Hypothyroid Phenotype Is Contributed by Mitochondrial Complex I Inactivation Due to Translocated Neuronal Nitric-oxide Synthase
2005; Elsevier BV; Volume: 281; Issue: 8 Linguagem: Inglês
10.1074/jbc.m512080200
ISSN1083-351X
AutoresMaría Clara Franco, Valeria G. Antico Arciuch, Jorge G. Peralta, Soledad Galli, Damián M. Levisman, Lidia M. López, Leonardo Romorini, Juan José Poderoso, Marı́a C. Carreras,
Tópico(s)Mitochondrial Function and Pathology
ResumoAlthough transcriptional effects of thyroid hormones have substantial influence on oxidative metabolism, how thyroid sets basal metabolic rate remains obscure. Compartmental localization of nitric-oxide synthases is important for nitric oxide signaling. We therefore examined liver neuronal nitric-oxide synthase-α (nNOS) subcellular distribution as a putative mechanism for thyroid effects on rat metabolic rate. At low 3,3′,5-triiodo-l-thyronine levels, nNOS mRNA increased by 3-fold, protein expression by one-fold, and nNOS was selectively translocated to mitochondria without changes in other isoforms. In contrast, under thyroid hormone administration, mRNA level did not change and nNOS remained predominantly localized in cytosol. In hypothyroidism, nNOS translocation resulted in enhanced mitochondrial nitric-oxide synthase activity with low O2 uptake. In this context, NO utilization increased active O2 species and peroxynitrite yields and tyrosine nitration of complex I proteins that reduced complex activity. Hypothyroidism was also associated to high phospho-p38 mitogen-activated protein kinase and decreased phospho-extracellular signal-regulated kinase 1/2 and cyclin D1 levels. Similarly to thyroid hormones, but without changing thyroid status, nitric-oxide synthase inhibitor Nω-nitro-l-arginine methyl ester increased basal metabolic rate, prevented mitochondrial nitration and complex I derangement, and turned mitogen-activated protein kinase signaling and cyclin D1 expression back to control pattern. We surmise that nNOS spatial confinement in mitochondria is a significant downstream effector of thyroid hormone and hypothyroid phenotype. Although transcriptional effects of thyroid hormones have substantial influence on oxidative metabolism, how thyroid sets basal metabolic rate remains obscure. Compartmental localization of nitric-oxide synthases is important for nitric oxide signaling. We therefore examined liver neuronal nitric-oxide synthase-α (nNOS) subcellular distribution as a putative mechanism for thyroid effects on rat metabolic rate. At low 3,3′,5-triiodo-l-thyronine levels, nNOS mRNA increased by 3-fold, protein expression by one-fold, and nNOS was selectively translocated to mitochondria without changes in other isoforms. In contrast, under thyroid hormone administration, mRNA level did not change and nNOS remained predominantly localized in cytosol. In hypothyroidism, nNOS translocation resulted in enhanced mitochondrial nitric-oxide synthase activity with low O2 uptake. In this context, NO utilization increased active O2 species and peroxynitrite yields and tyrosine nitration of complex I proteins that reduced complex activity. Hypothyroidism was also associated to high phospho-p38 mitogen-activated protein kinase and decreased phospho-extracellular signal-regulated kinase 1/2 and cyclin D1 levels. Similarly to thyroid hormones, but without changing thyroid status, nitric-oxide synthase inhibitor Nω-nitro-l-arginine methyl ester increased basal metabolic rate, prevented mitochondrial nitration and complex I derangement, and turned mitogen-activated protein kinase signaling and cyclin D1 expression back to control pattern. We surmise that nNOS spatial confinement in mitochondria is a significant downstream effector of thyroid hormone and hypothyroid phenotype. Hypothyroidism is a prevalent disorder associated to low oxygen utilization and low tissue proliferation rate (1Moro L. Marra E. Capuano F. Greco M. Endocrinology. 2004; 145: 5121-5128Crossref PubMed Scopus (24) Google Scholar). In addition to non-genomic effects (2Scanlan T.S. Suchland K.L. Hart M.E. Chiellini G. Huang Y. Kruzich P.J. Frascarelli S. 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In the last decade, the effects of nitric oxide (NO) 2The abbreviations used are: NO, nitric oxide; nNOS, eNOS, iNOS, and mtNOS, neuronal, endothelial, inducible, and mitochondrial nitric-oxide synthase; BMR, basal metabolic rate; T3, 3,3′,5-triiodo-l-thyronine; Mn-SOD, manganese superoxide dismutase; CAT, catalase; GPX, glutathione peroxidase; l-NAME, Nω-nitro-l-arginine methyl esther; Hsp, heat shock protein; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; l-NMMA, l-NG-methyl-l-arginine. expanded from the vascular system to the intracellular milieu. In this context, subcellular localization of nitric oxide-synthases (NOS) with effector molecules is an important regulatory mechanism for NO signaling (6Barouch L.A. Harrison R.W. Skaf M.W. Rosas G.O. Cappola T.P. Kobeissi Z.A. Hobai I.A. Lemmon C.A. Burnett A.L. O'Rourke B. Rodriguez E.R. Huang P.L. Lima J.A. Berkowitz D.E. Hare J.M. 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Poderoso J.J. Am. J. Physiol. 2001; 281: H2282-H2288PubMed Google Scholar), release of cytochrome c (11Ghafourifar P. Schenk U. Klein S.D. Richter C. J. Biol. Chem. 1999; 274: 31185-31188Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar), mitochondrial protein nitration (12Elfering S.L. Haynes V.L. Traaseth N.J. Ettl A. Giulivi C. Am. J. Physiol. 2004; 286: H22-H29Crossref PubMed Scopus (96) Google Scholar), liver and brain development (13Carreras M.C. Converso D.P. Lorenti A.S. Barbich M. Levisman D.M. Jaitovich A. Antico Arciuch V.G. Galli S. Poderoso J.J. Hepatology. 2004; 40: 157-166Crossref PubMed Scopus (56) Google Scholar, 14Riobo N.A. Melani M. Sanjuan N. Fiszman M.L. Gravielle M.C. Carreras M.C. Cadenas E. Poderoso J.J. J. Biol. Chem. 2002; 277: 42447-42455Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar), adaptation to cold environment (15Peralta J.G. Finocchietto P.V. Converso D. Schopfer F. Carreras M.C. Poderoso J.J. Am. J. Physiol. 2003; 284: H2375-H2383PubMed Google Scholar), and hypoxia (16Valdez L.B. Zaobornyj T. Alvarez S. Bustamante J. Costa L.E. Boveris A. Mol. Aspects Med. 2004; 25: 49-59Crossref PubMed Scopus (50) Google Scholar). Under physiological O2 levels and in vivo respiratory dynamics, NO inhibits mitochondrial state 3 and 4 O2 uptake (17Brookes P.S. Kraus D.W. Shiva S. Doeller J.E. Barone M.C. Patel R.P. Lancaster Jr., J.R. Darley-Usmar V. J. Biol. Chem. 2003; 278: 31603-31609Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). At 10–20 nm, matrix NO tonically modulates O2 uptake by reversible inhibition of cytochrome oxidase through heme iron nitrosylation at a-a3 subunit (18Poderoso J.J. Carreras M.C. Lisdero C. Riobo N. Schopfer F. Boveris A. Arch. Biochem. Biophys. 1996; 328: 85-92Crossref PubMed Scopus (676) Google Scholar) and promotes the formation of superoxide (O2·¯) and hydrogen peroxide (H2O2) (18Poderoso J.J. Carreras M.C. Lisdero C. Riobo N. Schopfer F. Boveris A. Arch. Biochem. Biophys. 1996; 328: 85-92Crossref PubMed Scopus (676) Google Scholar, 19Poderoso J.J. Lisdero C. Schopfer F. Riobo N. Carreras M.C. Cadenas E. Boveris A. J. Biol. Chem. 1999; 274: 37709-37716Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). At complexes I and II-III, mitochondrial production of O2 species depends on autooxidation of intermediary ubisemiquinone during electron transfer from reduced ubiquinol pools to cytochrome c1, with formation of O2·¯. This reaction is considerably amplified by NO inhibition of cytochrome oxidase or cytochrome b-c1 segment that augments the reduction on the side of substrates (18Poderoso J.J. Carreras M.C. Lisdero C. Riobo N. Schopfer F. Boveris A. Arch. Biochem. Biophys. 1996; 328: 85-92Crossref PubMed Scopus (676) Google Scholar, 19Poderoso J.J. Lisdero C. Schopfer F. Riobo N. Carreras M.C. Cadenas E. Boveris A. J. Biol. Chem. 1999; 274: 37709-37716Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). In mitochondria, O2·¯ is either dismutated to H2O2 by manganese superoxide dismutase (Mn-SOD) or reacts with NO to form peroxynitrite (ONOO–). Considering that, variations in matrix NO differently change the proportion of these active species (19Poderoso J.J. Lisdero C. Schopfer F. Riobo N. Carreras M.C. Cadenas E. Boveris A. J. Biol. Chem. 1999; 274: 37709-37716Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar); the aim of this work was to search for nNOS traffic to mitochondria and its consequences in O2 uptake, mitochondrial oxidant yield, and cell responses (13Carreras M.C. Converso D.P. Lorenti A.S. Barbich M. Levisman D.M. Jaitovich A. Antico Arciuch V.G. Galli S. Poderoso J.J. Hepatology. 2004; 40: 157-166Crossref PubMed Scopus (56) Google Scholar) as experimentally related to thyroid status. Materials—3,3′,5-triiodo-l-thyronine (T3), NG-methyl l-arginine (l-NMMA), Nω-nitro-l-arginine methyl ester (l-NAME), and LR-White acrylic resin were from Sigma-Aldrich. 2′,7′-dichlorofluorescein diacetate (DCFH-DA), hydroethidine (HE), 4-amino-5-methylamino-2′, 7′-difluorescein diacetate (DAF-FM), MitoTracker Red 580, SYBR Green, and 39-kDa subunit of Complex I and VIc subunit of Complex IV monoclonal antibodies were from Invitrogen-Molecular Probes. Leukemia virus reverse transcriptase (MMLV-RT) and Taq polymerase were from Promega Corp. (Madison, WI). MAPK antibodies were from Cell Signaling Technology (Beverly, MA). Monoclonal nitrotyrosine antibody was provided by Prof. Alvaro Estévez, University of Alabama at Birmingham. Monoclonal nNOS and polyclonal eNOS antibodies were from BD Transduction Laboratories. Polyclonal nNOS, iNOS, Hsp90, and cyclin D1 antibodies were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Methimazole was provided by Laboratorios Gador (Buenos Aires, Argentina). Animals and Treatments—Male Wistar rats (250–300 g) were housed in a temperature-controlled room with food and water ad libitum; National Institutes of Health criteria for animal research were followed after approval by the Ethics Committee of the University Hospital. Rats were divided into five groups (n = 12/group): hypothyroid, (0.02% methimazole (w/v) in drinking water for 28 days); hypothyroid+T3, (after 25 days of methimazole treatment received 15 μg of T3/kg of body weight for 3 days by intraperitoneal injection); hypothyroid+l-NAME, (0.75 mg/ml l-NAME in drinking water in the last 21 days of methimazole treatment); hyperthyroid, (intraperitoneal injection of 60 μgofT3/kg of body weight for 3 days); and control group. Blood samples were collected at the time the animals were killed for estimation of thyrotropin (TSH) level by radioimmunoassay (20Bianchi M.S. Catalano P.N. Bonaventura M.M. Silveyra P. Bettler B. Libertun C. Lux-Lantos V.A. Neuroendocrinology. 2004; 80: 129-142Crossref PubMed Scopus (17) Google Scholar). Basal Metabolic Rate—BMR was measured at 22 °C in a non-recirculating open flow system after 30 min of equilibration with an O2-CO2 analyzer in standard temperature and pressure dry conditions (15Peralta J.G. Finocchietto P.V. Converso D. Schopfer F. Carreras M.C. Poderoso J.J. Am. J. Physiol. 2003; 284: H2375-H2383PubMed Google Scholar). Isolation and Purification of Liver Mitochondria—The livers were excised in ice-cold homogenization medium, and mitochondria were isolated and purified as described (13Carreras M.C. Converso D.P. Lorenti A.S. Barbich M. Levisman D.M. Jaitovich A. Antico Arciuch V.G. Galli S. Poderoso J.J. Hepatology. 2004; 40: 157-166Crossref PubMed Scopus (56) Google Scholar). Minimal contamination was found (2–3%) by comparing activities of lactate dehydrogenase (cytosolic marker) and succinate-cytochrome c reductase (mitochondrial marker). RT-PCR—Total liver RNA was extracted with TRIzol, and RT-PCR was performed (21Cymeryng C.B. Lotito S.P. Colonna C. Finkielstein C. Pomeraniec Y. Grión N. Gadda L. Maloberti P. Podestá E.J. Endocrinology. 2002; 143: 1235-1242Crossref PubMed Scopus (35) Google Scholar). Primers for β2-microglobulin and NOS isoforms were as described (21Cymeryng C.B. Lotito S.P. Colonna C. Finkielstein C. Pomeraniec Y. Grión N. Gadda L. Maloberti P. Podestá E.J. Endocrinology. 2002; 143: 1235-1242Crossref PubMed Scopus (35) Google Scholar, 22Alvarez M. Depino A.M. Podhajcer O.L. Pitossi F.J. Anal. Biochem. 2000; 287: 87-94Crossref PubMed Scopus (16) Google Scholar, 23Ogilvie P. Achilling K. Billingsley M.L. Schmidt H. FASEB J. 1995; 9: 799-806Crossref PubMed Scopus (95) Google Scholar): for cyclin D1, sense (5′-GCGTACCCTGACACCAATCT-3′) and antisense (5′-GCTCCAGAGACAAGAAACGG-3′). Nested PCR for eNOS isoform (35 cycles) was done with 0.5 μlof PCR product in 30-μl final volume with inner primers, sense (5-ATGTGGCTGTCTGCATGGAT-3′) and antisense (5′-TTGCTGCACTTCTTTCCAG-3′). Quantitative Real-time PCR—Real-time nested PCR for nNOS isoform was done with 0.5 μl of a 1/10 dilution of PCR product in 25-μl final volume with inner primers: nNOS, sense (5′-TTCAACTACATCTGTAACCA-3′) and antisense (5′-TGAACTGCACATTGGCTGGA-3′). Real-time PCR reactions included 0.4 mm dNTPs, 1 μm specific primers, 4 mm MgCl2, 2.5 units of Taq DNA polymerase, and 1:30,000 SYBR Green. Real-time PCR reactions were performed in DNA Engine Opticon (MJ Research, Inc.) and consisted of an initial denaturing step (94 °C for 4 min), followed by 35 cycles (each of 94 °C for 1 min, 55 °C for 40 s, 72 °C for 1 min). Sample quantification was normalized to endogenous β2-microglobulin that was also quantified by real-time PCR following the same protocol as nNOS isoform. Each experiment included a DNA minus control and a standard curve. Immunoblotting for NOS, Hsp90, and Mitochondrial Protein Nitration—Proteins were electrophoresed on 7.5% SDS-polyacrylamide gel, electrotransferred to polyvinylidene difluoride membranes (13Carreras M.C. Converso D.P. Lorenti A.S. Barbich M. Levisman D.M. Jaitovich A. Antico Arciuch V.G. Galli S. Poderoso J.J. Hepatology. 2004; 40: 157-166Crossref PubMed Scopus (56) Google Scholar), incubated with anti-nNOS, anti-eNOS, anti-iNOS, anti-Hsp90, and anti-nitrotyrosine antibodies, and detected with the ECL system. Equal loading was controlled with the appropriated subcellular markers. Incubation of the anti-nitrotyrosine antibody with 10 mm nitrotyrosine prior to the membrane incubation was used to ensure the antibody specificity. NOS Activity in Subcellular Fractions—NOS activity was determined in mitochondrial and cytosolic fractions by conversion of [3H]l-arginine to [3H]l-citrulline (13Carreras M.C. Converso D.P. Lorenti A.S. Barbich M. Levisman D.M. Jaitovich A. Antico Arciuch V.G. Galli S. Poderoso J.J. Hepatology. 2004; 40: 157-166Crossref PubMed Scopus (56) Google Scholar). Immunoelectron Microscopy—Purified mitochondria were suspended in 4% paraformaldehyde and 0.5% glutaraldehyde, pH 7.4, for 2 h at 4 °C, washed overnight with 0.32 m sucrose at 4 °C, and then dehydrated in 70% ethanol and embedded in LR White (13Carreras M.C. Converso D.P. Lorenti A.S. Barbich M. Levisman D.M. Jaitovich A. Antico Arciuch V.G. Galli S. Poderoso J.J. Hepatology. 2004; 40: 157-166Crossref PubMed Scopus (56) Google Scholar). Ultrathin sections were obtained in 300-mesh nickel grids. Immunocytochemistry was performed using a primary mouse anti-C-terminal nNOS (1095–1289) at a dilution of 1:20 in phosphate-buffered saline, pH 7.4. Grids were washed in phosphate-buffered saline and counterstained with 1% uranyl acetate. Nonspecific background was blocked by incubation with 5% normal goat serum in phosphate-buffered saline at the beginning of the procedure. Positive control against 39-kDa subunit of complex I (inner membrane marker) and negative control in the absence of a primary antibody were included. Specimens were observed in a Zeiss EM-109-T transmission electron microscope at 80 kV. Detection of Mitochondrial NO—Mitochondria (1 mg of protein per ml) were incubated in phosphate-buffered saline for 30 min at 37 °C with 5% CO2, 10 μm DAF-FM, and 0.5 μm MitoTracker, and fluorescence was measured with an Ortho-Cytoron Absolute Cytometer (Johnson and Johnson) (24López-Figueroa M. Caamaño C. Morano M.I. Ronn L.C. Akil H. Watson S.J. Biochem. Biophys. Res. Commun. 2000; 272: 129-133Crossref PubMed Scopus (111) Google Scholar). Mitochondrial O2 Utilization and Electron Transfer Activity—O2 uptake was measured polarographically with a Clark-type electrode (10Carreras M.C. Peralta J.G. Converso D.P. Finocchietto P.V. Rebagliati I. Zaninovich A.A. Poderoso J.J. Am. J. Physiol. 2001; 281: H2282-H2288PubMed Google Scholar). To assess NO effects, mitochondria were incubated with 0.3 mm l-arginine (l-Arg) alone or plus 3 mm l-NMMA for 5 min at 37 °C (10Carreras M.C. Peralta J.G. Converso D.P. Finocchietto P.V. Rebagliati I. Zaninovich A.A. Poderoso J.J. Am. J. Physiol. 2001; 281: H2282-H2288PubMed Google Scholar). State 4 O2 uptake was determined with 6 mm malate-glutamate as substrate of complex I and state 3 active respiration by the addition of 0.2 mm adenosine diphosphate (ADP). Complex I activity (NADH: ubiquinone reductase) was measured by the rotenone-sensitive reduction of 50 μm 2,3-dimethoxy-6-methyl-1,4-benzoquinone with 1 mm KCN and 200 μm NADH as electron donor at 340 nm with a Hitachi U3000 spectrophotometer at 30 °C. Activity of complexes II-III was determined by cytochrome c reduction at 550 nm. Cytochrome oxidase activity (Complex IV) was determined by monitoring cytochrome c oxidation at 550 nm (∈550, 21 mm–1·cm–1); the reaction rate was measured as the pseudo-first order reaction constant (k′) and expressed as k′/min·mg of protein (13Carreras M.C. Converso D.P. Lorenti A.S. Barbich M. Levisman D.M. Jaitovich A. Antico Arciuch V.G. Galli S. Poderoso J.J. Hepatology. 2004; 40: 157-166Crossref PubMed Scopus (56) Google Scholar, 14Riobo N.A. Melani M. Sanjuan N. Fiszman M.L. Gravielle M.C. Carreras M.C. Cadenas E. Poderoso J.J. J. Biol. Chem. 2002; 277: 42447-42455Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Mitochondrial Production of H2O2 and O2·¯—H2O2 production rate was monitored spectrofluorometrically at complexes I or II-III (6 mm malate-glutamate or 10 mm succinate as substrate) in an F-2000 spectrofluorometer (Hitachi, Tokyo, Japan) as described (13Carreras M.C. Converso D.P. Lorenti A.S. Barbich M. Levisman D.M. Jaitovich A. Antico Arciuch V.G. Galli S. Poderoso J.J. Hepatology. 2004; 40: 157-166Crossref PubMed Scopus (56) Google Scholar). To determine O2·¯ at complexes I and II-III, mitochondria were subjected to three freeze/thaw cycles, and SOD-sensitive cytochrome c reduction was measured at 550 nm (0.1 mg of protein/ml and 10 μm SOD to subtract unspecific reduction). Mn-SOD, catalase, and glutathione peroxidase activities were determined in 7,000 g supernatants as described (13Carreras M.C. Converso D.P. Lorenti A.S. Barbich M. Levisman D.M. Jaitovich A. Antico Arciuch V.G. Galli S. Poderoso J.J. Hepatology. 2004; 40: 157-166Crossref PubMed Scopus (56) Google Scholar). Liver Cell Isolation and Detection of Intracellular Oxidants—Hepatocytes were isolated by two-step collagenase perfusion (25Berry M.N. Friend D.S. J. Cell Biol. 1969; 43: 506-520Crossref PubMed Scopus (3633) Google Scholar). Intracellular oxidants and mitochondrial (O2·¯) were detected by flow cytometry after incubating hepatocytes in phenol red-free Dulbecco's modified Eagle's medium with 5 μm DCFH-DA or 5 μm HE for 30 min at 37 °C with 5% CO2. Blue Native Polyacrylamide Gel Electrophoresis—To separate mitochondrial complexes, Blue Native-PAGE was performed according to Schägger (26Schägger H. Methods Cell Biol. 2001; 65: 231-244Crossref PubMed Google Scholar). Gels of first dimension were stained with Coomassie Blue and membranes incubated with antibodies against 3-nitrotyrosine. For second-dimension analysis, gel bands, corresponding to the complex I region derived from 5-mm-wide lanes, were excised and incubated for 2 h in cathode buffer (50 mm glycine and 7.5 mm imidazole, pH 7) supplemented with 1% SDS and 1% β-mercaptoethanol before electrophoresis on 10%-16.5% Tris/glycine gels (27Schagger H. von Jagow G. Anal. Biochem. 1991; 199: 223-231Crossref PubMed Scopus (1918) Google Scholar). Membranes were incubated with antibodies against 3-nitrotyrosine. Immunoprecipitation—For immunoprecipitations, 500 μg of mitochondrial proteins were incubated with 4 μg of antibodies against Complex I 39-kDa subunit or Complex IV VIc subunit and 30 μl of Protein A/G PLUS-agarose (Santa Cruz) at 4 °C; samples were blotted against polyclonal nNOS antibody. Protein loading was controlled by the respective mitochondrial complex antibodies. Preparation of Whole Liver Homogenates and Immunoblotting for MAPKs and Cyclin D1—To study MAPKs and cyclin D1, liver was homogenized in lysis buffer as described (13Carreras M.C. Converso D.P. Lorenti A.S. Barbich M. Levisman D.M. Jaitovich A. Antico Arciuch V.G. Galli S. Poderoso J.J. Hepatology. 2004; 40: 157-166Crossref PubMed Scopus (56) Google Scholar). Proteins were separated on 12% SDS-PAGE, and cyclin D1 and MAPKs were detected with specific antibodies. Metabolic Calculations—All experiments were done at 1 mg of mitochondrial protein per ml (n = 5). Mitochondrial H2O2 steady-state concentration ([H2O2]ss) was calculated according to Ref. 13Carreras M.C. Converso D.P. Lorenti A.S. Barbich M. Levisman D.M. Jaitovich A. Antico Arciuch V.G. Galli S. Poderoso J.J. Hepatology. 2004; 40: 157-166Crossref PubMed Scopus (56) Google Scholar as shown in Equation 1.[H2O2]ss=+d[H2O2]/dt/k1×[CAT]+k2×[GPX]Eq. 1 where + d[H2 O2]/dt is the rate of l-arginine-dependent H2O2 production in Ms–1 (Fig. 2A, upper panel), k1 = 4.6 × 107m–1s–1, and k2 = 5 × 107m–1s–1 · CAT and and GPX correspond to catalase and glutathione peroxidase concentrations determined spectrophotometrically as previously described (13Carreras M.C. Converso D.P. Lorenti A.S. Barbich M. Levisman D.M. Jaitovich A. Antico Arciuch V.G. Galli S. Poderoso J.J. Hepatology. 2004; 40: 157-166Crossref PubMed Scopus (56) Google Scholar). Mitochondrial (O2·¯) steady-state concentration ([(O2·¯)]ss) was calculated from Equations 2 and 3 as follows.1/2d[H2O2]/dt=-d[O2·¯]/dt-d[O2-]/dt=k3[O2·¯][SOD]+k4[O2·¯][NO]Eq. 2 [O2·¯]ss=1/2d[H2O2]/dtk3[SOD]+k4[NO]Eq. 3 k3 and k4 are, respectively, 2.3 × 109 and 1.9 × 1010m–1 s–1, and [SOD] was determined spectrophotometrically by inhibition of cytochrome c reduction (13Carreras M.C. Converso D.P. Lorenti A.S. Barbich M. Levisman D.M. Jaitovich A. Antico Arciuch V.G. Galli S. Poderoso J.J. Hepatology. 2004; 40: 157-166Crossref PubMed Scopus (56) Google Scholar). Matrix NO ([NO]ss) was calculated by the percentage of liver mitochondria state 3 respiratory rate NO-dependent inhibition as previously described (15Peralta J.G. Finocchietto P.V. Converso D. Schopfer F. Carreras M.C. Poderoso J.J. Am. J. Physiol. 2003; 284: H2375-H2383PubMed Google Scholar, 19Poderoso J.J. Lisdero C. Schopfer F. Riobo N. Carreras M.C. Cadenas E. Boveris A. J. Biol. Chem. 1999; 274: 37709-37716Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). [ONOO–] production rate was calculated as shown in Equation 4.d[ONOO-]/-dt=k4[NO][O2·¯]Eq. 4 Statistical Analysis—Data are mean ± S.E. One-way analysis of variance was utilized with post hoc Dunnett test, and regression analysis and significance were accepted at p 3-fold increased in hypothyroid liver compared with controls (Fig. 1B), whereas eNOS or iNOS mRNA did not change (data not shown). In addition, nNOS became distinctively enhanced in mitochondria (mtNOS), indicating a subsequent import of the overexpressed enzyme to the organelles; these findings were reverted by hormone replacement (Fig. 1C). To corroborate subcellular distribution in the groups, liver fractions were compared. Western blotting confirmed the increase of total nNOS expression represented by liver homogenates and the enrichment of the mitochondrial fraction in the hypothyroid group. Differentially, at high T3, nNOS expression had results similar to controls, but this condition retained the protein predominantly localized in cytosol (Fig. 1C). This effect was parallel to the increased expression of heat shock protein 90 (Hsp90), one of the most important chaperones associated to nNOS (Fig. 1F). Alternatively, modulation of mtNOS content in the studied groups was validated as well by immunoelectron microscopy with monoclonal nNOS antibodies (Fig. 1D). In agreement, Ca2+-dependent NOS activity was significantly increased in hypothyroid mitochondria but was normal to slightly reduced after T3 administration, with an opposite cytosolic pattern (Fig. 1E); Ca2+-independent NOS activity was not detected.TABLE 1Oxygen uptake rates depend on thyroid status and nitric oxide utilizationControlHypothyroidHypothyroid + T3HyperthyroidTSH (ng/ml)12 ± 2>80*11 ± 25 ± 1*Basal metabolic rate (ml of O2/min·kg0.75)17 ± 112 ± 1*18 ± 123 ± 1*Mitochondrial O2 uptake (ngat/min·mg of protein)State 4 uptakeBasal14.2 ± 1.311.5 ± 1.715.8 ± 1.716.9 ± 0.3+ 0.3 mm l-Arg13.8 ± 0.79.0 ± 0.5 †14.0 ± 1.714.8 ± 0.6+ 3 mm l-NMMA15.6 ± 0.812.4 ± 2.014.6 ± 1.816.2 ± 2State 3 uptakeBasal79 ± 649 ± 3*†89 ± 5102 ± 10+ 0.3 mm l-Arg72 ± 746 ± 2†*85 ± 5106 ± 10*+ 3 mm l-NMMA86 ± 862 ± 4*92 ± 5105 ± 8Calculated matrix (NO) (nm)23 ± 994 ± 754 ± 156 ± 3Enzyme activitiesComplex I (nmole/min·mg of protein)108 ± 1641 ± 15*85 ± 998 ± 9Complex II/III (nmole/min·mg of protein)66 ± 1962 ± 2063 ± 1669 ± 1Complex IV (k′/min·mg of protein)13.7 ± 0.312.3 ± 0.2*13.5 ± 0.414.6 ± 0.4 Open table in a new tab Subcellular nNOS Localization Modulates Mitochondrial Respiration—According to BMR, organelles from hypo- and hyperthyroid groups had the lowest and highest O2 uptake rates, respectively (Table 1). To discern the effects of mtNOS, mitochondria were supplemented with l-Arg alone or plus NOS inhibitor l-NMMA. The sum of the opposite effects of NOS substrate and inhibitor on basal O2 utilization determines mtNOS functional activity on respiration (28Valdez L.B. Zaobornyj T. Boveris A. Methods Enzymol. 2005; 396: 444-455Crossref PubMed Scopus (43) Google Scholar). Likewise, mtNOS-dependent inhibition of state 3 O2 uptake was increased ∼39% in hypothyroid samples, 18% in controls, and negligible in T3-treated mitochondria. In agreement, matrix NO estimated from l-Arg inhibition of O2 uptake (18Poderoso J.J. Carreras M.C. Lisdero C. Riobo N. Schopfer F. Boveris A. Arch. Biochem. Biophys. 1996; 328: 85-92Crossref PubMed Scopus (676) Google Scholar) was augmented by 4-fold in hypothyroidism and decreased by a half in hyperthyroidism (Table 1). These results demonstrate that (a) translocated nNOS is functionally active and mitochondria retain ex vivo the cofactors for catalytic activity and (b) NOS confinement to the small mitochondrial compartment amplifies NO effects on O2 uptake and BMR. We next examined the contribution of segmental activities to mitochondrial O2 uptake. Electron transfer rate at complex I was markedly decreased at hypothyroid status solely (∼ 60%), whereas cytochrome oxidase was less inhibited (∼ 11%), and complex II-III activity was not modified (Table 1). No significant thyroid effects on antioxidant Mn-SOD, catalase, or glutathione peroxidase activities were detected. Thyroid Status Defines Quality and Intensity of Mitochondrial Oxidant Production—In connection with O2 uptake rate, previous observations proposed a decreased mitochondrial H2O2 yield in rat hypothyroidism and an increased yield in hyperthyroidism (29Venditti P. De Rosa R. Di Meo S. Mol. Cell. Endocrinol. 2003; 205: 185-192Crossref PubMed Scopus (87) Google Scholar), though opposite results were reported as well (30Lopez-Torres M. Romero M. Barja G. Mol. Cell. Endocrinol. 2000; 168: 127-134Crossref PubMed Scopus (70) Google Scholar, 31Das K. Chainy G.B. Biochim. Biophys. Acta. 2001; 1537: 1-13Crossref PubMed Scopus (139) Google Scholar). It is shown here that basal production of H2O2 with substrates of complex I (malate-glutamate) or II (succinate) is not esse
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