FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2022820008> ?p ?o ?g. }

• W2022820008 endingPage "8685" @default.
• W2022820008 startingPage "8678" @default.
• W2022820008 abstract "Nitric oxide (NO) is an important signaling molecule, and a number of NO synthesis inhibitors and scavengers have been developed to allow study of NO functions and to reduce excess NO levels in disease states. We showed previously that cobinamide, a cobalamin (vitamin B12) precursor, binds NO with high affinity, and we now evaluated the potential of cobinamide as a NO scavenger in biologic systems. We found that cobinamide reversed NO-stimulated fluid secretion in Drosophila Malpighian tubules, both when applied in the form of a NO donor and when produced intracellularly by nitricoxide synthase. Moreover, feeding flies cobinamide markedly attenuated subsequent NO-induced increases in tubular fluid secretion. Cobinamide was taken up efficiently by cultured rodent cells and prevented NO-induced phosphorylation of the vasodilator-stimulated phosphoprotein VASP both when NO was provided to the cells and when NO was generated intracellularly. Cobinamide appeared to act via scavenging NO because it reduced nitrite and nitrate concentrations in both the fly and mammalian cell systems, and it did not interfere with cGMP-induced phosphorylation of VASP. In rodent and human cells, cobinamide exhibited toxicity at concentrations ≥50 μm with toxicity completely prevented by providing equimolar amounts of cobalamin. Combining cobalamin with cobinamide had no effect on the ability of cobinamide to scavenge NO. Cobinamide did not inhibit the in vitro activity of either of the two mammalian cobalamin-dependent enzymes, methionine synthase or methylmalonyl-coenzyme A mutase; however, it did inhibit the in vivo activities of the enzymes in the absence, but not presence, of cobalamin, suggesting that cobinamide toxicity was secondary to interference with cobalamin metabolism. As part of these studies, we developed a facile method for producing and purifying cobinamide. We conclude that cobinamide is an effective intra- and extracellular NO scavenger whose modest toxicity can be eliminated by cobalamin. Nitric oxide (NO) is an important signaling molecule, and a number of NO synthesis inhibitors and scavengers have been developed to allow study of NO functions and to reduce excess NO levels in disease states. We showed previously that cobinamide, a cobalamin (vitamin B12) precursor, binds NO with high affinity, and we now evaluated the potential of cobinamide as a NO scavenger in biologic systems. We found that cobinamide reversed NO-stimulated fluid secretion in Drosophila Malpighian tubules, both when applied in the form of a NO donor and when produced intracellularly by nitricoxide synthase. Moreover, feeding flies cobinamide markedly attenuated subsequent NO-induced increases in tubular fluid secretion. Cobinamide was taken up efficiently by cultured rodent cells and prevented NO-induced phosphorylation of the vasodilator-stimulated phosphoprotein VASP both when NO was provided to the cells and when NO was generated intracellularly. Cobinamide appeared to act via scavenging NO because it reduced nitrite and nitrate concentrations in both the fly and mammalian cell systems, and it did not interfere with cGMP-induced phosphorylation of VASP. In rodent and human cells, cobinamide exhibited toxicity at concentrations ≥50 μm with toxicity completely prevented by providing equimolar amounts of cobalamin. Combining cobalamin with cobinamide had no effect on the ability of cobinamide to scavenge NO. Cobinamide did not inhibit the in vitro activity of either of the two mammalian cobalamin-dependent enzymes, methionine synthase or methylmalonyl-coenzyme A mutase; however, it did inhibit the in vivo activities of the enzymes in the absence, but not presence, of cobalamin, suggesting that cobinamide toxicity was secondary to interference with cobalamin metabolism. As part of these studies, we developed a facile method for producing and purifying cobinamide. We conclude that cobinamide is an effective intra- and extracellular NO scavenger whose modest toxicity can be eliminated by cobalamin. Nitric oxide (NO) 1The abbreviations used are: NO, nitric oxide; BHK, baby hamster kidney cells; Deta-NONOate, (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; G-kinase, cGMP-dependent protein kinase; HPLC, high performance liquid chromatography; LPS, lipopolysaccharide; NOS, nitric-oxide synthase; OH-Cbl, hydroxocobalamin; PAPA-NONOate, (Z)-1-[N-(3-ammoniopropyl)-N-(n-propyl)amino]-diazen-1-ium-1,2-diolate; 8-pCPT-cGMP, 8-parachlorophenylthiocyclic GMP; VASP, vasodilator-stimulated phosphoprotein; VSV, vesicular stomatitis virus. has multiple cellular functions including regulation of cell growth, differentiation, and apoptosis, and many physiological roles including modulation of blood pressure, platelet aggregation, and synaptic plasticity (1Lloyd-Jones D.M. Bloch K.D. Annu. Rev. Med. 1996; 47: 365-375Crossref PubMed Scopus (215) Google Scholar, 2Ignarro L. Murad F. Adv. Pharmacol. 1995; 45: 105-234Google Scholar, 3Hölsher C. Trends Neurosci. 1997; 20: 298-303Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar). Studies of NO functions have been aided by pharmacologic agents that raise or lower NO levels (4Moncada S. Palmer R.M.J. Higgs E.A. Pharmacol. Rev. 1991; 43: 109-142PubMed Google Scholar). A number of disease states including sepsis and hepatic failure are characterized by abnormally high NO production, and removing the excess NO could have salutary effects (5Shah V. Lyford G. Gores G. Farrugia G. Gastroenterology. 2004; 126: 903-913Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar, 6Komeno M. Akimoto A. Fujita T. Aramaki T. Aoki M. Shimada T. Ohashi F. J. Vet. Med. Sci. 2004; 66: 53-57Crossref PubMed Scopus (8) Google Scholar, 7Pfeilschifter J. Eberhardt W. Beck K.F. Pflugers Arch. 2001; 442: 479-486Crossref PubMed Scopus (113) Google Scholar). One approach to lower NO concentrations is to reduce NO synthesis. Four nitric-oxide synthase (NOS) isoforms are present in mammals: neuronal NOS (nNOS or NOS I), inducible NOS (iNOS or NOS II), endothelial NOS (eNOS or NOS III), and a recently described mitochondrial NOS (mtNOS) (8Nedvetsky P.I. Sessa W.C. Schmidt H.H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16510-16512Crossref PubMed Scopus (66) Google Scholar, 9Elfering S.L. Sarkela T.M. Giulivi C. J. Biol. Chem. 2002; 277: 38079-38086Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar). nNOS and eNOS are constitutively expressed in many tissues and produce pico to nanomolar concentrations of NO in response to increased intracellular calcium (10Clancy R.M. Abramson S.B. Proc. Soc. Exp. Biol. Med. 1995; 210: 93-101Crossref PubMed Scopus (248) Google Scholar). iNOS is expressed in a variety of cell types, and its level can be induced many-fold by endotoxin (lipopolysaccharide, LPS) and tumor necrosis factor-α, with iNOS producing almost 1,000 times higher NO, i.e. nano to micromolar, concentrations than nNOS and eNOS (7Pfeilschifter J. Eberhardt W. Beck K.F. Pflugers Arch. 2001; 442: 479-486Crossref PubMed Scopus (113) Google Scholar, 11Poon B.Y. Raharjo E. Patel K.D. Tavener S. Kubes P. Circulation. 2003; 108: 1107-1112Crossref PubMed Scopus (66) Google Scholar, 12Morin M.J. Unno N. Hodin R.A. Fink M.P. Crit. Care Med. 1998; 26: 1258-1264Crossref PubMed Scopus (73) Google Scholar, 13Nathan C. FASEB J. 1992; 6: 3051-3064Crossref PubMed Scopus (4161) Google Scholar). Less is known about mtNOS, but it accounts for 50% of cellular NO production in rat liver, and low micromolar NO concentrations have been found in rat heart mitochondria (9Elfering S.L. Sarkela T.M. Giulivi C. J. Biol. Chem. 2002; 277: 38079-38086Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar, 14Saavedra-Molina A. Ramirez-Emiliano J. Clemente-Guerrero M. Perez-Vazquez V. Aguilera-Aguirre L. Gonzalez-Hernandez J.C. Amino Acids. 2003; 24: 95-102Crossref PubMed Scopus (22) Google Scholar). A large number of NOS inhibitors have been generated, most of which are arginine analogs including isothiourea derivatives, and because of the multiple biochemical roles of arginine, these agents can have affects other than NOS inhibition (15Garvey E.P. Oplinger J.A. Tanoury G.J. Sherman P.A. Fowler M. Marshall S. Harmon M.F. Paith J.E. Furfine E.S. J. Biol. Chem. 1994; 269: 26669-26676Abstract Full Text PDF PubMed Google Scholar, 16El Mabrouk M. Singh A. Touyz R.M. Schiffrin E.L. Life Sci. 2000; 67: 1613-1623Crossref PubMed Scopus (17) Google Scholar, 17Hallemeesch M.M. Cobben D.C. Soeters P.B. Deutz N.E. Clin. Nutr. 2002; 21: 111-117Abstract Full Text PDF PubMed Scopus (12) Google Scholar). Another approach to reduce NO levels is to use a NO scavenger. For example, the heme moiety of hemoglobin binds NO with great avidity, but heme and free extracellular hemoglobin can be highly toxic, particularly in whole animals (18Kim H.W. Greenburg A.G. Shock. 2002; 17: 423-426Crossref PubMed Scopus (21) Google Scholar). Thus, other NO scavengers have been considered including dithiocarbamate derivatives that chelate iron and thus bind NO, but these too can have adverse effects (19Menezes J. Hierholzer C. Watkins S.C. Lyons V. Peitzman A.B. Billiar T.R. Tweardy D.J. Harbrecht B.G. Am. J. Physiol. 1999; 277: G144-G151PubMed Google Scholar, 20Nadler E.P. Dickinson E.C. Beer-Stolz D. Alber S.M. Watkins S.C. Pratt D.W. Ford H.R. Am. J. Physiol. 2001; 281: G173-G181PubMed Google Scholar). Cobalamin (vitamin B12) is structurally similar to heme and also binds NO, but with considerably less efficiency than heme (21Greenberg S.S. Xie J. Zatarain J.M. Kapusta D.R. Miller M.J.S. J. Pharmacol. Exp. Ther. 1995; 273: 257-265PubMed Google Scholar). We found recently that the cobalamin precursor cobinamide, which lacks the dimethylbenzimidazole ribonucleotide tail of cobalamin (Fig. 1), has a more than 100 times greater affinity for NO than cobalamin; moreover, each cobinamide molecule can potentially neutralize two NO molecules compared with only one NO molecule for cobalamin (22Sharma V.S. Pilz R.B. Boss G.B. Magde D. Biochemistry. 2003; 42: 8900-8908Crossref PubMed Scopus (67) Google Scholar). We now present studies demonstrating the use of cobinamide as a NO scavenger, both in a Drosophila model and in cultured mammalian cells. Cobinamide became toxic to cells at concentrations considerably higher than would be needed to neutralize NO produced in vivo, and the toxicity was reversed fully by cobalamin; cobalamin did not affect NO scavenging by cobinamide, and thus the combination of these two corrinoids could be a very effective method to scavenge NO. Production and Analysis of Cobinamide—Approximately 200 mg of hydroxocobalamin (OH-Cbl, Sigma) was dissolved in 1 ml of concentrated HCl and heated at 65 °C for 8 min to hydrolyze the phosphoester bond linking the dimethylbenzimidazole ribonucleotide moiety to the corrin ring (Fig. 1 and Ref. 23Hayward G.C. Hill H.A.O. Pratt J.M. Vanston N.J. Williams A.R.W. J. Chem. Soc. 1965; : 6485-6493Crossref Google Scholar). The solution was cooled on ice and applied to a l-ml C18 solid phase extraction column (Fisher Scientific). The HCl and unreacted OH-Cbl were removed by batch elution in water and 10% acetone, respectively, and the diaquocobinamide (cobinamide) product was eluted in 20% acetone and concentrated under reduced pressure in a SpeedVac (Savant Industries). Purity of the cobinamide was confirmed both spectrophotometrically by comparison with published spectra (22Sharma V.S. Pilz R.B. Boss G.B. Magde D. Biochemistry. 2003; 42: 8900-8908Crossref PubMed Scopus (67) Google Scholar, 24Baldwin D.A. Betterton E.A. Pratt J.M. J. Chem. Soc. Dalton Trans. 1983; : 217-223Crossref Google Scholar, 25Ford S.H. Nichols A. Shambee M. J. Inorg. Biochem. 1991; 41: 235-244Crossref PubMed Scopus (11) Google Scholar) and by high performance liquid chromatography (HPLC) using a C18 reversed phase column eluted isocratically in 100 mm NaH2PO4, pH 4.0, and 15% methanol (v/v) (25Ford S.H. Nichols A. Shambee M. J. Inorg. Biochem. 1991; 41: 235-244Crossref PubMed Scopus (11) Google Scholar, 26Boss G.R. J. Biol. Chem. 1984; 259: 2936-2941Abstract Full Text PDF PubMed Google Scholar); the column effluent was monitored at multiple wavelengths by a diode array detector. The yield was generally >80%, or ∼160 mg of cobinamide was produced per batch. When stored at –20 °C, the cobinamide was spectroscopically and biologically stable for at least 1 month. Assessment of Malpighian Tubule Secretion in Drosophila melanogaster—An elegant in vitro method has been devised to measure fluid transport by the Malpighian tubules of D. melanogaster; tubular secretion is stimulated markedly by NO donors and LPS, the latter via induction of the Drosophila NOS gene (27Broderick K.E. MacPherson M.R. Regulski M. Tully T. Dow J.A. Davies S.A. Am. J. Physiol. 2003; 285: C1207-C1218Crossref Scopus (40) Google Scholar, 28Dow J.T. Davies S.A. Physiol. Rev. 2003; 83: 687-729Crossref PubMed Scopus (106) Google Scholar). Briefly, two pairs of Malpighian tubules, each with a ureter, were dissected from 10 wild type Oregon R adult flies anesthetized on ice. The tubules were mounted in liquid paraffin with the distal end of one tubule bathed in a 10-μl droplet of Schneider's Insect medium. Fluid secretion rates were determined at room temperature by measuring the size of drops formed at the end of the ureter every 10 min. Basal fluid secretion rates were measured for three 10-min intervals prior to adding 10 μm Deta-NONOate (Cayman Chemical Co.) or 1 μm LPS (Sigma) to the droplet of Schneider's medium; after three more 10-min intervals, 10 μm cobinamide, with or without 10 μm OH-Cbl, was added to some of the tubules for a further 30 min. Each data point represents the mean from at least 20 pairs of tubules analyzed on three separate occasions. In some experiments, flies were grown for 48 h on food supplemented with 250 μm cobinamide prior to measuring the rates of tubular secretion. The supplemented food was generated by liquifying standard fly food paste by heating to ∼40 °C and after adding cobinamide, allowing the food to cool to room temperature. Malpighian tubules were dissected from the flies, and rates of tubular secretion were measured in the absence and presence of 1 μm LPS as described above. Measurement of Cobinamide Uptake into Mammalian Cells—To study cobinamide uptake into mammalian cells, we radioactively labeled cobinamide with 14C by adding 2 molar equivalents of [14C]KCN (54 mCi/mmol, Moravek Biochemicals) to form [14C]dicyanocobinamide ([57Co]cobalamin is no longer commercially available). The binding affinity of cyanide for cobinamide is extremely high (Koverall –1022m–1) (29Pratt J.M. Inorganic Chemistry of Vitamin B12. Academic Press, London1972: 144Google Scholar), but to be sure no [14C]KCN remained, we lowered the pH to 6 to form HCN (pKa of KCN is 9.2) and then bubbled argon through the solution to remove any [14C]HCN. The specific activity of the [14C]dicyanocobinamide product was 104 mCi/mmol (using a molar extinction coefficient of 2.8 × 104 at 348 nm) (25Ford S.H. Nichols A. Shambee M. J. Inorg. Biochem. 1991; 41: 235-244Crossref PubMed Scopus (11) Google Scholar). In the uptake studies, ∼1 × 106 baby hamster kidney (BHK) cells cultured in Dulbecco's modified Eagles' medium (DMEM) containing 10% fetal bovine serum (FBS) were grown to subconfluence in 6-well cluster dishes. The cells were incubated at 37 °C for 5 min with [14C]dicyanocobinamide at concentrations ranging from 100 nm to 100 μm. At the end of the incubation, the cells were washed rapidly four times with ice-cold phosphate-buffered saline, harvested with a rubber policeman, and collected by centrifugation for 15 s at 10,000 × g. The cell pellet was dissolved in 500 μl of 0.1 n NaOH, and radioactivity in sample aliquots was measured by liquid scintillation counting. Cobinamide uptake was linear between 2 and 15 min of incubation and from 0.5 to 2 × 106 cells; counts obtained in 0-time samples were <10% of those obtained in the 2-min samples. Assessment of VASP Phosphorylation—Vasodilator-stimulated phosphoprotein (VASP) is expressed in a wide variety of cells (30Reinhard M. Jarchau T. Walter U. Trends Biochem. Sci. 2001; 26: 243-249Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar); it is phosphorylated in response to NO and other vasodilators such as cGMP, with phosphorylation conveniently quantified by Western blotting because it retards the gel mobility of the protein (31Zhuang S. Nguyen G.T. Chen Y. Gudi T. Eigenthaler M. Jarchau T. Walter U. Boss G.R. Pilz R.B. J. Biol. Chem. 2004; 279: 10397-10407Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Approximately 1 × 106 rat C6 glioma or CS-54 vascular smooth muscle cells in 12-well cluster dishes were transfected with 50 ng of an expression vector for VSV epitope-tagged VASP as described previously; C6 cells additionally received 25 ng of cGMP-dependent protein kinase (G-kinase) Iα expression vector (31Zhuang S. Nguyen G.T. Chen Y. Gudi T. Eigenthaler M. Jarchau T. Walter U. Boss G.R. Pilz R.B. J. Biol. Chem. 2004; 279: 10397-10407Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 32Gudi T. Huvar I. Meinecke M. Lohmann S.M. Boss G.R. Pilz R.B. J. Biol. Chem. 1996; 271: 4597-4600Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). The cells were cultured for 36 h in DMEM supplemented with 10% FBS and were treated during the last 30 min with the indicated concentrations of the NO donor PAPA-NONOate (Cayman Chemical Co.) or the membrane-permeable cGMP analog 8-pCPT-cGMP (Biolog, Inc.) in the case of C6 cells or the calcium ionophore A23187 (Calbiochem) in the case of CS-54 cells. Some of the cultures received cobinamide or human hemoglobin, prepared as described previously (33Danishpajooh I.O. Gudi T. Chen Y. Kharitonov V.G. Sharma V.S. Boss G.R. J. Biol. Chem. 2001; 276: 27296-27303Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar), simultaneously with the PAPA-NONOate. The cells were extracted in situ in a gel sample buffer containing 1% SDS, and lysates were subjected to PAGE and Western blotting. VASP was detected using a mouse anti-VSV monoclonal antibody (Sigma) as described previously (31Zhuang S. Nguyen G.T. Chen Y. Gudi T. Eigenthaler M. Jarchau T. Walter U. Boss G.R. Pilz R.B. J. Biol. Chem. 2004; 279: 10397-10407Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). The blots shown were reproduced at least three times. Measurement of Nitrite and Nitrate—NO has a very short half-life and is oxidized rapidly to nitrite and nitrate under physiological conditions. Hence, one of the most common methods for assessing NO production is to measure nitrite and nitrate concentrations, which is generally done using the Griess reagent (33Danishpajooh I.O. Gudi T. Chen Y. Kharitonov V.G. Sharma V.S. Boss G.R. J. Biol. Chem. 2001; 276: 27296-27303Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 34Idriss S.D. Gudi T. Casteel D.E. Kharitonov V.G. Pilz R.B. Boss G.R. J. Biol. Chem. 1999; 274: 9489-9493Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). We measured nitrite and nitrate concentrations in both the Drosophila Malpighian tubule secretion system and in the C6 and CS-54 cells. In the Drosophila system, 20 tubules were incubated in 100 μl of Schneider's medium, and the tubules were stimulated for 60 min with either 10 μm Deta-NONOate or 10 μm LPS, in the absence or presence of 10 μm cobinamide. The C6 and CS-54 cells were incubated for 30 min with 15 μm PAPA-NONOate and 300 nm A23187, respectively, in the absence or presence of 15 μm cobinamide. In all cases, the medium was collected at the end of the incubation period, and nitrite and nitrate in the medium were measured using a nitric oxide quantitation kit from Active Motif, which is an enhanced Griess reagent-based method. Because Deta-NONOate and PAPA-NONOate will continue to release NO even after the end of the incubation with the tubules and cells, all subsequent steps were performed at room temperature immediately after harvesting the medium. Assessment of Cobinamide Cytotoxicity—The effect of varying concentrations of cobinamide on the growth of BHK, CS-54, and C6 cells, and human foreskin fibroblasts and human umbilical venous endothelial cells was assessed by counting the number of cells daily for 3 days using a model ZM Coulter Counter (Coulter Electronics). All of the cells were grown in DMEM containing 10% FBS, except the umbilical endothelial cells, which were grown in M199 medium containing endothelial cell growth supplement and 20% FBS (35Kim S. Harris M. Varner J.A. J. Biol. Chem. 2000; 275: 33920-33928Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). Cells were plated in 6-well culture dishes at initial densities of 1.5–3 × 105 cells/well. Measurement of the Activity of Methionine Synthase and Methylmalonyl-CoA Mutase in Vitro—BHK cells were extracted as described previously at a density of ∼50 × 106/ml in a buffer containing 100 mm Tris-HCl, pH 7.4, 5 mm dithiothreitol, 1 mm EDTA, and a protease inhibitor mixture (34Idriss S.D. Gudi T. Casteel D.E. Kharitonov V.G. Pilz R.B. Boss G.R. J. Biol. Chem. 1999; 274: 9489-9493Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Methionine synthase and methylmalonyl-CoA mutase activities were measured in the extracts in the absence and presence of 1–200 μm cobinamide. Methionine synthase activity was measured at 37 °C according to the method of Weissbach et al. (36Weissbach H. Peterkofsky A. Redfield B.G. Dickerman H. J. Biol. Chem. 1963; 238: 3318-3324Abstract Full Text PDF PubMed Google Scholar), except the [14C]methyltetrahydrofolate substrate was separated from the [14C]methionine product by thin layer chromatography on cellulose acetate plates developed in butanol:acetic acid:water (4:1:5); the RF values for substrate and product were 0.26 and 0.44, respectively. For methylmalonyl-CoA mutase, the extracts were preincubated for 10 min at 37 °C with 5 μm deoxyadenosylcobalamin to convert apoenzyme to holoenzyme, followed by a 10-fold dilution in extract buffer. Enzyme activity was measured at 30 °C according to the method of Kikuchi et al. (37Kikuchi M. Hanamizu H. Narisawa K. Tada K. Clin. Chim. Acta. 1989; 184: 307-313Crossref PubMed Scopus (22) Google Scholar), with the methylmalonyl-CoA substrate separated from the succinyl-CoA product by HPLC on a C18 reverse phase column eluted in 100 mm sodium phosphate, pH 4.0, containing 15% methanol; amounts of substrate and product were determined by comparison with known standards. Both assays were linear with time from 5 to 15 min and with a protein concentration from 0.1 to 0.5 mg/ml. Assessment of the Activity of Methionine Synthase and Methylmalonyl-CoA Mutase in Vivo—The activities of methionine synthase and methylmalonyl-CoA mutase were assessed in intact BHK cells by following incorporation of [14C]formate into purine nucleotides and [14C]propionic acid into protein, respectively; both of these assays have been used previously as surrogate measurements of the in vivo activities of the enzymes (33Danishpajooh I.O. Gudi T. Chen Y. Kharitonov V.G. Sharma V.S. Boss G.R. J. Biol. Chem. 2001; 276: 27296-27303Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 38Willard H.F. Ambani L.M. Hart A.C. Mahoney M.J. Rosenberg L.E. Hum. Genet. 1976; 32: 277-283Crossref Scopus (90) Google Scholar). Briefly, about 1 × 106 BHK cells were incubated in 6-well cluster dishes with 10 μCi of [14C]formate for 90 min or 20 μCi of [14C]propionic acid for 16 h; cobinamide, at concentrations of 1–200 μm, was added 6.5 h before the formate label (for a total incubation time of 8 h) and simultaneously with the propionate label. At the end of the incubation, the cells incubated with [14C]formate were extracted in 0.4 n perchloric acid, heated to 100 °C for 70 min to convert purine nucleotides to bases, and applied to Dowex 50 cation exchange columns to separate the purine bases from unincorporated [14C]formate (39Boss G.R. Erbe R.W. J. Biol. Chem. 1982; 257: 4242-4247Abstract Full Text PDF PubMed Google Scholar). The cells incubated with [14C]propionic acid were extracted in ice-cold 10% trichloroacetic acid, heated to 80 °C for 30 min to solubilize precipitated nucleic acids, and after recooling to 4 °C, precipitated protein was collected on glass microfiber filters (39Boss G.R. Erbe R.W. J. Biol. Chem. 1982; 257: 4242-4247Abstract Full Text PDF PubMed Google Scholar). Both assays were linear with time and cell number from 0.5 to 2 × 106 cells/ml. Production and Analysis of Cobinamide—The standard method for producing diaquocobinamide starts with dicyanocobinamide, removing the cyanide by acid treatment, and exposure to strong light (23Hayward G.C. Hill H.A.O. Pratt J.M. Vanston N.J. Williams A.R.W. J. Chem. Soc. 1965; : 6485-6493Crossref Google Scholar). As mentioned earlier, cobinamide has a very high binding affinity for cyanide, and hence it is difficult to remove cyanide completely from the cobinamide preparations. Moreover, exposure to light over a prolonged period can potentially alter the corrin ring. Because we were interested in producing cyanide-free cobinamide for use in biological systems, we started with OH-Cbl as the initial substrate. The dimethylbenzimidazole ribonucleotide tail was removed by brief acid treatment, and the diaquocobinamide was purified by batch elution over a small sample preparation column as described under “Experimental Procedures.” Beginning with about 200 mg of OH-Cbl, we obtained ∼150–170 mg of high purity cobinamide. Fig. 2 shows the absorbance spectrum of a typical cobinamide preparation at pH 3 having a major peak of 348 nm and smaller relatively equal peaks at 494 and 520 nm (23Hayward G.C. Hill H.A.O. Pratt J.M. Vanston N.J. Williams A.R.W. J. Chem. Soc. 1965; : 6485-6493Crossref Google Scholar, 24Baldwin D.A. Betterton E.A. Pratt J.M. J. Chem. Soc. Dalton Trans. 1983; : 217-223Crossref Google Scholar); in preparations containing contaminants, the 494 and 520 nm peaks tend to either merge together into one broad peak, or the 520 peak became predominant, and a broad band at 455 nm became evident (25Ford S.H. Nichols A. Shambee M. J. Inorg. Biochem. 1991; 41: 235-244Crossref PubMed Scopus (11) Google Scholar). Further evidence for high purity of the preparations was that at pH 12 the A344/A356 ratio was 1.06, well within the range of 1.05–1.11 reported previously for pure dihydroxocobinamide (25Ford S.H. Nichols A. Shambee M. J. Inorg. Biochem. 1991; 41: 235-244Crossref PubMed Scopus (11) Google Scholar), and HPLC analyses of the cobinamide product yielded a single peak when monitored at multiple wavelengths between 300 and 600 nm (40Ford S.H. Nichols A. Gallery J.M. J. Chromatogr. 1991; 536: 185-191Crossref PubMed Scopus (11) Google Scholar). Efficacy of Cobinamide as a NO Scavenger in a Drosophila Fluid Secretion Model—It has become clear that Drosophila is an excellent model for human disease and drug discovery (41Tickoo S. Russell S. Curr. Opin. Pharmacol. 2002; 2: 555-560Crossref PubMed Scopus (54) Google Scholar, 42O'Kane C.J. Semin. Cell Dev. Biol. 2003; 14: 3-10Crossref PubMed Scopus (51) Google Scholar). The Malpighian tubules of D. melanogaster are the organs for fluid transport and osmoregulation in the insect, corresponding to vertebrate kidneys. Rates of tubular secretion can be measured in vitro after extracting tubules from flies; NO stimulates secretion via activation of soluble guanylate cyclase, thereby increasing the intracellular cGMP concentration and activating the cGMP/G-kinase transduction pathway (27Broderick K.E. MacPherson M.R. Regulski M. Tully T. Dow J.A. Davies S.A. Am. J. Physiol. 2003; 285: C1207-C1218Crossref Scopus (40) Google Scholar, 28Dow J.T. Davies S.A. Physiol. Rev. 2003; 83: 687-729Crossref PubMed Scopus (106) Google Scholar). We studied the effect of cobinamide on tubular fluid secretion stimulated both by a NO donor and by LPS, an inducer of the Drosophila NOS gene. In addition, to simulate conditions in a whole animal, we administered cobinamide to the flies via their food and then measured the effect of LPS on rates of tubular fluid secretion. In the experiments with a NO donor, we treated tubules with 10 μm Deta-NONOate, which caused a rapid and sustained increase in the rate of fluid secretion (Fig. 3A; Deta-NONOate was added to all tubules after a 30-min basal period, as indicated by the arrowhead). Adding 10 μm cobinamide to Deta-NONOate-treated tubules reduced the rate of fluid secretion significantly, almost returning to the basal unstimulated level (Fig. 3A; cobinamide was added at 60 min to some of the tubules as indicated by the arrow and the filled circles). Adding cobinamide alone to the tubules was without effect (data not shown). To increase endogenous NO production by the tubules, we used LPS and observed a marked stimulation of tubular secretion (Fig. 3B; LPS was added at 30 min as indicated by the arrowhead). As in the experiments with Deta-NONOate, cobinamide rapidly reduced tubular secretion, returning rates near to the basal state (Fig. 3B; cobinamide was added at 60 min to some of the tubules as indicated by the arrow and the filled circles). Thus, cobinamide scavenges both extracellularly administered and intracellularly produced NO in a Drosophila whole organ system. As part of these studies, we measured the amount of NO released by Deta-NONOate or produced by LPS-treated tubules by incubating tubules for 60 min in Schneider's medium and measuring the sum of nitrite and nitrate in the medium. In the absence of the two drugs, no nitrite or nitrate could be detected in the medium, implying that the basa" @default.
• W2022820008 created "2016-06-24" @default.
• W2022820008 creator A5001149126 @default.
• W2022820008 creator A5029901998 @default.
• W2022820008 creator A5033495704 @default.
• W2022820008 creator A5041732307 @default.
• W2022820008 creator A5062840115 @default.
• W2022820008 creator A5066259415 @default.
• W2022820008 creator A5076494612 @default.
• W2022820008 creator A5078807352 @default.
• W2022820008 date "2005-03-01" @default.
• W2022820008 modified "2023-09-27" @default.
• W2022820008 title "Nitric Oxide Scavenging by the Cobalamin Precursor Cobinamide" @default.
• W2022820008 cites W113622738 @default.
• W2022820008 cites W1498504603 @default.
• W2022820008 cites W1503677472 @default.
• W2022820008 cites W1587377302 @default.
• W2022820008 cites W1603343740 @default.
• W2022820008 cites W1737562535 @default.
• W2022820008 cites W1834041853 @default.
• W2022820008 cites W192516002 @default.
• W2022820008 cites W1968113859 @default.
• W2022820008 cites W1969990802 @default.
• W2022820008 cites W1976192877 @default.
• W2022820008 cites W1977328014 @default.
• W2022820008 cites W1980243862 @default.
• W2022820008 cites W1983713447 @default.
• W2022820008 cites W1983949602 @default.
• W2022820008 cites W1987184422 @default.
• W2022820008 cites W1988454624 @default.
• W2022820008 cites W1992184689 @default.
• W2022820008 cites W1998709931 @default.
• W2022820008 cites W2001462532 @default.
• W2022820008 cites W2004475073 @default.
• W2022820008 cites W2005053677 @default.
• W2022820008 cites W2005793069 @default.
• W2022820008 cites W2007868439 @default.
• W2022820008 cites W2014916185 @default.
• W2022820008 cites W2015183577 @default.
• W2022820008 cites W2019630878 @default.
• W2022820008 cites W2022601650 @default.
• W2022820008 cites W2023357543 @default.
• W2022820008 cites W2024283446 @default.
• W2022820008 cites W2024862370 @default.
• W2022820008 cites W2027647801 @default.
• W2022820008 cites W2034320740 @default.
• W2022820008 cites W2035655203 @default.
• W2022820008 cites W2036140137 @default.
• W2022820008 cites W2042151875 @default.
• W2022820008 cites W2058720586 @default.
• W2022820008 cites W2062909542 @default.
• W2022820008 cites W2067777527 @default.
• W2022820008 cites W2069661887 @default.
• W2022820008 cites W2070540043 @default.
• W2022820008 cites W2073126125 @default.
• W2022820008 cites W2073169036 @default.
• W2022820008 cites W2074744314 @default.
• W2022820008 cites W2075910416 @default.
• W2022820008 cites W2076665664 @default.
• W2022820008 cites W2079915768 @default.
• W2022820008 cites W2082972937 @default.
• W2022820008 cites W2089924484 @default.
• W2022820008 cites W2091030719 @default.
• W2022820008 cites W2092514228 @default.
• W2022820008 cites W2092945190 @default.
• W2022820008 cites W2107327355 @default.
• W2022820008 cites W2119016702 @default.
• W2022820008 cites W2124378315 @default.
• W2022820008 cites W2128454891 @default.
• W2022820008 cites W2138542942 @default.
• W2022820008 cites W2139449432 @default.
• W2022820008 cites W2140296767 @default.
• W2022820008 cites W2154360826 @default.
• W2022820008 cites W2155951706 @default.
• W2022820008 cites W2158272509 @default.
• W2022820008 cites W2160215508 @default.
• W2022820008 cites W2161784662 @default.
• W2022820008 cites W2171716368 @default.
• W2022820008 cites W2313326603 @default.
• W2022820008 cites W2320770893 @default.
• W2022820008 cites W2414769601 @default.
• W2022820008 cites W89635679 @default.
• W2022820008 doi "https://doi.org/10.1074/jbc.m410498200" @default.
• W2022820008 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/15632180" @default.
• W2022820008 hasPublicationYear "2005" @default.
• W2022820008 type Work @default.
• W2022820008 sameAs 2022820008 @default.
• W2022820008 citedByCount "56" @default.
• W2022820008 countsByYear W20228200082012 @default.
• W2022820008 countsByYear W20228200082013 @default.
• W2022820008 countsByYear W20228200082014 @default.
• W2022820008 countsByYear W20228200082015 @default.
• W2022820008 countsByYear W20228200082016 @default.
• W2022820008 countsByYear W20228200082017 @default.
• W2022820008 countsByYear W20228200082018 @default.
• W2022820008 countsByYear W20228200082019 @default.
• W2022820008 countsByYear W20228200082021 @default.
• W2022820008 countsByYear W20228200082022 @default.

• next