Investigation of the effect of hyperthermic treatment on mitochondrial oxidative phosphorylation system ; Hipertermijos poveikio mitochondrijų oksidacinio fosforilinimo sistemai tyrimas
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Investigation of the effect of hyperthermic treatment on mitochondrial oxidative phosphorylation system ; Hipertermijos poveikio mitochondrijų oksidacinio fosforilinimo sistemai tyrimas

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VYTAUTAS MAGNUS UNIVERSITY Rasa Ž ūkien ė INVESTIGATION OF THE EFFECT OF HYPERTHERMIC TREATMENT ON MITOCHONDRIAL OXIDATIVE PHOSPHORYLATION SYSTEM Summary of Doctoral Dissertation Physical sciences, Biochemistry (04 P) Kaunas, 2008 The research work was carried out at Vytautas Magnus University in 2002 – 2007. Research supervisor: Prof. habil. dr. Vida Mildažien ė (Vytautas Magnus University, Physical Sciences, Biochemistry – 04 P) The Committee of Doctoral Studies: Chairperson of Biochemistry science council: dr. Romualdas Meškys (Biochemijos institutas, fiziniai mokslai, biochemija – 04 P) Members: dr. (hb) Gintautas Saulis (Vytauto Didžiojo universitetas, biomedicinos mokslai, biologija – 01 B) dr. Jurgis Kadziauskas (Vilniaus universitetas, fiziniai mokslai, biochemija – 04 P) dr. (hb) Vytenis Arvydas Skeberdis (Kauno medicinos universitetas, Kardiologijos institutas, fiziniai mokslai, biochemija – 04 P) dr. Ramunė Mork ūnien ė (Kauno medicinos universitetas, fiziniai mokslai, biochemija – 04 P) Oponents: prof. habil. dr. Adolfas Toleikis (Kauno medicinos universitetas, fiziniai mokslai, biochemija – 04 P) prof. dr.(hb) Rimantas Daugelavi čius (Vilniaus universitetas, fiziniai mokslai, biochemija – 04 P) thThe doctoral dissertation will be defended on the 9 of July 2008 at 11 a.m.

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Published 01 January 2008
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VYTAUTAS MAGNUS UNIVERSITY          Rasa ūkienė 
INVESTIGATION OF THE EFFECT OF HYPERTHERMIC TREATMENT ON MITOCHONDRIAL OXIDATIVE PHOSPHORYLATION SYSTEM
Summary of Doctoral Dissertation Physical sciences, Biochemistry (04 P)
Kaunas, 2008
The research work was carried out at Vytautas Magnus University in 2002  2007.   Research supervisor:  Prof. habil. dr. Vida Mildaienė(Vytautas Magnus University, Physical Sciences, Biochemistry  04 P)   The Committee of Doctoral Studies:  Chairperson of Biochemistry science council:  dr.Romualdas Mekys(Biochemijos institutas, fiziniai mokslai, biochemija  04 P)  Members:  dr. (hb)Gintautas Saulis (Vytauto Didiojo universitetas, biomedicinos mokslai, biologija  01 B) dr.Jurgis Kadziauskas(Vilniaus universitetas, fiziniai mokslai, biochemija  04 P) dr. (hb)Vytenis Arvydas Skeberdis(Kauno medicinos universitetas, Kardiologijos institutas, fiziniai mokslai, biochemija  04 P) dr.Ramunė Morkūnienė medicinos universitetas, fiziniai mokslai, (Kauno biochemija  04 P)   Oponents:  prof. habil. dr.Adolfas Toleikis (Kauno medicinos universitetas, fiziniai mokslai, biochemija  04 P) prof. dr.(hb)Rimantas Daugelavičius (Vilniaus universitetas, fiziniai mokslai, biochemija  04 P)  The doctoral dissertation will be defended on the 9thof July 2008 at 11 a.m. in a public meeting of the Doctoral Committee at Vytautas Magnus University, Faculty of Natural Sciences, 101 audience room.  Address: Vileikos 8, LT  44404, Kaunas, Lietuva  The summary of the Doctoral Dissertation (in English) was dispatched on the 9thof June 2008.  The Doctoral Dissertation is deposited at the Library of Vytautas Magnus University.
                             
 
VYTAUTO DIDIOJO UNIVERSITETAS          Rasa ūkienė 
HIPERTERMIJOS POVEIKIO MITOCHONDRIJŲOKSIDACINIO FOSFORILINIMO SISTEMAI TYRIMAS
Daktaro disertacijos santrauka Fiziniai mokslai, biochemija (04 P)
Kaunas, 2008
Disertacija rengta 2002  2007 metais Vytauto Didiojo universitete  Mokslinis vadovas:  prof. habil. dr. Vida Mildaienė (Vytauto Didiojo universitetas, fiziniai mokslai, biochemija  04 P)    Disertacija ginama jungtinėje Vytauto Didiojo universiteto ir Biochemijos instituto Biochemijos mokslo krypties taryboje:  Pirmininkas dr. Romualdas Mekys (Biochemijos institutas, fiziniai mokslai, biochemija  04 P)  Nariai: dr. (hb) Gintautas Saulis (Vytauto Didiojo universitetas, biomedicinos mokslai, biologija  01 B) dr. Jurgis Kadziauskas (Vilniaus universitetas, fiziniai mokslai, biochemija  04 P) dr. (hb) Vytenis Arvydas Skeberdis (Kauno medicinos universitetas, Kardiologijos institutas, fiziniai mokslai, biochemija  04 P) dr. Ramunė Morkūnienė (Kauno medicinos universitetas, fiziniai mokslai, biochemija  04 P)   Oponentai:  prof. habil. dr. Adolfas Toleikis (Kauno medicinos universitetas, fiziniai mokslai, biochemija  04 P) prof. dr.(hb) Rimantas Daugelavičius (Vilniaus universitetas, fiziniai mokslai, biochemija  04 P)  Disertacija bus ginama vieame Biochemijos mokslo krypties tarybos posėdyje 2008 m. liepos mėn. 9 d. 11 val. Vytauto Didiojo universiteto Gamtos mokslų fakulteto 101 auditorijoje.  Adresas: Vileikos g. 8 - 101, LT  44404, Kaunas, Lietuva  Disertacijos santrauka isiuntinėta 2008 m. birelio mėn. 9 d. Disertacijągalima periūrėti Vytauto Didiojo universiteto bibliotekoje
I. INTRODUCTION  Although remarkable progress has been made in cancer therapy, many cancers, particularly solid cancers, are still untreatable by conventional therapies such as radiation, immunotherapy, surgery or chemotherapy. This implies the need to explore more effective ways for cancer treatment including the combinatory approaches. Hyperthermia is recognized for its synergistic action with the aforementioned therapeutic modalities and may be considered the fifth modality of treatment. The phenomenon of curing tumour after longer fever periods was described in ancient medical literature. Since 18th scientists recognized, that tumour tissues are c. sensitive to temporary heating (44onot cause injury in normal tissue.C) that did Hyperthermia is defined as a therapy in which tumour temperature is raised to values between 41°C and 45°C by external means. It can be applied locally/regionally or to the whole body depending from the stage of the cancer patients. For decades hyperthermia has been an area of laboratory investigation with moments of enthusiasm and disappointment, but now there is renewed interest. Its effectiveness as a cancer treatment has been demonstrated by many trials that have highlighted that hyperthermia improves cancer treatment results while decreasing the side effects of conventional therapies. The protocols for use of hyperthermic treatment are under continuous development that requires basement on the knowledge of molecular and cellular processes. Heat transfer and distribution inside a target tissue are irregular and exhibit spatial and temporal instabilities, there always exists a risk that certain areas of the target will survive the thermotherapeutic intervention to cause the recurrence of disease (Gellermann, 2005). It is important to establish thresholds for thermal damage in human tissues that vary among tissue species as well as among normal and diseased tissues (Park, 2005). The elucidation of the molecular mechanism of the cell response to moderate heating is of importance for understanding the events that occur in the cell upon use of heating for therapeutic purpose or during illnesses that are associated with fever. The death or survival of different cells upon hyperthermia is determined by the molecular mechanisms that are not yet well established. The events following the exposure of cell to heat involve complex interplay of multiple pathways and factors operating at different regulatory levels (inducing changes in metabolic activities, signal transduction, and gene expression). Despite physiological importance, the information about the molecular events induced by fever in various types of cells is scarce. Supra-physiological hyperthermia (usually in the range 42-45oC) is clinically applied for cancer treatment, although quite little is known about differences in cellular response to heating under fever compared to more severe hyperthermic conditions. Multiple mechanisms may be responsible for the higher sensitivity of tumour cells to temperature treatment as compared to normal cells. Also, it is still not clear why cells of some tumours are more sensitive to heating than other tumours, as well as why some normal types of cells are more sensitive (e.g. neurons, cardiomyocytes) that other normal cells. Many reports have demonstrated mitochondria to be major targets of hyperthermic stress inside eukaryotic cells. Mitochondria are highly dynamic organelles that frequently move inside cells and exhibit morphological as well as biochemical changes during physiological cell metabolism and stress responses (Jakobs, 2006).
 
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These organelles may capture a central directory in the cells fate when affected by physical, biochemical or environmental stress factors. However, the response of mitochondria from different normal tissues to heating was investigated only by a few research groups. It was shown that regardless to the mitochondrial origin, hyperthermia induces uncoupling of oxidative phosphorylation (increase in state 2 respiration) and decrease in the efficiency of phosphorylation (drop in the ADP/O ratio), but the effect of hyperthermia on state 3 respiration to certain extent is dependent on mitochondrial origin tissue. It was demonstrated that mitochondria are important in the development of heat stress induced apoptosis in certain tumour cell lines (Ko et al., 2000) as well as in cardiomyocytes (Qian et al., 2004). Experimental evidences indicated possible importance of Ca2+ overload and permeability transition pore (PTP) in the hyperthermic response, since heat stress induced apoptosis in tumour cells via mitochondrial damage related to depolarisation of mitochondrial membrane and release of cytochrome c. These pro-apoptotic events were stopped by cyclosporin A treatment, indicating possible importance of mitochondrial permeability transition (MPT) in the hyperthermic response. In this context, the synergy between effects of Ca2+overload and hyperthermia in mitochondria from normal tissues also become important. Therefore we have focused the study on more detailed analysis of the functional response of mitochondria from normal tissues to hyperthermia. Until now there is a lack of data on interdependence between mitochondrial response to Ca2+ overload and to hyperthermic treatment. Cardiomyocytes are the most heating-sensitive normal cells after neurons. The comparative assessment of hyperthermia induced changes in activity of heart and liver mitochondria is important for better understanding of factors that may determine different response of healthy tissues to fever or their different survival upon heating applied for killing the neibouring tumours, e.g. during whole body hyperthermia or termoablation surgery of solid hepatic tumours. Scientific novelty.We have demonstrated mutual negative synergy of the effects of hyperthermia and Ca2+ overload on respiration of heart mitochondria in state 2 and state 3. Modular kinetic analysis for the first time was applied to evaluate effects of hypertermia on oxidative phosphorylation in rat heart and liver mitochondria. It allowed to reveale that febrile temperature activates the respiratory and phosphorylation functions of heart mitochondria, but only one degree above the fever temperature (42oC) causes severe impairment of mitochondrial ability to maintain membrane potential and to synthesize ATP. We demonstrated that changes in mitochondrial functions induced by mild hyperthermia (42 ºC) are reversible but more severe hyperthermia (45 ºC) causes partially irreversible uncoupling and inhibition of mitochondrial respiration in state 3. We confirm data that hyperthermia remarkably (3,6-2,1 fold) activates ROS generation in heart mitochondria and present new observation that maximal increase in rate of H2O2peroxidation is observed in the fever temperature and lipid  production range. We show that the response of liver mitochondria to hyperthermia is to certain extent dependent on gender and temperature. For the first time the comparative DSC analysis of male rat liver and heart mitochondrial components phase transitions was performed and specific differences have been revealed. Our results indicate that male hepatocytes are more resistant to mild hyperthermia (up to 45oC), while female hepatocytes better retain viability under conditions of severe hyperthermia (50-60oC).    
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II. OBJECTIVE AND TASKS  The objective of the studywas to investigate and to compare the effects of mild (fever) and severe hyperthermia on functional properties of oxidative phosphorylation system in normal tissue mitochondria.  The specific tasks of the studywere following: 1. to compare the effect of moderate heating (42 and 45oC) on the rate of respiration and NAD(P)H fluorescence in isolated rat heart mitochondria incubated at physiological (1 µM) and supra-physiological (10 µM) Ca2+ concentrations; 2. to perform modular kinetic analysis of the effects on physiologically relevant fluxes of respiration, phosphorylation and proton leak in heart mitochondria induced by the febrile temperature (40 ºC) and the higher temperatures commonly used for hyperthermic treatment (42 and 45 ºC); 3. to assess reversibility of hyperthermia induced changes on heart mitochondrial respiration; 4. hyperthermia induced changes in the rate of ROS production and theto assess amount of lipid peroxidation marker MDA in rat heart mitochondria; 5. to perform the comparative DSC analysis of hyperthermia induced phase transitions of the components of male rat liver and heart mitochondria. 6. to determine the gender dependency of the effect of hyperthermia on oxidative phosphorylation and lipid peroxidation in rat liver mitochondria as well as on viability of isolated hepatocytes  III. ABBREVIATIONS:  AM  assay medium; BSA Bovine Serum Albumin; DMSO  Dimethylsulfoxide; DNP  Dinitrophenol; DSC  differential scanning calorimetry; DCF-DA  dichlorfluorescein diacetate; EGTA  Ethylene Glycol bis (β-Aminoethyl Ether)-N,N,N,N-Tetraacetic Acid,JO Flux through the respiratory module of oxidative  phosphorylation;JP flux through the phosphorylation module of oxidative phosphorylation;JL  proton leak flux oxidative phosphorylation; MDA  Malondialdehyde; MKA  Modular Kinetic Analysis; NAD  Nicotinamide Adenine Dinucleotide; NADH  Nicotinamide Adenine Dinucleotide, reduced form; NTA  Nitrilotriacetic Acid; PTP  Permeability Transition Pore; RCI Respiratory Control Index; ROS  reactive oxygen species, TPP+  Tetraphenyl-phosphonium ions; Tris  Tris-(hydroxymethyl)-Amino Methane; V2 Rate of mitochondrial respiration in state 2; V3 Rate of mitochondrial respiration in state 2;ΔΨ  mitochondrial membrane potential
 
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IV.MATERIALS AND METHODS  Isolation of mitochondria.Mitochondria were isolated from hearts and livers of Wistar rats (250-300 g) as described previously [Scholteet al., 1973], using isolation medium containing 160 mM KCl, 20 mM Tris,10 mM NaCl, 5 mM EGTA, 1 mg/ml BSA (pH 7.7). After isolation mitochondria were suspended in the suspension buffer (SB) containing 180 mM KCl, 20 mM Tris, 3 mM EGTA (pH 7.3) and stored on ice. The concentration of mitochondrial preparation was approximately 50 mg of mitochondrial protein per ml of stored suspension. Protein content was determined by modified buret method [Gornalet al., 1949].  Determination of dissolved molecular oxygen concentration. The concentration of molecular oxygen dissolved in the assay medium at different temperatures (37-47±0.1ºC) was determined polarographically using glucose oxidase catalyzed reaction between D-glucose and O2while the pH of the medium was strictly controlled at each temperature (pH 7.2). The molar ratio coefficient of the reaction between D-glucose and O2known concentration of dissolved oxygen at 37ºC.was defined in the medium with  Measurement of mitochondrial respiration andΔψ. Mitochondrial respiration and membrane potentialΔψ at different temperatures (37-47±0.1oC) was measured simultaneously in a closed, stirred and thermostated 1.5 ml glass vessel equipped with Clark-type oxygen electrode and TPP+(tetraphenylphosphonium)-sensitive electrode. Δψwas calculated from the distribution of TPP+using the Nernst equation and a TPP+ binding correction factor of 0.16μprotein. The assay medium (AM) containedl/mg  30 mM Tris, 5 mM KH2PO4, 110 mM KCl, 10 mM NaCl, 1 mM EGTA, 5 mM NTA, 1 mM dithiothreitol, 50 mM creatine, 5.17 mM MgCl2 (1 mM free Mg2+), and 0.875 mM CaCl2 (1 µM free Ca2+), pH 7.2. Excess of creatine kinase (0.1 mg/ml) was added to maintain steady state respiration. The experiments were performed using 1 mM pyruvate plus 1 mM malate as oxidizable substrate. Mitochondria (0.3 mg protein/ml) were incubated in the assay medium with the respiratory substrate (state 2) for 3 min at 37, 40, 42 or 45oC before the state 3 respiration was initiated by addition of 1 mM ATP. The rate of mitochondrial respiration in state 2 (V2), state 3 (V3) and the respiratory control index (RCI = V3/V2) are defined according to the conventional terminology. Mitochondrial swelling at different temperatures was determined spectrophotometrically in the same medium.  Measurement of NAD(P)H fluorescence.NAD(P)H fluorescence was measured in the same vessel as mitochondrial respiration. Experimental setup for NAD(P)H measurements consists of a Lumatec SUV-DC light source with a lens Computar (f=25 mm, 1:1.8), a liquid wave guide and an intensified CCD-camera CPL-22B by Canadian Photonics Labs. The excitation and emission wavelengths were 347±4 nm and 467±3 nm (after passing the band pass filters), respectively.  Modular kinetic analysis. Mitochondrial oxidative phosphorylation system was conceptually divided into three functional modules, interacting through the linking intermediate membrane potential (Δψ): (i) the respiratory module that producesΔψ (comprised of the substrate carriers, dehydrogenases, respiratory chain complexes), (ii)
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the phosphorylation module that consumesΔψ(comprised of the ATP synthase, adenine nucleotide translocator and phosphate carrier), and (iii) the proton leak module that also consumesΔψ (comprisedof the passive permeability of the mitochondrial inner membrane to protons, and any cation cycling reactions). The dependence of the flux through the respiratory module (JO) onΔψ was determined by gradually inhibiting the flux through the phosphorylation module with the inhibitor of adenine nucleotide translocator carboxyatractyloside (0-1.75 nmol/mg protein) and this way modulating the value ofΔψ, and concomitantly measuring the respiration fluxJOandΔψcorresponding to each new steady state. The dependence of the flux through the phosphorylation module (JP) onΔψ was determined by titrating the flux through the respiratory module with the respiratory chain inhibitor rotenone (0-0.06 nmol/mg protein), and concomitantly measuring the respiration flux (JO) andΔψ to each new corresponding steady state. The phosphorylation fluxJPcalculated by subtracting proton leak fluxwas JL from the respiration fluxJO at the same value ofΔψ (i.e.JP =JO JL). The dependence of the flux through the proton leak module (JL) onΔψ determined by was titrating the flux through the respiratory module with rotenone (0-0.08 nmol/mg protein) when the flux through the phosphorylation module was fully blocked by addition of excess oligomycin (2 µg/mg mitochondrial protein). The experiments of modular kinetic analysis were paired experiments: the measurements were performed at 37 and 40ºC, or at 37 and 42 ºC using the same mitochondrial preparation.  Evaluation of lipid peroxidation by the assessment of malondialdehyde (MDA) concentration.Mitochondria (5 mg of mitochondrial protein) were incubated 3 min in 1 ml AM supplied with substrate (pyruvate + malate, 0.1 or 0.5 mM each) in a thermostated poliarographic chamber (2 metabolic state). After incubation the medium with mitochondria was collected, transfered to the glass vial with 1 ml ice-cold 0.5 % TBA solution in 20% trichloracetic acid and vortexed. The vial was sealed and boiled in water bath for 30 min. The solution was cooled down and centrifuged for 10 min at 10000 x g, 4°C. The absorbance of supernatant was measured at 532 nm and 600 nm. MDA concentration was calculated according to the equation cMDA=(A532-A600)/0.156·L, εMDA=1.56×105M-1.cm-1width of cuvette in cm. Blank sample was used for, L  the zero calibration.  Assessment of intramitochondrial H2O2. The mitochondrial suspension was loaded with H2DCF-DA dissolved in DMSO at a final concentration of 5μM (30 min on ice, dark). After incubation, mitochondrial suspension was diluted 1:50 with SB and centrifuged 10 min at 6800 x g, 4°C. Mitochondria were resuspended in SB and the protein concentration was determined (approx. 50 mg of protein per ml of medium). Parallely, the protein concentration was determined in non-stained mitochondria. The fluorescence was measured by thermostated fluorometer Tecan GENios ProTM (Tecan Group Ltd., Menedorf, Switzerland) with XFluorTM (Tecan Group Ltd., software Menedorf, Switzerland), excitation at 485±20 nm, emission at 535±25 nm. Measurement was performed in the fluorometric semi-micro cuvette filled with 1.5 ml AM supplied with substrate (pyruvate + malate, 0.1 M each). Measurement was started immediately after mitochondrial suspension containing 0.5 mg of protein was added to the cuvette, closed and mixed. H2O2generation was registered for 3 min, then 100 µM H2O2added for positive control. Negative control was performed with non-stainedwas
 
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mitochondria. H2oxidation rate was calculated in relative fluorescence units perDCF minute per mg of protein. Each measurement was performed at 37, 40, 42 and 45 °C and repeated 3 times with 3 independent mitochondrial preparations.  Mitochondrial transition measurements by differential scanning calorimetry (DSC).Phase transitions of mitochondria isolated from heart and liver were performed by MicroCal VP-DSC calorimeter (MicroCal, LLC, Northampton, USA) equipped with 0.5 ml sample and reference cells. Prior to measurements the baseline (medium against medium) was registered. The medium used was SB (see Isolation of mitochondria). Mitochondrial suspension was diluted with vacuum degassed SB and scanned from 30 up to 75 ºC against SB, scan rate 1ºC/min. Date were registered and analysed with specialized software Origin 7.0 (MicroCal, LLC, Northampton, JAV), calculating heat capacity (Cp).  Data presentation and statistical analysis. The results are presented as means ±   SEM (n=  3).Statistical significance of the temperature effects was evaluated using Students t-test. The differences were assumed to be statistically significant when p<0.05.  
 
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V. RESULTS AND DISCUSSIONS  1. Investigation of the effect of hyperthermia on oxidative phosphorylation in isolated heart mitochondria  Cardiac dysfunction is supposed to be the main cause of heat related death. Substantial injury of cardiomyocytes by heat treatment is associated with destructive changes in mitochondrial morphology and function that are followed by impairment of cellular energy metabolism leading to severe cell injury and death. The experimental evidences for Ca2+ oxidative stress and facilitated PTP opening in cardiac overload, mitochondria isolated from heated cardiomyocytes were obtained. However, the question about possible contribution of changes in cytoplasmic and mitochondrial Ca2+ concentration to hyperthermic injury of cell still remains the matter of debate. We aimed to investigate how the response of mitochondria to heat treatment is modulated by extramitochondrial concentration of Ca2+ions.  1.1. on state 2 and state 3 respiration rate withThe effect of hyperthermia different substrates and Ca2+concentrations  We compared temperature effects (37, 42 and 45oC) on mitochondrial respiration at 1 µM and 10 µM Ca2+ in the medium. The first Ca concentrations2+ concentration may be considered as physiological or optimal for stimulation of respiration with most substrates, whereas the second concentration is supra-physiologi l, relevant to C2+ ca a overload condition and pronounced inhibition of mitochondrial respiration. The results (Fig. 1) indicate that the effect of temperature on oxygen consumption depends on the metabolic state of mitochondria and, to some extent  on the substrate for respiration. Increase of temperature above the physiological value (from 37 to 42 and 45oC) significantly increased state 2 respiration both at 1 µM and 10 µM Ca2+, indicating strong uncoupling. This effect was more pronounced at the higher temperature range  increase of temperature by 5 degrees (from 37 to 42oC) induced rise in state 2 respiration rate by 74% at 1 µM Ca2+, whereas further elevation of temperature by 3 degrees (from 42 to 45oC) resulted in almost 3 times higher respiration with pyruvate + malate. The corresponding numbers for glutamate + malate were 23% (from 37 to 42oC) and 57% (from 42 to 45oC) activation of state 2 respiration. Thus, the respiration at 45oC was not several-fold higher than at 37oC, as it was for pyruvate + malate (Fig 1A), i.e., temperature increased state 2 respiration with glutamate + malate much less than with pyruvate + malate. This difference implies that uncoupling is not the only temperature effect on oxidation of different substrates. To elucidate possible effects on the respiratory chain in mitochondria oxidizing pyruvate +malate and glutamate + malate, we determined temperature induced changes in the uncoupled respiration with both substrates. The results showed that the rise of temperature from 37 to 42oC did not affect the rate of pyruvate + malate oxidation in mitochondria uncoupled by DNP - it was 403±32 and 404±40 (1 µM Ca2+) and 261±14 and 271±33 nmol O/min per mg protein (10 µM Ca2+) at 37 and 42oC, respectively. The uncoupled respiration with glutamate + malate was increased with the same rise in temperature from 383±33 to 468±26 (1 µM Ca2+and from 255±29 to 302±6 nmol) O/min per mg protein (10 µM Ca2+). However, the uncoupled respiration with both substrates substantially decreased at 45oC: the rate with pyruvate + malate was only
 
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