Artigo Acesso aberto Revisado por pares

Bupivacaine Myotoxicity Is Mediated by Mitochondria

2002; Elsevier BV; Volume: 277; Issue: 14 Linguagem: Inglês

10.1074/jbc.m108938200

ISSN

1083-351X

Autores

William Irwin, Éric Fontaine, Laura Agnolucci, Daniele Penzo, Romeo Betto, Susan Bortolotto, Carlo Reggiani, G. Salviati, Paolo Bernardi,

Tópico(s)

Intensive Care Unit Cognitive Disorders

Resumo

We have investigated the effects of the myotoxic local anesthetic bupivacaine on rat skeletal muscle mitochondria and isolated myofibers from flexor digitorum brevis, extensor digitorum longus, soleus, and from the proximal, striated portion of the esophagus. In isolated mitochondria, bupivacaine caused a concentration-dependent mitochondrial depolarization and pyridine nucleotide oxidation, which were matched by an increased oxygen consumption at bupivacaine concentrations of 1.5 mm or less at pH 7.4, whereas respiration was inhibited at higher concentrations. As a consequence of depolarization, bupivacaine caused the opening of the permeability transition pore (PTP), a cyclosporin A-sensitive inner membrane channel that plays a key role in many forms of cell death. In intact flexor digitorum brevis fibers bupivacaine caused mitochondrial depolarization and pyridine nucleotides oxidation that were matched by increased concentrations of cytosolic free Ca2+, release of cytochrome c, and eventually, hypercontracture. Both mitochondrial depolarization and cytochrome c release were inhibited by cyclosporin A, indicating that PTP opening rather than bupivacaine as such was responsible for these events. Similar responses to bupivacaine were observed in the soleus, which is highly oxidative. In contrast, fibers from the esophagus (which we show to be more fatigable than flexor digitorum brevis fibers) and from the highly glycolytic extensor digitorum longus didn't undergo pyridine nucleotide oxidation upon the addition of bupivacaine and were resistant to bupivacaine toxicity. These results suggest that active oxidative metabolism is a key determinant in bupivacaine toxicity, that bupivacaine myotoxicity is a relevant model of mitochondrial dysfunction involving the PTP and Ca2+ dysregulation, and that it represents a promising system to test new PTP inhibitors that may prove relevant in spontaneous myopathies where mitochondria have long been suspected to play a role. We have investigated the effects of the myotoxic local anesthetic bupivacaine on rat skeletal muscle mitochondria and isolated myofibers from flexor digitorum brevis, extensor digitorum longus, soleus, and from the proximal, striated portion of the esophagus. In isolated mitochondria, bupivacaine caused a concentration-dependent mitochondrial depolarization and pyridine nucleotide oxidation, which were matched by an increased oxygen consumption at bupivacaine concentrations of 1.5 mm or less at pH 7.4, whereas respiration was inhibited at higher concentrations. As a consequence of depolarization, bupivacaine caused the opening of the permeability transition pore (PTP), a cyclosporin A-sensitive inner membrane channel that plays a key role in many forms of cell death. In intact flexor digitorum brevis fibers bupivacaine caused mitochondrial depolarization and pyridine nucleotides oxidation that were matched by increased concentrations of cytosolic free Ca2+, release of cytochrome c, and eventually, hypercontracture. Both mitochondrial depolarization and cytochrome c release were inhibited by cyclosporin A, indicating that PTP opening rather than bupivacaine as such was responsible for these events. Similar responses to bupivacaine were observed in the soleus, which is highly oxidative. In contrast, fibers from the esophagus (which we show to be more fatigable than flexor digitorum brevis fibers) and from the highly glycolytic extensor digitorum longus didn't undergo pyridine nucleotide oxidation upon the addition of bupivacaine and were resistant to bupivacaine toxicity. These results suggest that active oxidative metabolism is a key determinant in bupivacaine toxicity, that bupivacaine myotoxicity is a relevant model of mitochondrial dysfunction involving the PTP and Ca2+ dysregulation, and that it represents a promising system to test new PTP inhibitors that may prove relevant in spontaneous myopathies where mitochondria have long been suspected to play a role. Bupivacaine is a local anesthetic that induces rapid degeneration of skeletal muscle fibers (1.Milburn A. J. Neurocytol. 1976; 5: 425-446Crossref PubMed Scopus (26) Google Scholar, 2.Hall-Craggs E.C. Br. J. Exp. Pathol. 1980; 61: 139-149PubMed Google Scholar). As is the case for muscular dystrophies, the pathogenesis of bupivacaine-induced muscle cell death remains unclear. Solving this problem is of interest for the understanding of degenerative muscular diseases because the sequence of fiber breakdown induced by bupivacaine is similar to that of progressive muscular dystrophy (3.Nonaka I. Takagi A. Ishiura S. Nakase H. Sugita H. Acta Neuropathol. (Berl.). 1983; 60: 167-174Crossref PubMed Scopus (119) Google Scholar). It is also striking that the same types of muscle fibers are spared by both Duchenne's muscular dystrophy and bupivacaine toxicity (4.Porter J.D. Edney D.P. McMahon E.J. Burns L.A. Investig. Ophthalmol. Vis. Sci. 1988; 29: 163-174PubMed Google Scholar, 5.Kaminski H.J. al Hakim M. Leigh R.J. Katirji M.B. Ruff R.L. Ann. Neurol. 1992; 32: 586-588Crossref PubMed Scopus (101) Google Scholar). It has been suggested that bupivacaine may disrupt Ca2+homeostasis in vivo, triggering Ca2+-activated cellular death pathways that include proteolysis (4.Porter J.D. Edney D.P. McMahon E.J. Burns L.A. Investig. Ophthalmol. Vis. Sci. 1988; 29: 163-174PubMed Google Scholar, 6.Steer J.H. Mastaglia F.L. Papadimitriou J.M. Van Bruggen I. J. Neurol. Sci. 1986; 73: 205-217Abstract Full Text PDF PubMed Scopus (24) Google Scholar). This suggestion is supported by the findings that (i) bupivacaine affects sarcoplasmic reticulum function in vitro (3.Nonaka I. Takagi A. Ishiura S. Nakase H. Sugita H. Acta Neuropathol. (Berl.). 1983; 60: 167-174Crossref PubMed Scopus (119) Google Scholar), (ii) extracellular Ca2+ omission delays the morphological changes (6.Steer J.H. Mastaglia F.L. Papadimitriou J.M. Van Bruggen I. J. Neurol. Sci. 1986; 73: 205-217Abstract Full Text PDF PubMed Scopus (24) Google Scholar) and decreases the protein degradation rate (7.Steer J.H. Mastaglia F.L. J. Neurol. Sci. 1986; 75: 343-351Abstract Full Text PDF PubMed Scopus (7) Google Scholar) that are observed in isolated rat soleus muscle exposed to bupivacaine, and (iii) bupivacaine uncouples isolated rat liver and heart mitochondria (8.Dabadie P. Bendriss P. Erny P. Mazat J.P. FEBS Lett. 1987; 226: 77-82Crossref PubMed Scopus (75) Google Scholar, 9.van Dam K. Shinohara Y. Unami A. Yoshida K. Terada H. FEBS Lett. 1990; 277: 131-133Crossref PubMed Scopus (33) Google Scholar, 10.Terada H. Shima O. Yoshida K. Shinohara Y. J. Biol. Chem. 1990; 265: 7837-7842Abstract Full Text PDF PubMed Google Scholar, 11.Sun X. Garlid K.D. J. Biol. Chem. 1992; 267: 19147-19154Abstract Full Text PDF PubMed Google Scholar, 12.Schönfeld P. Sztark F. Slimani M. Dabadie P. Mazat J.P. FEBS Lett. 1992; 304: 273-276Crossref PubMed Scopus (33) Google Scholar) and decreases mitochondrial membrane potential and oxygen consumption both in cultured fibroblasts (13.Grouselle M. Tueux O. Dabadie P. Georgescaud D. Mazat J.P. Biochem. J. 1990; 271: 269-272Crossref PubMed Scopus (66) Google Scholar, 14.Sztark F. Tueux O. Erny P. Dabadie P. Mazat J.P. Anesth. Analg. 1994; 78: 335-339Crossref PubMed Scopus (37) Google Scholar) and Ehrlich tumor cells (15.Floridi A. Barbieri R. Pulselli R. Fanciulli M. Arcuri E. Oncol. Res. 1994; 6: 593-601PubMed Google Scholar, 16.Pulselli R. Arcuri E. Paggi M.G. Floridi A. Oncol. Res. 1996; 8: 267-271PubMed Google Scholar). Mitochondrial dysfunction results in ATP depletion (14.Sztark F. Tueux O. Erny P. Dabadie P. Mazat J.P. Anesth. Analg. 1994; 78: 335-339Crossref PubMed Scopus (37) Google Scholar) and in turn is expected to have a major impact on intracellular Ca2+ homeostasis (17.Bernardi P. Physiol. Rev. 1999; 79: 1127-1155Crossref PubMed Scopus (1347) Google Scholar). The importance of mitochondria in the pathways to cell death is largely recognized even if the exact mechanism(s) in specific experimental paradigms may not be easy to identify (18.Bernardi P. Scorrano L. Colonna R. Petronilli V. Di Lisa F. Eur. J. Biochem. 1999; 264: 687-701Crossref PubMed Scopus (660) Google Scholar). A potential mechanism is represented by the opening of the PTP, 1The abbreviations used are: PTPmitochondrial permeability transition poreCsAcyclosporin AFDBflexor digitorum brevisEDLextensor digitorum longusPNpyridine nucleotidesTMRMtetramethylrhodamine methyl esterRh123rhodamine 123Δψmitochondrial membrane potential differenceBAPTA1,2-bis-(2-aminophenoxy)ethane-N, N, N′N′-tetraacetic acidMHCmyosin heavy chainMOPS4-morpholinepropanesulfonic acid a high conductance channel located in the inner mitochondrial membrane that can be inhibited by CsA (19.Crompton M. Biochem. J. 1999; 341: 233-249Crossref PubMed Scopus (2119) Google Scholar). PTP opening in vitro leads to collapse of the protonmotive force, disruption of ionic homeostasis, mitochondrial swelling, and release of cytochrome c (20.Petronilli V. Nicolli A. Costantini P. Colonna R. Bernardi P. Biochim. Biophys. Acta. 1994; 1187: 255-259Crossref PubMed Scopus (194) Google Scholar). This sequence of events has drawn considerable attention to the PTP as a potential effector in the pathways to cell death through at least three mechanisms, i.e. decreased levels of cellular ATP (21.Imberti R. Nieminen A.L. Herman B. Lemasters J.J. J. Pharmacol. Exp. Ther. 1993; 265: 392-400PubMed Google Scholar, 22.Pastorino J.G. Snyder J.W. Serroni A. Hoek J.B. Farber J.L. J. Biol. Chem. 1993; 268: 13791-13798Abstract Full Text PDF PubMed Google Scholar, 23.Duchen M.R. McGuinness O. Brown L.A. Crompton M. Cardiovasc. Res. 1993; 27: 1790-1794Crossref PubMed Scopus (265) Google Scholar), increase of cytosolic Ca2+ (24.Duchen M.R. J. Physiol. (Lond.). 1999; 516: 1-17Crossref Scopus (533) Google Scholar), and release of apoptotic factors such as cytochrome c (25.Pastorino J.G. Chen S.T. Tafani M. Snyder J.W. Farber J.L. J. Biol. 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Nature. 2001; 412: 95-99Crossref PubMed Scopus (1404) Google Scholar). mitochondrial permeability transition pore cyclosporin A flexor digitorum brevis extensor digitorum longus pyridine nucleotides tetramethylrhodamine methyl ester rhodamine 123 mitochondrial membrane potential difference 1,2-bis-(2-aminophenoxy)ethane-N, N, N′N′-tetraacetic acid myosin heavy chain 4-morpholinepropanesulfonic acid With the long term goal of defining the role of mitochondria in the pathways to muscle cell death (30.Bernardi P. Ital. J. Neurol. Sci. 1999; 20: 395-400Crossref PubMed Scopus (38) Google Scholar), we have investigated the effects of bupivacaine both on rat skeletal muscle mitochondria and on isolated mouse FDB, EDL, soleus, and esophagus fibers. We found that bupivacaine causes depolarization, PN oxidation, and PTP opening in isolated skeletal muscle mitochondria. Measurements on the isolated FDB fibers indicated that bupivacaine also induced mitochondrial depolarization that was significantly delayed by CsA in situ, indicating that depolarization was due to PTP opening rather than to the uncoupling effects of bupivacaine as such. Consistent with these data, bupivacaine caused CsA-inhibitable release of cytochrome c in situ. Fibers from glycolytic, non-resistant to fatigue muscles such as EDL and esophagus, were instead strikingly resistant to bupivacaine toxicity, suggesting that bupivacaine toxicity selectively affects oxidative muscles. Thus, bupivacaine toxicity is a relevant model of mitochondrial dysfunction involving the PTP and Ca2+ dysregulation and represents a promising system to test new PTP inhibitors that may prove relevant in spontaneous myopathies where mitochondria have long been suspected to play a role, like Duchenne's muscular dystrophy (31.Wrogemann K. Pena S.D. Lancet. 1976; 1: 672-674Abstract PubMed Scopus (383) Google Scholar). Rat skeletal muscle mitochondria were prepared according to Madsen et al. (32.Madsen K. Ertbjerg P. Pedersen P.K. Anal. Biochem. 1996; 237: 37-41Crossref PubMed Scopus (20) Google Scholar) with slight modifications. Albino Wistar rats weighing 250–350 g were killed by decapitation, and the gastrocnemius muscles were rapidly excised and transferred into the isolation medium (150 mm sucrose, 75 mm KCl, 50 mm Tris-HCl, 1 mmKH2PO4, 5 mm MgCl2, 1 mm EGTA, pH 7.4). Muscles were minced with scissors and trimmed clean of visible fat and connective tissues. Muscle pieces were transferred to 30 ml of isolation medium supplemented with 0.2% bovine serum albumin and 0.2 mg ml−1 Nagarse (Fluka, Buchs). After 1 min, muscles were homogenized using a motor-driven Plexiglas/Plexiglas potter, transferred to 120 ml of isolation medium supplemented with 0.2% bovine serum albumin, and centrifuged at 700 × g for 10 min. The supernatant was decanted and centrifuged at 10,000 × g for 10 min. The resulting pellet was resuspended in a medium containing 250 mmsucrose, 0.1 mm EGTA-Tris, 10 mm Tris-HCl, pH 7.4, and centrifuged at 7,000 × g for 6 min. The final mitochondrial pellet was resuspended in 0.5 ml of the same medium at a final protein concentration of about 20 mg ml−1. All procedures were carried out at 0–4 °C. Mitochondrial oxygen consumption was measured polarographically at 25 °C using a Clark-type electrode. Measurements of membrane potential and PN oxidation-reduction status were carried out fluorimetrically with a PerkinElmer 650–40 spectrofluorimeter equipped with magnetic stirring and thermostatic control. Membrane potential was measured in the presence of 0.1 μm Rh123 as described (33.Fontaine E. Eriksson O. Ichas F. Bernardi P. J. Biol. Chem. 1998; 273: 12662-12668Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar) (excitation-emission, 548–573 nm). The PN oxidation-reduction status was evaluated based on endogenous NAD(P)H fluorescence (excitation-emission, 345–450 nm). Isolated muscle fibers were prepared from FDB, EDL, soleus muscle, and from the upper region of the esophagus of C57BL/10ScSn mice according to (34.Liu Y. Carroll S.L. Klein M.G. Schneider M.F. Am. J. Physiol. 1997; 272: C1919-C1927Crossref PubMed Google Scholar) with slight modifications. Mice were killed by cervical dislocation, and the muscles were incubated for 1 h at 4 °C in Tyrode solution containing 135 mm NaCl, 4 mmKCl, 1 mm CaCl2, 1 mmMgCl2, 0.33 mm KH2PO4, 10 mm glucose, and 10 mm Hepes, pH 7.3, supplemented with 0.3% collagenase type I and 0.2% bovine serum albumin. The temperature was raised to 37 °C, and the incubation was continued for a further 1 h. The muscle mass was then removed and washed twice in Tyrode solution, and single myocytes were dispersed by passing the muscle repeatedly through a wide-pore Pasteur pipette. Myocytes suspended in Tyrode solution were plated on glass coverslips and allowed to attach for at least 1 h before the experiment. Myocytes were then rinsed and placed in 1 ml of Tyrode solution and loaded with the indicated concentrations of TMRM for 20 min at 37 °C. Myocytes were then placed on the stage of the confocal microscope, maintaining the temperature at 37 °C. In some experiments, myocytes were also loaded with 2 μmFluo-3-AM. Imaging was performed with either a real time confocal system (Nikon, RCM 8000) on a Nikon Diaphot-300 microscope with a ×40 1.3 NA oil immersion objective or on a Zeiss Axiovert 100TV inverted fluorescence microscope. For the Nikon setup excitation wavelength/detection filter were 488/525 ± 25 nm bandpass and 568/585 longpass for Fluo-3 and TMRM, respectively. In some experiments, Fluo-3 and TMRM fluorescence emissions were collected simultaneously by using two separate color channels on the detector assembly. In most of the experiments sequential confocal images were acquired and stored typically at 60-s intervals for 20–45 min. The time course of Δψ and [Ca2+] (measured in arbitrary fluorescence units) were performed using the Nikon RCM8000 real time confocal system data acquisition software. Skeletal fibers were identified as regions of interest, and background was identified as an area without cells. For the Zeiss setup, a 10× objective was used. TMRM was excited with 546 ± 5 nm, and the emission was monitored at 580 ± 15 nm with a 560-nm dichroic mirror. NAD(P)H was excited at 365 ± 15 nm, and the emission was monitored at 460 ± 25 nm. The data was analyzed with the MetaMorph MetaFluor Imaging Software. Cytochrome c release was monitored exactly as described in Petronilli et al. (35.Petronilli V. Penzo D. Scorrano L. Bernardi P. Di Lisa F. J. Biol. Chem. 2001; 276: 12030-12034Abstract Full Text Full Text PDF PubMed Scopus (430) Google Scholar). FDB fibers were treated with vehicle or with 1 mm bupivacaine as specified in the legend to Fig. 8 and then washed. Fibers were fixed for 30 min at room temperature with 3.7% (v/v) ice-cold formaldehyde, permeabilized for 20 min with 0.01% (v/v) ice-cold Nonidet P-40, and incubated for 15 min with a 0.5% solution of bovine serum albumin and then for 15 min at 37 °C with a mouse monoclonal anti-cytochrome c antibody (Pharmingen, San Diego, CA, clone 6H2.B4) and with an affinity-purified rabbit antibody against the rat bc1 complex (a generous gift of Prof. Roberto Bisson, Padova). Fibers were then sequentially incubated for 15 min at 37 °C with tetramethylrhodamine B isothiocyanate-conjugated goat anti-mouse IgG and with fluorescein isothiocyanate-conjugated goat anti-rabbit IgG. Cellular fluorescence images were acquired with a Nikon Eclipse E600 microscope equipped with a Bio-Rad MRC-1024 laser- scanning confocal imaging system. For cytochrome c and bc1 complex detection, red and green channel images were acquired simultaneously using two separate color channels on the detector assembly of the Nikon Eclipse E600 microscope equipped with 488/522 ± 25 nm bandpass and 568/605 longpass filter settings, and a ×60 1.4 NA oil immersion objective (Nikon). Using the Bio-Rad LaserSharp analysis program, a set of lines was drawn across the cells, and the fluorescence intensity of each pixel along the lines in both the green and the red channel was measured. The localization index, LI, is defined as the ratio of the standard deviation of the fluorescence intensity divided by the total fluorescence for each channel, (S.D./Σ)red/(S.D./Σ)green. A punctate distribution (which is typical of mitochondria) results in a higher S.D., and normalization allows correction for different fluorescence intensities in the two channels. The normal localization index must be 1, which indicates that the bc1 complex and cytochrome c have the same pattern of intracellular distribution. A localization index <1 indicates that the distribution of cytochrome c is more homogeneous than that of the bc1 complex, i.e. that cytochrome c has diffused away from mitochondria (35.Petronilli V. Penzo D. Scorrano L. Bernardi P. Di Lisa F. J. Biol. Chem. 2001; 276: 12030-12034Abstract Full Text Full Text PDF PubMed Scopus (430) Google Scholar). Fiber typing of FDB, EDL, esophagus, and soleus muscles was based on electrophoretic separation of MHC isoforms, which can be used as molecular markers of fiber type (36.Schiaffino S. Reggiani C. Physiol. Rev. 1996; 76: 371-423Crossref PubMed Scopus (1277) Google Scholar). Muscle samples were immersed in Laemmli solution (37.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207538) Google Scholar), and MHC isoforms were resolved by the SDS-PAGE method as described (38.Talmadge R.J. Roy R.R. J. Appl. Physiol. 1993; 75: 2337-2340Crossref PubMed Scopus (725) Google Scholar). Briefly, 8% polyacrylamide slab gels containing 30% glycerol were run for 24 h at 275 V in the cold room (4 °C). The gels were removed and stained with Coomassie Brilliant Blue G. Densitometry was performed using one-dimensional Image Analysis Software (Kodak Digital Science). Muscle fatigability was used as an index of their dependence on glycolytic or oxidative metabolic supply (39.Fitts R.H. Physiol. Rev. 1994; 74: 49-94Crossref PubMed Scopus (0) Google Scholar, 40.Westerblad H. Allen D.G. Bruton J.D. Andrade F.H. Lannergren J. Acta Physiol. Scand. 1998; 162: 253-260Crossref PubMed Scopus (151) Google Scholar). The muscles were mounted in a myograph and perfused with oxygenated Krebs solution (temperature, 20 °C). The muscles were allowed to equilibrate for 10 min, then the frequency (Fmax) at which maximum isometric tension was obtained was identified. Fatigue was induced by repetitive stimulation at Fmax. The muscles were stimulated to contract isometrically for 0.5 s every other second (duty cycle 25%). The ratio between the tension developed after 30 s and after 60 s of stimulation and the initial tension (time zero) was taken as a fatigue index. Bupivacaine and collagenase were purchased from Sigma; Rh123, TMRM, and Fluo-3 AM were purchased from Molecular Probes; and CsA was a gift from Novartis (Basel, Switzerland). All other chemicals were of the highest purity commercially available. Bupivacaine is an uncoupler in isolated liver and heart mitochondria (8.Dabadie P. Bendriss P. Erny P. Mazat J.P. FEBS Lett. 1987; 226: 77-82Crossref PubMed Scopus (75) Google Scholar) and causes complex effects on respiration in cultured cells (13.Grouselle M. Tueux O. Dabadie P. Georgescaud D. Mazat J.P. Biochem. J. 1990; 271: 269-272Crossref PubMed Scopus (66) Google Scholar, 14.Sztark F. Tueux O. Erny P. Dabadie P. Mazat J.P. Anesth. Analg. 1994; 78: 335-339Crossref PubMed Scopus (37) Google Scholar, 15.Floridi A. Barbieri R. Pulselli R. Fanciulli M. Arcuri E. Oncol. Res. 1994; 6: 593-601PubMed Google Scholar, 16.Pulselli R. Arcuri E. Paggi M.G. Floridi A. Oncol. Res. 1996; 8: 267-271PubMed Google Scholar). The PTP is a voltage-dependent channel that can be opened by depolarization with uncouplers and respiratory inhibitors (41.Bernardi P. J. Biol. Chem. 1992; 267: 8834-8839Abstract Full Text PDF PubMed Google Scholar). We have therefore first characterized the effects of bupivacaine on rat skeletal muscle mitochondria in the presence of CsA (to prevent PTP opening). The experiments of Fig. 1 document a biphasic effect of bupivacaine on the rate of oxygen consumption and on the PN oxidation-reduction status. Respiration increased linearly up to a concentration of about 1.5 mm bupivacaine, and it then declined as the concentration was increased further (Fig. 1, triangles). The reduction levels of PN were a mirror image of the respiratory changes, with increased oxidation matching uncoupling and increased reduction matching respiratory inhibition (Fig. 1, squares). Fig. 2, panel A, reports the effects of increasing bupivacaine concentrations on oxygen consumption by skeletal muscle mitochondria at pH 7.4 (triangles) and pH 7.0 (squares). Although the overall pattern remained biphasic, at pH 7.4, the concentrations of bupivacaine required for 50% stimulation and inhibition of respiration were ∼0.75 and 2.2 mm, respectively. At pH 7.0 these values became ∼1.5 and 4.2 mm bupivacaine, i.e. about twice the values obtained at pH 7.4. When the data were replotted as a function of the calculated concentration of the de-protonated form of bupivacaine (BPo, p Ka 8.1), it became apparent that the effects of the drug correlated with BPo (Fig. 2, panel B). Although the p Ka of bupivacaine in the membrane is not known, these findings suggest that BPo is responsible for both uncoupling and inhibition of respiration and indicate that the concentration of BPorequired for maximal stimulation of respiration may be as low as 0.25 mm (Fig. 2B). We next investigated the effects of bupivacaine on the membrane potential (Fig. 3, panel A) and respiration (Fig. 3, panel B) maintained by isolated skeletal muscle mitochondria. Mitochondria were energized with complex I substrates and loaded with a small amount of Ca2+ in the presence of Pi, an optimal condition to reveal PTP opening by depolarization (42.Petronilli V. Cola C. Bernardi P. J. Biol. Chem. 1993; 268: 1011-1016Abstract Full Text PDF PubMed Google Scholar). It can be seen that the addition of 1 mm bupivacaine was readily followed by a fast but partial mitochondrial depolarization followed within a few minutes by a further depolarization (Fig. 3, panel A, trace a). These changes were matched by a transient stimulation of respiration followed by respiratory inhibition (panel B, trace a). The nature of these complex changes was elucidated when the experiment was repeated in the presence of CsA. Under these conditions, the addition of 1 mm bupivacaine was only followed by the fast, partial depolarization (panel A, trace b), whereas uncoupling was not followed by respiratory inhibition (panel B, trace b). Thus, both the late depolarization and the delayed inhibition of respiration were due to PTP opening. Indeed, inhibition of respiration could be prevented by the addition of exogenous NADH (panel B, trace b) (43.Vinogradov A. Scarpa A. Chance B. Arch. Biochem. Biophys. 1972; 152: 646-654Crossref PubMed Scopus (103) Google Scholar). Thus, by acting on three key sites of PTP regulation (i.e. membrane potential (41.Bernardi P. J. Biol. Chem. 1992; 267: 8834-8839Abstract Full Text PDF PubMed Google Scholar), NAD(P)H levels (44.Haworth R.A. Hunter D.R. J. Membr. Biol. 1980; 54: 231-236Crossref PubMed Scopus (118) Google Scholar), and electron flux through complex I (33.Fontaine E. Eriksson O. Ichas F. Bernardi P. J. Biol. Chem. 1998; 273: 12662-12668Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar)), bupivacaine is a trigger for PTP opening in isolated skeletal muscle mitochondria. We next addressed the question of whether bupivacaine could decrease the mitochondrial membrane potential per se and/or through PTP opening in FDB muscle fibers. Fig. 4, panel A, shows the typical preparation used for these experiments as a fluorescence image after TMRM loading. Panel B documents that within 40 min of the addition of 1 mmbupivacaine, the TMRM signal was largely lost, and the fiber shortened considerably (panel B). The experiment of Fig. 5 illustrates the time course of the fluorescence changes of single TMRM-loaded FDB fibers elicited by the addition of 1 mm bupivacaine. The signal decreased within about 30 min (squares, trace a), and depolarization was prevented by CsA (closed circles, trace b), indicating that PTP opening rather than bupivacaine as such was responsible for complete depolarization under these conditions (Fig. 5). Finally, Fig. 5 shows that mitochondrial depolarization was favored by Ca2+. Indeed, it could be delayed but not prevented by treatment with dantrolene, an inhibitor of Ca2+ release from the sarcoplasmic reticulum (triangles, trace c) or BAPTA-AM, a permeant Ca2+ chelator (open circles, trace d). The role of intracellular Ca2+ in bupivacaine toxicity was investigated further in direct measurements with the fluorescent indicator Fluo-3. The experiments of Fig. 6, panel A, show that mitochondrial depolarization induced by bupivacaine (circles, trace a) was mirrored by an increase of cytosolic Ca2+ concentraton ([Ca2+]c; squares, trace b), which was observed also in nominally Ca2+-free media (not shown). Fig. 6, panel B, shows that in a Ca2+-free medium, ryanodine was able to prevent the rise of [Ca2+]c (squares, trace b) but not mitochondrial depolarization (circles, trace a). These experiments suggest that the latter is the cause rather than the consequence of dysregulation of Ca2+homeostasis.Figure 5Effects of CsA, dantrolene, and BAPTA-AM on bupivacaine-dependent changes of mitochondrial TMRM fluorescence in isolated FDB muscle fibers. Experimental conditions were as in Fig. 4. In all cases, 1 mmbupivacaine was added at T = 4 min. Additions of CsA (2 μm, closed circles, trace b), dantrolene (0.1 mm, triangles, trace c), or BAPTA-AM (5 μm, open circles, trace d), were made at T = 0 min (not shown). Squares (trace a), only bupivacaine was added. Values on the ordinate refer to the normalized TMRM fluorescence signals.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 6Effect of bupivacaine on [Ca2+]c and mitochondrial TMRM fluorescence in FDB fibers. The experimental conditions were as in Fig. 4. The incubation medium was complete Tyrode in panel A and Ca2+-free Tyrode supplemented with 100 μmryanodine in panel B. Isolated FDB fibers were stained with 10 nm TMRM and 2 μm Fluo-3 AM, and the fluorescence of TMRM (circles, traces a) and Fluo-3 (squares, traces b) was followed over time by laser confocal microscopy. Where indicated, 2 mmbupivacaine was added. Values on the ordinate refer to the normalized TMRM or Fluo-3 fluorescence signals. In both panels an asterisk denotes the onset of hypercontracture.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The depolarization due to the addition of bupivacaine before onset of the PTP opening was clearly detectable in iso

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