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• W2034991285 abstract "Mitochondrial permeability transition (MPT), which contributes substantially to the regulation of normal mitochondrial metabolism, also plays a crucial role in the initiation of cell death. It is known that MPT is regulated in a tissue-specific manner. The importance of MPT in the pancreatic β-cell is heightened by the fact that mitochondrial bioenergetics serve as the main glucose-sensing regulator and energy source for insulin secretion. In the present study, using MIN6 and INS-1 β-cells, we revealed that both Ca2+-phosphate- and oxidant-induced MPT is remarkably different from other tissues. Ca2+-phosphate-induced transition is accompanied by a decline in mitochondrial reactive oxygen species production related to a significant potential dependence of reactive oxygen species formation in β-cell mitochondria. Hydroperoxides, which are indirect MPT co-inducers active in liver and heart mitochondria, are inefficient in β-cell mitochondria, due to the low mitochondrial ability to metabolize them. Direct cross-linking of mitochondrial thiols in pancreatic β-cells induces the opening of a low conductance ion permeability of the mitochondrial membrane instead of the full scale MPT opening typical for liver mitochondria. Low conductance MPT is independent of both endogenous and exogenous Ca2+, suggesting a novel type of nonclassical MPT in β-cells. It results in the conversion of electrical transmembrane potential into ΔpH instead of a decrease in total protonmotive force, thus mitochondrial respiration remains in a controlled state. Both Ca2+- and oxidant-induced MPTs are phosphate-dependent and, through the “phosphate flush” (associated with stimulation of insulin secretion), are expected to participate in the regulation in β-cell glucose-sensing and secretory activity. Mitochondrial permeability transition (MPT), which contributes substantially to the regulation of normal mitochondrial metabolism, also plays a crucial role in the initiation of cell death. It is known that MPT is regulated in a tissue-specific manner. The importance of MPT in the pancreatic β-cell is heightened by the fact that mitochondrial bioenergetics serve as the main glucose-sensing regulator and energy source for insulin secretion. In the present study, using MIN6 and INS-1 β-cells, we revealed that both Ca2+-phosphate- and oxidant-induced MPT is remarkably different from other tissues. Ca2+-phosphate-induced transition is accompanied by a decline in mitochondrial reactive oxygen species production related to a significant potential dependence of reactive oxygen species formation in β-cell mitochondria. Hydroperoxides, which are indirect MPT co-inducers active in liver and heart mitochondria, are inefficient in β-cell mitochondria, due to the low mitochondrial ability to metabolize them. Direct cross-linking of mitochondrial thiols in pancreatic β-cells induces the opening of a low conductance ion permeability of the mitochondrial membrane instead of the full scale MPT opening typical for liver mitochondria. Low conductance MPT is independent of both endogenous and exogenous Ca2+, suggesting a novel type of nonclassical MPT in β-cells. It results in the conversion of electrical transmembrane potential into ΔpH instead of a decrease in total protonmotive force, thus mitochondrial respiration remains in a controlled state. Both Ca2+- and oxidant-induced MPTs are phosphate-dependent and, through the “phosphate flush” (associated with stimulation of insulin secretion), are expected to participate in the regulation in β-cell glucose-sensing and secretory activity. Mitochondrial permeability transition (MPT) 1The abbreviations used are: MPT, mitochondrial permeability transition; ROS, reactive oxygen species; DCF, dichlorofluorescein; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; BCECF, 2′,7′-bis(carboxyethyl)-5,6-carboxyfluorescein; PhArs, phenylarsine oxide; NEM, N-ethylmaleimide. is a permeability increase of the inner mitochondrial membrane to solutes of molecular mass up to ∼1500 Da, which is caused by an opening of specific nonselective proteinaceous pores in the inner mitochondrial membrane (1Gunter T.E. Pfeiffer D.R. Am. J. Physiol. 1990; 258: C755-C786Crossref PubMed Google Scholar, 2Bernardi P. Physiol. Rev. 1999; 79: 1127-1155Crossref PubMed Scopus (1342) Google Scholar). Increased permeability of the inner membrane is initiated by an increased level of intramitochondrial Ca2+ and is regulated by multiple effectors, including inorganic phosphate, the redox state of pyridine nucleotides and thiols (oxidative stress), membrane potential, cyclosporin A, and other factors (3Zoratti M. Szabo I. Biochim. Biophys. Acta. 1995; 1241: 139-176Crossref PubMed Scopus (2194) Google Scholar). MPT pores are thought to have at least two open conformations and a variable degree of reversibility. Most importantly, MPT in its different forms contributes to both normal cell physiology (regulation of oxidative phosphorylation and Ca2+ metabolism) and to regulatory processes leading to apoptosis (2Bernardi P. Physiol. Rev. 1999; 79: 1127-1155Crossref PubMed Scopus (1342) Google Scholar, 3Zoratti M. Szabo I. Biochim. Biophys. Acta. 1995; 1241: 139-176Crossref PubMed Scopus (2194) Google Scholar, 4Halestrap A.P. McStay G.P. Clarke S.J. Biochimie (Paris). 2002; 84: 153-166Crossref PubMed Scopus (629) Google Scholar). Specific features of MPT show significant tissue-specific variability (5Brustovetsky N. Dubinsky J.M. J. Neurosci. 2000; 20: 8229-8237Crossref PubMed Google Scholar, 6Brustovetsky N. Dubinsky J.M. J. Neurosci. 2000; 20: 103-113Crossref PubMed Google Scholar, 7Andreyev A. Fiskum G. Cell Death Differ. 1999; 6: 825-832Crossref PubMed Scopus (164) Google Scholar, 8Fontaine E. Eriksson O. Ichas F. Bernardi P. J. Biol. Chem. 1998; 273: 12662-12668Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar). The most significant and well documented deviation from classical MPT described in liver mitochondria was observed in brain tissue (9Berman S.B. Watkins S.C. Hastings T.G. Exp. Neurol. 2000; 164: 415-425Crossref PubMed Scopus (104) Google Scholar). Brain mitochondria demonstrate high resistance to MPT opening (10Schild L. Keilhoff G. Augustin W. Reiser G. Striggow F. FASEB J. 2001; 15: 565-567Crossref PubMed Scopus (81) Google Scholar), low sensitivity to cyclosporin A (5Brustovetsky N. Dubinsky J.M. J. Neurosci. 2000; 20: 8229-8237Crossref PubMed Google Scholar), different responses to MPT modifiers, and a distinctive composition of Ca2+-phosphate complexes sequestered in the matrix (11Panov A.V. Andreeva L. Greenamyre J.T. Arch. Biochem. Biophys. 2004; 424: 44-52Crossref PubMed Scopus (52) Google Scholar). Mitochondria from skeletal muscle, unlike hepatocyte mitochondria, show the dependence of MPT on electron flux through respiratory complex 1 (12Chauvin C. De Oliveira F. Ronot X. Mousseau M. Leverve X. Fontaine E. J. Biol. Chem. 2001; 276: 41394-41398Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Most importantly, basal endogenous MPT activity (transient pore opening), which affects Ca2+ signaling, oxidative phosphorylation, and ROS production, is different in different cell types, which can be attributed to the variable cellular redox states (13Kowaltowski A.J. Smaili S.S. Russell J.T. Fiskum G. Am. J. Physiol. 2000; 279: C852-C859Crossref PubMed Google Scholar). Recent studies in pancreatic β-cells demonstrate a dual role for MPT. Studies examining the role of cytokines in the pancreatic β-cell indicated, by means of mitochondrial depolarization and cytochrome c release, that MPT is a common effector of both apoptosis and necrosis (14Barbu A. Welsh N. Saldeen J. Mol. Cell. Endocrinol. 2002; 190: 75-82Crossref PubMed Scopus (112) Google Scholar, 15Contreras J.L. Smyth C.A. Bilbao G. Young C.J. Thompson J.A. Eckhoff D.E. Transplantation. 2002; 74: 1252-1259Crossref PubMed Scopus (102) Google Scholar). Thus, MPT is implicated in β-cell death, which is a key factor in the etiology of type 1 diabetes and a potentially important contributor to some forms of type 2 diabetes (16Mandrup-Poulsen T. Diabetes. 2001; 50: 58-63Crossref PubMed Google Scholar). With respect to type 2 diabetes, inhibition of MPT opening with cyclosporin A was found to suppress glucose-induced insulin secretion, suggesting that a basal level of MPT transient opening (flickering) is important for β-cell secretory function (17Dufer M. Krippeit-Drews P. Lembert N. Idahl L.A. Drews G. Mol. Pharmacol. 2001; 60: 873-879PubMed Google Scholar). Previous studies examining MPT in β-cells were performed using intact cell preparations, and the manipulation of the mitochondrial functional state, specifically controlling the levels of MPT effectors, is extremely difficult. A better understanding of MPT in the β-cell requires a more detailed investigation at the mitochondrial level. Permeabilized clonal pancreatic β-cells allow for direct experimental access to mitochondria within the cell and, therefore, provide an appropriate system for detailed investigation of mitochondrial bioenergetics under conditions inducing MPT. The pancreatic mouse β-cell line, MIN6, was the primary model used for this study as it has been shown to retain insulin-secretory responses to glucose and other secretagogues and has been used extensively in studies of β-cell metabolism (18Soejima A. Inoue K. Takai D. Kaneko M. Ishihara H. Oka Y. Hayashi J.I. J. Biol. Chem. 1996; 271: 26194-26199Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 19Minami K. Yano H. Miki T. Nagashima K. Wang C.Z. Tanaka H. Miyazaki J.I. Seino S. Am. J. Physiol. 2000; 279: E773-E781Crossref PubMed Google Scholar, 20Ishihara H. Asano T. Tsukuda K. Katagiri H. Inukai K. Anai M. Kikuchi M. Yazaki Y. Miyazaki J.I. Oka Y. Diabetologia. 1993; 36: 1139-1145Crossref PubMed Scopus (342) Google Scholar). Key findings were verified by using another widely used rat β-cell line, INS-1 (21Poitout V. Olson L.K. Robertson R.P. Diabetes Metab. Rev. 1996; 22: 7-14Google Scholar). We demonstrate that mitochondria from both MIN6 and INS-1 cells exhibited novel MPT characteristics as follows: (i) Ca2+-phosphate-induced MPT is associated with reduced (instead of enhanced) mitochondrial ROS formation; (ii) initiation of MPT with hydroperoxides does not happen in intact β-cell mitochondria but requires exogenous glutathione peroxidase mimetic activity; and (iii) nonclassical MPT, caused by thiol cross-linking, is entirely independent of mitochondrial Ca2+ and causes the interconversion of electrical and chemical components of the mitochondrial protonmotive force. The role of inorganic phosphate (Pi) in MPT in the β-cell is of particular interest, as the stimulation of insulin secretion is associated with a significant drop in the Pi level (“phosphate flush”). Here we demonstrate that inorganic phosphate in the physiological range enhances the effect of Ca2+ on the MPT, while suppressing the effects of thiol oxidants. This supports a potential link between insulin-secretory activity and Ca2+- and oxidant-induced MPT in the β-cell. Reagents—Clonal pancreatic β-cells MIN6 (a gift from Dr. S. Seino, Chiba University, Japan) and INS-1 (a gift from Dr. C. Wollheim, University Medical Center, Geneva, Switzerland, passage number 68) were used in this study. Ca-Green 5N and Amplex Red were purchased from Molecular Probes (Eugene, OR), and all other chemicals were obtained from Sigma. Growth and Permeabilization of Cells—MIN6 cells were cultured in Dulbecco's modified Eagle's medium containing 25 mm glucose and supplemented with 10% fetal bovine serum, 1 mm pyruvate, 100 units/ml penicillin, and 100 μg/ml streptomycin. For INS-1 cells RPMI 1640 medium supplemented with 10% fetal bovine serum, 10 mm HEPES, 2 mm glutamine, 1 mm pyruvate, 0.05 mm β-mercaptoethanol, 100 units/ml penicillin, and 100 μg/ml streptomycin was used. After 4–6 days of growth, with daily medium change, trypsinized cells were washed in Ca2+-free Krebs-Ringer buffer (KRB buffer, 120 mm NaCl, 1.0 mm MgCl2, 24 mm NaHCO3, and 10 mm HEPES, pH 7.3) and permeabilized essentially as described by Civelek et al. (22Civelek V.N. Deeney J.T. Shalosky N.J. Tornheim K. Hansford R.G. Prentki M. Corkey B.E. Biochem. J. 1996; 318: 615-621Crossref PubMed Scopus (78) Google Scholar). Briefly, ∼2 × 107 cells were suspended in 0.7 ml of KRB buffer containing 80 μg/ml saponin. After incubation at room temperature for 5 min, the cells were centrifuged (735 × g for 3 min) at 4 °C and washed in cold KRB buffer. At this point more than a half of the total cellular protein and more than 80% of soluble malate dehydrogenase was found in the supernatant. Finally, the permeabilized cells were suspended in cold 0.25 m sucrose containing 10 mm HEPES, pH 7.3, and stored at 0–4 °C until required. All experiments were performed in the MIN6 line origin, and the main findings were validated by using INS-1 β-cells. Respiration Measurements—Mitochondrial O2 consumption was measured using a Clark-type electrode coupled to an Oxygraph unit (Hansatech, Pentney, UK). Permeabilized cells were suspended at a concentration of 0.6–0.9 mg of protein/ml in incubation medium containing 0.25 m sucrose, 10 mm HEPES, 1 mm MgCl2,20 μm EGTA, 0.1% bovine serum albumin, varying concentrations of Pi, pH 7.3 (adjusted with KOH). Glycerol 3-phosphate (7.5 mm), 10 mm succinate, 5/5 mm glutamate/malate were used as respiratory substrates. Oxygen kinetic traces were treated as described by Estabrook (23Estabrook R.W. Methods Enzymol. 1967; 10: 41-47Crossref Scopus (1896) Google Scholar), and respiration rates were converted into molar oxygen units using O2 solubility in sucrose medium, as reported by Reynafarje et al. (24Reynafarje B. Costa L.E. Lehninger A.L. Anal. Biochem. 1985; 145: 406-418Crossref PubMed Scopus (196) Google Scholar). Mitochondrial Membrane Potential Monitoring—Mitchondrial membrane potential was monitored by observing safranin O fluorescence (25Akerman K.E. Wikstrom M.K. FEBS Lett. 1976; 68: 191-197Crossref PubMed Scopus (671) Google Scholar, 26Vercesi A.E. Bernardes C.F. Hoffmann M.E. Gadelha F.R. Docampo R. J. Biol. Chem. 1991; 266: 14431-14434Abstract Full Text PDF PubMed Google Scholar) in suspensions of permeabilized cells. Measurements were performed using a FluoroCount plate reader (Packard Instrument Co.) at excitation/emission wavelengths of 530/590 nm. A decrease in fluorescence corresponded to an increase in mitochondrial membrane potential. Incubation medium for permeabilized cells was essentially identical to that used for respiratory assays but was further supplemented with 2.5 μm safranin. NAD(P)H Assay and Intramitochondrial pH Measurement—The extent of mitochondrial NAD(P)H reduction in permeabilized cells was estimated by fluorescence at excitation/emission wavelengths of 360/460 nm according to Ref. 27Sekine N. Cirulli V. Regazzi R. Brown L.J. Gine E. Tamarit-Rodriguez J. Girotti M. Marie S. MacDonald M.J. Wollheim C.B. J. Biol. Chem. 1994; 269: 4895-4902Abstract Full Text PDF PubMed Google Scholar. To estimate dynamics of intramitochondrial pH, permeabilized cells were loaded with BCECF-AM essentially as described previously (28Kowaltowski A.J. Cosso R.G. Campos C.B. Fiskum G. J. Biol. Chem. 2002; 277: 42802-42807Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). Epifluorescent imaging of individual BCECF-loaded cells was performed using an Olympus IX70 inverted epifluorescence microscope with a 40× oil immersion objective, in combination with an Ultrapix camera and a PC computer with Merlin imaging software (LSR Inc., UK). BCECF fluorescence from the cell suspension was monitored with a FluoroCount plate reader at excitation/emission wavelengths of 490/530 nm. Fluorometric Determination of Reactive Oxygen Species and Cytochrome c Assay—ROS production was assayed as hydrogen peroxide formation by monitoring fluorescein or Amplex Red oxidation in the presence of horseradish peroxidase (29Cocco T. Di Paola M. Papa S. Lorusso M. Free Radic. Biol. Med. 1999; 27: 51-59Crossref PubMed Scopus (193) Google Scholar, 30Starkov A.A. Polster B.M. Fiskum G. J. Neurochem. 2002; 83: 220-228Crossref PubMed Scopus (208) Google Scholar). Appearance of dichlorofluorescein (DCF) from nonfluorescent reduced form was monitored at excitation/emission wavelengths 485/530 nm. DCF was obtained from the stable compound DCF diacetate by alkaline hydrolysis (31Black M.J. Brandt R.B. Anal. Biochem. 1974; 58: 246-254Crossref PubMed Scopus (110) Google Scholar). For Amplex Red measurement, wavelengths of 550/590 nm were used. The high concentrations (1.55 units/ml) and the low Km value of peroxidase helps to circumvent interference from endogenous hydrogen peroxidemetabolizing enzymes (32Kwong L.K. Sohal R.S. Methods Enzymol. 2002; 349: 341-346Crossref PubMed Scopus (6) Google Scholar). Cytochrome c release from the intermembrane mitochondrial space was estimated by assaying it in the incubation medium, using a sandwich enzyme immunoassay kit from R & D Systems (Minneapolis, MN) (30Starkov A.A. Polster B.M. Fiskum G. J. Neurochem. 2002; 83: 220-228Crossref PubMed Scopus (208) Google Scholar). Insulin Secretion Assay—MIN6 cells cultured in 12-well plates were washed and preincubated for two sequential 30-min periods in a modified glucose-free Krebs-Ringer buffer (KRB buffer, 115 mm NaCl, 5.0 mm KCl, 2.5 mm CaCl2, 1.0 mm MgCl2, 24 mm NaHCO3, and 10 mm HEPES, pH 7.3) with 0.1% bovine serum albumin, followed by incubation for 1 h in the same buffer containing 0 or 16.7 mm glucose in the absence or presence of 5 μm cyclosporin A. Insulin secretion in response to glucose was quantified using a radioimmunoassay (Linco Research, St. Charles, MO) according to the manufacturer's instructions. Statistical Analysis—Data were analyzed using an unpaired two-tailed Student's t test. Statistical significance was assumed at p < 0.05. Calcium Phosphate-induced MPT and Mitochondrial ROS Production—Because experiments in permeabilized β-cells were supplemented with respiratory substrate but not ATP, only mitochondrial Ca2+ stores were available under these experimental conditions. This was verified by using inhibitors of mitochondrial and endoplasmic reticulum Ca2+ uptake as shown in Fig. 1. Glycerol 3-phosphate was used as the main respiratory substrate for two reasons. First, because of the high glycerol-3-phosphate dehydrogenase activity in pancreatic β-cells (27Sekine N. Cirulli V. Regazzi R. Brown L.J. Gine E. Tamarit-Rodriguez J. Girotti M. Marie S. MacDonald M.J. Wollheim C.B. J. Biol. Chem. 1994; 269: 4895-4902Abstract Full Text PDF PubMed Google Scholar), this substrate exhibits the highest respiratory control ratio, and as such, it is possible to observe the full range of mitochondrial functional states. Second, glycerol 3-phosphate is a physiologically important mitochondrial substrate in β-cells where the glycerophosphate shuttle ensures coupling between glycolysis and mitochondrial oxidation (27Sekine N. Cirulli V. Regazzi R. Brown L.J. Gine E. Tamarit-Rodriguez J. Girotti M. Marie S. MacDonald M.J. Wollheim C.B. J. Biol. Chem. 1994; 269: 4895-4902Abstract Full Text PDF PubMed Google Scholar) and takes part in the activation of insulin secretion (33Eto K. Tsubamoto Y. Terauchi Y. Sugiyama T. Kishimoto T. Takahashi N. Yamauchi N. Kubota N. Murayama S. Aizawa T. Akanuma Y. Aizawa S. Kasai H. Yazaki Y. Kadowaki T. Science. 1999; 283: 981-985Crossref PubMed Scopus (396) Google Scholar). MIN6 cells exhibit high activity of glycerol-3-phosphate dehydrogenase similar to that in primary β-cells (34Ishihara H. Nakazaki M. Kanegae Y. Inukai K. Asano T. Katagiri H. Yazaki Y. Kikuchi M. Miyazaki J. Saito I. Oka Y. Diabetes. 1996; 45: 1238-1244Crossref PubMed Scopus (30) Google Scholar). Glycerol-3-phosphate dehydrogenase is activated by divalent cations, including the main MPT-inducer Ca2+ (35Beleznai Z. Szalay L. Jancsik V. Eur. J. Biochem. 1988; 170: 631-636Crossref PubMed Scopus (20) Google Scholar). Therefore, to ensure independence of glycerol-3-phosphate dehydrogenase activity on the experimental variation in Ca2+ concentration, the medium contained a high concentration of glycerol 3-phosphate (7.5 mm) and a physiological level of magnesium (1 mm). The presence of magnesium also maintained optimal mitochondrial coupling and did not qualitatively change mitochondrial responses to MPT inducers. The suitability of permeabilized β-cells for studying MPT is demonstrated by Fig. 2A, showing gradual acceleration of respiration, decreased membrane potential, and exhaustion of Ca2+ loading capacity of MIN6 cell mitochondria upon titration with Ca2+, which are typical characteristics of MPT. At moderate Ca2+ loads (<60–80 μm), MPT was reversible as indicated by the restoration of membrane potential and state 4 respiration rate upon addition of 1 μm cyclosporin A or 1 mm EGTA (data not shown). Ca2+ exerted these effects in the presence of 2.5 mm Pi (Fig. 2A), which was the first MPT co-inducer described (3Zoratti M. Szabo I. Biochim. Biophys. Acta. 1995; 1241: 139-176Crossref PubMed Scopus (2194) Google Scholar). Because of the fundamental importance of Pi in MPT regulation, it is of interest that initiation and cessation of insulin secretion in β-cells is accompanied by the decrease and restoration, respectively, of the cellular Pi concentration (36Giroix M.H. Sener A. Bailbe D. Leclercq-Meyer V. Portha B. Malaisse W.J. Biochem. Med. Metab. Biol. 1993; 50: 301-321Crossref PubMed Scopus (38) Google Scholar, 37Corkey B.E. Deeney J.T. Glennon M.C. Matschinsky F.M. Prentki M. J. Biol. Chem. 1988; 263: 4247-4253Abstract Full Text PDF PubMed Google Scholar, 38Trus M. Warner H. Matschinsky F. Diabetes. 1980; 29: 1-14Crossref PubMed Scopus (53) Google Scholar, 39Bukowiecki L. Trus M. Matschinsky F.M. Freinkel N. Biochim. Biophys. Acta. 1979; 583: 370-377Crossref PubMed Scopus (24) Google Scholar, 40Johnson R.C. Freinkel N. Biochem. Biophys. Res. Commun. 1985; 129: 862-867Crossref PubMed Scopus (4) Google Scholar). This so-called phosphate flush amounts to 40–50% of the initial cytosolic Pi level (2–4 mm) (38Trus M. Warner H. Matschinsky F. Diabetes. 1980; 29: 1-14Crossref PubMed Scopus (53) Google Scholar, 39Bukowiecki L. Trus M. Matschinsky F.M. Freinkel N. Biochim. Biophys. Acta. 1979; 583: 370-377Crossref PubMed Scopus (24) Google Scholar). Therefore, we investigated the concentration range in which Pi regulates MPT in β-cell mitochondria in greater detail. Sustained activation of mitochondrial respiration with Ca2+, reflecting MPT opening at the different Pi levels, is shown in Fig. 2B. Here MPT-stimulated mitochondrial oxygen consumption in β-cells is highly sensitive to Pi concentrations in the 0.5–5 mm range, suggesting that Pi-dependent MPT provides an additional link between insulin-secreting activity and mitochondrial functional state in β-cells. Increased mitochondrial production of reactive oxygen species (ROS) is considered an essential step in the mechanism of Ca2+-phosphate-induced MPT. This was deduced from the stimulation of ROS generation in the liver mitochondria respiratory chain by Ca2+ and Pi (42Kowaltowski A.J. Castilho R.F. Grijalba M.T. Bechara E.J. Vercesi A.E. J. Biol. Chem. 1996; 271: 2929-2934Abstract Full Text PDF PubMed Scopus (172) Google Scholar, 43Kowaltowski A.J. Castilho R.F. Vercesi A.E. FEBS Lett. 1996; 378: 150-152Crossref PubMed Scopus (225) Google Scholar, 44Kowaltowski A.J. Castilho R.F. Vercesi A.E. FEBS Lett. 2001; 495: 12-15Crossref PubMed Scopus (713) Google Scholar). The mitochondrial production of ROS could be of even greater importance in β-cells, which contain only low levels of antioxidant enzymes (45Robertson R.P. Harmon J. Tran P.O. Tanaka Y. Takahashi H. Diabetes. 2003; 52: 581-587Crossref PubMed Scopus (688) Google Scholar, 46Tiedge M. Lortz S. Drinkgern J. Lenzen S. Diabetes. 1997; 46: 1733-1742Crossref PubMed Google Scholar, 47Lenzen S. Drinkgern J. Tiedge M. Free Radic. Biol. Med. 1996; 20: 463-466Crossref PubMed Scopus (932) Google Scholar). Consequently, we monitored the production of H2O2 (estimated by Amplex Red fluorescence) which accompanied Ca2+-phosphate-stimulated MPT (estimated by stimulation of mitochondrial respiration). We found that Ca2+ in the presence of Pi causes MPT opening (Fig. 3B) but inhibited, rather than stimulated, H2O2 production (Fig. 3A). Similar results were observed when ROS production was monitored with another ROS-sensitive probe (DCF) or when the order of the addition of Ca2+ and phosphate were reversed (data not shown). Thus the relationship between Ca2+ load and mitochondrial ROS production in MIN6 β-cells is distinct from that seen in liver (42Kowaltowski A.J. Castilho R.F. Grijalba M.T. Bechara E.J. Vercesi A.E. J. Biol. Chem. 1996; 271: 2929-2934Abstract Full Text PDF PubMed Scopus (172) Google Scholar, 43Kowaltowski A.J. Castilho R.F. Vercesi A.E. FEBS Lett. 1996; 378: 150-152Crossref PubMed Scopus (225) Google Scholar) but resembles the inhibitory effect of Ca2+ on ROS generation reported in brain mitochondria (30Starkov A.A. Polster B.M. Fiskum G. J. Neurochem. 2002; 83: 220-228Crossref PubMed Scopus (208) Google Scholar). This duality of function can arise because of the variety of ways mitochondrial ROS can be induced. Mitochondrial formation of ROS results from a highly reduced state of the respiratory chain, caused by either hyperpolarization of mitochondrial membrane potential (typically complex I) or by substantial inhibition of respiratory flux (complex III) (48Kushnareva Y. Murphy A.N. Andreyev A. Biochem. J. 2002; 368: 545-553Crossref PubMed Scopus (547) Google Scholar, 49Liu Y. Fiskum G. Schubert D. J. Neurochem. 2002; 80: 780-787Crossref PubMed Scopus (974) Google Scholar, 50Votyakova T.V. Reynolds I.J. J. Neurochem. 2001; 79: 266-277Crossref PubMed Scopus (510) Google Scholar, 51Miwa S. Brand M.D. Biochem. Soc. Trans. 2003; 31: 1300-1301Crossref PubMed Google Scholar). It is thought that the most physiologically relevant mitochondrial ROS production is from complex I, in particular driven by reverse electron transfer (51Miwa S. Brand M.D. Biochem. Soc. Trans. 2003; 31: 1300-1301Crossref PubMed Google Scholar). This pathway is very sensitive to membrane potential and, hence, Ca2+-induced mitochondrial depolarization. Therefore, we considered if this mechanism applies to our experimental system. The ability of the respiratory substrate glycerol 3-phosphate to support reverse electron transfer is not well documented. It has been observed in insect flight muscle mitochondria (52Chance B. Hollunger G. J. Biol. Chem. 1961; 236: 1534-1543Abstract Full Text PDF PubMed Google Scholar, 53Miwa S. St Pierre J. Partridge L. Brand M.D. Free Radic. Biol. Med. 2003; 35: 938-948Crossref PubMed Scopus (265) Google Scholar) but could not be detected in mitochondria from mouse brain and kidney (54Kwong L.K. Sohal R.S. Arch. Biochem. Biophys. 1998; 350: 118-126Crossref PubMed Scopus (214) Google Scholar). This discrepancy was attributed to the tissue-specific difference between the electron transfer mechanisms of succinate and glycerol-3-phosphate dehydrogenases (54Kwong L.K. Sohal R.S. Arch. Biochem. Biophys. 1998; 350: 118-126Crossref PubMed Scopus (214) Google Scholar, 55Drahota Z. Chowdhury S.K. Floryk D. Mracek T. Wilhelm J. Rauchova H. Lenaz G. Houstek J. J. Bioenerg. Biomembr. 2002; 34: 105-113Crossref PubMed Scopus (85) Google Scholar). In permeabilized β-cells we found that succinate (classical substrate for reverse electron transfer) and glycerol 3-phosphate cause a similar increase of the NAD(P)H fluorescence, which is negated by the uncoupler FCCP (Fig. 4). This provides evidence for reverse electron transport from glycerol 3-phosphate to complex I, which can support the generation of ROS at this site. Indeed, glycerol 3-phosphate-dependent ROS production is inhibited by rotenone (inhibitor of complex I and reverse electron transfer) to 35 ± 3% (n = 3) of the initial rate (data not shown). This mechanism of ROS formation explains the inhibitory rather than stimulatory action of Ca2+ and Pi on mitochondrial oxygen radicals in MIN6 cells by Ca2+-induced mitochondrial depolarization. In addition, the direct inhibitory action of Ca2+ on the mitochondrial H2O2-producing site was reported recently (56Zoccarato F. Cavallini L. Alexandre A. J. Biol. Chem. 2004; 279: 4166-4174Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). The effect of Ca2+ and Pi on ROS production, supported by NAD-linked respiratory substrates (glutamate/malate), was also investigated. They were shown not to stimulate mitochondrial H2O2 production (data not shown). MPT in the Presence of Hydroperoxides—In liver and heart mitochondria, hydroperoxides and other oxidants serve as potent co-inducers of MPT initiated by Ca2+ (1Gunter T.E. Pfeiffer D.R. Am. J. Physiol. 1990; 258: C755-C786Crossref PubMed Google Scholar, 3Zoratti M. Szabo I. Biochim. Biophys. Acta. 1995; 1241: 139-176Crossref PubMed Scopus (2194) Google Scholar). To test for the occurrence of Ca2+ prooxidant-induced MPT in MIN6 cells, we used tert-butyl and cumene hydroperoxides as models for naturally produced peroxides. Neither compound had a significant effect on membrane potential (Fig. 5), respiration, or Ca2+ retention in mitochondria nor did they affect Ca2+-phosphate-induced mitochondrial permeabilization (data not shown). We attribute this to the fact that the effect of peroxides on MPT is mediated by the glutathione peroxidase/glutathione reductase system, which results in glutathione and NAD(P)H oxidation, an oxidative shift in the mitochondrial redox state and finally oxidation of critical thiol groups governing MPT opening (4Halestrap A.P. McStay G.P. Clarke S.J. Biochimie (Paris). 2002; 84: 153-166Crossref PubMed Scopus (629) Google Scholar, 57Kowaltowski A.J. Ve" @default.
• W2034991285 created "2016-06-24" @default.
• W2034991285 creator A5031528167 @default.
• W2034991285 creator A5034890894 @default.
• W2034991285 creator A5080928485 @default.
• W2034991285 creator A5088312569 @default.
• W2034991285 date "2004-10-01" @default.
• W2034991285 modified "2023-09-30" @default.
• W2034991285 title "The Characterization of Mitochondrial Permeability Transition in Clonal Pancreatic β-Cells" @default.
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