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• W2106894101 abstract "Tetrahydrobiopterin (BH4) is a key redox-active cofactor in endothelial isoform of NO synthase (eNOS) catalysis and is an important determinant of NO-dependent signaling pathways. BH4 oxidation is observed in vascular cells in the setting of the oxidative stress associated with diabetes. However, the relative roles of de novo BH4 synthesis and BH4 redox recycling in the regulation of eNOS bioactivity remain incompletely defined. We used small interference RNA (siRNA)-mediated “knockdown” GTP cyclohydrolase-1 (GTPCH1), the rate-limiting enzyme in BH4 biosynthesis, and dihydrofolate reductase (DHFR), an enzyme-recycling oxidized BH4 (7,8-dihydrobiopterin (BH2)), and studied the effects on eNOS regulation and biopterin metabolism in cultured aortic endothelial cells. Knockdown of either DHFR or GTPCH1 attenuated vascular endothelial growth factor (VEGF)-induced eNOS activity and NO production; these effects were recovered by supplementation with BH4. In contrast, supplementation with BH2 abolished VEGF-induced NO production. DHFR but not GTPCH1 knockdown increased reactive oxygen species (ROS) production. The increase in ROS production seen with siRNA-mediated DHFR knockdown was abolished either by simultaneous siRNA-mediated knockdown of eNOS or by supplementing with BH4. In contrast, addition of BH2 increased ROS production; this effect of BH2 was blocked by BH4 supplementation. DHFR but not GTPCH1 knockdown inhibited VEGF-induced dephosphorylation of eNOS at the inhibitory site serine 116; these effects were recovered by supplementation with BH4. These studies demonstrate a striking contrast in the pattern of eNOS regulation seen by the selective modulation of BH4 salvage/reduction versus de novo BH4 synthetic pathways. Our findings suggest that the depletion of BH4 is not sufficient to perturb NO signaling, but rather that concentration of intracellular BH2, as well as the relative concentrations of BH4 and BH2, together play a determining role in the redox regulation of eNOS-modulated endothelial responses. Tetrahydrobiopterin (BH4) is a key redox-active cofactor in endothelial isoform of NO synthase (eNOS) catalysis and is an important determinant of NO-dependent signaling pathways. BH4 oxidation is observed in vascular cells in the setting of the oxidative stress associated with diabetes. However, the relative roles of de novo BH4 synthesis and BH4 redox recycling in the regulation of eNOS bioactivity remain incompletely defined. We used small interference RNA (siRNA)-mediated “knockdown” GTP cyclohydrolase-1 (GTPCH1), the rate-limiting enzyme in BH4 biosynthesis, and dihydrofolate reductase (DHFR), an enzyme-recycling oxidized BH4 (7,8-dihydrobiopterin (BH2)), and studied the effects on eNOS regulation and biopterin metabolism in cultured aortic endothelial cells. Knockdown of either DHFR or GTPCH1 attenuated vascular endothelial growth factor (VEGF)-induced eNOS activity and NO production; these effects were recovered by supplementation with BH4. In contrast, supplementation with BH2 abolished VEGF-induced NO production. DHFR but not GTPCH1 knockdown increased reactive oxygen species (ROS) production. The increase in ROS production seen with siRNA-mediated DHFR knockdown was abolished either by simultaneous siRNA-mediated knockdown of eNOS or by supplementing with BH4. In contrast, addition of BH2 increased ROS production; this effect of BH2 was blocked by BH4 supplementation. DHFR but not GTPCH1 knockdown inhibited VEGF-induced dephosphorylation of eNOS at the inhibitory site serine 116; these effects were recovered by supplementation with BH4. These studies demonstrate a striking contrast in the pattern of eNOS regulation seen by the selective modulation of BH4 salvage/reduction versus de novo BH4 synthetic pathways. Our findings suggest that the depletion of BH4 is not sufficient to perturb NO signaling, but rather that concentration of intracellular BH2, as well as the relative concentrations of BH4 and BH2, together play a determining role in the redox regulation of eNOS-modulated endothelial responses. Regulation of endothelial nitric oxide (NO) 2The abbreviations used are: NO, nitric oxide; eNOS, endothelial isoform of nitric-oxide synthase; BH4, 5,6,7,8-tetrahydrobiopterin; BH2, 7,8-dihydrobiopterin; siRNA, small interfering RNA; GTPCH-1, guanosine triphosphate cyclohydrolase-1; DHFR, dihydrofolate reductase; ROS, reactive oxygen species; BAEC, bovine aortic endothelial cell; DAHP, 2,4-diamino-6-hydroxypyrimidine; VEGF, vascular endothelial growth factor; ANOVA, analysis of variance. 2The abbreviations used are: NO, nitric oxide; eNOS, endothelial isoform of nitric-oxide synthase; BH4, 5,6,7,8-tetrahydrobiopterin; BH2, 7,8-dihydrobiopterin; siRNA, small interfering RNA; GTPCH-1, guanosine triphosphate cyclohydrolase-1; DHFR, dihydrofolate reductase; ROS, reactive oxygen species; BAEC, bovine aortic endothelial cell; DAHP, 2,4-diamino-6-hydroxypyrimidine; VEGF, vascular endothelial growth factor; ANOVA, analysis of variance. production represents a critical mechanism for the modulation of vascular homeostasis. NO is released by endothelial cells in response to diverse humoral, neural, and mechanical stimuli (1Arnal J.F. Dinh-Xuan A.T. Pueyo M. Darblade B. Rami J. Cell Mol. Life Sci. 1999; 55: 1078-1087Crossref PubMed Scopus (260) Google Scholar, 2Fleming I. Busse R. Am. J. Physiol. 2003; 284: R1-R12PubMed Google Scholar, 3Loscalzo J. Welch G. Prog. Cardiovasc. Dis. 1995; 38: 87-104Crossref PubMed Scopus (506) Google Scholar, 4Shaul P.W. J. Physiol. 2003; 547: 21-33Crossref PubMed Scopus (135) Google Scholar). Endothelial cell-derived NO activates guanylate cyclase in vascular smooth muscle cells, leading to increased levels of cGMP and to smooth muscle relaxation. Blood platelets represent another key target for the actions of endothelium-derived NO (5Loscalzo J. Circ. Res. 2001; 88: 756-762Crossref PubMed Scopus (517) Google Scholar): platelet aggregation is inhibited by NO-induced guanylate cyclase activation. Many other effects of NO have been identified in cultured vascular cells and in vascular tissues, including the regulation of apoptosis, cell adhesion, angiogenesis, thrombosis, vascular smooth muscle proliferation, and atherogenesis, among other cellular responses and (patho)physiological processes. The endothelial isoform of NO synthase (eNOS) is a membrane-associated homodimeric 135-kDa protein that is robustly expressed in endothelial cells (2Fleming I. Busse R. Am. J. Physiol. 2003; 284: R1-R12PubMed Google Scholar, 4Shaul P.W. J. Physiol. 2003; 547: 21-33Crossref PubMed Scopus (135) Google Scholar, 6Dudzkinski D. Igarashi J. Greif D. Michel T. Annu. Rev. Pharmacol. Toxicol. 2006; 46: 235-276Crossref PubMed Scopus (318) Google Scholar, 7Michel T. Feron O. J. Clin. Invest. 1997; 100: 2146-2152Crossref PubMed Scopus (846) Google Scholar). Similar to all the mammalian NOS isoforms, eNOS functions as an obligate homodimer that includes a cysteine-complex Zn2+ (zinc-tetrathiolate) at the dimer interface (8Raman C.S. Li H. Martasek P. Kral V. Masters B.S. Poulos T.L. Cell. 1998; 95: 939-950Abstract Full Text Full Text PDF PubMed Scopus (571) Google Scholar, 9Hemmens B. Goessler W. Schmidt K. Mayers B. J. Biol. Chem. 2000; 275: 35786-35791Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 10Kolodziejski P.J. Rashid M.B. Eissa N.T. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 14263-14268Crossref PubMed Scopus (32) Google Scholar). eNOS is a Ca2+/calmodulin-dependent enzyme that is activated in response to the stimulation of a variety of Ca2+-mobilizing cell surface receptors in vascular endothelium and in cardiac myocytes. The activity of eNOS is also regulated by phosphorylation at multiple sites (11Shaul P.W. Annu. Rev. Physiol. 2002; 64: 749-774Crossref PubMed Scopus (468) Google Scholar) that are differentially modulated following the activation of cell surface receptors by agonists such as insulin and vascular endothelial growth factor (VEGF) (12He H. Venema V.J. Gu X. Venema R.C. Marrero M.B. Caldwell R.B. J. Biol. Chem. 1999; 274: 25130-25135Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar). The phosphorylation of eNOS at Ser-1179 activates eNOS, but phosphorylation at Thr-497 or Ser-116 is associated with inhibition of eNOS activity (13Fulton D. Gratton J.P. McCabe T.J. Fontana J. Fujio Y. Walsh K. Franke T.F. Papapetropoulos A. Sessa W.C. Nature. 1999; 399: 597-601Crossref PubMed Scopus (2214) Google Scholar, 14Michell B.J. Griffiths J.E. Mitchelhill K.I. Rodriguez-Crespo I. Tiganis T. Bozinovski S. de Montellano P.R. Kemp B.E. Pearson R.B. Curr. Biol. 1999; 9: 845-848Abstract Full Text Full Text PDF PubMed Scopus (412) Google Scholar, 15Dimmeler S. Fleming I. Fisslthaler B. Hermann C. Busse R. Zeiher A.M. Nature. 1999; 399: 601-605Crossref PubMed Scopus (3024) Google Scholar, 16Kou R. Greif D. Michel T. J. Biol. Chem. 2002; 277: 29669-29673Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 17Harris M.B. Ju H. Venema V.J. Liang H. Zou R. Michell B.J. Chen Z.P. Kemp B.E. Venema R.C. J. Biol. Chem. 2001; 276: 16587-16591Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar). eNOS is reversibly targeted to plasmalemmal caveolae as a consequence of the protein's N-myristoylation and thiopalmitoylation. The generation of NO by eNOS requires several redox-active cofactors, including nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), calmodulin, and tetrahydrobiopterin (BH4), which have key roles in the electron flow required for eNOS catalysis. If the flow of electrons within eNOS is disrupted, the enzyme is uncoupled from NO production and other redox-active products are generated, including hydrogen peroxide and superoxide anion radical (18Vasquez-Vivar J. Kalyanaraman B. Martasek P. Hogg N. Masters B.S. Karoui H. Tordo P. Pritchard Jr., K.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9220-9225Crossref PubMed Scopus (1220) Google Scholar, 19Wever R.M. van Dam T. van Rijn H.J. de Groot F. Rabelink T.J. Biochem. Biophys. Res. Commun. 1997; 237: 340-344Crossref PubMed Scopus (245) Google Scholar). In vascular disease states such as diabetes, endothelial dysfunction is characterized by a decrease in NO bioactivity and by a concomitant increase in superoxide formation, while eNOS mRNA and protein levels are maintained or even increased. “Uncoupled” eNOS generates reactive oxygen species (ROS), shifting the nitroso-redox balance and having adverse consequences in the vascular wall (20Heller R. Werner-Felmayer G. Wener E.R. Eur. J. Clin. Pharmacol. 2006; 62: 21-28Crossref Scopus (36) Google Scholar). Several enzymes expressed in vascular tissues contribute to the production and efficient degradation of ROS, and an enhanced activity of oxidant enzymes and/or reduced activity of antioxidant enzymes may cause oxidative stress. Various agonists, pathological conditions, and therapeutic interventions lead to modulated expression and function of oxidant and antioxidant enzymes. However, the intimate relationship between intracellular redox state, eNOS regulation, and NO bioavailability remains incompletely characterized. BH4 is a key redox-active cofactor for activity of all NOS enzymes (21Cosentino F. Luscher T.F. Cardiovasc. Res. 1999; 43: 274-278Crossref PubMed Scopus (147) Google Scholar). The exact role of BH4 in NOS catalysis is not yet completely defined, but this cofactor appears to facilitate electron transfer from the eNOS reductase domain and maintains the heme prosthetic group of the enzyme in its redox-active form (18Vasquez-Vivar J. Kalyanaraman B. Martasek P. Hogg N. Masters B.S. Karoui H. Tordo P. Pritchard Jr., K.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9220-9225Crossref PubMed Scopus (1220) Google Scholar, 22Stuehr D.J. Biochim. Biophys. Acta. 1999; 1411: 217-230Crossref PubMed Scopus (806) Google Scholar, 23Stuehr D. Pou S. Rosen G.M. J. Biol. Chem. 2001; 276: 14533-14536Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar). Moreover, BH4 promotes formation of active NOS homodimers (24Tzeng E. Billiar T.R. Robbins P.D. Loftus M. Stuehr D.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11771-11775Crossref PubMed Scopus (175) Google Scholar) and inhibits the formation of hydrogen peroxide or superoxide by uncoupled eNOS (18Vasquez-Vivar J. Kalyanaraman B. Martasek P. Hogg N. Masters B.S. Karoui H. Tordo P. Pritchard Jr., K.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9220-9225Crossref PubMed Scopus (1220) Google Scholar, 19Wever R.M. van Dam T. van Rijn H.J. de Groot F. Rabelink T.J. Biochem. Biophys. Res. Commun. 1997; 237: 340-344Crossref PubMed Scopus (245) Google Scholar). It has been reported that the endothelial dysfunction associated with diabetes is accompanied a decrease in the abundance of bioactive BH4. Supplementation with BH4 has been shown to improve endothelial function in the models of diabetes and hypertension (25Moens A.L. Kass D.A. Arterioscler. Thromb. Vasc. Biol. 2006; 26: 2439-2444Crossref PubMed Scopus (157) Google Scholar, 26Katusic Z.S. Am. J. Physiol. 2001; 28: H981-H986Google Scholar, 27Channon K.M. Trends Cardiovasc. Med. 2004; 14: 323-327Crossref PubMed Scopus (174) Google Scholar). Moreover, BH4 oxidation is seen in vascular cells in the setting of oxidative stress associated with diabetes (28Meininger C.J. Marinos R.S. Hatakeyama K. Martinez-Zaguilan R. Rojas J.D. Kelly K.A. Wu G. Biochem. J. 2000; 349: 353-356Crossref PubMed Scopus (163) Google Scholar) and hypertension (29Landmesser U. Dikalov S. Price S.R. McCann L. Fukai T. Holland S.M. Mitch W.E. Harrison D.G. J. Clin. Invest. 2003; 111: 1201-1209Crossref PubMed Scopus (1353) Google Scholar). BH4 can be formed either by a de novo biosynthetic pathway or by a salvage pathway. Guanosine triphosphate cyclohydrolase-1 (GTPCH1) catalyzes the conversion of GTP to dihydroneopterin triphosphate. BH4 is generated by further steps catalyzed by 6-pyruvoyltetrahydropterin synthase and sepiapterin reductase (30Thorny B. Auerbach G. Blau N. Biochem. J. 2000; 347: 1-16Crossref PubMed Google Scholar). GTPCH1 appears to be the rate-limiting enzyme in BH4 biosynthesis; overexpression of GTPCH1 is sufficient to augment BH4 levels in cultured endothelial cells (31Cai S. Alp N.J. McDonald D. Smith I. Kay J. Canevari L. Healeas S. Cardiovasc. Res. 2002; 55: 838-849Crossref PubMed Scopus (99) Google Scholar). On the other hand, dihydrofolate reductase (DHFR) catalyzes the regeneration of BH4 from its oxidized form, 7,8-dihydrobiopterin (BH2), in several cell types (30Thorny B. Auerbach G. Blau N. Biochem. J. 2000; 347: 1-16Crossref PubMed Google Scholar, 32Werner-Felmayer G. Golderer G. Werner E.R. Curr. Drug Metab. 2002; 3: 159-173Crossref PubMed Scopus (141) Google Scholar). DHFR is mainly involved in folate metabolism and converts inactive BH2 back to BH4 and plays an important role in the metabolism of exogenously administered BH4. However, the relative contributions of endothelial GTPCH1 and DHFR to the modulation of eNOS-dependent pathways are incompletely understood. In these studies, we have used siRNA-mediated “knockdown” of GTPCH1 and DHFR to explore the relative roles of BH4 synthesis and recycling in the modulation of eNOS bioactivity, as well as in the regulation of NO-dependent signaling pathways in endothelial cells. Materials—Fetal bovine serum was from HyClone Laboratories (Logan, UT). Lipofectamine™ 2000, Amplex™ Red reagent, and most cell culture reagents were from Invitrogen. Polyclonal antibodies against phospho-Akt (Ser-473), Akt, and phospho-eNOS (Ser-1179) were from Cell Signaling Technology, Inc. (Danvers, MA). Polyclonal antibody against phospho-Ser-116-eNOS was from Upstate Biotechnology (Lake Placid, NY). Monoclonal antibodies against eNOS and DHFR were from BD Transduction Laboratories (Lexington, KY). Polyclonal antibody against GTPCH1 was from Novus Biologicals (Littleton, CO). The SuperSignal chemiluminescence detection reagents and secondary antibodies conjugated with horseradish peroxidase were from Pierce. Tetrahydro-l-biopterin (BH4), 7,8-dihydro-l-biopterin (BH2), and l-biopterin were from Cayman Chemical (Ann Arbor, MI). VEGF was from Calbiochem. Protein concentrations were determined using protein assay kits from Bio-Rad Laboratories (Philadelphia, PA). Determinations of protein abundance using immunoblot analyses were quantitated using a ChemiImager HD4000 (AlphaInnotech, San Leandro, CA). All other reagents were from Sigma. Culture and Treatment of Cells—Bovine aortic endothelial cells (BAECs) were obtained from Cambrex (Walkersville, MD) and maintained in culture on gelatin-coated 100-mm culture dishes with Dulbecco's modified Eagle's medium supplemented with fetal bovine serum (10% v/v) as described previously (33Michel T. Li G.K. Busconi L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6252-6256Crossref PubMed Scopus (305) Google Scholar). BAECs are used for experiments between passages 6 and 8, and serum-starved overnight before experiments. Treatments with VEGF and preparation of cell lysates were performed as described previously (34Kou R. Michel T. J. Biol. Chem. 2007; 282: 32719-32729Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), with corresponding vehicle treatments as controls. siRNA Preparation and Transfection—Consistent with previous work (35Gonzalez E. Kou R. Lin A.J. Golan D.E. Michel T. J. Biol. Chem. 2002; 277: 39554-39560Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar), we designed an siRNA duplex targeting bovine GTPCH1 mRNA (GTPCH1 siRNA, sequence 5′-CCGUGACGAGCACAAUGUU-3′, corresponding to bases 856–874 from the open reading frame of the bovine GTPCH1 mRNA; GenBank™ accession number XM_001251704.2). DHFR siRNA (sequence 5′-CCCAGAACAUGGGCAUCGGC-3′, corresponding to bases 527–546 from the open reading frame of the human DHFR mRNA; GenBank™ accession number NM_000791.3) was characterized in an earlier report (36Tai N. Schmitz J.C. Chen T.M. Chu E. Biochem. J. 2004; 378: 999-1006Crossref PubMed Scopus (25) Google Scholar). The siRNA duplex oligonucleotides were from Ambion, Inc. (Austin, TX). The nonspecific control siRNA 5′-AUUGUAUGCGAUCGCAGAC-dTdT-3′ was from Dharmacon, Inc. (Lafayette, CO). We found that optimal conditions for siRNA knockdown involved transfecting BAECs when cells were at 50–70% confluence, and transfected cells were maintained in Dulbecco's modified Eagle's medium/10% fetal bovine serum; transfections with siRNA (30–45 nm) used Lipofectamine™ 2000 (0.15%, v/v) and followed protocols provided by the manufacturer (Invitrogen). Fresh medium was added 5 h after transfection, and experiments were typically conducted 48 h after transfection. Measurement of Intracellular Levels of Biopterins—Oxidized and reduced forms of biopterins were analyzed by the differential oxidation method of Fukushima and Nixon (37Fukushima T. Nixon J.C. Anal. Biochem. 1980; 102: 176-188Crossref PubMed Scopus (657) Google Scholar). The whole procedure was performed in the dark. BAECs were washed and suspended in cold extract buffer (0.1 m phosphoric acid, 5 mm dithiothreitol), and protein concentration was measured using the Bio-Rad protein assay. Proteins were removed by adding 35 μl of 2 m trichloroacetic acid to 300 μl of the extracts, followed by centrifugation. To determine total biopterins (BH4, BH2, and biopterin) by acid oxidation, 100 μl of cell extract was mixed with 15 μl of 0.2 m trichloroacetic acid and 15 μl of 1% iodine in 2% KI in 0.2 m trichloroacetic acid. To determine BH2 and biopterin by alkali oxidation, 15 μl of 1 m NaOH was added to 100 μl of extract, followed by addition of 15 μl of 1% iodine/2% KI in 3 m NaOH. After incubation at room temperature for 1 h in the dark, excess iodine was reduced by adding 25 μl of fresh ascorbic acid (20 mg/ml). After centrifugation, 10 μl of supernatant was injected into a high-performance liquid chromatography system (Agilent 1100 series, Agilent Technologies, Palo Alto, CA) equipped with a 150- × 0.32-mm ODS Hypersil column (Thermo Scientific, Waltham, MA), and couple to a helium-cadmium laser-induced highly sensitive fluorescent detector (325 nm laser, series 56, Melles Griot, Carlsbad, CA; ZETALIF detector, Model LIF-SA-03, Picometrics, Ramonville, France), as described in Ref. 38Levy B.D. Bonnans C. Silverman E.S. Palmer L.J. Marigowda G. Israel E. Am. J. Respir. Crit. Care Med. 2005; 172: 824-830Crossref PubMed Scopus (219) Google Scholar. The mobile phase was methanol:doubly distilled H2O (5:95, v/v) with a flow rate of 400 μl/min that was reduced to 4 μl/min with a precolumn flow splitter (100:1, series 620, Analytical Scientific Instruments, El Sobrante, CA) before laser-induced fluorescence detection. The criteria used for identification of biopterin were fluorescence, and retention time was compared with the standards. BH4 concentration, expressed as picomoles per milligram of protein, was calculated by subtracting BH2 plus biopterin from total biopterins. eNOS Activity Assay—eNOS activity was quantified as the formation of l-[3H]citrulline from l-[3H]arginine as described before (16Kou R. Greif D. Michel T. J. Biol. Chem. 2002; 277: 29669-29673Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). Briefly, the reactions are initiated by adding l-[3H]arginine (10 μCi/ml, diluted with unlabeled l-arginine to give a final concentration of 10 μm) plus various drug treatments; each treatment was performed in triplicate cultures, which were analyzed in duplicate. Measurement of NO–2—NO production from cells was measured by using an NO sensor (BioStat, ESA, Inc., Chelmsford, MA). Cell culture medium was replaced with Dulbecco's phosphate-buffered saline, and various drugs were added as indicated. After incubation for varying times, aliquots were removed and added to the reagent solution (100 mm H2SO4, 100 mm NaI) with the NO sensor. The amount of redox current generated is directly proportional to the NO–2 concentration in the solution. The absolute NO–2 concentrations were calculated against a standard curve prepared by using NaNO2 solution. Cells were then harvested to determine protein concentration, permitting NO–2 production to be reported as picomoles per mg of protein. Measurement of H2O2—H2O2 production from cells was quantitated using the Amplex™ Red fluorescence assay using previously described methods (39Rinaldi M. Moroni P. Paapa M.J. Bannerman D.D. Vet. Immunol. Imuunopathol. 2007; 115: 107-125Crossref PubMed Scopus (76) Google Scholar). Cell culture medium was replaced with Amplex Red reaction mixture, which consists of 50 μm Amplex Red, 0.1 unit/ml horseradish peroxidase, and 10 units/ml superoxide dismutase diluted in Dulbecco's phosphate-buffered saline (10 units/ml superoxide dismutase is sufficient to convert all cell-derived superoxide to hydrogen peroxide; data not shown). After incubation for varying times as indicated, aliquots were withdrawn and fluorescence was quantitated using a FluoroMax-2 spectrofluorometer (JY-Spex, Edison, NJ) at an excitation wavelength of 530 nm and an emission wavelength of 590 nm. The absolute H2O2 concentrations were calculated against a standard curve prepared by using H2O2 solution. Cells were then harvested to determine protein concentration, permitting H2O2 production to be reported as picomoles per mg of protein. Immunoblot Analysis—BAEC lysates were prepared using a cell lysis buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1% Nonidet P-40, 0.025% sodium deoxycholate, 1 mm EDTA, 2 mm Na3VO4, 1 mm NaF, 2 μg/ml leupeptin, 2 μg/ml antipain, 2 μg/ml soybean trypsin inhibitor, and 2 μg/ml lima trypsin inhibitor). Immunoblot analyses of endothelial protein expression and phosphorylation were assessed as described previously in detail (34Kou R. Michel T. J. Biol. Chem. 2007; 282: 32719-32729Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Quantitative densitometric analyses of immunoblots were performed using a ChemiImager HD4000 (AlphaInnotech). Statistical Analysis—All experiments were performed at least three times. Mean values for individual experiments are presented as mean ± S.E. Statistical differences were assessed by ANOVA or t test when appropriate. A p value of less than 0.05 was considered significant. siRNA-mediated Down-regulation of DHFR and GTPCH1 Expression in BAECs—To selectively knockdown the expression of DHFR and GTPCH1 protein in BAECs, we transfected siRNA duplex-targeting constructs, as described in detail under “Experimental Procedures.” After validating the specificity of these DHFR and GTPCH1 siRNA constructs and optimizing experimental conditions for optimal protein knockdown, we determined the effects of “double knockdown” of these proteins transfecting both DHFR and GTPCH1 siRNA-targeting constructs in BAECs (Fig. 1). We analyzed immunoblots probed for DHFR, GTPCH1, or β-actin in cells transfected with DHFR siRNA and/or GTPCH1 siRNA constructs; the same quantity of siRNA was used in each transfection using nonspecific siRNA as indicated. DHFR protein expression was efficiently and specifically knocked down by transfection with DHFR siRNA, co-transfected either with control siRNA or GTPCH1-specific siRNA. Similarly, GTPCH1 expression was efficiently knocked down by transfection with GTPCH1 siRNA along with either control siRNA or DHFR-specific siRNA. Co-transfection of the DHFR siRNA along with GTPCH1 siRNA targeting constructs led to marked knockdown of both proteins. Under all experimental conditions, the levels of β-actin protein in cell lysates remained unchanged; as can be seen in this and subsequent figures and as noted in a prior report (36Tai N. Schmitz J.C. Chen T.M. Chu E. Biochem. J. 2004; 378: 999-1006Crossref PubMed Scopus (25) Google Scholar), expression of other endothelial proteins was not affected by transfection of DHFR and/or GTPCH1 siRNA-targeting constructs. Intracellular Levels of BH4 and BH2 in siRNA-transfected BAECs—We next determined the intracellular concentrations of biopterins following siRNA-mediated knockdown of DHFR or GTPCH1 to determine the effects of these knockdowns on pterin metabolites (Table 1). As would be anticipated from its critical role in pterin synthesis, GTPCH1 knockdown markedly decreased the intracellular concentration of total biopterins, including BH4 and BH2 (82 ± 1% decrease compared with control siRNA-transfected cells, n = 3, p < 0.01), whereas the ratio of BH4 to BH2 was not affected (Fig. 2). On the other hand, DHFR knockdown significantly decreased the intracellular BH4 level (56 ± 10% decrease in BH4 concentration compared with control siRNA-transfected cells, n = 3, p < 0.01), with no significant change in the amount of total cellular biopterins (Table 1). As shown in Fig. 2, the percentage of BH4 relative to total cellular biopterins was 79 ± 9% in control siRNA-transfected cells; 28 ± 11% in DHFR-knockdown cells (p < 0.01 compared with control cells); and 74 ± 9% in GTPCH1-knockdown cells (p not significantly different from control), respectively. These data indicate that DHFR siRNA-mediated suppression of the BH4 recycling pathway results in a relative increase in cellular BH2 levels. Because a number of studies have shown that administration of BH4 acutely improve endothelial function, we examined the effects of supplementation of BAECs with BH4 and BH2 on the intracellular levels of biopterins. As shown in Table 1, supplementation with BH4 (10 μm for 1 h) induced marked increases in total intracellular biopterins in treated cells. The decrease in BH4 levels seen with siRNA-mediated DHFR knockdown was recovered to the same levels as control cells following supplementation with BH4, as shown in Fig. 2 and Table 1. Supplementation with BH2 (10 μm, 1 h) also yielded marked increases in the levels of total biopterins (Table 1). However, as shown in Fig. 2, the proportion of BH4 in the BH2-treated cells was significantly and uniformly decreased compared with BH4-supplemented cells (n = 3, p < 0.01), independent of siRNA transfection (Table 1).TABLE 1Intracellular levels of biopterins in siRNA-transfected endothelial cells supplemented with BH4 or BH2SupplementBiopterins concentrationControl siRNADHFR siRNAGTPCH1 siRNA[Total biopterin][BH4][Total biopterin][BH4][Total biopterin][BH4]pmol/mg proteinVehicle3.8 ± 0.53.0 ± 0.34.6 ± 1.31.3 ± 0.5ap < 0.01 versus the BH4 concentration in control siRNA-transfected cells supplemented with vehicle (p values determined by ANOVA).0.7 ± 0.1bp < 0.01 versus total biopterins concentration in control siRNA-transfected cells incubated with vehicle.0.5 ± 0.1ap < 0.01 versus the BH4 concentration in control siRNA-transfected cells supplemented with vehicle (p values determined by ANOVA).BH422.5 ± 3.3bp < 0.01 versus total biopterins concentration in control siRNA-transfected cells incubated with vehicle.18.7 ± 3.6ap < 0.01 versus the BH4 concentration in control siRNA-transfected cells supplemented with vehicle (p values determined by ANOVA).20.4 ± 3.1bp < 0.01 versus total biopterins concentration in control siRNA-transfected cells incubated with vehicle.14.4 ± 2.3ap < 0.01 versus the BH4 concentration in control siRNA-transfected cells supplemented with vehicle (p values determined by ANOVA).23.4 ± 6.6bp < 0.01 versus total biopterins concentration in control siRNA-transfected cells incubated with vehicle.20.2 ± 5.3ap < 0.01 versus the BH4 concentration in control siRNA-transfected cells supplemented with vehicle (p values determined by ANOVA).BH217.7 ± 3.6bp < 0.01 versus total biopterins concentration in control siRNA-transfected cells incubated with vehicle.6.6 ± 3.519.1 ± 2.8bp < 0.01 versus total biopterins concentration in control siRNA-transfected cells incubated with vehicle.2.5 ± 2.020.1 ± 3.2bp < 0.01 versus total biopterins con" @default.
• W2106894101 created "2016-06-24" @default.
• W2106894101 creator A5050132863 @default.
• W2106894101 creator A5055920645 @default.
• W2106894101 creator A5084285813 @default.
• W2106894101 date "2009-05-01" @default.
• W2106894101 modified "2023-09-28" @default.
• W2106894101 title "Tetrahydrobiopterin Recycling, a Key Determinant of Endothelial Nitric-oxide Synthase-dependent Signaling Pathways in Cultured Vascular Endothelial Cells" @default.
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