A Transverse Tubule NADPH Oxidase Activity Stimulates Calcium Release from Isolated Triads via Ryanodine Receptor Type 1 S -Glutathionylation
2006; Elsevier BV; Volume: 281; Issue: 36 Linguagem: Inglês
10.1074/jbc.m600451200
ISSN1083-351X
AutoresCecilia Hidalgo, Gina Sánchez, Genaro Barrientos, Paula Aracena-Parks,
Tópico(s)Redox biology and oxidative stress
ResumoWe report here the presence of an NADPH oxidase (NOX) activity both in intact and in isolated transverse tubules and in triads isolated from mammalian skeletal muscle, as established by immunochemical, enzymatic, and pharmacological criteria. Immunohistochemical determinations with NOX antibodies showed that the gp91phox membrane subunit and the cytoplasmic regulatory p47phox subunit co-localized in transverse tubules of adult mice fibers with the α1s subunit of dihydropyridine receptors. Western blot analysis revealed that isolated triads contained the integral membrane subunits gp91phox and p22phox, which were markedly enriched in isolated transverse tubules but absent from junctional sarcoplasmic reticulum vesicles. Isolated triads and transverse tubules, but not junctional sarcoplasmic reticulum, also contained varying amounts of the cytoplasmic NOX regulatory subunits p47phox and p67phox. NADPH or NADH elicited superoxide anion and hydrogen peroxide generation by isolated triads; both activities were inhibited by NOX inhibitors but not by rotenone. NADH diminished the total thiol content of triads by one-third; catalase or apocynin, a NOX inhibitor, prevented this effect. NADPH enhanced the activity of ryanodine receptor type 1 (RyR1) in triads, measured through [3H]ryanodine binding and calcium release kinetics, and increased significantly RyR1 S-glutathionylation over basal levels. Preincubation with reducing agents or NOX inhibitors abolished the enhancement of RyR1 activity produced by NADPH and prevented NADPH-induced RyR1 S-glutathionylation. We propose that reactive oxygen species generated by the transverse tubule NOX activate via redox modification the neighboring RyR1 Ca2+ release channels. Possible implications of this putative mechanism for skeletal muscle function are discussed. We report here the presence of an NADPH oxidase (NOX) activity both in intact and in isolated transverse tubules and in triads isolated from mammalian skeletal muscle, as established by immunochemical, enzymatic, and pharmacological criteria. Immunohistochemical determinations with NOX antibodies showed that the gp91phox membrane subunit and the cytoplasmic regulatory p47phox subunit co-localized in transverse tubules of adult mice fibers with the α1s subunit of dihydropyridine receptors. Western blot analysis revealed that isolated triads contained the integral membrane subunits gp91phox and p22phox, which were markedly enriched in isolated transverse tubules but absent from junctional sarcoplasmic reticulum vesicles. Isolated triads and transverse tubules, but not junctional sarcoplasmic reticulum, also contained varying amounts of the cytoplasmic NOX regulatory subunits p47phox and p67phox. NADPH or NADH elicited superoxide anion and hydrogen peroxide generation by isolated triads; both activities were inhibited by NOX inhibitors but not by rotenone. NADH diminished the total thiol content of triads by one-third; catalase or apocynin, a NOX inhibitor, prevented this effect. NADPH enhanced the activity of ryanodine receptor type 1 (RyR1) in triads, measured through [3H]ryanodine binding and calcium release kinetics, and increased significantly RyR1 S-glutathionylation over basal levels. Preincubation with reducing agents or NOX inhibitors abolished the enhancement of RyR1 activity produced by NADPH and prevented NADPH-induced RyR1 S-glutathionylation. We propose that reactive oxygen species generated by the transverse tubule NOX activate via redox modification the neighboring RyR1 Ca2+ release channels. Possible implications of this putative mechanism for skeletal muscle function are discussed. The NADPH oxidases (NOX) 3The abbreviations used are: NOX, NADPH oxidase; BSA, bovine serum albumin; CICR, Ca2+-induced Ca2+ release; DHPR, dihydropyridine receptor(s); DPI, diphenyleneiodonium; E-C, excitation-contraction; RyR1, ryanodine receptor type 1; ROS, reactive oxygen species; PBS, phosphate-buffered saline; SH, sulfhydryl; SOD, superoxide dismutase; SR, sarcoplasmic reticulum; T-tubule, transverse tubule; MOPS, 4-morpholinepropanesulfonic acid.3The abbreviations used are: NOX, NADPH oxidase; BSA, bovine serum albumin; CICR, Ca2+-induced Ca2+ release; DHPR, dihydropyridine receptor(s); DPI, diphenyleneiodonium; E-C, excitation-contraction; RyR1, ryanodine receptor type 1; ROS, reactive oxygen species; PBS, phosphate-buffered saline; SH, sulfhydryl; SOD, superoxide dismutase; SR, sarcoplasmic reticulum; T-tubule, transverse tubule; MOPS, 4-morpholinepropanesulfonic acid. are flavoprotein enzymes that use NADPH as electron donor to mediate the univalent reduction of molecular oxygen to superoxide anion (1Lambeth J.D. Nat. Rev. Immunol. 2004; 4: 181-189Crossref PubMed Scopus (2406) Google Scholar), a free radical that by spontaneous or enzymatically catalyzed dismutation is readily converted into H2O2. The phagocytic NOX isoform (NOX2) was first discovered as a pivotal component of the neutrophil respiratory burst (2Vignais P.V. Cell Mol. Life Sci. 2002; 59: 1428-1459Crossref PubMed Scopus (620) Google Scholar, 3Quinn M.T. Gauss K.A. J. Leukocyte Biol. 2004; 76: 760-781Crossref PubMed Scopus (380) Google Scholar). The functional NOX2 enzyme is composed of two integral plasma membrane subunits, gp91phox and p22phox, which make up cytochrome b558, plus three cytosolic regulatory subunits: p40phox, p47phox, and p67phox (2Vignais P.V. Cell Mol. Life Sci. 2002; 59: 1428-1459Crossref PubMed Scopus (620) Google Scholar, 4Babior B.M. Am. J. Med. 2000; 109: 33-44Abstract Full Text Full Text PDF PubMed Scopus (867) Google Scholar). A variety of tissues, including endothelial cells (5Jones S.A. O'Donnell V.B. 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Cell Mol. Life Sci. 2002; 59: 1428-1459Crossref PubMed Scopus (620) Google Scholar, 22Bokoch G.M. Diebold B.A. Blood. 2002; 100: 2692-2696Crossref PubMed Scopus (279) Google Scholar). Regulation of the NOX4 isoform has been recently characterized as unusual among the NOX family, since this isoform displays constitutive activity (i.e. without relying on cytosolic components, such as Rac) and generates large amounts of hydrogen peroxide (23Martyn K.D. Frederick L.M. von Loehneysen K. Dinauer M.C. Knaus U.G. Cell. Signal. 2006; 18: 69-82Crossref PubMed Scopus (605) Google Scholar). The NOX5 isoform contains EF-hand motifs in its structure, a structural feature that makes it Ca2+-dependent (24Banfi B. Tirone F. Durussel I. Knisz J. Moskwa P. Molnar G.Z. Krause K.H. Cox J.A. J. Biol. Chem. 2004; 279: 18583-18591Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar). Homologues of the p47phox and p67phox cytosolic components (NOXO1 and NOXA1), which selectively regulate NOX1 (25Banfi B. Clark R.A. Steger K. Krause K.H. J. Biol. Chem. 2003; 278: 3510-3513Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar, 26Takeya R. Ueno N. Kami K. Taura M. Kohjima M. Izaki T. Nunoi H. Sumimoto H. J. Biol. Chem. 2003; 278: 25234-25246Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar) and NOX3 (27Cheng G. Ritsick D. Lambeth J.D. J. Biol. Chem. 2004; 279: 34250-34255Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar), have been also described. Both the NOX2 and the NOX4 isoforms are present in cardiac muscle cells (28Byrne J.A. Grieve D.J. Bendall J.K. Li J.M. Gove C. Lambeth J.D. Cave A.C. Shah A.M. Circ. Res. 2003; 93: 802-805Crossref PubMed Scopus (354) Google Scholar). NADPH addition to microsomes isolated from heart muscle significantly enhances, via NOX activation, both RyR2 S-glutathionylation and Ca2+-induced Ca2+ release (CICR) (29Sánchez G. Pedrozo Z. Domenech R.J. Hidalgo C. Donoso P. J. Mol. Cell Cardiol. 2005; 39: 982-991Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Skeletal muscle homogenates also contain all of the NOX2 protein components and display an activity that generates superoxide anion at the expense of NADH oxidation (30Javesghani D. Magder S.A. Barreiro E. Quinn M.T. Hussain S.N. Am. J. Respir. Crit. Care Med. 2002; 165: 412-418Crossref PubMed Scopus (164) Google Scholar). In addition, human skeletal muscle tissue exhibits transcripts for the NOX2 and NOX4 isoforms (31Cheng G. Cao Z. Xu X. van Meir E.G. Lambeth J.D. Gene (Amst.). 2001; 269: 131-140Crossref PubMed Scopus (691) Google Scholar). All of these findings provide experimental support to earlier proposals suggesting the presence of NOX in skeletal muscle cells (32Brotto M.A. Nosek T.M. J. Appl. Physiol. 1996; 81: 731-737Crossref PubMed Scopus (97) Google Scholar, 33Reid M.B. J. Appl. Physiol. 2001; 90: 724-731Crossref PubMed Scopus (13) Google Scholar). We have found recently that tetanic field stimulation of rat skeletal muscle cells in primary culture increases NOX-dependent ROS production and elicits ryanodine-sensitive Ca2+ signals that are significantly decreased by NOX inhibitors. 4Espinosa, A., Leiva, A., Peña, A., Müller, A., Debandi, A., Hidalgo, C., Carrasco, M. A., and Jaimovich, E. (2006) J. Cell Physiol., in press.4Espinosa, A., Leiva, A., Peña, A., Müller, A., Debandi, A., Hidalgo, C., Carrasco, M. A., and Jaimovich, E. (2006) J. Cell Physiol., in press. These results suggest a role for activity-dependent NOX as a source of RyR-modifying ROS in skeletal muscle. In this work, we investigated the membrane origin of the skeletal muscle NOX and the effects of NOX-generated ROS on RyR1 activity and RyR1 S-glutathionylation levels. For these experiments, we used either intact fibers isolated from adult mouse skeletal muscle or three different membrane fractions isolated from rabbit skeletal muscle: transverse tubules (T-tubules), triads (which contain 10-15% T-tubules attached to junctional sarcoplasmic reticulum (SR) vesicles), and junctional (heavy) SR vesicles devoid of T-tubules but enriched in RyR1 channels. Immunohistochemistry with specific antibodies showed that the NOX membrane gp91phox subunit and the cytoplasmic p47phox regulatory subunit co-localized with the α1s subunit of dihydropyridine receptors (DHPR) in T-tubules of adult mice fibers. Western blot analysis showed that the NOX integral membrane components gp91phox and p22phox were markedly enriched in T-tubules and also present in triads but were absent from heavy SR vesicles. Triads displayed NADPH-dependent generation of superoxide anion and H2O2 that was significantly decreased by NOX inhibitors. In addition to promoting NOX activity, NADPH enhanced significantly RyR1 S-glutathionylation and increased RyR1 activity. Possible physiological consequences of these findings, in terms of potential cross-talk between redox and calcium signaling cascades that may lead to enhanced Ca2+ release in skeletal muscle, are discussed. Materials—All reagents used were of analytical grade. Ryanodine, bovine serum albumin, bovine liver catalase, β-mercaptoethanol, and protease inhibitors (leupeptin, pepstatin A, benzamidine, and phenylmethylsulfonyl fluoride) were from Sigma. Bovine erythrocyte superoxide dismutase (SOD), rotenone, NADH, NADPH, lucigenin, 2,4-dithiothreitol, diphenyleneiodonium (DPI), 5,5′-dithiobis-(2-nitrobenzoic) acid, and NG-nitro-l-arginine methyl ester were from Calbiochem. [3H]Ryanodine was from PerkinElmer Life Sciences, apocynin was from Aldrich, and 10-acetyl-3,7-dihydrophenoxazine (Amplex Red reagent) was from Molecular Probes, Inc. (Eugene, OR). Polyvinylidene difluoride membranes were from Bio-Rad. Antibodies—The mouse monoclonal anti-gp91phox, anti-p22phox, anti-p47phox, and anti-p67phox antibodies used for Western blot assays were a kind gift from Dr. Mark T. Quinn (Veterinary Molecular Biology, Montana State University, Bozeman, MT); these antibodies were raised as described previously (34Burritt J.B. Quinn M.T. Jutila M.A. Bond C.W. Jesaitis A.J. J. Biol. Chem. 1995; 270: 16974-16980Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). The goat polyclonal anti-gp91phox and anti-p47phox antibodies used for immunohistochemical experiments, as well as the antibodies against RyR or horseradish peroxidase-conjugated anti-IgG (anti-mouse or anti-rabbit) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The anti-glutathione (anti-GSH) antibody was from Virogen (Watertown, MA). Mouse monoclonal antibodies against the α1s subunit of DHPR were from Affinity BioReagents (Golden, CO), and Alexa Fluor secondary antibodies were from Molecular Probes. Fiber Isolation and Immunohistochemistry—Adult AJ mice were anesthetized with ketamine, following a procedure approved by the local animal ethics committee. The flexor digitorum longus or tibialis anterioris muscles were dissected out, placed in Krebs solution (135 mm NaCl, 5.9 mm KCl, 1.2 mm MgCl2, 1.5 mm CaCl2, 11.5 mm Hepes, pH 7.4, 11.5 mm glucose) and immediately fixed in 4% paraformaldehyde in PBS (134 mm NaCl, 2.7 mm KCl, 17 mm Na2HPO4, 2.3 mm KH2PO4, pH 7.4) for 30 min at room temperature. After three washes with PBS, muscle fibers were obtained by gentle mechanical dissociation with a surgical tool. Isolated fibers were permeabilized with 0.05% Triton X-100 added to PBS containing 1% bovine serum albumin (BSA) to block nonspecific binding sites. After a 10-h incubation, the Triton X-100 solution was replaced by PBS plus 1% BSA; antibodies against gp91phox, p47phox, or the α1s subunit of DHPR were added at a 1:100 dilution; and the fibers were incubated for 10 h at room temperature. Fibers were washed three times with PBS plus 1% BSA and were incubated for 1 h with Alexa Fluor secondary antibodies (1:500). After five washes with PBS plus 1% BSA, fibers were placed on glass coverslips to record fluorescent images in an inverted confocal microscope (Carl Zeiss LSM 5 Pascal, Oberkochen, Germany). Fluorescent images were analyzed with the Image J 1.32j software; the percentage of colocalization was estimated using the colocalization finder plugin, version 1.1. Control experiments with the secondary antibodies did not show detectable fluorescence. Membrane Isolation—Membrane preparations from rabbit fast skeletal muscle were isolated as described previously (35Hidalgo C. González M.E. Lagos R. J. Biol. Chem. 1983; 259: 13937-13945Abstract Full Text PDF Google Scholar, 36Hidalgo C. Jorquera J. Tapia V. Donoso P. J. Biol. Chem. 1993; 268: 15111-15117Abstract Full Text PDF PubMed Google Scholar). Triad-enriched SR vesicles isolated from rabbit muscle (hereafter referred to as triads) contain 10-15% attached T-tubules, as evidenced by their density of [3H]ouabain binding sites. Membrane fractions were stored in liquid nitrogen and were used within 3 days; longer storage resulted in significant deterioration of NOX activity. Protein was determined (37Hartree E.F. Anal. Biochem. 1972; 48: 422-427Crossref PubMed Scopus (4493) Google Scholar) using commercial BSA as a standard. Western Blotting—Triad vesicles (10 μg) were incubated in nonreducing loading buffer (6 m urea, 1% SDS, 0.02% bromphenol blue, 50 mm NaH2PO4, 17mm Na2HPO4) plus 5 mm N-ethylmaleimide at 60 °C for 20 min. Proteins were separated in 3.5-8% gradient gels by SDS-PAGE under nonreducing conditions, as described elsewhere (38Aracena P. Sanchez G. Donoso P. Hamilton S.L. Hidalgo C. J. Biol. Chem. 2003; 278: 42927-42935Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). For RyR1 detection, proteins were transferred to polyvinylidene difluoride membranes at 350 mA for 3 h at 4°C; for smaller molecular weight proteins, the transfer times were only 1 h under the same conditions. Membranes were blocked with 5% BSA in Tris-saline buffer (140 mm NaCl, 20 mm Tris-HCl, pH 7.6) plus 0.2% Tween 20 and were probed with specific antibodies directed to RyR, GSH, gp91phox, p22phox, p47phox, or p67phox. Horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG was used as secondary antibody. Blots were developed using an ECL kit (Pierce). Membranes were stripped between blots by 30-min incubation at 50 °C with a solution containing 100 mm β-mercaptoethanol, 2% SDS, 62.5 mm Tris-HCl, pH 6.7. Determination of NADPH Oxidase Activity—To determine NOX activity, we measured the oxidation of both NADH and NADPH as well as the generation of superoxide anion and H2O2. Oxidation of NADH or NADPH by triads (0.1 mg/ml) was monitored at 340 nm (ϵ = 6,250 m-1 cm-1) in a Hewlett-Packard ChemStation UV-visible spectrophotometer (Waldbronn, Germany). Superoxide generation was measured by the lucigenin-derived chemiluminiscence method in a Berthold FB 12 luminometer; SR vesicles (0.2 mg/ml) were incubated at 25 °C with 100 mm MOPS-Tris, pH 7.0, 5 μm lucigenin, and variable concentrations of NADPH, as specified in each case. Chemiluminiscence was expressed as nmol of superoxide ion generated/mg of protein/min; calibration was done using hypoxanthine and xanthine oxidase as described (30Javesghani D. Magder S.A. Barreiro E. Quinn M.T. Hussain S.N. Am. J. Respir. Crit. Care Med. 2002; 165: 412-418Crossref PubMed Scopus (164) Google Scholar). Hydrogen peroxide generation was measured at 37 °C or at 25 °C in a FluoroMax 2 spectrofluorotometer (ISA Jobinybon-Spex) with λex = 527 nm, λem = 583 nm, using Amplex Red as described (39Zhou M. Diwu Z. Panchuk-Voloshina N. Haugland R.P. Anal. Biochem. 1997; 253: 162-168Crossref PubMed Scopus (1087) Google Scholar). The effects of DPI (10 μm), apocynin (4 mm), rotenone (200 nm), NG-nitro-l-arginine methyl ester (2 mm), and SOD (300 units/ml) on superoxide anion or H2O2 generation were also tested, as indicated in each case. Nonenzymatic controls were performed using vesicles heat-denatured for 10 min at 100 °C; this procedure essentially abolished H2O2 generation and reduced by >90% NADH/NADPH oxidation and superoxide anion generation. To calculate specific activities, the activity displayed by heat-denatured vesicles was subtracted from the total activity. Detection of Endogenous or NADPH-promoted RyR1 S-Glutathionylation—To detect endogenous RyR1 S-glutathionylation, triad vesicles (10 μg) were separated by SDS-PAGE under nonreducing conditions as described above. After electrophoresis and transfer to polyvinylidene difluoride membranes, proteins were probed with anti-GSH antibody (1:10,000). Following ECL detection of the antigen-antibody reaction, membranes were stripped and probed with anti-RyR antibody. Blots were quantified by densitometric analysis (Quantity One software; Bio-Rad). Results were expressed as the ratio of anti-GSH/anti-RyR band densities. To detect NADPH-promoted S-glutathionylation, triad vesicles (1 mg/ml) were incubated at 25 °C for 10 min in a solution containing 0.1-0.5 mm NADPH, 100 mm MOPS-Tris, pH 7.0. The effect of apocynin (8 mm) was tested in some experiments. Labeling of Triad Proteins with [35S]GSH—Vesicles were incubated for 10 min at 37 °C in a solution containing 100 mm MOPS-Tris, pH 7.2, 0.1 mm NADPH plus [35S]GSH added as tracer. The reaction was terminated by the addition of nonreducing sample buffer plus 5 mm N-ethylmaleimide. Triad proteins (10 μg) were separated in 3.5-8% nonreducing gels as above. Gels were stained with Coomassie Blue, and the 35S radioactivity incorporated into RyR1 was determined in a Molecular Imager FX system (Bio-Rad) using a CP Phosphor Screen (Eastman Kodak Co.) essentially as reported earlier (38Aracena P. Sanchez G. Donoso P. Hamilton S.L. Hidalgo C. J. Biol. Chem. 2003; 278: 42927-42935Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Titration of Total Sulfhydryl Content—Triads were incubated at 0.1 mg/ml at 37 °C for 10 min with or without 0.1 mm NADH; samples were taken at different times to determine their total sulfhydryl content. For this purpose, triads were incubated for an additional 30-min period at 25 °C with 0.1 mm 5,5′-dithiobis-(2-nitrobenzoic) acid in 50 mm Tris-base (40Ellman G.L. Arch. Biochem. Biophys. 1959; 82: 70-77Crossref PubMed Scopus (20884) Google Scholar). Alternatively, triads (0.1 mg/ml) were incubated with 100 μm H2O2 for 10 min at 37 °C prior to sulfhydryl titration as above. The effects of apocynin (2 mm) or catalase (300 units/ml) were tested in some experiments. Detection of NADPH-dependent S-S Cross-link Formation between RyR1 Subunits—Triads were incubated for 10 min at 37 °C with 0.1 mm NADPH or with 0.25 mm diamide. To analyze RyR cross-linked products, the procedure described in detail elsewhere (41Aghdasi B. Zhang J.Z. Wu Y. Reid M.B. Hamilton S.L. J. Biol. Chem. 1997; 272: 3739-3748Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar) was followed without substantial modifications. [3H]Ryanodine Binding—To measure initial rates of [3H]ryanodine binding, triads (0.2 mg/ml) were incubated at pCa 5 under high ionic strength conditions (500 mm KCl, 20 mm MOPS-Tris, pH 7.2). Total binding, determined in the presence of 5 nm [3H]ryanodine, was measured at 37 °C for 10 min, taking samples at 5-, 7-, and 10-min intervals; nonspecific binding was determined in the additional presence of 10 μm ryanodine. To test the effects of NADH, NADPH, GSH, or DPI, vesicles were preincubated with these reagents for 2 min at 25 °C. The effects of SOD plus catalase or of H2O2 were tested in some experiments. Calcium Release Kinetics—Calcium release kinetics was determined as described in detail elsewhere (38Aracena P. Sanchez G. Donoso P. Hamilton S.L. Hidalgo C. J. Biol. Chem. 2003; 278: 42927-42935Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Briefly, triads were actively loaded with calcium by incubation for 10 min at 25 °C in a solution containing 100 mm KCl, 2 mm ATP (sodium salt), 3 mm MgCl2, 20 mm MOPS/Tris, pH 7.20, 10 mm phosphocreatine, and 15 units/ml creatine kinase. Vesicles were then mixed (1:10) in a stopped flow fluorescence spectrophotometer with a releasing solution containing 100 mm KCl, 7 mm ATP-Na, 5.56 mm MgCl2, 0.165 mm CaCl2, 20 mm MOPS/Tris, pH 7.20, plus 1 μm Calcium Green-5N. Upon mixing, free concentrations were 1.4 mm ATP, 0.348 mm Mg2+, and 0.019 mm Ca2+ (pCa 4.7). To test the effects of NADPH, GSH, or DPI on calcium release kinetics, vesicles were incubated with these reagents for 10 min at 25 °C during active loading as above. Statistical Analysis—Data are presented as mean ± S.E. of at least three independent determinations. For statistical comparisons, depending on the number of determinations, an analysis of variance test was performed either with Dunnett post-test analysis using the GraphPad® Prism software or the Holm-t test using the Primer of Bioestatistics software. Values were considered significantly different if p was <0.05. Occurrence of NADPH Oxidase Subunits in Transverse Tubules of Mouse Skeletal Muscle Fibers—Rat skeletal muscle contains mRNA and proteins for the NOX gp91phox, p22phox, p47phox, and p67phox subunits localized in close proximity to the sarcolemma, albeit their precise membrane location was not specified (30Javesghani D. Magder S.A. Barreiro E. Quinn M.T. Hussain S.N. Am. J. Respir. Crit. Care Med. 2002; 165: 412-418Crossref PubMed Scopus (164) Google Scholar). Our earlier observations 5J. L. Fernandez and C. Hidalgo, unpublished observations. indicating that isolated T-tubules but not SR vesicles oxidized NADH at high rates suggested a T-tubular location for this NADH oxidase activity. In this work, we detected the presence of the NOX subunits gp91phox and p47phox in skeletal muscle fibers from adult mice (Fig. 1). Over 95% colocalization of these two NOX subunits with the α1S DHPR subunit was found in confocal serial images, strongly suggesting that the skeletal muscle NOX is localized in the T-tubule system. Presence of NOX Protein Subunits in Isolated Triads and T-tubule Vesicles—Western blot analysis of isolated triads, T-tubules, and heavy SR vesicles revealed that antibodies against the NOX integral membrane subunit gp91phox or p22phox recognized protein bands in triads but not in heavy SR vesicles; these two protein bands were markedly enriched in isolated T-tubules (Fig. 2). The enrichment in gp91phox and p22phox in T-tubules relative to triads was consistently observed in all preparations studied. Antibodies against the NOX cytosolic components p47phox and p67phox also recognized protein bands in T-tubules and triads but not in heavy SR vesicles (Fig. 2). In contrast to the bands recognized by antibodies against gp91phox and p22phox, these two regulatory components were not enriched in the isolated T-tubule preparation illustrated in Fig. 2. Furthermore, some isolated T-tubule preparations had somewhat lower p47phox and p67phox contents than triads, suggesting that these regulatory subunits were lost to varying degrees during T-tubule isolation. This finding may explain why our attempts to measure NADH/NADPH oxidation and the coupled ROS generation by isolated T-tubule vesicles yielded variable results (see below). The presence of the two NOX integral subunits gp91phox and p22phox in triads but not in heavy SR vesicles plus their marked enrichment in T-tubules reinforce the proposal that the skeletal NOX enzyme is a T-tubule enzyme. NADPH Oxidase Activity in Triads—As described below, isolated triad vesicles, which are composed by junctional T-tubule and SR membranes, contain a NOX activity that can oxidize both NADH- and NADPH-generating superoxide anion and H2O2. Isolated triads have the capacity to promote significant oxidation of 0.1 mm NADPH or NADH, as illustrated in Fig. 3A. On average, at 37 °C, triads oxidized NADPH at a rate of 25.1 ± 1.4 nmol/mg/min (n = 3) and NADH at a rate of 20.5 ± 1.3 nmol/mg/min (n = 7). Lowering the reaction temperature to 25 °C decreased NADPH oxidation to 14 ± 1.2 nmol/mg/min (n = 3). The addition to triads of increasing NADPH concentrations, from 100 μm to1mm, immediately elicited superoxide anion generation; at 25 °C, the reaction followed a linear time course for up to 20 min (Fig. 3B, left), and at 100 μm NADPH, the average rate was 2.01 ± 0.12 nmol/mg/min (n = 10). Increasing [NADPH] to 1 mm produced a hyperbolic increase in the rate of superoxide anion generation, with an apparent Km of 204 ± 49 μm and a Vmax of 6.25 ± 0.06 nmol/mg/min. The addition of 10 μm DPI or SOD (300 units/ml) inhibited markedly the rate of superoxide anion generation induced by 100 μm NADPH; 4 mm apocynin exerted a less pronounced inhibitory effect, whereas rotenone (200 nm) had no effect (Fig. 3B, right). Preincubation with 2 mm NG-nitro-l-arginine methyl ester, a nitric-oxide synthase inhibitor, did not produce detectable effects on superoxide anion generation (data not shown). These results make unlikely mitochondrial contribution or nitric-oxide synthase as sources of superoxide anion. The addition of 0.1 mm NADPH or NADH to triad vesicles induced H2O2 generation after a short delay (Fig. 4A, left). The average H2O2-generating activities at 37 °C (calculated from the linear part of the curves) were 3.3 ± 0.6 (n = 4) and 3.6 ± 0.4 (n = 6) (in nmol/mg/min) for triads incubated with 0.1 mm NADPH or NADH, respectively (Fig. 4A, right). At 25 °C, triads incubated with 0.1 mm NADPH generated 1.18 nmol of H2O2/mg
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