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• W2149092212 abstract "In brown adipose tissue (BAT) adrenaline promotes a rise of the cytosolic Ca2+ concentration from 0.05 up to 0.70 μm. It is not known how the rise of Ca2+ concentration activates BAT thermogenesis. In this report we compared the effects of Ca2+ in BAT and liver mitochondria. Using electron microscopy and immunolabeling we identified a sarco/endoplasmic reticulum (ER) Ca2+-ATPase bound to the inner membrane of BAT mitochondria. A Ca2+-dependent ATPase activity was detected in BAT mitochondria when the respiratory substrates malate and pyruvate were included in the medium. ATP and Ca2+ enhanced the amount of heat produced by BAT mitochondria during respiration. The Ca2+ concentration needed for half-maximal activation of the ATPase activity and rate of heat production were the same and varied between 0.1 and 0.2 μm. Heat production was partially inhibited by the proton ionophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone and abolished by thapsigargin, a specific ER Ca2+-ATPase inhibitor, and by both rotenone and KCN, two substances that inhibit the electron transfer trough the mitochondrial cytochrome chain. In liver mitochondria Ca2+ did not stimulate the ATPase activity nor increase the rate of heat production. Thapsigargin had no effect on liver mitochondria. In conclusion, this is the first report of a Ca2+-ATPase in mitochondria that is BAT-specific and can generate heat in the presence of Ca2+ concentrations similar to those noted in the cell during adrenergic stimulation. In brown adipose tissue (BAT) adrenaline promotes a rise of the cytosolic Ca2+ concentration from 0.05 up to 0.70 μm. It is not known how the rise of Ca2+ concentration activates BAT thermogenesis. In this report we compared the effects of Ca2+ in BAT and liver mitochondria. Using electron microscopy and immunolabeling we identified a sarco/endoplasmic reticulum (ER) Ca2+-ATPase bound to the inner membrane of BAT mitochondria. A Ca2+-dependent ATPase activity was detected in BAT mitochondria when the respiratory substrates malate and pyruvate were included in the medium. ATP and Ca2+ enhanced the amount of heat produced by BAT mitochondria during respiration. The Ca2+ concentration needed for half-maximal activation of the ATPase activity and rate of heat production were the same and varied between 0.1 and 0.2 μm. Heat production was partially inhibited by the proton ionophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone and abolished by thapsigargin, a specific ER Ca2+-ATPase inhibitor, and by both rotenone and KCN, two substances that inhibit the electron transfer trough the mitochondrial cytochrome chain. In liver mitochondria Ca2+ did not stimulate the ATPase activity nor increase the rate of heat production. Thapsigargin had no effect on liver mitochondria. In conclusion, this is the first report of a Ca2+-ATPase in mitochondria that is BAT-specific and can generate heat in the presence of Ca2+ concentrations similar to those noted in the cell during adrenergic stimulation. BAT 3The abbreviations used are: BAT, brown adipose tissue; MOPS, 4-morpholinepropanesulfonic acid; ER, endoplasmic reticulum; SERCA, sarco/endoplasmic reticulum Ca2+-ATPase; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; UCP1, uncoupling protein 1.3The abbreviations used are: BAT, brown adipose tissue; MOPS, 4-morpholinepropanesulfonic acid; ER, endoplasmic reticulum; SERCA, sarco/endoplasmic reticulum Ca2+-ATPase; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; UCP1, uncoupling protein 1. is capable of rapidly converting fat stores to heat and has been used as a model system for the understanding of nonshivering heat production and mechanisms of energy wasting to control obesity (1Nichols D. Locke R.M. Physiol. Rev. 1984; 64: 1-64Crossref PubMed Scopus (1338) Google Scholar, 2Janský L. Physiol. Rev. 1995; 75: 237-259Crossref PubMed Scopus (121) Google Scholar, 3Skulachev V.P. Biochim. Biophys. Acta. 1998; 1363: 100-124Crossref PubMed Scopus (812) Google Scholar, 4Boss O. Muzzin P. Giacobino J.P. Eur. J. Endocrinol. 1998; 130: 1-9Crossref Scopus (227) Google Scholar, 5Lowell B.B. Splegelman B.M. Nature. 2000; 404: 652-660Crossref PubMed Scopus (1294) Google Scholar, 6Nicholls D.G. Rial E. J. Bioenerg. Biomembr. 1999; 31: 399-406Crossref PubMed Scopus (163) Google Scholar, 7Ribeiro M.O. Carvalho S.D. Schultz J.J. Chiellini G. Scanlan T.S. Bianco A.C. Brent G.A. J. Clin. Invest. 2001; 108: 97-105Crossref PubMed Scopus (216) Google Scholar, 8Bachman E.S. Dhillon H. Zhang C. Cinti S. Bianco A.C. Kobilka B.K. Lowell B. Science. 2002; 297: 843-845Crossref PubMed Scopus (625) Google Scholar, 9Cannon B. Nedergaard J. Physiol. Rev. 2004; 84: 277-35964Crossref PubMed Scopus (4456) Google Scholar). The signal that activates the rate of heat production in BAT cells is the rise of the cytosolic Ca2+ concentration from a basal level of 0.05 μm up to the range of 0.2–0.7 μm. This is promoted by α1- and β3-adrenergic receptors located in the cell membrane. Activation of α1-adrenoreceptors leads to the release of Ca2+ from intracellular stores into the cytosol, whereas β3-adrenergic receptors promote the release of free fatty acids and increase the effect of Ca2+ release induced by α1-adrenoreceptors (10Leaver E.V. Pappone P. Am. J. Physiol. 2002; 282: C1016-C1024Crossref PubMed Scopus (35) Google Scholar, 11Breitwieser G.E. Am. J. Physiol. 2002; 282: C980-C1981Crossref Scopus (4) Google Scholar). At present we do not know how the rise of the cytosolic Ca2+ concentration activates the rate of heat production in BAT cells. In a previous report (12de Meis L. J. Biol. Chem. 2003; 278: 41856-41861Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar) we identified a sarco/ER Ca2+ ATPase (SERCA 1) in vesicles derived from BAT ER. In this report we show that the rate of heat produced by BAT mitochondria is enhanced when the Ca2+ concentration in the medium is raised to a level similar to that observed in BAT cells during adrenergic stimulation. This effect is not observed in liver mitochondria, a tissue that is not specialized in heat production. Isolation of Mitochondria from Rat BAT and Liver—Adult male Wistar rats were killed by decapitation. Mitochondria were isolated as previously described (13da-Siva W.S. Gómez-Puyou A. Gómez-Puyou M.T. Moreno-Sanchez R. De Felice F.G. de Méis L. Oliveira M.F. Galina A. J. Biol. Chem. 2004; 279: 39846-39855Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar). Briefly, interscapular BAT and liver were removed and homogenized in a mixture containing 0.32 m sucrose, 1 mm EDTA, 10 mm MOPS/Tris buffer, pH 7.4, and 0.2 mg ml of fatty acid-free bovine serum albumin. The homogenate was centrifuged at 1,330 × g for 30 min. The supernatant was carefully removed and centrifuged at 21,200 × g for 10 min. The pellet was re-suspended in 15% Percoll. A discontinuous density gradient was prepared manually by layering 3-ml fractions of the re-suspended pellet on two preformed layers consisting of 3.5 ml of 23% Percoll above 3.5 ml of 40% Percoll. Tubes were centrifuged for 5 min at 37,700 × g. The material equilibrating near the interface between 23 and 40% Percoll layer was removed and gently diluted with the isolation buffer described above. The pellet was re-suspended in the isolation buffer. After centrifugation at 6,900 × g for 10 min, the supernatant was decanted and the pellet re-suspended in the same buffer using a fine Teflon pestle. Protein was determined by the Folin-Lowry method using serum albumin as standard (14Lowry O.H. Rosenbrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). Vesicles Derived from BAT Endoplasmic Reticulum—These were prepared as previously described (12de Meis L. J. Biol. Chem. 2003; 278: 41856-41861Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Transmission Electron Microscopy and Immunolabeling—BAT was chopped to 1-mm3 pieces and fixed in 0.7% glutaraldehyde (v/v), 0.1% picric acid, 1% sucrose, 2% paraformaldehyde, and 5 mm CaCl2 in 0.1 m cacodylate buffer (pH 7.2), dehydrated in ethanol and embedded in Unicryl (Ted Pella, Redding, CA). Ultrathin sections were quenched in 50 mm NH4Cl for 30 min and incubated in the presence of monoclonal anti-Serca-1 antibodies (clone IIH11) from Affinity BioReagents, Inc., Brazil. After several washes in phosphate-buffered saline/1% albumin, sections were incubated in the presence of 10 nm of gold-labeled goat anti-mouse IgG (BB International, UK), washed, and observed in a JEOL 1210 electron microscope. This method allows an adequate diffusion of the antibody but decreases the preservation of the material; therefore, it decreases the quality of the image. Isolated mitochondria were centrifuged at 150 × g for 15 min. The pellet was chopped into 1-mm3 pieces and treated as described above. Gel Electrophoresis and Western Blot—Samples were separated in a 7.5% polyacrylamide gel according to Laemmli (15Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206281) Google Scholar). Electrotransfer of protein from the gel to polyvinylidene difluoride membrane was performed for 15 min at 250 mA per gel in 25 mm Tris, 192 mm glycine, and 20% methanol using a Mini Trans-Blot cell from Bio-Rad. Membranes were blocked with 3% nonfat dry milk in Tris-buffered saline with 0.1% Tween 20 for 1 h at room temperature. Membranes were then washed and incubated for 1 h with monoclonal antibody anti-SERCA 1 at room temperature. The membranes were washed, and blots were revealed using an ECL detection kit from Amersham Biosciences (16Reis M. Farage M. de Meis L. Mol. Membr. Biol. 2002; 19: 301-310Crossref PubMed Scopus (30) Google Scholar). Monoclonal antibody for SERCA 1 (clone VE121G9) was obtained from Affinity BioReagents, Inc. Determination of ΔΨ—Mitochondrial membrane potential was measured by using the fluorescence signal of the cationic dye safranine O, which is accumulated and quenched inside energized mitochondria (17Wieckowski M.R. Wojczak L. FEBS Lett. 1998; 423: 339-342Crossref PubMed Scopus (108) Google Scholar). Fluorescence was detected with an excitation wavelength of 495 nm (slit 5 nm) and an emission wavelength of 586 nm. Other details were as described previously (13da-Siva W.S. Gómez-Puyou A. Gómez-Puyou M.T. Moreno-Sanchez R. De Felice F.G. de Méis L. Oliveira M.F. Galina A. J. Biol. Chem. 2004; 279: 39846-39855Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar). ATPase Activity—This was assayed as previously described (18Grubmeyer C. Penefsky H.S. J. Biol. Chem. 1981; 256: 3718-3727Abstract Full Text PDF PubMed Google Scholar) using [γ-32P]ATP. Measurements were performed at 35 °C, and the reaction was arrested with trichloroacetic acid, final concentration 5% (w/v). In all experiments the amount of ATP cleaved never exceeded 20% of the total amount of ATP added in the assay medium. Heat of Reaction—This was measured using an OMEGA Isothermal Titration Calorimeter from MicroCal, Inc. (Northampton, MA). The calorimeter sample cell (1.5 ml) was filled with reaction medium, and the reference cell was filled with Milli-Q water. After equilibration at 35 °C, the reaction was started by injecting mitochondria into the sample cell, and the heat change was recorded for 30 min. The volume of mitochondria suspension injected in the sample cell varied between 0.03 and 0.05 ml. The heat change measured during the initial 5 min after mitochondria injection was discarded to avoid artifacts such as heat derived from the dilution of the mitochondria suspension in the reaction medium and binding of ions to mitochondria. The duration of these events is <1 min (12de Meis L. J. Biol. Chem. 2003; 278: 41856-41861Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 19de Meis L. J. Biol. Chem. 2001; 276: 25078-25087Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Negative heat values indicate that the reaction is exothermic, and positive values indicate that it is endothermic. The enthalpy of buffer protonation (ΔHp) was measured at 35 °C by measuring the heat released following the addition of known amounts of HCl to the assay medium, and the value found was –3.8 kcal/mol. The concentration of the different magnesium complexes and ionic species of ATP, ADP, Pi, and Ca2+ were calculated as previously described (20Schwartzenbach G. Senn H. Anderegg G. Helv. Chim. Acta. 1957; 40: 1886-1900Crossref Scopus (216) Google Scholar, 21Fabiato A. Fabiato F. J. Physiol. (Paris). 1979; 75: 463-505PubMed Google Scholar, 22Sorenson M.M. Coelho H.S.L. Reuben J.P. J. Membr. Biol. 1986; 90: 219-230Crossref PubMed Scopus (72) Google Scholar), and from these values the fraction of ATP cleaved that generates free protons at pH 7.0, was estimated to be <30%. Thus, the heat derived from buffer protonation during ATP cleaved was ∼1 mcal/μmol of ATP cleaved. Ca2+ Uptake—This was measured by the filtration method (23Chiesi M. Inesi G. J. Biol. Chem. 1979; 254: 10370-10377Abstract Full Text PDF PubMed Google Scholar). For 45Ca uptake, trace amounts of 45Ca were included in the assay medium. The reaction was arrested by filtering samples of the assay medium through Millipore filters. After filtration, the filters were washed five times with 5 ml of 3 mm La(NO3)3, and the radioactivity remaining on the filters was counted using a liquid scintillation counter. Oxygen Uptake Measurements—Oxygen consumption was measured at 35 °C in a Strathkelvin oxymeter as previously described (13da-Siva W.S. Gómez-Puyou A. Gómez-Puyou M.T. Moreno-Sanchez R. De Felice F.G. de Méis L. Oliveira M.F. Galina A. J. Biol. Chem. 2004; 279: 39846-39855Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar). Determination of Mitochondrial Hydrogen Peroxide Generation—Mitochondrial H2O2 production was assessed by the scopoletin oxidation method (13da-Siva W.S. Gómez-Puyou A. Gómez-Puyou M.T. Moreno-Sanchez R. De Felice F.G. de Méis L. Oliveira M.F. Galina A. J. Biol. Chem. 2004; 279: 39846-39855Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 24Boveris A. Martino E. Stoppani A.O. Anal. Biochem. 1977; 80: 145-158Crossref PubMed Scopus (105) Google Scholar). Experimental Conditions—These were similar to those found in the cell, i.e., pH 7.0–7.4, 2 mm Pi, 4 mm MgCl2, and 100 mm KCl and different EGTA and CaCl2 concentrations. The free Ca2+ concentration in the medium was calculated as described previously (20Schwartzenbach G. Senn H. Anderegg G. Helv. Chim. Acta. 1957; 40: 1886-1900Crossref Scopus (216) Google Scholar, 21Fabiato A. Fabiato F. J. Physiol. (Paris). 1979; 75: 463-505PubMed Google Scholar, 22Sorenson M.M. Coelho H.S.L. Reuben J.P. J. Membr. Biol. 1986; 90: 219-230Crossref PubMed Scopus (72) Google Scholar). In most experiments ATP was included in the medium. In addition to being utilized by mitochondrial ATPases, ATP impair the uncoupling protein 1 (UCP1) found in BAT mitochondria. In all experiments we compared the effects obtained with BAT and liver mitochondria. The aim was to evaluate whether or not the effects observed were specific for BAT mitochondria. All experiments were performed at 35 °C. Transmission Electron Microscopy and Immunolabeling—In a previous work (12de Meis L. J. Biol. Chem. 2003; 278: 41856-41861Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar) we found that vesicles derived from BAT ER retain a membrane-bound Ca2+-ATPase that in Western blot reacted with antibodies for SERCA 1. This work began with an attempt to visualize in electron microscopy the BAT SERCA 1 attached to the membrane of the ER (25de Meis L. Oliveira G.M. Arruda A.P. Santos R. Costa R.M. Benchimol M. IUBMB Life. 2005; 57: 337-345Crossref PubMed Scopus (22) Google Scholar). When ultrathin sections were analyzed, to our surprise, we found that, in addition to the ER, the anti-SERCA 1 antibodies also reacted with BAT mitochondrial cristae. This could be visualized using 10 nm gold-labeled anti-mouse IgG. Labeling could be clearly identified in sections of whole tissue (Fig. 1) and in isolated mitochondria, where controls with (Fig. 2, c and d) and without anti-SERCA (Fig. 2, a and b) were compared. The presence of SERCA in BAT mitochondria was confirmed in different preparations examined.FIGURE 2Isolated mitochondria. Immunolabeling using anti-SERCA 1 antibody (c and d) and the corresponding controls (a and b) with gold-labeled goat anti-mouse IgG but without anti-SERCA 1. Intense labeling was observed on the mitochondria cristae (c and arrowheads in d). No labeling was detected in the control without anti-SERCA 1. Bars: 500 nm in a and c and 50 nm in b and d.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Western blot analysis of BAT mitochondria preparations revealed a faint band with the same mobility than SERCA 1 (Fig. 3). This band was detected in most, but not all BAT mitochondria preparations tested. In control experiments, the band was not detected when antibodies for SERCA 2 were used (data not shown). We were not able to identify a SERCA in liver mitochondria, suggesting that the presence of Ca2+-ATPase in mitochondria is a special feature of BAT cells. These findings led us to compare the effect of Ca2+ in mitochondria isolated from rats BAT and liver. Membrane Potential (ΔΨ)—In agreement with the bibliography (13da-Siva W.S. Gómez-Puyou A. Gómez-Puyou M.T. Moreno-Sanchez R. De Felice F.G. de Méis L. Oliveira M.F. Galina A. J. Biol. Chem. 2004; 279: 39846-39855Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar) we found that a mixture of pyruvate and malate (Fig. 4A) and ATP (Fig. 4B) promoted the formation of a membrane potential in liver mitochondria. The gradient formed by ATP was enhanced by the subsequent addition of pyruvate and malate (Fig. 4C). The liver mitochondrial ΔΨ was collapsed by Ca2+ (2 μm) and FCCP (1 μm). Contrasting with liver, BAT mitochondria were unable to form a measurable ΔΨ when ATP, pyruvate, and malate were added to the medium (Fig. 4D). These data show that the proton permeability of liver and BAT mitochondrial membrane are different. The Effect of Ca2+ on Mitochondrial ATPase Activity—In the absence of respiratory substrates, both liver and mitochondria catalyzed the hydrolysis of ATP, and the addition of 2 μm Ca2+ promoted a small increase of the ATPase activity in both mitochondrial preparations (Table 1). For liver mitochondria the addition of respiratory substrate to the medium did not lead to a significant change of the ATPase activity measured in the presence and absence of Ca2+ (Table 1). However, in BAT mitochondria, the addition of pyruvate and malate promoted a 2-fold increase of the Mg2+-dependent and a 10-fold increase of the Ca2+-dependent ATPase activities (Fig. 5 and Table 1). The BAT ATPase activity measured in the presence of respiratory substrates varied significantly among the different mitochondria preparations tested (see ±S.E. in Table 1), however, in all of them there was a significant increment of the ATPase activity when Ca2+ was added to the medium. Despite the Ca2+-ATPase activity enhancement, very little Ca2+ was accumulated by BAT mitochondria, no more than 3–5 nmol/mg after 20-min incubation at 35 °C (data not shown).TABLE 1ATPase activity and heat production by BAT and liver mitochondriaAdditionsTissueATPase activityHeat productionMg2+Mg2+ + Ca2+Ca2+-dependentMg2+Mg2+ + Ca2+Ca2+-dependentμmol Pi/mg 20 minmcal/mg 20 minATPBAT0.79 ± 0.11 (10)0.83 ± 0.10 (10)0.11 ± 0.04 (10)-18 (2)-19 (2)-1 (2)Pyruvate + malate-95 ± 12 (4)-161 ± 12 (4)-71 ± 16 (4)ATP + pyruvate + malate1.69 ± 0.37 (9)2.83 ± 0.47 (9)1.11 ± 0.42 (9)-119 ± 20 (16)-295 ± 38 (16)-176 ± 32 (16)ATPLiver1.14 ± 0.08 (24)1.43 ± 0.11 (24)0.30 ± 0.04 (24)-47 (2)-74 (2)-27 (2)Pyruvate + malate-44 (2)-27 (2)ATP + pyruvate + malate1.36 ± 0.20 (7)1.54 ± 0.18 (7)0.22 ± 0.06 (7)-42 ± 6 (6)-21 ± 2 (6) Open table in a new tab Heat Production—A small amount of heat was produced when either BAT or liver mitochondria were incubated with ATP in the absence of respiratory substrate (Fig. 6 and Table 1). The heat produced by BAT mitochondria increased when pyruvate and malate were included in the assay medium (Table 1). A surprising new finding was that Ca2+ enhanced the rate of heat produced by BAT mitochondria with pyruvate and malate. The activation of heat production by Ca2+ was further enhanced when ATP was included in the media containing pyruvate and malate (Table 1 and Fig. 6). The increment promoted by ATP and Ca2+ exceeded the sum of the heat produced by the single addition of either ATP or respiratory substrates, indicating that, in the presence of pyruvate and malate, ATP magnifies the thermal effect of Ca2+. The amount of heat produced by liver mitochondria was smaller than that measured with BAT mitochondria in all the conditions tested in Table 1, i.e. only ATP, only respiratory substrates and a mixture of both ATP and respiratory substrates. In media containing ATP and no respiratory substrates, Ca2+ promoted a small increase in the rate of heat production. However, different from BAT, when respiratory substrates were used, Ca2+ decreased the rate of heat production in liver mitochondria, and ATP did not modify the effect of Ca2+ (Table 1). Ca2+ Dependence—In three experiments the Ca2+ concentration needed for half-maximal activation of both Ca2+-dependent ATPase activity and Ca2+-dependent heat production by BAT mitochondria was found to vary between 0.1 and 0.2 μm. This was measured using media containing ATP, pyruvate, and malate. When extrapolated to living cells, the data of Fig. 7 indicate that the concentration of Ca2+ needed to enhance heat production of isolated BAT mitochondria is in the same range as the cytosolic Ca2+ concentration measured in BAT cells during adrenergic stimulation (10Leaver E.V. Pappone P. Am. J. Physiol. 2002; 282: C1016-C1024Crossref PubMed Scopus (35) Google Scholar, 11Breitwieser G.E. Am. J. Physiol. 2002; 282: C980-C1981Crossref Scopus (4) Google Scholar). Oxygen Consumption—In the absence of ATP, BAT mitochondria consumed oxygen at a fast rate when pyruvate and malate were added to the medium, indicating a lack of respiratory control. The addition of either ADP (Fig. 8A) or Ca2+ (Fig. 8B and Table 2) did not alter the rate of oxygen consumption. In the presence of ATP, however (Fig. 8C), the rate of oxygen consumption elicited by the respiratory substrates decreased, indicating that ATP restored the respiratory control of BAT mitochondria. The lack of respiratory control of BAT mitochondria, and its restoration by ATP, has already been described, and it is attributed to the binding of ATP to UCP1 (6Nicholls D.G. Rial E. J. Bioenerg. Biomembr. 1999; 31: 399-406Crossref PubMed Scopus (163) Google Scholar, 28Klingenberg M. Biochemistry. 1988; 27: 781-791Crossref PubMed Scopus (71) Google Scholar, 29Klingenberg M. Huang S.-G. Biochim. Biophys. Acta. 1999; 1415: 271-296Crossref PubMed Scopus (313) Google Scholar, 30Nicholls D.G. Biochem. Soc. Trans. 2001; 29: 751-755Crossref PubMed Google Scholar, 31Jaburek M. Garlid K.D. J. Biol. Chem. 2003; 278: 25825-25831Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 32Mitchell P. Moyle J. Eur. J. Biochem. 1969; 7: 471-484Crossref PubMed Scopus (400) Google Scholar, 33Nicholls D.G. Biochim. Biophys. Acta. 2005; 1710: 63-66Crossref PubMed Scopus (18) Google Scholar). The effect of ATP in Fig. 8C contrasts with the experiment of Fig. 4D where ATP was not able to restore the ΔΨ measured with safranine. The mitochondria protonmotive force (Δp), as defined by Mitchell (34Andrienko T. Kuznetsov A.V. Kaambre T. Usson Y. Orosco A. Appaix F. Tiivel T. Sikk P. Vendelin M. Margreiter R. Saks V.A. J. Exp. Biol. 2003; 206: 2059-2072Crossref PubMed Scopus (71) Google Scholar, 35Shkryl V.M. Shirokova N. J. Biol. Chem. 2006; 281: 1547-1554Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar), involves two different parameters, an electrochemical and a pH difference between the two sides of the membrane: Δp =ΔΨ+ 2.2(RT/F) ΔpH. Therefore, in the experimental conditions shown in Fig. 8C, BAT mitochondria were able to form a ΔpH but not a ΔΨ.TABLE 2Oxygen consumption and heat production by BAT and liver mitochondriaAdditionsHeat releasedO2 consumedRatiomcal/mg 20 minμmol/mg 20 minkcal/mol O2BAT mitochondria Mg2+-95 ± 12 (4)1.76 ± 0.17 (7)-54 Mg2+ + Ca2+-161 ± 12 (4)1.79 ± 0.19 (5)-90 ATP + Mg2+-119 ± 20 (16)0.39 ± 0.05 (8)-305 ATP + Mg2+ + Ca2+-295 ± 38 (16)2.22 ± 0.34 (8)-133Liver mitochondria EGTA + Mg2+-440.27 ± 0.03 (4)-163 Mg2+ + Ca2+-270.10 ± 0.02 (4)-270 ATP + Mg2+-42 ± 6 (6)0.51 ± 0.03 (6)-82 ATP + Mg2+ + Ca2+-21 ± 2 (6)0.20 ± 0.02 (5)-105 Open table in a new tab In Fig. 8C, the rate of oxygen consumption decreased with ATP, but the subsequent addition of Ca2+ sharply accelerated the rate of both oxygen utilization and heat released (Tables 1 and 2). A different pattern was observed with liver mitochondria (Fig. 8D and Table 2). The addition of pyruvate and malate elicited the consumption of oxygen (state 2), a small amount of ADP (50 μm) increased the rate of oxygen utilization (state 3), and at prolonged incubation intervals the rate decreased to a value similar to that observed before the addition of ADP (state 4). Different from BAT, in liver mitochondria ATP accelerated and Ca2+ decreased the rate of oxygen consumption (Fig. 8, compare C, E, and F). The heat generated during respiration depends, at least, on two different parameters, (i) the rate of oxygen consumption and (ii) the amount of heat produced during the utilization of each oxygen molecule (ratio of kilocalories/moles of O2 in Table 2). In BAT mitochondria, the effect of ATP varied depending on the addition of Ca2+. In the absence of Ca2+, ATP slowed down the rate of O2 consumption induced by pyruvate and malate but increased the heat/O2 ratio. This indicates that most of the energy derived from each oxygen molecule consumed was converted into heat. When Ca2+ was included, ATP accelerated the rate of O2 utilization, but from the parcel of energy released, only a fraction was converted in heat and the rest was converted in another form of energy not detected by the methods used. Effect of FCCP—The aim of these experiments was to verify if the effect of Ca2+ and ATP on heat formation were related to a formation of ΔpH in BAT mitochondria. The proton ionophore FCCP promoted a 50% decrease of the heat produced, but the activation by Ca2+ was not modified and the total amount of heat released was still larger than that of liver mitochondria (Fig. 9). The degree of inhibition did not vary when the FCCP concentration was raised from 1 up to 5 μm. This suggests that the gradient was not an absolute requirement for heat production as in the case of ATP synthesis from ADP and Pi, but it enhanced the effects of ATP and Ca2+. In liver mitochondria FCCP did not inhibit, but on the contrary, promoted a small increase of heat production (Fig. 9C). Inhibition of Heat Production by Drugs—Thapsigargin is a specific inhibitor of the various SERCA isoforms. The BAT ATPase activity measured in the presence of Mg2+ was not modified by thapsigargin. However, both the Ca2+-dependent ATPase activity and the heat produced by BAT mitochondria in the presence of ATP, pyruvate, and malate were inhibited by thapsigargin (Figs. 10 and 11A and Table 3). The concentration of thapsigargin needed for half-maximal inhibition of BAT mitochondria was found to vary between 1 and 2 μm. This is two to three orders of magnitude higher than the concentration needed to inhibit the SERCA isoforms found in vesicles derived from the ER of BAT and skeletal muscle (12de Meis L. J. Biol. Chem. 2003; 278: 41856-41861Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Contrasting with BAT, the small amount of heat produced by liver mitochondria was not impaired by thapsigargin (Fig. 11B and Table 3).FIGURE 11Effect of thapsigargin on the rate of heat production by BAT (A) and liver mitochondria (B). In A the assay medium composition was 50 mm MOPS/Tris buffer, pH 7.4, 4 mm MgCl2, 100 mm KCl, 2 mm Pi, 1 mm ATP, 1 mm pyruvate, 1 mm malate, 0.1 mm EGTA, and 0.1 mm CaCl2 (free Ca2+ = 3.9 μm). The reaction was started by the addition of 50 μg/ml BAT without thapsigargin (○) and 2 μm (•), 4 μm (▵), or 10 μm thapsigargin (▴). In B the assay medium was the same as in A, but the EGTA concentration was raised to 10 mm and no CaCl2 was added to the medium. The reaction was started by 50 μg/ml liver mitochondria without thapsigargin (○) and with 5 μm thapsigargin (▴).View Large Image Figure ViewerDownload Hi-res image Download (PPT)TABLE 3Inhibition of heat release by thapsigarginMitochondriaMg2+Mg2++Ca2+Ca2+-dependentmcal/mg 20 minBAT-154 ± 63 (4)-312 ± 50 (7)-174 ± 52 (4)BAT + 5 μm-11 ± 11 (4)-16 ± 7 (6)-15 ± 13 (4)thapsigargrinLiver-35 ± 3 (3)-14 ± 2 (3)Liver + 5 μm-39 ± 3 (3)-10 ± 4 (3)thapsigargrin Open table in a new tab Rotenone and KCN inhibit the electron flow trough the mitochondrial cytochrome chain. Both drugs strongly inhibited the heat produced by BAT mitochondria (Fig. 12), indicating that the energy-transducing system responsible for heat production depends on the electrons transfer trough the mitochondrial cytochromes chain. In addition to heat production, rotenone promoted a 60% inhibition of the mitochondria ATPase activity (average of two experiments, data not shown). Reactive Oxygen Species and Endoplasmic Reticulum Ca2+-ATPase Contamination—BAT mitochondria did not generate a measurable formation of reactive oxygen species in media containing ATP, pyruvate, malate, and Ca2+ (data not shown). This was tested in three different BAT mitochondrial preparations. The Ca2+-ATPase activity measured in the BAT mitochondrial fraction was not due to a contamination with vesicles derived from the ER because: (i) in a previous report (12de Meis L. J. Biol. Chem. 2003; 278: 41856-41861Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar) it was shown that the thapsigargin concentration needed for half-maximal inhibition of the ER Ca2+-ATPase was 1 nm, whereas for BAT mitochondria, half-maximal inhibition was only attained with 1–2 μm thapsigargin (Fig. 10A), i.e. a three orders of magnitude higher concentration than that needed for the ER; (ii) the Ca2+-ATPase activity, Ca2+ transport, and heat production by vesicles derived from BAT ER were not inhibited by FCCP, rotenone, and KCN as observed for BAT mitochondria (data not shown). Several reports (34Andrienko T. Kuznetsov A.V. Kaambre T. Usson Y. Orosco A. Appaix F. Tiivel T. Sikk P. Vendelin M. Margreiter R. Saks V.A. J. Exp. Biol. 2003; 206: 2059-2072Crossref PubMed Scopus (71) Google Scholar, 35Shkryl V.M. Shirokova N. J. Biol. Chem. 2006; 281: 1547-1554Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar) have described a close association between the ER and mitochondria and that Ca2+ can be transferred from the ER to the mitochondria matrix. However, as far as we know, there are no reports describing a Ca2+-ATPase located within the mitochondria. The data presented indicate that the Ca2+ released in the cytosol of adipocytes during adrenergic stimulation interact directly with the mitochondria activating the rate of heat production. The main findings that correlate the Ca2+-ATPase visualized in Figs. 1 and 2 with the enhancement of heat production are (i) a Ca2+-dependent ATPase activity was detected in BAT mitochondria when respiration was activated with pyruvate and malate (Fig. 5); (ii) the Ca2+ concentration needed for half-maximal activation of both the Ca2+-dependent ATPase activity and Ca2+-dependent heat production were the same (Fig. 7); and (iii) both activities were inhibited by thapsigargin (Figs. 10 and 11). We do not know why the concentration of thapsigargin needed to inhibit the mitochondrial Ca2+-ATPase is much higher than that needed to inhibit the various SERCA isoforms. Thapsigargin is a highly hydrophobic substance. One possibility that may account for this discrepancy is that the Ca2+-ATPase of the ER is exposed in the outer surface of vesicles (25de Meis L. Oliveira G.M. Arruda A.P. Santos R. Costa R.M. Benchimol M. IUBMB Life. 2005; 57: 337-345Crossref PubMed Scopus (22) Google Scholar, 36Inesi G. Asai I. Arch. Biochem. Biophys. 1968; 126: 469-474Crossref PubMed Scopus (48) Google Scholar, and 37Inesi G. Annu. Rev. Physiol. 1985; 47: 573-601Crossref PubMed Google Scholar) and is readily accessible to the thapsigargin in solution, whereas in BAT the Ca2+-ATPase is buried inside the mitochondria. Thus, a large amount of thapsigargin can be trapped by the extensive mitochondrial membrane surface and a high concentration of the drug would be needed to reach the Ca2+-ATPase located in the mitochondria inner space. In liver and Trypanosome brucei mitochondria, high concentrations of thapsigargin (15–20 μm) was found to promote Ca2+ release and collapse of the membrane potential. In concentrations lower than 10 μm, thapsigargin had no effect on both types of mitochondria (38Vercesi A.E. Moreno S.N. Bernardes C.F. Meinicke A.R. Fernandes E.C. Docampo R. J. Biol. Chem. 1993; 268: 8564-8568Abstract Full Text PDF PubMed Google Scholar). During the past decade (1Nichols D. Locke R.M. Physiol. Rev. 1984; 64: 1-64Crossref PubMed Scopus (1338) Google Scholar, 2Janský L. Physiol. Rev. 1995; 75: 237-259Crossref PubMed Scopus (121) Google Scholar, 9Cannon B. Nedergaard J. Physiol. Rev. 2004; 84: 277-35964Crossref PubMed Scopus (4456) Google Scholar), heat production by BAT has been correlated with thermogenin or uncoupling protein 1 (UCP1). This protein is inserted in the mitochondria membrane, and when activated, it would act as an H+ pore. During proton leakage osmotic energy would be converted into heat and the decrease of the gradient promoted by the leakage would accelerate the various reactions involved in O2 consumption with more heat production. The leakage of H+ through the UCP1 is impaired by ATP and activated by fatty acids (9Cannon B. Nedergaard J. Physiol. Rev. 2004; 84: 277-35964Crossref PubMed Scopus (4456) Google Scholar, 26Rial E. Poustie A. Nicholls D.G. Eur. J. Biochem. 1983; 137: 197-203Crossref PubMed Scopus (147) Google Scholar, 27LaNoue K. Strzeleski T. Strzelecka D. Koch C. J. Biol. Chem. 1986; 261: 296-305Abstract Full Text PDF Google Scholar, 28Klingenberg M. Biochemistry. 1988; 27: 781-791Crossref PubMed Scopus (71) Google Scholar, 29Klingenberg M. Huang S.-G. Biochim. Biophys. Acta. 1999; 1415: 271-296Crossref PubMed Scopus (313) Google Scholar, 30Nicholls D.G. Biochem. Soc. Trans. 2001; 29: 751-755Crossref PubMed Google Scholar). In conditions of maximal heat production shown in Table 1 and Fig. 6, UCP1 was probably inactive, because ATP was included in the medium to maximize the rate of heat production elicited by Ca2+ (Table 1). Furthermore, fatty acids were not added to the assay medium, and the solutions used to isolate mitochondria contained an excess of fatty acid-free albumin to avoid fatty acid contamination. Recent studies (39Echtay K.S. Roussel D. St-Pierre J. Jekabsons M.B. Cadenas S. Stuart J.A. Harper J.A. Roebuck S.J. Morrison A. Pickering S. Clapham J.C. Brand M.D. Nature. 2002; 415: 96-99Crossref PubMed Scopus (1135) Google Scholar, 40Murphy M.P. Echtay K.S. Blaikie F.H. Asin-Cayuela J. Cocheme H.M. Green K. Buckingham J.A. Taylor E.R. Hurrell F. Hughes G. Miwa S. Cooper C.E. Svistunenko D.A. Smith R.A. Brand M.D. J. Biol. Chem. 2003; 278: 48534-48540Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar) indicate that the various UCP isoforms are involved in the regulation of the membrane potential and prevention of reactive oxygen species formation and not with heat production. The experiments described in this report suggest that in rat BAT mitochondria the presence of an H+ gradient is not an absolute requirement for heat production, because a large amount of heat could be detected even after the addition of the proton ionophore FCCP (Fig. 9). Finally, on the basis of the data presented, we hypothesize that BAT mitochondria would be able to synthesize ATP during the electron flux through the cytochromes without the need to form a ΔΨ or ΔpH, and the ATP synthesized would then be cleaved by the Ca2+-ATPase before leaving the mitochondria. Thus, heat would be derived from the continuous synthesis and hydrolysis of ATP in the mitochondria matrix. We are grateful to Valdecir A. Suzano and Antônio Carlos Miranda for technical assistance." @default.
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• W2149092212 title "Identification of a Ca2+-ATPase in Brown Adipose Tissue Mitochondria" @default.
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