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Effects of lipophilic dications on planar bilayer phospholipid membrane and mitochondria
Biochimica et Biophysica Acta 1767 (2007) 1164 – 1168 www.elsevier.com/locate/bbabio
Effects of lipophilic dications on planar bilayer phospholipid membrane and mitochondria Inna I. Severina a , Mikhail Yu. Vyssokikh a , Antonina V. Pustovidko a , Ruben A. Simonyan a , Tatiana I. Rokitskaya a , Vladimir P. Skulachev a,b,⁎ a
Department of Bioenergetics, A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119992, Russia b Faculty of Bioengineering and Bioinformatics, Moscow State University, Moscow 119992, Russia Received 22 April 2007; received in revised form 28 May 2007; accepted 31 May 2007 Available online 12 June 2007
Abstract In this paper, we studied effects of phosphonium dications P2C5 and P2C10 on bilayer planar phospholipid membrane (BLM) and rat liver mitochondria. In line with our previous observations [M.F. Ross, T. Da Ros, F.H. Blaikie, T.A. Prime, C.M. Porteous, I.I. Severina, V.P. Skulachev, H.G. Kjaergaard, R.A. Smith, M.P. Murphy, Accumulation of lipophilic dications by mitochondria and cells, Biochem. J. 400 (2006) 199–208], we showed both P2C5 and P2C10 are cationic penetrants for BLM. They generated transmembrane diffusion potential (ΔΨ), the compartment with a lower dication concentration positive. However, the ΔΨ values measured proved to be lower that the Nernstian. This fact could be explained by rather low BLM conductance for the cations at their small concentrations and by induction of some BLM damage at their large concentrations. The damage in question consisted in appearance of non-Ohmic current/voltage relationships which increased in time. Such a nonOhmicity was especially strong at ΔΨ N 100 mV. Addition of penetrating lipophilic anion TPB, which increases the BLM conductance for lipophilic cations, yielded the Nernstian ΔΨ, i.e. 30 mV per ten-fold dication gradient. In the State 4 mitochondria, dications stimulated respiration and lowered ΔΨ. Moreover, they inhibited the State 3 respiration with succinate or glutamate and malate (but not with TMPD and ascorbate) in an uncoupler-sensitive fashion. Effect on the in State 4 mitochondria, similarly to that on BLM, was accounted for by a timedependent membrane damage. On the other hand, the State 3 effect was most probably due to inhibition of the respiratory chain Complex I and/or Complex III. The damaging and inhibitory activities of lipophilic dications should be taken into account when one considers a possibility to use them as a vehicle to target antioxidants or other compounds to mitochondria. © 2007 Elsevier B.V. All rights reserved. Keywords: Dication; Lipophilic cation; Planar phospholipids membrane; Mitochondria; Membrane potential; Damaging effect
1. Introduction Lipophilic penetrating cations have been introduced as tool to monitor membrane potential across mitochondrial and bacterial membranes [1–3]. It was also suggested that these cations can be Abbreviations: ΔΨ, transmembrane electric potential difference; BLM, bilayer planar phospholipid membrane; BSA, bovine serum albumin; DNP, 2,4p-dinitrophenol; P2C5 and P2C10, bistriphenylphosphonium with five or ten methylene linker, respectively; TPMP, N,N′-tetramethyl-p-phenylenediamine; TPB, tetraphenyl borate; TPP, tetraphenylphosphonium ⁎ Corresponding author. Department of Bioenergetics, A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Leninskie Gory 1, Moscow 119992, Russia. Tel.: +7 4959395530; fax: +7 495 9390338. E-mail address: [email protected] (V.P. Skulachev). 0005-2728/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.bbabio.2007.05.010
used as “electric locomotives” to accumulate inside mitochondria electroneutral residues attached to these cations [4–8]. Such a principle was recently successfully employed by Murphy and coworkers to target antioxidants like CoQ or vitamin E to mitochondria [7–9] (for reviews, see refs. 10,11). According to the Nernst equation, the accumulation coefficient for monocations in energized mitochondria (ΔΨ about 180 mV) should be around 103. For dications, it should increase up to 106. This means that lipophilic dications might be much more powerful “locomotives” than monocations [12]. Investigation of such a possibility revealed the following. At ΔΨ lower than 90–100 mV, lipophilic dications P2C5 and P2C10, as had been expected, were accumulated by mitochondria much faster than the monocation TPP. However, a ΔΨ increase above this threshold
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resulted in negligible further increase in the dication uptake. The reason for such a phenomenon remained obscure [12]. In this work, we studied the possible mechanism of nonNernstian behaviour of the dications, using BLM and rat liver mitochondria as experimental systems. It was found that there are at least two reasons for the above phenomenon occurring at high dication concentrations. One of them is a damage to phospholipid bilayer, resulting in a non-Ohmic increase in its conductance in response to a ΔΨ increase. This damage develops in time. In mitochondria, it is revealed as an increase in State 4 respiration and a lowering of ΔΨ. The second reason consists in a dication-induced inhibition of the State 3 respiration at levels of Complex I and/or Complex III. 2. Materials and methods 2.1. Chemicals MOPS (3-[N-Morpholino]propanesulfonic acid), bovine serum albumin (fraction V), rotenone, succinate, malate, glutamate, oligomycin, ascorbic acid, TMPD, DNP, ADP, magnesium and calcium chlorides were from Sigma. TPB was from Fluka; TPP was from Aldrich. P2C5 and P2C10 were generous gifts of Dr. M.P. Murphy. Bilayer planar phospholipid membrane (BLM) was formed on a 1-mm aperture in a Teflon septum separating the experimental chamber into two compartments. To form BLM, E. coli phospholipids were used (57% phosphatidylethanolamine, 15% phosphatidylglycerol, 10% cardiolipin, 18% others). Phospholipids were dissolved in decane (20 mg/ml). Electric parameters were measured with AgCl electrodes and a VA-J-5 electrometer (for details, see [13]).
2.2. Isolation of mitochondria Rat liver mitochondria were purified by differential centrifugation. Rats were killed by cervical dislocation according to recommendation of the University Ethics Committee. The liver was cooled for 1–2 min in ice-cold isolation medium (IM) containing 250 mM sucrose, 10 mM MOPS–KOH, pH 7.4, 1 mM EGTA, 0.1% BSA. After washing out blood from the liver surface, the tissue was cut into 2- to 3-mm pieces, using scissors on a cooled Petri dish. The cut tissue was washed 3–5 times with IM until free from blood and then homogenized by the Potter glass-Teflon homogenizer at 160 rpm for 2 min at 4 °C in 8 ml IM per g tissue. After 10 min centrifugation at 500 g, 4 °C, the supernatant was filtered through cheese-cloth and centrifuged once more at 9000 g, 0 min, 4 °C. A Beckman J2-20 centrifuge and a cooled JA-20 rotor were used. Supernatant was discarded and the tube was cleaned from fat. The pellet was rinsed by gentle stirring with 1–2 ml IM (to remove fluffy layer from the top of the pellet) and resuspended in 1 ml IM supplemented with 0.3% BSA, using a glass stick, and pipetting by a 1-ml Gilson sampler for 1 min. The suspension was diluted with IM to the final volume 8 ml per g protein and centrifuged again under the same conditions. The final pellet was resuspended in one or two drops of IM supplemented by 0.3% BSA with a glass stick and 250 μM Gilson Pipetman, the final volume being about 100–200 μl. Oxygen consumption rate was measured by a Clark electrode and an oxygraph (“Oroboros”, Austria). Protein concentration was 0.4 mg/ml, the chamber volume being 2 ml. The incubation medium composition was identical to that of IM. As respiration substrates, 5 mM succinate in presence of 1 μM rotenone, 4 mM glutamate and 1 mM malate, or 5 mM ascorbate with 0.1 mM TMPD were employed. The State 3 respiration was induced by adding 1 mM ADP in the presence of 5 mM potassium phosphate and 2 mM MgCl2. The uncoupled state was achieved by adding DNP.
2.3. Mitochondrial membrane potential measurements Safranine O was used as a membrane potential probe [14]. The 555- to 523-nm light absorption was measured with an Aminco DW-2000 spectrophotometer
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(dual-wavelength regime). Protein concentration was 0.7 mg/ml; safranin O concentration was 15 μM. Mitochondrial swelling was measured by a decrease in the light scattering at 520 nm, using the Aminco DW-2000 spectrophotometer and stirred cuvettes. Rat liver mitochondria were incubated at 25 °C (final protein concentration, 0.5 mg/ml). Protein concentration was determined with bicinchoninic acid according to the producer's instructions (Pierce, USA), using BSA (2 mg/ml) as a standard protein solution.
3. Results and discussion In the first series of experiments, a bilayer planar phospholipid membrane (BLM) was employed. It was shown that P2C5 and P2C10 are penetrating dications, since trans-BLM dication concentration asymmetry was found to result in generation of an electric potential difference (ΔΨ) across the BLM, the compartment with lower cation concentration positive (Fig. 1A–C). The ΔΨ in question was generated due to electrogenic transmembrane diffusion of a dication from the compartment with a higher dication level to that with a lower dication level. Such conclusion was confirmed by the observation that under optimal conditions the ΔΨ value was equal to 30 mV per ten-fold difference in the dication concentration, which exactly corresponds to the Nernst equation. Under the same conditions, ten-fold gradient of monocation TPP resulted in generation of a 60 mV ΔΨ (Fig. 1C). To make the above-mentioned quantitative relationships reproducible, a penetrating anion (TPB) should be added, a fact which is in line with our previous finding that TPB can operate as a carrier for phosphonium cation derivatives [2,15]. Without TPB, the P2C5 and P2C10 gradients generated diffusion potentials of a magnitude that usually proved to be lower than predicted by the Nernst equation. This was most probably due to rather low phosphonium cation conductance which was comparable to the intrinsic (without these cations) BLM conductance (for details, see ref. [6]). The problem could not be solved by increasing the dication concentration since the linear ΔΨ/[dication] relationship was observed at low [dication] values only. In these experiments, the dication concentrations should, in fact, not exceed some critical level. This level was 1 × 10− 5 M or 1 × 10− 4 M in samples without or with 1 × 10− 7 M TPB, respectively. Above the levels mentioned, a further increase in the dication concentration caused no additional ΔΨ increase (Fig. 1B and C). The observed deviation from the Nernstian behavior might be explained assuming that intrinsic membrane conductance increases due to membrane damage by increasing dication concentration. Apparently, the damage takes some time since without TPB (i.e. when the dication conductance was rather low), the dication gradient-supported ΔΨ, initially rather high, lowers during 2–3 min, whereas such a lowering was not observed within this period of time if TPB was present (Fig. 1A). Consistent with the above assumption, in experiments where the Nernstian ΔΨ values were obtained in samples without TPB, this took place immediately after the dication addition. Then these values lowered very fast. The dication-induced conductance rise was shown to increase not only in time but also at high ΔΨ. In fact, the
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Fig. 1. Effects of P2C5, P2C10, and TPP on BLM. (A) Kinetics of generation of electric potential difference across BLM by transmembrane gradient of P2C5. Initial P2C5 concentration in both compartments was 1 × 10− 6M. When indicated, both compartments were supplemented with 1 × 10− 7M TPB. Arrow, addition of 1 × 10− 5M P2C5 to the right-hand compartment, which resulted in positive charging of the left-hand compartment (ordinate). Incubation mixture, 50 mM Tris–HCl (pH 7.4). (B) Generation of electric potential difference across BLM by transmembrane gradients of TPP+ (circles), P2C5 (triangles), and P2C10 (squares). In the left-hand compartment, the TPP, P2C5, or P2C10 concentration was 1 × 10− 6M; in the right-hand compartment, the cation concentrations varied from 1 × 10− 6M to 1 × 10− 4M. Panel C, as B but both compartments were supplemented with 1 × 10− 7M TPB. (D) Current as a function of applied voltage. 1 × 10− 6M TPP or 3 × 10− 5M P2C5 in both compartments. Incubation mixture, 50 mM Tris–HCl, pH 7.4. (E) Current as a function of applied voltage after incubation of BLM with 5 × 10− 5M P2C5 (in both compartments) for 20 or 40 min. Incubation mixture, 10 mM Tris–HCl, 10 mM MES, 10 mM β-alanine, 100 mM KCl, pH 7.0.
current–voltage relationship proved to be non-Ohmic, the nonOhmicity being increased at ΔΨ N 100 mV. For monocation TPP, the relationship was exactly Ohmic (see the linear dependence of current upon voltage, Fig. 1D). Non-Ohmicity was much bigger when the time of treatment of BLM with dication increased from 20 to 40 min (Fig. 1E). The BLM data suggest that a damaging effect of dications can also be present in mitochondria, where phospholipid bilayer occupies some membrane areas. As one can see in Fig. 2A, addition of 5 μM P2C10 stimulated the State 4 respiration. Such stimulation did not disappear in time and, therefore, could not be explained by a dication influx, which should be transient.
Rather, dications caused an increase in the membrane leakage. Addition of uncouplers (DNP) resulted in further respiration increase, which was higher in a sample with dication (apparently due to a dication-induced increase in the membrane permeability for the uncoupler anion [16–18]). As to oligomycin, it was without effect on the dication-induced respiration increase (not shown). Fig. 2B shows the dependence of the State 4 respiration with succinate as a substrate upon concentrations of dications and TPP. The damaging effect on mitochondria, like that on BLM, was stronger with P2C5 and especially with P2C10 than with TPP. Similar relationships were revealed with glutamate and malate or ascorbate and TMPD.
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Fig. 2. Effects of P2C5, P2C10, and TPP on respiration of rat liver mitochondria. (A) Effect of P2C10 on the kinetics of the State 4 respiration. Substrate, 2.5 mM succinate with 2 μM rotenone. Additions, 5 × 10− 6M P2C10 and 2 × 10− 5M DNP. (B) Stimulation of the State 4 respiration by P2C5, P2C10, and TPP (symbols as in Fig. 1B) as a function of the cation concentration; substrate, succinate with rotenone. (C) Kinetics of the State 3 respiration; substrate, 2.5 mM succinate with 2 μM rotenone. Additions, 5 × 10− 4M ADP, 5 × 10− 6M 2PC5 (each addition), 2 × 10− 5M DNP. (D–F) Effects of P2C5, P2C10 and TPP on the State 3 respiration as a function of the cation concentration; substrates, succinate with rotenone (D), glutamate and malate (E) and ascorbate and TMPD (F).
In Fig. 2C–F, effects of dications and TPP on the State 3 respiration are shown. It was found that all three cations tested decrease the respiration rate (for kinetics of the P2C5 effect, see Fig. 2C). One can see that DNP abolished such an inhibition
(most probably, due to a ΔΨ collapse entailed by release of the dication from mitochondria). The inhibitory effect was especially strong with P2C10, the other two cations being less damaging for the respiratory chain. The inhibition could be
Fig. 3. Effects of cations on the safranine O response of rat liver mitochondria in the State 4. Substrate, 2.5 μM succinate with 2 μM rotenone. Additions, 30 μM P2C5 (A), 6 μM P2C10 (B), 25 μM TPP (C), 50 μM CaCl2, 50 μM DNP (A–D).
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observed with succinate (Fig. 2D) as well as with NAD substrates (Fig. 2E). The State 3 respiration with ascorbate and TMPD was resistant to dications and TPP (Fig. 2F). Thus, the respiratory chain Complexes I–III, rather than Complex IV, are targets for the inhibitory activity of the phosphonium cations. Fig. 3 illustrates the effect of the studied cations as well as of Ca2+ on mitochondrial ΔΨ monitored with safranine O response. The dications and TPP induced the permanent decrease in this response, whereas the action of Ca2+ was transient. The difference in effects of dications and TPP consisted in that in the former (but not in the latter) case fast initial decrease in ΔΨ was followed by further slow ΔΨ decrease. This was consistent with the BLM data (Fig. 1A and F) indicating that dication-induced damage to the membrane develops in time. These data can explain the paradoxical behavior of dications in mitochondria, namely, failure to increase the intramitochondrial level of these compounds by a ΔΨ increase above some critical level [12]. Apparently, accumulation of dications results in membrane damage and, hence, in membrane conductance increase, which prevents further accumulation. This may result in some balance between (i) ΔΨ increase favorable for the dication uptake and (ii) a dication-induced conductance increase that is unfavorable for the uptake. Such damage is rather delicate. As was shown by our experiments, light scattering of mitochondria was not decreased by the highest (3 × 10− 5 M) concentrations of P2C10 used in our experiments. Under the same conditions, 1 × 10− 4 M Ca2+ produced a high amplitude mitochondrial swelling. The above reasoning is in line with our data published in the preceding paper [12]. In that paper, it was reported that a TPB addition allowed P2C5 to accumulate inside mitochondria at ΔΨ levels higher than the ΔΨ threshold observed without TPB. Such an effect may well be a consequence of a TPB-induced rise in the mitochondria membrane conductance for P2C5. Apparently, the dication damage-induced conductance depends on ΔΨ, resulting in an non-Ohmicity of the membrane. Such an assumption explains why in mitochondria the dication accumulation is arrested at high ΔΨ even at low [dication] in the medium [12]. It may be noted in this context that non-Ohmic effects have been shown to be inherent in certain situations when the mitochondrial membrane conductance rises [see, e.g., ref. 19]. It should be stressed that in mitochondria additional unfavorable dication effects other that the membrane damage may also be involved, namely, an inhibition of the respiratory chain Complexes I and III by high concentrations of dications (see Fig. 2C–E). This inhibition might also lower the ΔΨ generation, especially when the membrane leakage increases. All the above complications should be taken into account when a practical use of lipophilic dications is considered [20]. Acknowledgements The authors are very grateful to Dr. M.P. Murphy for samples of dications. This study was supported by grant “Leading Scientific Schools” 5952.2006.4, Ministry of Education and
Science, RF and grant of Russian Foundation for Basic Research (RFBR-05-04-49700). References [1] E.A. Liberman, V.P. Topali, L.M. Tsofina, A.A. Jasaitis, V.P. Skulachev, Mechanisms of coupling of oxidative phosphorylation and the membrane potential of mitochondria, Nature 222 (1969) 1076–1078. [2] L.L. Grinius, A.A. Jasaitis, Yu.L. Kadziauskas, E.A. Liberman, V.P. Skulachev, V.P. Topali, L.M Tsofina, M.A. Vladimirova, Conversion of biomembrane-produced energy into electric form: I. Submitochondrial particles, Biochim. Biophys. Acta 216 (1970) 1–12. [3] E.A. Liberman, V.P. Skulachev, Conversion of biomembrane-produced energy into electric form: IV. General discussion, Biochim. Biophys. Acta 216 (1970) 30–42. [4] S.E. Severin, V.P. Skulachev, L.S. Yagizhinsky, A possible role of carnitine in transport of fatty acid through the mitochondrial membrane, Biokhimiya 35 (1970) 1250–1257 (Russ.). [5] D.O. Levitsky, V.P. Skulachev, Carnitine: the carrier transporting fatty acyl into mitochondria by means of electrochemical gradient of H+, Biochim. Biophys. Acta 275 (1972) 33–50. [6] V.P. Skulachev, Membrane Bioenergetics, Springer-Verlag, Berlin, Heidelberg, 1988. [7] R.A. Smith, C.M. Porteous, C.V. Coulter, M.P. Murphy, Selective targeting of an antioxidant to mitochondria, Eur. J. Biochem. 263 (1999) 709–716. [8] M.P. Murphy, R.A. Smith, Drug delivery to mitochondria: the key to mitochondrial medicine, Adv. Drug Deliv. Rev. 41 (2000) 235–250. [9] G.F. Kelso, C.M. Porteous, C.V. Coulter, G. Hughes, W.K. Porteous, E.C. Ledgerwood, R.A. Smith, M.P. Murphy, Selective targeting of a redoxactive ubiquinone to mitochondria within cells: antioxidant and antiapoptotic properties, J. Biol. Chem. 276 (2001) 4588–4596. [10] M.F. Ross, G.F. Kelso, F.H. Blaikie, A.M. James, H.M. Cocheme, A. Filipovska, T. Da Ros, T.R. Hurd, R.A. Smith, M.P. Murphy, Lipophilic triphenylphosphonium cations as tools in mitochondrial bioenergetics and free radical biology, Biochemistry (Moscow) 70 (2005) 222–230. [11] V.P. Skulachev, How to clean the dirtiest place in the cell: cationic antioxidants as intramitochondrial ROS scavengers, IUBMB-Life 57 (2005) 305–310. [12] M.F. Ross, T. Da Ros, F.H. Blaikie, T.A. Prime, C.M. Porteous, I.I. Severina, V.P. Skulachev, H.G. Kjaergaard, R.A. Smith, M.P. Murphy, Accumulation of lipophilic dications by mitochondria and cells, Biochem. J. 400 (2006) 199–208. [13] I.I. Severina, Nystatin-induced increase in photocurrent in the system “bacteriorhodopsin proteoliposome/bilayer planar membrane”, Biochim. Biophys. Acta 681 (1982) 311–317. [14] K.E.O. Akerman, M.K.F. Wikstrom, Safranine as a probe of the mitochondrial membrane potential, FEBS Lett. 68 (1976) 191–197. [15] L.E. Bakeeva, L.L. Grinius, A.A. Jasaitis, V.V. Kuliene, D.O. Levitsky, E.A. Liberman, I.I. Severina, V.P. Skulachev, Conversion of biomembrane-produced energy into electric form: II. Intact mitochondria, Biochim. Biophys. Acta 216 (1970) 12–21. [16] I. Ahmed, G. Krishnamoorthy, Enhancement of transmembrane proton conductivity of protonophores by membrane-permeant cations, Biochim. Biophys. Acta 1024 (1990) 298–306. [17] P. Schonfeld, Anion permeation limits the uncoupling activity of fatty acids in mitochondria, FEBS Lett. 303 (1992) 190–192. [18] V.I. Dedukhova, E.N. Mokhova, A.A. Starkov, Yu.N. Leikin, Carboxyatractylate inhibits the potentiating effect of lipophylic cation TPP+ on uncoupling activity of fatty acid, Biochem. Mol. Biol. Intern. 30 (1993) 1161–1167. [19] V. Beck, M. Jaburek, E.P. Breen, R.K. Porter, P. Jezek, E.E. Pohl, A new automated technique for the reconstitution of hydrophobic proteins into planar bilayer membranes. Studies of human recombinant uncoupling protein 1, Biochim. Biophys. Acta 1757 (2006) 474–479. [20] M.P. Murphy, R.A.J. Smith, Targeting antioxidants to mitochondria by conjugation to lipophilic cations, Annu. Rev. Pharmacol. Toxicol. 47 (2007) 629–656.
Effects of lipophilic dications on planar bilayer phospholipid membrane and mitochondria Inna I. Severina a , Mikhail Yu. Vyssokikh a , Antonina V. Pustovidko a , Ruben A. Simonyan a , Tatiana I. Rokitskaya a , Vladimir P. Skulachev a,b,⁎ a
Department of Bioenergetics, A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119992, Russia b Faculty of Bioengineering and Bioinformatics, Moscow State University, Moscow 119992, Russia Received 22 April 2007; received in revised form 28 May 2007; accepted 31 May 2007 Available online 12 June 2007
Abstract In this paper, we studied effects of phosphonium dications P2C5 and P2C10 on bilayer planar phospholipid membrane (BLM) and rat liver mitochondria. In line with our previous observations [M.F. Ross, T. Da Ros, F.H. Blaikie, T.A. Prime, C.M. Porteous, I.I. Severina, V.P. Skulachev, H.G. Kjaergaard, R.A. Smith, M.P. Murphy, Accumulation of lipophilic dications by mitochondria and cells, Biochem. J. 400 (2006) 199–208], we showed both P2C5 and P2C10 are cationic penetrants for BLM. They generated transmembrane diffusion potential (ΔΨ), the compartment with a lower dication concentration positive. However, the ΔΨ values measured proved to be lower that the Nernstian. This fact could be explained by rather low BLM conductance for the cations at their small concentrations and by induction of some BLM damage at their large concentrations. The damage in question consisted in appearance of non-Ohmic current/voltage relationships which increased in time. Such a nonOhmicity was especially strong at ΔΨ N 100 mV. Addition of penetrating lipophilic anion TPB, which increases the BLM conductance for lipophilic cations, yielded the Nernstian ΔΨ, i.e. 30 mV per ten-fold dication gradient. In the State 4 mitochondria, dications stimulated respiration and lowered ΔΨ. Moreover, they inhibited the State 3 respiration with succinate or glutamate and malate (but not with TMPD and ascorbate) in an uncoupler-sensitive fashion. Effect on the in State 4 mitochondria, similarly to that on BLM, was accounted for by a timedependent membrane damage. On the other hand, the State 3 effect was most probably due to inhibition of the respiratory chain Complex I and/or Complex III. The damaging and inhibitory activities of lipophilic dications should be taken into account when one considers a possibility to use them as a vehicle to target antioxidants or other compounds to mitochondria. © 2007 Elsevier B.V. All rights reserved. Keywords: Dication; Lipophilic cation; Planar phospholipids membrane; Mitochondria; Membrane potential; Damaging effect
1. Introduction Lipophilic penetrating cations have been introduced as tool to monitor membrane potential across mitochondrial and bacterial membranes [1–3]. It was also suggested that these cations can be Abbreviations: ΔΨ, transmembrane electric potential difference; BLM, bilayer planar phospholipid membrane; BSA, bovine serum albumin; DNP, 2,4p-dinitrophenol; P2C5 and P2C10, bistriphenylphosphonium with five or ten methylene linker, respectively; TPMP, N,N′-tetramethyl-p-phenylenediamine; TPB, tetraphenyl borate; TPP, tetraphenylphosphonium ⁎ Corresponding author. Department of Bioenergetics, A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Leninskie Gory 1, Moscow 119992, Russia. Tel.: +7 4959395530; fax: +7 495 9390338. E-mail address: [email protected] (V.P. Skulachev). 0005-2728/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.bbabio.2007.05.010
used as “electric locomotives” to accumulate inside mitochondria electroneutral residues attached to these cations [4–8]. Such a principle was recently successfully employed by Murphy and coworkers to target antioxidants like CoQ or vitamin E to mitochondria [7–9] (for reviews, see refs. 10,11). According to the Nernst equation, the accumulation coefficient for monocations in energized mitochondria (ΔΨ about 180 mV) should be around 103. For dications, it should increase up to 106. This means that lipophilic dications might be much more powerful “locomotives” than monocations [12]. Investigation of such a possibility revealed the following. At ΔΨ lower than 90–100 mV, lipophilic dications P2C5 and P2C10, as had been expected, were accumulated by mitochondria much faster than the monocation TPP. However, a ΔΨ increase above this threshold
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resulted in negligible further increase in the dication uptake. The reason for such a phenomenon remained obscure [12]. In this work, we studied the possible mechanism of nonNernstian behaviour of the dications, using BLM and rat liver mitochondria as experimental systems. It was found that there are at least two reasons for the above phenomenon occurring at high dication concentrations. One of them is a damage to phospholipid bilayer, resulting in a non-Ohmic increase in its conductance in response to a ΔΨ increase. This damage develops in time. In mitochondria, it is revealed as an increase in State 4 respiration and a lowering of ΔΨ. The second reason consists in a dication-induced inhibition of the State 3 respiration at levels of Complex I and/or Complex III. 2. Materials and methods 2.1. Chemicals MOPS (3-[N-Morpholino]propanesulfonic acid), bovine serum albumin (fraction V), rotenone, succinate, malate, glutamate, oligomycin, ascorbic acid, TMPD, DNP, ADP, magnesium and calcium chlorides were from Sigma. TPB was from Fluka; TPP was from Aldrich. P2C5 and P2C10 were generous gifts of Dr. M.P. Murphy. Bilayer planar phospholipid membrane (BLM) was formed on a 1-mm aperture in a Teflon septum separating the experimental chamber into two compartments. To form BLM, E. coli phospholipids were used (57% phosphatidylethanolamine, 15% phosphatidylglycerol, 10% cardiolipin, 18% others). Phospholipids were dissolved in decane (20 mg/ml). Electric parameters were measured with AgCl electrodes and a VA-J-5 electrometer (for details, see [13]).
2.2. Isolation of mitochondria Rat liver mitochondria were purified by differential centrifugation. Rats were killed by cervical dislocation according to recommendation of the University Ethics Committee. The liver was cooled for 1–2 min in ice-cold isolation medium (IM) containing 250 mM sucrose, 10 mM MOPS–KOH, pH 7.4, 1 mM EGTA, 0.1% BSA. After washing out blood from the liver surface, the tissue was cut into 2- to 3-mm pieces, using scissors on a cooled Petri dish. The cut tissue was washed 3–5 times with IM until free from blood and then homogenized by the Potter glass-Teflon homogenizer at 160 rpm for 2 min at 4 °C in 8 ml IM per g tissue. After 10 min centrifugation at 500 g, 4 °C, the supernatant was filtered through cheese-cloth and centrifuged once more at 9000 g, 0 min, 4 °C. A Beckman J2-20 centrifuge and a cooled JA-20 rotor were used. Supernatant was discarded and the tube was cleaned from fat. The pellet was rinsed by gentle stirring with 1–2 ml IM (to remove fluffy layer from the top of the pellet) and resuspended in 1 ml IM supplemented with 0.3% BSA, using a glass stick, and pipetting by a 1-ml Gilson sampler for 1 min. The suspension was diluted with IM to the final volume 8 ml per g protein and centrifuged again under the same conditions. The final pellet was resuspended in one or two drops of IM supplemented by 0.3% BSA with a glass stick and 250 μM Gilson Pipetman, the final volume being about 100–200 μl. Oxygen consumption rate was measured by a Clark electrode and an oxygraph (“Oroboros”, Austria). Protein concentration was 0.4 mg/ml, the chamber volume being 2 ml. The incubation medium composition was identical to that of IM. As respiration substrates, 5 mM succinate in presence of 1 μM rotenone, 4 mM glutamate and 1 mM malate, or 5 mM ascorbate with 0.1 mM TMPD were employed. The State 3 respiration was induced by adding 1 mM ADP in the presence of 5 mM potassium phosphate and 2 mM MgCl2. The uncoupled state was achieved by adding DNP.
2.3. Mitochondrial membrane potential measurements Safranine O was used as a membrane potential probe [14]. The 555- to 523-nm light absorption was measured with an Aminco DW-2000 spectrophotometer
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(dual-wavelength regime). Protein concentration was 0.7 mg/ml; safranin O concentration was 15 μM. Mitochondrial swelling was measured by a decrease in the light scattering at 520 nm, using the Aminco DW-2000 spectrophotometer and stirred cuvettes. Rat liver mitochondria were incubated at 25 °C (final protein concentration, 0.5 mg/ml). Protein concentration was determined with bicinchoninic acid according to the producer's instructions (Pierce, USA), using BSA (2 mg/ml) as a standard protein solution.
3. Results and discussion In the first series of experiments, a bilayer planar phospholipid membrane (BLM) was employed. It was shown that P2C5 and P2C10 are penetrating dications, since trans-BLM dication concentration asymmetry was found to result in generation of an electric potential difference (ΔΨ) across the BLM, the compartment with lower cation concentration positive (Fig. 1A–C). The ΔΨ in question was generated due to electrogenic transmembrane diffusion of a dication from the compartment with a higher dication level to that with a lower dication level. Such conclusion was confirmed by the observation that under optimal conditions the ΔΨ value was equal to 30 mV per ten-fold difference in the dication concentration, which exactly corresponds to the Nernst equation. Under the same conditions, ten-fold gradient of monocation TPP resulted in generation of a 60 mV ΔΨ (Fig. 1C). To make the above-mentioned quantitative relationships reproducible, a penetrating anion (TPB) should be added, a fact which is in line with our previous finding that TPB can operate as a carrier for phosphonium cation derivatives [2,15]. Without TPB, the P2C5 and P2C10 gradients generated diffusion potentials of a magnitude that usually proved to be lower than predicted by the Nernst equation. This was most probably due to rather low phosphonium cation conductance which was comparable to the intrinsic (without these cations) BLM conductance (for details, see ref. [6]). The problem could not be solved by increasing the dication concentration since the linear ΔΨ/[dication] relationship was observed at low [dication] values only. In these experiments, the dication concentrations should, in fact, not exceed some critical level. This level was 1 × 10− 5 M or 1 × 10− 4 M in samples without or with 1 × 10− 7 M TPB, respectively. Above the levels mentioned, a further increase in the dication concentration caused no additional ΔΨ increase (Fig. 1B and C). The observed deviation from the Nernstian behavior might be explained assuming that intrinsic membrane conductance increases due to membrane damage by increasing dication concentration. Apparently, the damage takes some time since without TPB (i.e. when the dication conductance was rather low), the dication gradient-supported ΔΨ, initially rather high, lowers during 2–3 min, whereas such a lowering was not observed within this period of time if TPB was present (Fig. 1A). Consistent with the above assumption, in experiments where the Nernstian ΔΨ values were obtained in samples without TPB, this took place immediately after the dication addition. Then these values lowered very fast. The dication-induced conductance rise was shown to increase not only in time but also at high ΔΨ. In fact, the
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Fig. 1. Effects of P2C5, P2C10, and TPP on BLM. (A) Kinetics of generation of electric potential difference across BLM by transmembrane gradient of P2C5. Initial P2C5 concentration in both compartments was 1 × 10− 6M. When indicated, both compartments were supplemented with 1 × 10− 7M TPB. Arrow, addition of 1 × 10− 5M P2C5 to the right-hand compartment, which resulted in positive charging of the left-hand compartment (ordinate). Incubation mixture, 50 mM Tris–HCl (pH 7.4). (B) Generation of electric potential difference across BLM by transmembrane gradients of TPP+ (circles), P2C5 (triangles), and P2C10 (squares). In the left-hand compartment, the TPP, P2C5, or P2C10 concentration was 1 × 10− 6M; in the right-hand compartment, the cation concentrations varied from 1 × 10− 6M to 1 × 10− 4M. Panel C, as B but both compartments were supplemented with 1 × 10− 7M TPB. (D) Current as a function of applied voltage. 1 × 10− 6M TPP or 3 × 10− 5M P2C5 in both compartments. Incubation mixture, 50 mM Tris–HCl, pH 7.4. (E) Current as a function of applied voltage after incubation of BLM with 5 × 10− 5M P2C5 (in both compartments) for 20 or 40 min. Incubation mixture, 10 mM Tris–HCl, 10 mM MES, 10 mM β-alanine, 100 mM KCl, pH 7.0.
current–voltage relationship proved to be non-Ohmic, the nonOhmicity being increased at ΔΨ N 100 mV. For monocation TPP, the relationship was exactly Ohmic (see the linear dependence of current upon voltage, Fig. 1D). Non-Ohmicity was much bigger when the time of treatment of BLM with dication increased from 20 to 40 min (Fig. 1E). The BLM data suggest that a damaging effect of dications can also be present in mitochondria, where phospholipid bilayer occupies some membrane areas. As one can see in Fig. 2A, addition of 5 μM P2C10 stimulated the State 4 respiration. Such stimulation did not disappear in time and, therefore, could not be explained by a dication influx, which should be transient.
Rather, dications caused an increase in the membrane leakage. Addition of uncouplers (DNP) resulted in further respiration increase, which was higher in a sample with dication (apparently due to a dication-induced increase in the membrane permeability for the uncoupler anion [16–18]). As to oligomycin, it was without effect on the dication-induced respiration increase (not shown). Fig. 2B shows the dependence of the State 4 respiration with succinate as a substrate upon concentrations of dications and TPP. The damaging effect on mitochondria, like that on BLM, was stronger with P2C5 and especially with P2C10 than with TPP. Similar relationships were revealed with glutamate and malate or ascorbate and TMPD.
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Fig. 2. Effects of P2C5, P2C10, and TPP on respiration of rat liver mitochondria. (A) Effect of P2C10 on the kinetics of the State 4 respiration. Substrate, 2.5 mM succinate with 2 μM rotenone. Additions, 5 × 10− 6M P2C10 and 2 × 10− 5M DNP. (B) Stimulation of the State 4 respiration by P2C5, P2C10, and TPP (symbols as in Fig. 1B) as a function of the cation concentration; substrate, succinate with rotenone. (C) Kinetics of the State 3 respiration; substrate, 2.5 mM succinate with 2 μM rotenone. Additions, 5 × 10− 4M ADP, 5 × 10− 6M 2PC5 (each addition), 2 × 10− 5M DNP. (D–F) Effects of P2C5, P2C10 and TPP on the State 3 respiration as a function of the cation concentration; substrates, succinate with rotenone (D), glutamate and malate (E) and ascorbate and TMPD (F).
In Fig. 2C–F, effects of dications and TPP on the State 3 respiration are shown. It was found that all three cations tested decrease the respiration rate (for kinetics of the P2C5 effect, see Fig. 2C). One can see that DNP abolished such an inhibition
(most probably, due to a ΔΨ collapse entailed by release of the dication from mitochondria). The inhibitory effect was especially strong with P2C10, the other two cations being less damaging for the respiratory chain. The inhibition could be
Fig. 3. Effects of cations on the safranine O response of rat liver mitochondria in the State 4. Substrate, 2.5 μM succinate with 2 μM rotenone. Additions, 30 μM P2C5 (A), 6 μM P2C10 (B), 25 μM TPP (C), 50 μM CaCl2, 50 μM DNP (A–D).
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observed with succinate (Fig. 2D) as well as with NAD substrates (Fig. 2E). The State 3 respiration with ascorbate and TMPD was resistant to dications and TPP (Fig. 2F). Thus, the respiratory chain Complexes I–III, rather than Complex IV, are targets for the inhibitory activity of the phosphonium cations. Fig. 3 illustrates the effect of the studied cations as well as of Ca2+ on mitochondrial ΔΨ monitored with safranine O response. The dications and TPP induced the permanent decrease in this response, whereas the action of Ca2+ was transient. The difference in effects of dications and TPP consisted in that in the former (but not in the latter) case fast initial decrease in ΔΨ was followed by further slow ΔΨ decrease. This was consistent with the BLM data (Fig. 1A and F) indicating that dication-induced damage to the membrane develops in time. These data can explain the paradoxical behavior of dications in mitochondria, namely, failure to increase the intramitochondrial level of these compounds by a ΔΨ increase above some critical level [12]. Apparently, accumulation of dications results in membrane damage and, hence, in membrane conductance increase, which prevents further accumulation. This may result in some balance between (i) ΔΨ increase favorable for the dication uptake and (ii) a dication-induced conductance increase that is unfavorable for the uptake. Such damage is rather delicate. As was shown by our experiments, light scattering of mitochondria was not decreased by the highest (3 × 10− 5 M) concentrations of P2C10 used in our experiments. Under the same conditions, 1 × 10− 4 M Ca2+ produced a high amplitude mitochondrial swelling. The above reasoning is in line with our data published in the preceding paper [12]. In that paper, it was reported that a TPB addition allowed P2C5 to accumulate inside mitochondria at ΔΨ levels higher than the ΔΨ threshold observed without TPB. Such an effect may well be a consequence of a TPB-induced rise in the mitochondria membrane conductance for P2C5. Apparently, the dication damage-induced conductance depends on ΔΨ, resulting in an non-Ohmicity of the membrane. Such an assumption explains why in mitochondria the dication accumulation is arrested at high ΔΨ even at low [dication] in the medium [12]. It may be noted in this context that non-Ohmic effects have been shown to be inherent in certain situations when the mitochondrial membrane conductance rises [see, e.g., ref. 19]. It should be stressed that in mitochondria additional unfavorable dication effects other that the membrane damage may also be involved, namely, an inhibition of the respiratory chain Complexes I and III by high concentrations of dications (see Fig. 2C–E). This inhibition might also lower the ΔΨ generation, especially when the membrane leakage increases. All the above complications should be taken into account when a practical use of lipophilic dications is considered [20]. Acknowledgements The authors are very grateful to Dr. M.P. Murphy for samples of dications. This study was supported by grant “Leading Scientific Schools” 5952.2006.4, Ministry of Education and
Science, RF and grant of Russian Foundation for Basic Research (RFBR-05-04-49700). References [1] E.A. Liberman, V.P. Topali, L.M. Tsofina, A.A. Jasaitis, V.P. Skulachev, Mechanisms of coupling of oxidative phosphorylation and the membrane potential of mitochondria, Nature 222 (1969) 1076–1078. [2] L.L. Grinius, A.A. Jasaitis, Yu.L. Kadziauskas, E.A. Liberman, V.P. Skulachev, V.P. Topali, L.M Tsofina, M.A. Vladimirova, Conversion of biomembrane-produced energy into electric form: I. Submitochondrial particles, Biochim. Biophys. Acta 216 (1970) 1–12. [3] E.A. Liberman, V.P. Skulachev, Conversion of biomembrane-produced energy into electric form: IV. General discussion, Biochim. Biophys. Acta 216 (1970) 30–42. [4] S.E. Severin, V.P. Skulachev, L.S. Yagizhinsky, A possible role of carnitine in transport of fatty acid through the mitochondrial membrane, Biokhimiya 35 (1970) 1250–1257 (Russ.). [5] D.O. Levitsky, V.P. Skulachev, Carnitine: the carrier transporting fatty acyl into mitochondria by means of electrochemical gradient of H+, Biochim. Biophys. Acta 275 (1972) 33–50. [6] V.P. Skulachev, Membrane Bioenergetics, Springer-Verlag, Berlin, Heidelberg, 1988. [7] R.A. Smith, C.M. Porteous, C.V. Coulter, M.P. Murphy, Selective targeting of an antioxidant to mitochondria, Eur. J. Biochem. 263 (1999) 709–716. [8] M.P. Murphy, R.A. Smith, Drug delivery to mitochondria: the key to mitochondrial medicine, Adv. Drug Deliv. Rev. 41 (2000) 235–250. [9] G.F. Kelso, C.M. Porteous, C.V. Coulter, G. Hughes, W.K. Porteous, E.C. Ledgerwood, R.A. Smith, M.P. Murphy, Selective targeting of a redoxactive ubiquinone to mitochondria within cells: antioxidant and antiapoptotic properties, J. Biol. Chem. 276 (2001) 4588–4596. [10] M.F. Ross, G.F. Kelso, F.H. Blaikie, A.M. James, H.M. Cocheme, A. Filipovska, T. Da Ros, T.R. Hurd, R.A. Smith, M.P. Murphy, Lipophilic triphenylphosphonium cations as tools in mitochondrial bioenergetics and free radical biology, Biochemistry (Moscow) 70 (2005) 222–230. [11] V.P. Skulachev, How to clean the dirtiest place in the cell: cationic antioxidants as intramitochondrial ROS scavengers, IUBMB-Life 57 (2005) 305–310. [12] M.F. Ross, T. Da Ros, F.H. Blaikie, T.A. Prime, C.M. Porteous, I.I. Severina, V.P. Skulachev, H.G. Kjaergaard, R.A. Smith, M.P. Murphy, Accumulation of lipophilic dications by mitochondria and cells, Biochem. J. 400 (2006) 199–208. [13] I.I. Severina, Nystatin-induced increase in photocurrent in the system “bacteriorhodopsin proteoliposome/bilayer planar membrane”, Biochim. Biophys. Acta 681 (1982) 311–317. [14] K.E.O. Akerman, M.K.F. Wikstrom, Safranine as a probe of the mitochondrial membrane potential, FEBS Lett. 68 (1976) 191–197. [15] L.E. Bakeeva, L.L. Grinius, A.A. Jasaitis, V.V. Kuliene, D.O. Levitsky, E.A. Liberman, I.I. Severina, V.P. Skulachev, Conversion of biomembrane-produced energy into electric form: II. Intact mitochondria, Biochim. Biophys. Acta 216 (1970) 12–21. [16] I. Ahmed, G. Krishnamoorthy, Enhancement of transmembrane proton conductivity of protonophores by membrane-permeant cations, Biochim. Biophys. Acta 1024 (1990) 298–306. [17] P. Schonfeld, Anion permeation limits the uncoupling activity of fatty acids in mitochondria, FEBS Lett. 303 (1992) 190–192. [18] V.I. Dedukhova, E.N. Mokhova, A.A. Starkov, Yu.N. Leikin, Carboxyatractylate inhibits the potentiating effect of lipophylic cation TPP+ on uncoupling activity of fatty acid, Biochem. Mol. Biol. Intern. 30 (1993) 1161–1167. [19] V. Beck, M. Jaburek, E.P. Breen, R.K. Porter, P. Jezek, E.E. Pohl, A new automated technique for the reconstitution of hydrophobic proteins into planar bilayer membranes. Studies of human recombinant uncoupling protein 1, Biochim. Biophys. Acta 1757 (2006) 474–479. [20] M.P. Murphy, R.A.J. Smith, Targeting antioxidants to mitochondria by conjugation to lipophilic cations, Annu. Rev. Pharmacol. Toxicol. 47 (2007) 629–656.