by DZ, the regulation of mPTP opening strongly correlated with its impact on ROS production. DZ suppressed the mPTP opening when glutamate was used as a substrate. However, an mPTP opening was promoted by DZ in mitochondria, which respired on succinate. In the presence of rotenone strong mPTP inhibition was observed (Fig. 6C). While it was shown in the literature that rotenone could inhibit the mPTP independently of ROS formation [36], activation of the mKATP channel highly strengthened this effect (Fig. 6C). Obtained results indicated an inhibitory effect of the mKATP channels opening on ROS production under the conditions which allowed only for the forward electron transport in the respiratory chain. Suppression of ROS formation resulted in the mPTP inhibition in mitochondria respiring on glutamate and succinate in the presence of rotenone (Fig. 6A,C). However under the conditions when RET was observed, the mPTP was highly activated by DZ, which correlated with the stimulation of ROS production.

In line with these observations, with succinate as substrate, in the absence of rotenone, glibenclamide reduced the mPTP activity (Fig. 6E,F). Similarly to the effects of DZ, the opposing effect of glibenclamide on the mPTP activity in mitochondria respiring on succinate could be explained mainly by the regulation of ROS production. Thus, we can assume that glibenclamide could inhibit the mPTP opening in those cases when it reduced ROS formation simultaneously with the reduction of Ca²⁺ uptake, which caused an inhibition of the mPTP opening (Fig. 6E,F).