1Departamento de Fisiologia e Biofísica, Instituto de Ciências Biomédicas, and 2Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, 05508-900 São Paulo, Brazil
Submitted 13 March 2003 ; accepted in final form 26 August 2003
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ABSTRACT |
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kidney mitochondria; K+ transport; ischemic preconditioning; uncoupling
The concomitant activity of K+ uptake through mitoKATP and removal by the K+/H+ exchanger would be expected to result in a decrease of the inner membrane electrochemical H+ gradient, which could hamper oxidative phosphorylation. However, mitoKATP activity was found to be compatible with the maintenance of oxidative phosphorylation because K+ transport rates through this channel in all mammalian tissues studied to date are very limited (4, 28). Indeed, mitoKATP activation seems to be directly involved in the regulation of more effective oxidative phosphorylation, because the small increments in K+ transport promoted by this channel are accompanied by substantial increases in mitochondrial matrix volume, resulting in more efficient respiration (21), oxidative phosphorylation (11), and less ATP loss when respiration is inhibited (6). MitoKATP may also be involved in the regulation of mitochondrial reactive oxygen species generation (7, 12, 14, 29).
The specific effects of mitoKATP on mitochondrial structure and function gained interest when Garlid et al. (18) demonstrated that diazoxide (DZX), a KATP agonist used at concentrations that activate mitoKATP but not plasma membrane KATP channels, protected heart tissue against ischemic damage. The protective effect of mitoKATP opening was later confirmed by independent groups, both in heart tissue and in brain (10, 18). MitoKATP was also shown to participate in heart ischemic preconditioning (the protective effect of short, nondeleterious ischemic episodes on a longer, potentially damaging ischemic event), because this process was inhibited by mitoKATP antagonists such as 5-hydroxydecanoate and glibenclamide (3, 20, 24). The cardioprotective effect of mitoKATP is probably related to the ability of this channel to prevent ATP loss during ischemia and increase the efficiency of postischemic oxidative phosphorylation (6, 11).
Renal tissue is also protected by ischemic preconditioning (30, 37), suggesting that mitoKATP may be present in this tissue. However, to our knowledge, no previous attempt to characterize mitoKATP and establish its regulatory properties in renal mitochondria has been made. In this manuscript, we describe a K+ transport pathway inhibited by adenine nucleotides in kidney mitochondria and measure ATP-sensitive K+ transport rates and the effects of this transport on mitochondrial function.
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MATERIALS AND METHODS |
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Mitochondrial swelling. Changes in light scattering, reflecting changes in mitochondrial volume (5, 24, 36), were followed using a temperature-controlled Hitachi F4500 spectrofluorometer, operating with continuous stirring at excitation and emission wavelengths of 520 nm, with 2.5-nm slits.
Mitochondrial membrane potential estimation. Mitochondrial membrane potentials were estimated by following safranin O (5 µM) fluorescence (1) at 495-nm excitation and 586-nm emission on a Hitachi F4500 spectrofluorometer. A calibration curve was constructed using the K+ ionophore valinomycin (0.1 µg/ml) and known concentrations of K+, assuming matrix K+ concentration to be 150 mM (1).
Measurement of mitochondrial respiration. Respiration was measured using a computer-interfaced Clark-type oxygen electrode from Hansatech equipped with magnetic stirring. Oxygen solubility at 37°C was taken to be 203 nmol/ml. When fixed potentials were needed (Fig. 3), 0.1 µg/ml valinomycin and known K+ concentrations were used to manipulate , calculated using the Nernst equation, assuming matrix K+ concentration to be
150 mM (27).
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ATP measurements. ATP concentrations were determined by light emission at 560 nm on a Hitachi F4500 spectrofluorometer using a commercial luciferin-luciferase kit (Promega FF2021). Light emission during the first 120 s following the addition of luciferin-luciferase was integrated, and data were calibrated using known concentrations of ATP.
Reagents. Safranin O, EGTA, succinate, BSA, FCCP, rotenone, valinomycin, cyclosporin A, oligomycin, glibenclamide, diazoxide, 5-hydroxydecanoic acid, ADP, ATP, GTP, and antimycin A were purchased from Sigma.
Data analysis. Replicate experiments were performed in mitochondria isolated from separate animals, each representing an n of 1. Data are expressed as means ± SE. The inhibition of mitochondrial swelling promoted by ADP and ATP was normalized to control and analyzed by comparison to one using t-tests for a single sample. The remaining swelling data were analyzed with one-way ANOVA multiple t-tests for planned comparisons between mean values. Planned comparisons were DZX + ATP vs. ATP; glibenclamide + DZX + ATP vs. DZX + ATP; 5-hydroxydecanoate + DZX + ATP vs. DZX + ATP; and GTP + ATP vs. ATP. and oxygen consumption variation data (Fig. 2) were compared with zero, and ATP measurements were normalized to control and compared with one using t-tests for a single sample.
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RESULTS |
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The inhibitory effect of ATP on mitochondrial swelling in K+ salts was reversed by GTP (P < 0.005), a physiological mitoKATP activator (33), and DZX (P < 0.001), a pharmacological mitoKATP agonist that acts selectively on mitochondrial channels at the concentration used (19). As previously described (24), GTP and DZX had no effect in the absence of ATP and/or K+ (data not shown). The DZX effect could be prevented by the concomitant presence of glibenclamide (P < 0.005), a mitoKATP antagonist (24), demonstrating that renal ATP-sensitive K+ transport is also regulated by sulphonylureas, and must, therefore, be mediated by a channel similar to previously described mitoKATPs (4, 19, 24, 32, 33). Another effective inhibitor of the DZX-induced swelling was 5-hydroxydecanoate (P < 0.02), which is thought to be specific for mitoKATP (24). These inhibitors were also ineffective in the absence of ATP and DZX (data not shown), as previously seen (24).
Effect of ATP-sensitive K+ transport on respiration and . With the use of safranin O fluorescence to measure
, we found an increase of 11.4 ± 1.5 mV (n = 4; P < 0.005 vs. 0 mV) when 200 µM ATP was added to the reaction medium to inhibit mitoKATP (Fig. 2A). An increment of 16.2 ± 4.9 mV (n = 5; P < 0.03 vs. 0 mV; P > 0.05 vs. the previous group) in
after a 200 µM ATP addition was measured in a similar reaction mixture containing fatty acid-free BSA (1 g/l), demonstrating that the ATP effect was not due to an inhibition of fatty acid-stimulated uncoupling (26). Cyclosporin A, an inhibitor of the mitochondrial permeability transition (38), also did not eliminate the ATP effect (data not shown), suggesting that our
increases were not due to prevention of permeability transition by ATP. In addition, 200 µM ATP did not significantly increase
(0.8 ± 2.0 mV, n = 4; P > 0.05 vs. 0 mV) when added to a reaction medium that did not contain K+. Thus K+ transport through an ATP-sensitive pathway results in
decreases of
10 mV. In all experiments, the proton ionophore FCCP was added at the end of the run to dissipate
and ensure that safranin O fluorescence returned to similar levels.
Associated with the changes in , we found that mitochondria incubated in K+ salts in the absence of ATP presented higher respiratory rates than those in which K+ transport was inhibited by ATP (Fig. 2B), in a manner unaltered by the presence of BSA (not shown). With the use of succinate as a respiratory substrate, ATP-sensitive increments in respiration observed in K+-rich media were 11.9 ± 1.4 nmol O2·min1 ·mg protein1 (n = 10; P < 0.001 vs. 0), which corresponds to an estimated K+ transport rate of 143 nmol O2·min1·mg protein1, considering that K+/H+ exchange is electroneutral and 6 protons are pumped per oxygen atom consumed (4).
A verification of the compatibility of our data for and respiration rate changes promoted by mitoKATP was conducted by measuring oxygen consumption rates in the presence of fixed
s, as described in MATERIALS AND METHODS and shown in Fig. 3. By fitting the
data obtained with respiratory rates lower than the maximal rates, we found that respiration and
are related by a slope of 1.067 ± 0.012 nmol O2·mV1·min1·mg protein1, a result fully compatible with the finding that mitoKATP alters respiration and
by 1.04 ± 0.18 nmol O2·mV1·min1·mg protein1.
Effect of K+ transport on the reverse activity of the F0F1 ATP synthase. Opening ATP-sensitive K+ channels in heart mitochondria changes ADP and ATP transport across mitochondrial membranes, increases the efficiency of oxidative phosphorylation, and preserves tissue ATP levels during ischemia (6, 11). To verify whether the same effect was present in kidney, we incubated renal mitochondria in the presence of antimycin A (a complex III inhibitor), to simulate an ischemic condition, and measured ATP loss promoted by hydrolysis through the reverse activity of the F0F1 ATP synthase. Initially, ATP hydrolysis by the F0F1 ATP synthase was measured by following the generated by proton pumping by this protein. We observed that the
sustained by ATP hydrolysis was lower and more rapidly lost when K+ transport was stimulated by the presence of DZX (a representative trace of 6 independent experiments is shown in Fig. 4). The addition of 2 µg/ml oligomycin completely abolished
generation by ATP in the presence or absence of DZX (not shown), confirming that the
generated was related to ATP hydrolysis by the F0F1 ATP synthase. We also measured ATP concentrations in the reaction buffer after 120-s incubation in the presence or absence of DZX. We found that ATP concentrations in the DZX group were 1.86 ± 0.21 times greater (n = 8; P = 0.005 vs. 1) than those found in the control group, confirming that DZX decreases mitochondrial ATP hydrolysis by the F0F1 ATP synthase in the absence of respiration.
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DISCUSSION |
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ATP-sensitive K+ transport in renal mitochondria results in mild respiratory rate enhancement and a partial decrease in (Fig. 2), as would be expected for the uptake of a cation into the negatively charged mitochondrial matrix.
Changes promoted by ATP in renal mitochondria (1020 mV) were somewhat greater than those described in heart (12 mV; 28) and in brain (36 mV; 4). On the other hand, kidney mitoKATP transport rates (
140 nmol·min1·mg protein1), estimated by measuring respiratory rate differences, were larger than those found in heart (
30 nmol·min1·mg protein1; 28) and slightly lower than brain K+ transport rates (
170 nmol·min1·mg protein1; 4), suggesting that renal tissue expresses intermediate mitoKATP protein levels. In kidney and all other tissues studied to date (4, 24), the respiration and
effects of mitoKATP activation were small and certainly insufficient to hamper oxidative phosphorylation or Ca2+ uptake, indicating that large-scale
and respiratory regulation must not be the central role of mitoKATP. On the other hand, a major role of mitoKATP may be to regulate mitochondrial volume, which can change up to 20% in response to ATP-regulated K+ uptake rates as limited as those found in heart (7, 24, 28).
In addition to altering volume, respiration, and in respiring mitochondria, DZX-stimulated K+ transport in renal mitochondria was associated with lower ATP hydrolysis through the reverse activity of the F0F1 ATP synthase under nonrespiring conditions (Fig. 4). We previously obtained a similar result in heart mitochondria (6), and there is evidence that this effect is linked to volume changes (11). Indeed, we found that perfused hearts subjected to ischemia presented higher ATP levels when treated with DZX, atractyloside (an inhibitor of nucleotide transport into the mitochondrial matrix), or oligomycin (which inhibits the mitochondrial F0F1 ATP synthase). This is strong evidence that limitation of ATP hydrolysis by the F0F1 ATP synthase can be relevant for the maintenance of high-energy phosphate levels during ischemia and that mitoKATP may contribute to ischemic protection by preventing ATP hydrolysis secondarily to changes in mitochondrial volume (6, 11).
The reproduction of this finding in kidney mitochondria suggests that this channel may also have a protective effect in renal ischemia. Unfortunately, although we attempted to obtain a protective effect against ischemic damage using DZX in renal tissue and cell lines, we have not yet been able to confirm this hypothesis due to the toxic effect of this drug on these models (Cancherini, Trabuco, Kowaltowski, and Rebouças, unpublished observations). Pinacidil, another mitoKATP agonist frequently used to study the protective effects of this channel (2, 8, 25, 34), previously failed to protect the kidney against ischemia (30).
Although a possible protective role for renal mitoKATP under pathological conditions remains to be verified, our data clearly indicate that this channel may have important functions under physiological conditions. We found that renal mitoKATP can increase mitochondrial matrix volume by stimulating K+ uptake rates (Fig. 1). This provides a regulated mechanism through which renal mitochondria can swell and counteracts the contraction promoted by the K+/H+ exchanger (16), which appears to be highly active in this tissue (results not shown). MitoKATP activation thus allows for the maintenance of mitochondrial volume when K+ leak through the inner membrane is decreased due to oxidative phosphorylation (28). In fact, the maintenance of adequate matrix volume and a narrow intermembrane space is important to ensure the preferential transport of creatine phosphate in relation to ATP across mitochondrial membranes in heart (11). Matrix swelling can also significantly activate the electron transport chain (21).
The concomitant activity of both mitoKATP and the K+/H+ exchanger also results in a small decrease in mitochondrial and increased respiration, which may be another important function for this channel. Although insufficient to hamper oxidative phosphorylation or Ca2+ uptake (28),
mild uncoupling
such as that promoted by mitoKATP may be important to generate heat, regulate metabolism, and even prevent reactive oxygen species generation by mitochondria (12, 35). Indeed, uncoupling proteins, whose sole known function is to promote mild uncoupling, exist ubiquitously (13, 26), attesting to the importance of limited physiological
regulation.
In conclusion, we characterized a controlled K+ import pathway in renal mitochondria similar to mitoKATP previously described in other tissues. These putative renal mitoKATP channels regulate mitochondrial volume, respiration, , and F0F1 ATP synthase activity. The possible importance of the regulation of these mitochondrial functions in cellular physiology and pathology remains to be determined.
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DISCLOSURES |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked advertisement
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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