ISSN 0006-3509, Biophysics, 2008, Vol. 53, No. 6, pp. 515–518. © Pleiades Publishing, Inc., 2008. Original Russian Text © S.V. Murzaeva, N.V. Belosludtseva, L. Gavrovskaya, G.D. Mironova, 2008, published in Biofizika, 2008, Vol. 53, No. 6, pp. 962–966.
CELL BIOPHYSICS
The Effect of Taurine on the Ion Transport System in Mitochondria S. V. Murzaevaa, 1, N. V. Belosludtsevaa, L. Gavrovskayab, and G. D. Mironovaa a
Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, ul. Institutskaya 3, Pushchino, Moscow oblast, 142290 Russia b Federal Institution Research Institute of Experimental Medicine, Russian Academy of Medical Sciences, ul. Akademika Pavlova 12, St.-Petersburg, 197376 Russia Received June 26, 2008
Abstract—The effect of taurine on the ATP-dependent mitochondrial swelling that characterizes the activity of mitochondrial ATP-dependent K+ channel and the formation of Ca2+-dependent pores, different in sensitivity to cyclosporin A, has been studied in rat liver mitochondria. It has been shown that taurine in micromolar concentrations (0.5–125 µM) stimulates the energy-dependent swelling of mitochondria. Taurine in physiological concentrations (0.5–20 mM) has no effect on the ATP-dependent swelling and the formation of cyclosporin Ainsensitive Pal/Ca2+-activated pore in mitochondria. Taurine in these concentrations increased the rate of cyclosporin A-sensitive swelling of mitochondria induced by Ca2+ and Pi and reduced the Ca2+ capacity of mitochondria. The different effects of physiological taurine concentrations on the ATP-dependent transport of K+ and Ca2+ ions in mitochondrial membranes as compared with cell membranes are discussed. Key words: mitochondria, mito-KATP , cyclosporin A-sensitive Ca2+-induced pore, palmitate/Ca2+-activated pore, cyclosporin A, taurine DOI: 10.1134/S0006350908060080 1
Taurine is a natural aminoethane sulfonic acid, which is abundant in free state in animal tissues, particularly heart, skeletal muscles, central nervous system, and white blood cells [1, 2]. Taurine is involved in many physiological processes and reveals osmoregulatory and antioxidant effects. Besides, it participates in the regulation of conductivity of Ca2+, Zn2+, Mg2+, Na+ and K+ ions across biological membranes and has a positive effect on total metabolism, arterial pressure, sugar content in blood, cardiac rhythm disturbance, etc. The biotropicity of taurine at medical treatment of heart pathologies and decrease of concentration of this amino acid in cardiomyocytes at myocardial infarction [3, 4] have initiated a series of studies in respect of the effect of taurine on the ATP-sensitive potassium channels of cell membranes (cyto-KATP). These channels are known to have cardioprotective properties which protect heart from ischemic injury [5]. It was shown that taurine in dose-dependent manner blocked cyto-KATP in the membranes of rabbit and pig cardiomyocytes [6, 7], oocytes and β-cells of rat pancreas [8]. The mechanism of taurine action is associated with regulatory interaction with the benzamide-binding site of subunit SUR1 on the receptor side sensitive to sulfonyl ureas [8, 9]. 1 The
corresponding author; e-mail:
[email protected]
Abbreviations: MPT, mitochondrial permeability transition pore.
The cardioprotective properties of taurine, as a natural regulator, arouse interest in the study of the mechanism of its action on the mitochondrial ATP-sensitive K+ channel (mito-KATP). It is suggested that the activation of mito-KATP is the key mechanism of animals' adaptation to hypoxia and heart protection at ischemic injury of myocardium [5, 10]. At our laboratory, the search of the natural mito-KATP modulators protecting heart from ischemic injury and myocardial infarction and the modulators of mitochondrial Ca2+-dependent pores is carried out [11–15]. As is known, the opening of Ca2+-dependent pores induces the release of proapoptotic proteins such as cytochrome c, etc., from mitochondria, indicating their involvement in apoptosis [16, 17]. In this connection, taurine is also interesting as a natural metabolite. The recent studies demonstrate that taurine prevents ischemia-induced apoptosis in the cultures of cardiomyocytes [18] and apoptosis induced by various unfavorable factors such as oxidative and photochemical stresses, etc., in different cell cultures [19, 20]. The effect of taurine on the mitochondrial ATP-sensitive potassium channel and on the formation of Ca2+dependent pores in the inner mitochondrial membrane has not been studied yet. In the present study we have investigated the effect of taurine in physiological concentrations on ATP-dependent K+ transport in mitochondria and formation of Ca2+-dependent pores in mitochondrial membrane.
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MURZAEVA et al. Swelling rate, % 140 (a) 120 100 80 60 40 20 0 0 0.05 0.5 5 10 125 Taurine, µM
(b)
1 2.5 10 20 mM
Fig. 1. The effect of taurine on the energy-dependent swelling of rat liver mitochondria. The reaction medium for incubation of mitochondria: KCl, 50 mM; NaH2PO4, 5 mM; MgCl2, 0.5 mM; HEPES, 5 mM (pH 7.4); EGTA, 0.1 mM; succinate, 5 × 10–3 M; rotenone, 10–1 M; protein 0.132 mg/ml. Time of incubation, 3 min; temperature, 26.7°C. The average results of a series of experiments (n = 5) are presented. The rates in the control (160– 260 ∆A520 min–1 mg–1 protein) were taken as 100%.
MATERIALS AND METHODS Mitochondria were isolated from the liver of sexually mature white rats, 220–250 g, by the standard method of differential centrifugation [13]. The isolation medium contained: mannitol, 210 mM; sucrose, 70 mM; EDTA, 1 mM; Hepes-KOH, 10 mM (pH 7.4). The mitochondrial protein concentration was assayed by the Bradford method [21]. The ATP-dependent K+ transport in mitochondria, which reflects the activity of mitochondrial ATP-sensitive potassium channel, was assayed by the method of energy-dependent mitochondrial swelling by absorption at 520 nm with succinate as a respiration substrate in the presence of rotenone. The measurements were made in a Shimadzu UV2401PC spectrophotometer (Japan) in a thermostatically controlled cuvette under continuous stirring at 26.7°C. The incubation medium contained: KCl, 50 mM; MgCl2, 0.5 mM; KH2PO4, 5 mM; EGTA, 0.1 µM; Hepes-KOH, 5 mM (pH 7.4); rotenone, 2 µM; the mitochondrial protein concentration was 0.125 mg/ml. The reaction was started by adding 5 mM succinate; incubation time was 3 min. The energy-dependent mitochondrial swelling was examined at addition of taurine in the presence and absence of ATP. The amount of Ca2+ necessary for the formation of cyclosporine A-sensitive Ca2+-induced pore (the mitochondrial permeability transition pore (MPT pore)) was assessed after Ca2+ overload of rat liver mitochondria. The measurements were made in a thermostatically controlled cuvette under continuous stirring at 26°C, using a Ca2+-selective electrode. Mitochondria were incubated in a medium containing: mannitol, 210 mM;
sucrose, 70 mM; KH2PO4, 1 mM; EGTA, 5 µM; succinate, 5 mM; rotenone, 1 µM; Hepes-KOH, 10 mM (pH 7.4). The mitochondrial protein concentration in the cuvette was 1 mg/ml. CaCl2 (20 µM) was added into the incubation medium every 60 s. After several additions of Ca2+, the concentration of external Ca2+ in the incubation medium with mitochondria abruptly increased, indicating massive release of the ion from the organelles due to formation of the MPT pore in the inner mitochondrial membrane. The Ca2+ amount necessary for massive release of the ion from the organelles was used to estimate the formation of MPT pore in mitochondria, defined as Ca2+ capacity. Mitochondrial swelling observed at the induction of MPT pore and cyclosporin A-insensitive palmitate/Ca2+-activated pore was determined by absorption at 540 nm in a thermostatically controlled cuvette under continuous stirring at 25°C in spectrometric fiber optic system “Ocean Optics USB 2000” (Ocean Optics Inc., USA). The incubation medium contained mannitol, 210 mM; sucrose, 70 mM; EGTA, 5 µM; succinate, 5 mM; rotenone, 1 µM; Hepes-KOH, 10 mM (pH 7.4). In experiments on the formation of palmitate/Ca2+-activated pore, 1 µM cyclosporine A was added to the incubation medium. The mitochondrial protein concentration in the cuvette was 0.4 mg/ml. The rate of mitochondrial swelling (Vmax = ∆A540 min–1 mg–1 protein) was calculated by the change of absorption in the first 30 s after the beginning of high-amplitude swelling. The following reagents were used in the work: cytochrome c, ATP, ADP, Hepes, Tris, sucrose, KH2PO4, NaH2PO4, EGTA, EDTA, palmitic acid, succinate, cyclosporin A, rotenone (Sigma, USA), mannitol, KCl, and CaCl2 (Merck, Germany). RESULTS AND DISCUSSION The effect of taurine on the ATP-dependent K+ transport in mitochondria. According to the literature data, taurine in dose-dependent manner blocks of the cyto-KATP channels in the range of concentrations 10–5– 10–1 M. Physiological concentrations of taurine (10 and 20 mM) are the most effective: they block the potassium channels in cardiomyocytes for 50 and 100%, respectively [2, 6–8, 10, 11]. Based on these data, in this study we have used the range of taurine concentrations from micro- to millimolar values. Figure 1a shows that the low taurine concentrations of 0.05–125 µM activate the energy-dependent swelling of mitochondria. It should be noted that the degree of activation varies in different experiments and depends on the rate of mitochondrial swelling in the control. The maximal activation (up to 40%) was observed at the low rates: 150–180 ∆A520 min–1 mg–1 protein; the stimulating effects at the high rates (above) were minimal. Figure 1b shows that millimolar taurine concentrations have actually no effect on the energy-dependent BIOPHYSICS
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THE EFFECT OF TAURINE ON THE ION TRANSPORT SYSTEM IN MITOCHONDRIA
mitochondrial swelling. Thus, physiological taurine concentrations (10 and 20 mM), which blocked cytoKATP , were inefficient in our experiments. For more complete estimation of taurine effect on mito-KATP in our experiments, the energy-dependent mitochondrial swelling was studied in the presence of the potassium channel inhibitors. The potassium channel identification in the presence of ATP showed that ATP inhibited the energy-dependent mitochondrial swelling by 50–60% in the concentration of 40–50 µM and by 100% in the concentration of above 300 µM, indicating the functioning of the ATP-sensitive potassium channel in mitochondria. As follows from the data in Table 1, the effect of taurine in all of the tested concentrations has been shown neither at 57% inhibition of mito-KATP in the presence of ATP (50 µM) nor at 100% inhibition of the potassium channel by 300 µM of ATP. Hence, the inhibitor of mitochondrial potassium channel (ATP) completely suppresses the activating effect of taurine at low concentrations. The comparison of these results with the data on taurine effect on the energy-dependent swelling of mitochondria in the absence of ATP suggests that stimulation of the mitochondrial swelling by low taurine concentrations is associated with the potassium channel opening. These results have been confirmed by using of other inhibitors of the ATP-sensitive potassium channels, glibenclamid and 5-hydroxydecanoate, which completely eliminate the activating effect of micromolar taurine concentrations (the data not presented). In conclusion of this section of the study concerning the effect of different taurine concentrations on the energy-dependent mitochondrial swelling, it should be noted that our results contradict the literature data on the effect of taurine on ATP-dependent potassium cell channels (cyto-KATP) [2, 6–8]. In the works of reference, taurine in physiological concentrations is a blocker of cyto-KATP . Our measurements of the energydependent mitochondrial swelling, reflecting the functioning of mito-KATP and confirmed by the inhibition of swelling by ATP, showed that taurine in micromolar concentrations (0.5–125 µM) activated the ATP-sensitive potassium channel in mitochondria, while physiological taurine concentrations (0.5–20 mM) had no effect on this channel. It may be suggested that such difference in the action of taurine on mito-KATP is due to different mechanisms of regulation of cell and mitochondrial ATP-sensitive potassium channels conditioned by their differing structural organizations [5, 11, 22]. The effect of taurine on the Ca2+-activated pores in mitochondria. In the present work we have also studied the effect of taurine on the formation of two types of mitochondrial pores different in sensitivity to cyclosporin A: (1) cyclosporin A-sensitive pore, which is supposed to be a protein megachannel (the mitochondrial permeability transition pore (MPT)) consisting of BIOPHYSICS
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Table 1. The effect of taurine on the energy-dependent swelling of rat liver mitochondria in the presence of ATP Conditions
V of swelling*, %
Control ATP, 50 µM ATP, 50 µM; Taurine, 0.5 µM ATP, 50 µM; Taurine, 125 mM ATP, 50 µM; Taurine, 20 mM ATP, 300 µM ATP, 300 µM; Taurine, 0.5 µM ATP, 300 µM; Taurine, 20 mM Taurine, 0.5 µM Taurine, 125 µM Taurine, 20 mM
100 ± 15 43 ± 12 (–57) 42 ± 10 (–58) 37 ± 5 (–63) 36 ± 13 (–64) 0.0 (–100) 0.0 (–100) 0.0 (–100) 117 ± 21 130 ± 11 100 ± 5
* ∆A520 min–1 mg–1 protein. Reaction medium and conditions are as in Figure 1, except for those specified in the Table. The mean values are presented, ± error of the mean (n = 5). The mean values of inhibition are given in brackets, %.
Table 2. The effect of taurine on the formation of MPT (the mitochondrial permeability transition pore) and the Pal/Ca2+activated pores in rat liver mitochondria Vmax of swelling, % Taurin, mM 0.0 0.5 1.0 10
Pi/Ca2+-activated pore (MPT)
Palmitate/Ca2+-activated pore
100 ± 2 121 ± 3 131 ± 4 129 ± 3
100 ± 1 100 ± 2 101 ± 2 102 ± 3
Note: Incubation medium contained: mannitol, 210 mM; sucrose, 70 mM; EGTA, 5 µm; succinate, 5 mM; rotenone, 1 µM; Hepes-KOH, 10 mM (pH 7.4); in experiments on induction of the Pal/Ca2+-activated pore, 1 µM of cycloprosin A was added to the medium. The MPT pore was induced by 1 mM Pi and 75 µM CaCl2; the Pal/Ca2+-activated pore was induced by 15 µM palmitic acid and 30 µM CaCl2. The mean values are given, ± error of the mean (n = 4).
adenylatetranslocator, porin, cyclophilin D, and a number of other membrane mitochondrial proteins [23, 24] and (2) cyclosporin A-insensitive Ca2+-dependent pore induced by palmitic acid. The studies of our laboratory showed that the Pal/Ca2+-activated pore differs from the MPT pore in the properties and the mechanism of formation. In particular, it is insensitive to the known modulators of MPT pore, can close spontaneously, and probably is of lipid nature [12–15]. It has been established that the opening of Ca2+-dependent pores of both types results in the release from mitochondria of proapoptotic proteins, indicating their involvement in apoptosis.
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to hypoxia or prevention of apoptosis. However, this assumption needs further research.
100 80
REFERENCES
60 40 20 0
0 10 Taurine, µM
Fig. 2. The effect of taurine on the Ca2+ capacity of rat liver mitochondria. The incubation medium contained: mannitol, 210 mM; sucrose, 70 mM; EGTA, 5 µM; succinate, 5 mM; rotenone, 1 µM; KH2PO4, 1 mM; Hepes-KOH, 10 mM (pH 7.4). The mean values are presented (n = 4).
Table 2 presents the data on the effect of taurine on the rate of mitochondrial swelling conditioned by the opening of these pores. As follows from the Table, taurine (0.5–10 mM) increases the rates of the cyclosporin A-sensitive mitochondrial swelling induced by Ca2+ (75 µM) and Pi (1 mM) by 20–30% but has no effect on the swelling induced by palmitic acid (15 µM) and Ca2+ (30 µM). These results demonstrate that taurine in physiological concentrations stimulates the formation of MPT pore but has no effect on the opening of cyclosporin A-insensitive palmitate/Ca2+-activated pore in mitochondria. It is interesting that the formation of cyclosporin A-sensitive MPT pore in the presence of 10 mM taurine needs less Ca2+ (by 20%) as compared with the control (Fig. 2). Thus, physiological concentrations of taurine, on the one hand, stimulated the opening of the MPT pore conducting calcium ions and, on the other hand, reduced the Ca2+ capacity of mitochondria. The decrease of the Ca2+ capacity of mitochondria under the influence of taurine can be considered as the limitation of Ca2+ entry into mitochondrial matrix, which may play a role in apoptosis prevention and taurine involvement in the anti-ischemic effect. As regards the cyclosporine A-insensitive Ca2+-dependent pore, it is insensitive to taurine, thereby confirming its distinction from the MPT pore. Based on the study of taurine effect on the ATP-sensitive mitochondrial swelling associated with the entry of potassium and calcium into mitochondrial matrix, we come to a conclusion that taurine probably acts as a modulator of the mito-KATP channel and the MPT pore, which may contribute to its positive role at adaptation
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