RESUMO
Articular cartilage comprises collagens, proteoglycans, and glycosaminoglycans (GAGs) together with water, in hyaline matrixes. Articular cartilage is resistant to proteolytic solubilization for comprehensive GAG analyses partly because of assemblies of collagen fibers with thermolabile hydrogen bonds. In this study, we used the heat-stable protease thermolysin to digest collagen in solid articular cartilage at 70⯰C and compared the efficiencies of collagen digestion and GAG extraction to those with collagenase digestion at 50⯰C. Overnight digestion with thermolysin completely solubilized cartilage, whereas collagenase with >10-times higher proteolytic activity digested <20% of collagen. Following thermolysin treatments, almost all GAGs were extracted from the cartilage, whereas only 56% of GAGs were extracted after collagenase digestion. Disaccharide analyses of extracted GAG chains revealed >98% extraction efficiencies of several GAG classes from thermolysin-treated cartilage, compared with <60% extraction efficiencies using collagenase, depending on GAG classes. These results indicate that thermolysin allows complete GAG extraction from solid articular cartilage and that complete solubilization is required for accurate and reproducible analyses of cartilage GAGs. Hence, thermolysin offers a tool for complete solubilization of cartilage prior to comprehensive GAGomic analysis, and is likely applicable to other collagen-rich tissues such as ligaments, skin, and blood vessels.
Assuntos
Cartilagem/química , Dissacarídeos , Glicosaminoglicanos , Termolisina/química , Animais , Bovinos , Dissacarídeos/análise , Dissacarídeos/química , Glicosaminoglicanos/análise , Glicosaminoglicanos/química , Temperatura AltaRESUMO
We developed a method using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) with a selected reaction monitoring (SRM) mode for simultaneous quantitative analysis of glycosaminoglycans (GAGs). Using one-shot analysis with our MS/MS method, we demonstrated the simultaneous quantification of a total of 23 variously sulfated disaccharides of four GAG classes (8 chondroitin/dermatan sulfates, 1 hyaluronic acid, 12 heparan sulfates, and 2 keratan sulfates) with a sensitivity of less than 0.5 pmol within 20 min. We showed the differences in the composition of GAG classes and the sulfation patterns between porcine articular cartilage and yellow ligament. In addition to the internal disaccharides described above, some saccharides derived from the nonreducing terminal were detected simultaneously. The simultaneous quantification of both internal and nonreducing terminal saccharides could be useful to estimate the chain length of GAGs. This method would help to establish comprehensive "GAGomic" analysis of biological tissues.
Assuntos
Cartilagem Articular/metabolismo , Cromatografia Líquida/métodos , Glicosaminoglicanos/análise , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodos , Animais , Sulfatos de Condroitina/análise , Dermatan Sulfato/análogos & derivados , Dermatan Sulfato/análise , Heparitina Sulfato/análise , Ácido Hialurônico/análise , Sulfato de Queratano/análise , SuínosRESUMO
Liver damage has been induced in animal experiments using carbon tetrachloride (CCl4), a potent hepatotoxin. CCl4 is activated by cytochrome P450 2E1, which results in the formation of various metabolites including phosgene. Although D-dopachrome tautomerase (DDT) is abundant in the liver, its role currently remains unclear. The biological activity of DDT, for which the N-terminal proline is a key site, has been detected in various tissues. We herein incidentally detected a 333 Da modification to the N-terminal proline of DDT in rat livers damaged by CCl4. We identified that this modification as glutathionyl carbonylated group, which was formed by condensation of phosgene and reduced glutathione (GSH). We examined other glutathionyl-carbonylated proteins using two dimensional-polyacrylamide gel electrophoresis, mass spectrometry, and Western blotting for GSH, and detected only one glutathionyl-carbonylated protein, macrophage migration inhibitory factor (MIF). DDT belongs to the MIF family of proteins, and amino acid sequence identity between DDT and MIF is 33%. We concluded that MIF family proteins are major targets for glutathionyl carbonylation.
Assuntos
Fatores Inibidores da Migração de Macrófagos , Fosgênio , Ratos , Animais , Tetracloreto de Carbono/toxicidade , Fatores Inibidores da Migração de Macrófagos/genética , Fatores Inibidores da Migração de Macrófagos/química , Fatores Inibidores da Migração de Macrófagos/metabolismo , DDT , Fígado/metabolismo , Prolina , Oxirredutases Intramoleculares/genéticaRESUMO
OBJECTIVE: Osteoarthritis (OA) is more prevalent in females. We hypothesized that changes in articular cartilage (AC) constituents with aging may cause differences. Herein, we aimed to compare the changes in AC constituents with aging in male and female normal rats. DESIGN: The glycosaminoglycan (GAG) and collagen (COL) contents of the AC in knee, hip, and shoulder joints of male and female rats were quantified and compared between age groups and sexes. RESULTS: The amount of GAG was decreased in multiple joints in both males and females with aging. In females, it had a significant decrease in all joints measured. The decrease in GAG with aging was more severe in females than in males. Even in young rats, the amount of knee joint GAG was significantly less in females than in males. The amount of COL in the AC was unchanged with aging in both sexes. CONCLUSIONS: The drastic GAG decrease with aging in female normal rats may explain the higher prevalence and more severe OA in females.
Assuntos
Cartilagem Articular , Osteoartrite , Envelhecimento , Animais , Colágeno , Feminino , Glicosaminoglicanos , Articulação do Joelho , Masculino , RatosRESUMO
Arginine (Arg)-specific ADP-ribosylation is one of the posttranslational modifications of proteins and is thought to play an important role in reversibly regulating functions of the target proteins in eukaryotes. However, the physiological target protein has not been established. We examined the fragmentation pattern of both ADP-ribosyl-Arg (ADP-R-Arg) and Arg-ADP-ribosylated peptides by quadrupole tandem mass spectrometry and found a specific cleavage of ADP-R-Arg into N-(ADP-ribosyl)-carbodiimide (ADP-R-carbodiimide) and ornithine. Based on this specific fragmentation pattern, we successfully identified the modification site and sequence of Arg-ADP-ribosylated peptide using a two-step collision and showed that ADP-R-carbodiimide is an excellent marker ion for precursor ion scanning of Arg-ADP-ribosylated peptide. We propose that a combination of the precursor ion scanning with ADP-R-carbodiimide as a marker ion and two-step collision is useful in searching for physiological target proteins of Arg-ADP-ribosylation.
Assuntos
Adenosina Difosfato Ribose/análogos & derivados , Métodos Analíticos de Preparação de Amostras/métodos , Peptídeos/química , Proteínas/química , Análise de Sequência de Proteína/métodos , Espectrometria de Massas em Tandem/métodos , Adenosina Difosfato Ribose/análise , Ornitina/análise , Poli Adenosina Difosfato Ribose/química , Processamento de Proteína Pós-TraducionalRESUMO
OBJECT: Indoleamine 2,3-dioxygenase (IDO), a kynurenine pathway (KP) enzyme catalyzing oxidation of the essential amino acid tryptophan (Trp), is thought to be involved in the immune resistance of malignant tumors through T-cell inactivation caused by Trp depletion and metabolite accumulation. Human malignant gliomas may use this strategy to escape immune attack. The object of this study was to investigate the possibility of IDO-dependent Trp depletion by malignant gliomas and the practicability of using an IDO inhibitor together with anticancer drugs to reserve Trp without decreasing the cytotoxicity of the drugs. METHODS: The authors studied expression of IDO and other KP enzymes and the effects of an IDO inhibitor, 1-methyl L-tryptophan (1MT), on Trp metabolism and cytotoxicity of anticancer drugs, together with direct measurement of KP metabolites, in cultured human malignant glioma cells. RESULTS: Upon interferon-gamma (IFN-gamma) stimulation, the glioma cells greatly increased their IDO mRNA expression concomitant with depletion of Trp. The IDO inhibitor 1MT successfully prevented Trp consumption by the stimulated glioma cells. Combining 1MT with anticancer drugs (temozolomide, bischloroethylnitrosourea [BCNU], etoposide and cisplatin) did not interfere with the drugs' suppression of growth of LN229 glioma cells but rather increased their inhibitory effects on IDO activity. CONCLUSIONS: These findings suggest that the robust IDO expression with rapid consumption of Trp in human glioma cells induced by IFN-gamma could lead to immune resistance in glioma cells. Indoleamine 2,3-dioxygenase inhibitors that prevent Trp depletion could be used with anticancer drugs to improve therapeutic effects.
Assuntos
Antineoplásicos/administração & dosagem , Glioma/tratamento farmacológico , Indolamina-Pirrol 2,3,-Dioxigenase/antagonistas & inibidores , Triptofano/análogos & derivados , Triptofano/metabolismo , Sistemas de Liberação de Medicamentos , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/análise , Interferon gama/farmacologia , Triptofano/administração & dosagem , Triptofano/farmacologia , Células Tumorais CultivadasRESUMO
NAD+ is mainly synthesized from nicotinamide (Nam) by the rate-limiting enzyme Nam phosphoribosyltransferase (Nampt) and degraded to Nam by NAD+-degrading enzymes in mammals. Numerous studies report that tissue NAD+ levels decrease during aging and age-related diseases and suggest that NAD+ replenishment promotes healthy aging. Although increased expression of Nampt might be a promising intervention for healthy aging, forced expression of Nampt gene, inducing more than 10-fold increases in the enzyme protein level, has been reported to elevate NAD+ levels only 40-60% in mammalian cells. Mechanisms underlying the limited increases in NAD+ levels remain to be determined. Here we show that Nampt is inhibited in cells and that enhanced expression of Nampt activates NAD+ breakdown. Combined with the measurement of each cell's volume, we determined absolute values (µM/h) of the rates of NAD+ synthesis (RS) and breakdown (RB) using a flux assay with a 2H (D)-labeled Nam, together with the absolute NAD+ concentrations in various mammalian cells including primary cultured cardiomyocytes under the physiological conditions and investigated the relations among total cellular Nampt activity, RS, RB, and the NAD+ concentration. NAD+ concentration was maintained within a narrow range (400-700 µM) in the cells. RS was much smaller than the total Nampt activity, indicating that NAD+ synthesis from Nam in the cells is suppressed. Forced expression of Nampt leading to 6-fold increase in total Nampt activity induced only a 1.6-fold increase in cellular NAD+ concentration. Under the conditions, RS increased by 2-fold, while 2-fold increase in RB was also observed. The small increase in cellular NAD+ concentration is likely due to both inhibited increase in the NAD+ synthesis and the activation of its breakdown. Our findings suggest that cellular NAD+ concentrations do not vary dramatically by the physiological fluctuation of Nampt expression and show the tight link between the NAD+ synthesis and its breakdown.
Assuntos
NAD/metabolismo , Nicotinamida Fosforribosiltransferase/metabolismo , Animais , Tamanho Celular , Células Cultivadas , Humanos , Marcação por Isótopo , Cinética , Camundongos , Miócitos Cardíacos/metabolismo , NAD/biossíntese , Ratos , Espectrometria de Massas em TandemRESUMO
We have expanded a liquid chromatographic-tandem mass spectrometric method that measures 3-hydroxykynurenine and 3-hydroxyanthranilic acid in addition to tryptophan and kynurenine both intra- and extracellularly. After reversed phase HPLC separation, the compounds were detected in the MS positive multiple reaction monitoring mode. We found a good linear response for each tryptophan metabolite. The lower limit of quantification for each compound ranged from 0.01 to 0.1 microM. The extraction efficiencies from spiked cell samples and culture medium ranged between 83 and 111% and the overall coefficient of variation of analyses was less than 7%. Using our method, we found tryptophan metabolites in the cells and the culture medium of LN229 human glioma cells were stimulated by interferon-gamma, a known inducer of indoleamine 2,3-dioxygenase. The intracellular concentrations of kynurenine, 3-hydroxykynurenine and 3-hydroxyanthranilic acid were higher than those in the medium. This is the first report of a method for the simultaneous determination of tryptophan and its metabolic products both intra- and extracellularly.
Assuntos
Cromatografia Líquida/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodos , Triptofano/análise , Linhagem Celular , Padrões de Referência , Sensibilidade e EspecificidadeRESUMO
Arginine-specific ADP-ribosylation is one of the posttranslational modifications of proteins by transferring one ADP-ribose moiety of NAD to arginine residues of target proteins. This modification, catalyzed by ADP-ribosyltransferase (Art), is reversed by ADP-ribosylarginine hydrolase (AAH). In this study, we describe a new method combining an anti-ADP-ribosylarginine antibody (alphaADP-R-Arg Ab) and AAH for detection of the target protein of ADP-ribosylation. We have raised alphaADP-R-Arg Ab with ADP-ribosylated histone and examined the reactivity of the antibody with proteins treated by Art and/or AAH, as well as in situ ADP-ribosylation system with mouse T cells. Our results indicate that the detection of ADP-ribosylated protein with alphaADP-R-Arg Ab and AAH is a useful tool to explore the target proteins of ADP-ribosylation. We applied the method to search endogenously ADP-ribosylated protein in the rat, and detected possible target proteins in the skeletal muscle, which has high Art activity.
Assuntos
Adenosina Difosfato Ribose/análise , Adenosina Difosfato Ribose/metabolismo , Anticorpos/imunologia , Arginina/análise , Arginina/metabolismo , N-Glicosil Hidrolases/metabolismo , Adenosina Difosfato Ribose/imunologia , Animais , Arginina/imunologia , Galinhas , Camundongos , RatosRESUMO
Mono-ADP-ribosylation is a post-translational modification that regulates the functions of target proteins or peptides by attaching an ADP-ribose moiety. Here we report the purification, molecular cloning, characterization and tissue-specific distribution of novel arginine-specific Arts (ADP-ribosyltransferases) from chicken. Arts were detected in various chicken tissues as GPI (glycosylphosphatidylinositol)-anchored forms, and purified from the lung membrane fraction. By molecular cloning based on the partial amino acid sequence using 5'- and 3'-RACE (rapid amplification of cDNA ends), two full-length cDNAs of chicken GPI-anchored Arts, cgArt1 (chicken GPI-anchored Art1) and cgArt2, were obtained. The cDNA of cgArt1 encoded a novel polypeptide of 298 amino acids which shows a high degree of identity with cgArt2 (82.9%), Art6.1 (50.2%) and rabbit Art1 (42.1%). In contrast, the nucleotide sequence of cgArt2 was identical with that of Art7 cloned previously from chicken erythroblasts. cgArt1 and cgArt2 proteins expressed in DT40 cells were shown to be GPI-anchored Arts with a molecular mass of 45 kDa, and these Arts showed different enzymatic properties from the soluble chicken Art, Art6.1. RNase protection assays and real-time quantitative PCR revealed distinct expression patterns of the two Arts; cgArt1 was expressed predominantly in the lung, spleen and bone marrow, followed by the heart, kidney and muscle, while cgArt2 was expressed only in the heart and skeletal muscle. Thus GPI-anchored Arts encoded by the genes cgArt1 and cgArt2 are expressed extensively in chicken tissues. It may be worthwhile determining the functional roles of ADP-ribosylation in each tissue.
Assuntos
ADP Ribose Transferases/química , ADP Ribose Transferases/metabolismo , Arginina/química , Glicosilfosfatidilinositóis/química , ADP Ribose Transferases/isolamento & purificação , Sequência de Aminoácidos , Animais , Sequência de Bases , Galinhas , Clonagem Molecular , Regulação da Expressão Gênica , Dados de Sequência Molecular , Especificidade por Substrato , Distribuição TecidualRESUMO
Because the aberrantly activated phosphoinositide 3-kinase (PI3K)/Akt pathway renders tumor cells resistant to cytotoxic insults, including those related to anticancer drugs, inhibition of the pathway may possibly restore or augment the effectiveness of chemotherapy. Using the human malignant glioma cell lines U87, A172, LN18, and LN229, we examined effects of the PI3K inhibitor LY294002 on both apoptosis and cytotoxicity induced by chemotherapeutic agents, including antimicrotubule agents vincristine and paclitaxel, an alkylating agent 1,3-bis(2-chloroethyl)-1-nitrosourea, a topoisomerase II inhibitor etoposide, and a DNA cross-linking agent cisplatin (cis-diamminedichloroplatinum), and we compared the LY294002-induced enhancement of effects of those agents. Ten to 20 micro M LY294002 augmented both apoptosis and caspase 3-like activity caused by antimicrotubule agents to a larger extent than induced by 1,3-bis(2-chloroethyl)-1-nitrosourea, etoposide, and cisplatin in all four malignant glioma cell lines examined. The same doses of LY294002 enhanced cytotoxicity more efficiently with antimicrotubule agents than with other chemotherapeutic agents. Quantitative analyses using a modified isobologram and median effect plot method revealed that enhancement by LY294002 of vincristine- or paclitaxel-induced cytotoxicity was synergistic, whereas enhancement by the PI3K inhibitor of the other chemotherapeutic agent-induced cytotoxicity was additive. Our study indicates that the synergistic augmentation of the cytotoxicity by LY294002 occurs specifically with antimicrotubule agents, at least partially through an increase in caspase 3-dependent apoptosis, and we suggest that inhibitors of the PI3K/Akt pathway in combination with antimicrotubule agents may induce cell death effectively and be a potent modality to treat patients with malignant gliomas.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Cromonas/farmacologia , Inibidores Enzimáticos/farmacologia , Glioma/tratamento farmacológico , Morfolinas/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Carmustina/administração & dosagem , Carmustina/farmacologia , Cromonas/administração & dosagem , Cisplatino/administração & dosagem , Cisplatino/farmacologia , Sinergismo Farmacológico , Inibidores Enzimáticos/administração & dosagem , Etoposídeo/administração & dosagem , Etoposídeo/farmacologia , Glioma/enzimologia , Humanos , Microtúbulos/efeitos dos fármacos , Morfolinas/administração & dosagem , Paclitaxel/administração & dosagem , Paclitaxel/farmacologia , Células Tumorais Cultivadas , Vincristina/administração & dosagem , Vincristina/farmacologiaRESUMO
OBJECT: The phosphatase and tensin homolog deleted from chromosome 10 (PTEN) functions as a tumor suppressor by negatively regulating the growth/survival signals of the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway. The PI3-K/Akt pathway in PTEN-deficient tumors may be one of the key targets for anticancer therapy. The authors examined the effects of the PI3-K inhibitor 2-(4-morpholinyl)-8-phenylchromone (LY294002) on human malignant glioma cells, and compared these effects on PTEN-deficient cells with those on PTEN-wild-type (PTEN-wt) cells. METHODS: Using human malignant glioma cell lines, including the PTEN-deficient cells A172 and U87MG and the PTEN-wt cells LN18 and LN229, the effects of LY294002 on cell growth, apoptosis, and chemotherapeutic agent-induced cytotoxicity were evaluated. The LY294002 inhibited the growth of U87MG cells associated with reduced phosphatidylinositol 3,4,5,-trisphosphate and phosphorylated Akt, and also induced growth inhibition in three other cell lines. Although LY294002 caused apoptosis in all four cell lines, apoptosis seemed to contribute to only a small portion of growth inhibition induced by LY294002. There was no link between the status of PTEN and the median inhibitory concentration values for LY294002 or between the gene status and the extent of LY294002-induced apoptosis. The LY294002 significantly augmented the cytotoxicity induced by etoposide in PTEN-deficient cells, but not in PTEN-wt cells. Enhancement of 1,3-bis(2-chloroethyl)-1-nitrosourea- and cisplatin-induced cytotoxicity by LY294002 was not linked to the status of PTEN. No marked difference in the amounts of phosphorylated Akt was found between PTEN-deficient and PTEN-wt cells. CONCLUSIONS: The findings show that PI3-K is a possible target for therapy in patients with gliomas, and PI3-K inhibitors in combination with chemotherapeutic agents could be potent therapeutic modalities for patients with malignant gliomas.
Assuntos
Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Cromonas/farmacologia , Inibidores Enzimáticos/farmacologia , Glioma/fisiopatologia , Inibidores do Crescimento/farmacologia , Morfolinas/farmacologia , Monoéster Fosfórico Hidrolases/farmacologia , Proteínas Serina-Treonina Quinases , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/fisiologia , Proteínas Supressoras de Tumor/farmacologia , Humanos , Técnicas In Vitro , PTEN Fosfo-Hidrolase , Monoéster Fosfórico Hidrolases/deficiência , Proteínas Proto-Oncogênicas/farmacologia , Proteínas Proto-Oncogênicas c-akt , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Proteínas Supressoras de Tumor/deficiênciaRESUMO
To determine the rates of cellular NAD⺠synthesis and breakdown, incorporation of stable isotope-labeled precursors into NAD⺠should be quantified. Although with ²H (D)-labeled precursors [2,4,5,6-D4]nicotinamide ([D4]Nam) and [2,4,5,6-D4]nicotinic acid ([D4]NA), [D3]NAD⺠is formed in human cells, why only three of four D atoms from [D4]Nam and [D4]NA are present in NAD⺠remains unknown. Using a liquid chromatography-tandem mass spectrometry, we tested the involvement of D/¹H (H) exchange at the redox site of NADâº/NADH (C-4 carbon of the pyridine ring) by oxidoreductases exhibiting opposite stereospecificity for the coenzymes in the 1-Da mass decrease in the cellular NAD⺠formation. In all cells examined, [Nam-D3]NADâº, but not [Nam-D4]NADâº, was obtained after the incubation with the D4-labeled precursors, whereas [Nam-D4]NADâº, but not [Nam-D3]NADâº, was synthesized from the same precursors with purified recombinant NAD⺠biosynthetic enzymes. [D4]Nam group of [Nam-D4]NAD⺠was converted to [D3]Nam group via [D4]NADH by in vitro sequential reduction and oxidation with oxidoreductases exhibiting opposite stereospecificity for the coenzymes. Furthermore, using [2,5,6-D3]Nam, which has H instead of D at the C-4 carbon, as a precursor of NAD⺠in the cells, the 1-Da mass decrease in the nucleotide was not observed. Based on these observations, we conclude that following the synthesis of [Nam-2,4,5,6-D4]NADâº, cellular redox reactions of NADâº/NADH convert [Nam-2,4,5,6-D4]NAD⺠to [Nam-2,5,6-D3]NADâº. Quantification of [Nam-2,5,6-D]NAD⺠and [2,5,6-D3]Nam would successfully determine the rate of the NAD⺠turnover and provide clues to understand regulatory mechanisms of cellular NAD⺠concentrations.
Assuntos
NAD/química , Niacinamida/química , Ácidos Nicotínicos/química , Carbono/química , Cromatografia Líquida , Coenzimas , Células Hep G2 , Humanos , Hidrogênio/química , Oxirredução , Espectrometria de Massas em TandemRESUMO
Nicotinamide (Nam) phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme in mammalian NAD synthesis, catalyzing nicotinamide mononucleotide (NMN) formation from Nam and 5-phosphoribosyl 1-pyrophosphate (PRPP). NAMPT has also been described as an adipocytokine visfatin with a variety of actions, although physiological significance of this protein remains unclear. It has been proposed that possible actions of visfatin are mediated through the extracellular formation of NMN. However, we did not detect NMN in mouse blood plasma, even with a highly specific and sensitive liquid chromatography/tandem mass spectrometry. Furthermore, there is no or little ATP, the activator of NAMPT, in extracellular spaces. We thus questioned whether visfatin catalyzes the in situ formation of NMN under such extracellular milieus. To address this question, we here determined K(m) values for the substrates Nam and PRPP in the NAMPT reaction without or with ATP using a recombinant human enzyme and found that 1 mM ATP dramatically decreases K(m) values for the substrates, in particular PRPP to its intracellular concentration. Consistent with the kinetic data, only when ATP is present at millimolar levels, NAMPT efficiently catalyzed the NMN formation at the intracellular concentrations of the substrates. Much lower concentrations of Nam and almost the absence of PRPP and ATP in the blood plasma suggest that NAMPT should not efficiently catalyze its reaction under the extracellular milieu. Indeed, NAMPT did not form NMN in the blood plasma. From these kinetic analyses of the enzyme and quantitative determination of its substrates, activator, and product, we conclude that visfatin does not participate in NMN formation under the extracellular milieus. Together with the absence of NMN in the blood plasma, our conclusion does not support the concept of "NAMPT-mediated systemic NAD biosynthesis." Our study would advance current understanding of visfatin physiology.
Assuntos
Nicotinamida Fosforribosiltransferase/metabolismo , Plasma/enzimologia , Plasma/metabolismo , Animais , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Niacinamida/metabolismo , Mononucleotídeo de Nicotinamida/metabolismo , Compostos Organofosforados/metabolismoRESUMO
Nicotinamide N-methyltransferase (NNMT) catalyzes the N-methylation of nicotinamide. NNMT is strongly expressed in tumor cells and an increase in NNMT activity may reduce cellular nicotinamide level and thereby promote cell survival in the cells. However, there has been no report of a relationship between NNMT activity and nicotinamide level in tumor cells. We report herein that human glioma cells produce relatively large amounts of NNMT and that when these cells are cultured in the presence of interferon-gamma (IFN-gamma) their 1-methylnicotinamide levels increase. To clarify the mechanisms by which IFN-gamma increases 1-methylnicotinamide levels in these cells, we measured NNMT activity and the levels of NNMT expression, nicotinamide and nicotinamide adenine dinucleotide (NAD(+)) in the presence and absence of IFN-gamma. We also examined whether addition of exogenous 1-methylnicotinamide directly affects cell viability and/or the cellular levels of 1-methylnicotinamide, nicotinamide and NAD(+). While addition of 1-methylnicotinamide increased the total amount of cellular 1-methylnicotinamide present, it did not affect nicotinamide or NAD(+) levels, or cell viability. Conversely, IFN-gamma significantly increased NNMT activity and the nicotinamide cellular concentration, while leaving NNMT expression and the NAD(+) cellular concentration unchanged. Therefore, the increase in the 1-methylnicotinamide level found when IFN-gamma is present in culture may be a consequence of increases in both the nicotinamide concentration and NNMT activity, whereas, 1-methylnicotinamide did not influence nicotinamide levels, NAD(+) levels, or cell viability per se. These results suggest that an increase in NNMT activity does not always reduce cellular nicotinamide concentration in tumor cells.
Assuntos
Antineoplásicos/farmacologia , Glioma/enzimologia , Interferon gama/farmacologia , Niacinamida/metabolismo , Nicotinamida N-Metiltransferase/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Ativação Enzimática , Glioma/metabolismo , Humanos , Nicotinamida N-Metiltransferase/efeitos dos fármacos , Proteínas Recombinantes , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Células Tumorais CultivadasRESUMO
NAD plays critical roles in various biological processes through the function of SIRT1. Although classical studies in mammals showed that nicotinic acid (NA) is a better precursor than nicotinamide (Nam) in elevating tissue NAD levels, molecular details of NAD synthesis from NA remain largely unknown. We here identified NA phosphoribosyltransferase (NAPRT) in humans and provided direct evidence of tight link between NAPRT and the increase in cellular NAD levels. The enzyme was abundantly expressed in the small intestine, liver, and kidney in mice and mediated [(14)C]NAD synthesis from [(14)C]NA in human cells. In cells expressing endogenous NAPRT, the addition of NA but not Nam almost doubled cellular NAD contents and decreased cytotoxicity by H(2)O(2). Both effects were reversed by knockdown of NAPRT expression. These results indicate that NAPRT is essential for NA to increase cellular NAD levels and, thus, to prevent oxidative stress of the cells. Kinetic analyses revealed that NAPRT, but not Nam phosphoribosyltransferase (NamPRT, also known as pre-B-cell colony-enhancing factor or visfatin), is insensitive to the physiological concentration of NAD. Together, we conclude that NA elevates cellular NAD levels through NAPRT function and, thus, protects the cells against stress, partly due to lack of feedback inhibition of NAPRT but not NamPRT by NAD. The ability of NA to increase cellular NAD contents may account for some of the clinically observed effects of the vitamin and further implies a novel application of the vitamin to treat diseases such as those associated with the depletion of cellular NAD pools.
Assuntos
NAD/metabolismo , Niacina/metabolismo , Pentosiltransferases/metabolismo , Animais , Células HeLa , Humanos , Cinética , Camundongos , Camundongos Endogâmicos BALB C , Modelos Biológicos , Dados de Sequência Molecular , Estresse Oxidativo , RNA Interferente Pequeno/metabolismo , Ratos , Distribuição TecidualRESUMO
Liver cirrhosis remains a difficult-to-treat disease with a substantial morbidity and mortality rate. There is an emerging body of data purporting a pivotal role of the activated p38 mitogen-activated protein kinase (MAPK) in the process of cirrhosis. Several anticirrhotic agents have been developed over the past few years, and most of them exert their effects by indirectly inhibiting the p38 pathway. Effect of a selective p38 inhibitor is yet to be reported. In this study, we evaluated the salutary effect of FR-167653 (FR), a selective p38 inhibitor, in a carbon tetrachloride (CCl(4))-induced rat cirrhotic model. Twenty rats were assigned into four groups: Sham, olive oil only; Control, CCl(4) in olive oil; FR50, FR 50 mg/kg/day and CCl(4); and FR100, FR 100 mg/kg/day and CCl(4). FR dose-dependently inhibited activation of p38 and had an ameliorating effect on cirrhosis formation. Significant dose-dependent reduction in alpha-smooth muscle actin immunostaining and hydroxyproline content of the liver was noticed in the FR-treated rats. Also densitometric analysis showed a significant reduction in azan-stained area in the FR-treated rats. These fibrotic changes were observed in the myofibroblasts including the hepatic stellate cells and portal fibroblasts. mRNA expression of runt-related protein 2 (Runx2), a profibrogenic transcription factor, was significantly low in FR-treated livers, indicating that Runx2 might be a key downstream regulator of the p38 pathway. A similar reduction in expression of Smad4 and tissue inhibitor of metalloproteinase-1 was noticed in the FR-treated rats. In conclusion, FR treatment exerted a significant beneficial effect in a CCl(4)-induced rat cirrhotic model. The ameliorating effect of FR could be partially attributable to an inhibition of the Smad4/p38/Runx2 axis in the cirrhotic liver.
Assuntos
Subunidade alfa 1 de Fator de Ligação ao Core/fisiologia , Regulação para Baixo/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Cirrose Hepática Experimental/tratamento farmacológico , Pirazóis/farmacologia , Piridinas/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Animais , Tetracloreto de Carbono/toxicidade , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Imuno-Histoquímica , Cirrose Hepática Experimental/induzido quimicamente , Cirrose Hepática Experimental/patologia , Masculino , Modelos Biológicos , RNA Mensageiro/metabolismo , Distribuição Aleatória , Ratos , Ratos Sprague-DawleyRESUMO
We have developed a liquid chromatographic-tandem mass spectrometric method that is sensitive and specific and that simultaneously measures cellular NAD(+) and related compounds. Using this method, NAD(+), NAAD, NMN, NAMN, NAM, NA, ADPR, and 5'AMP were first separated over a reverse-phase high-performance liquid chromatography resin in a mobile ammonium formate-methanol linear gradient. Then each compound was ionized at an electrospray source and detected in the positive multiple reaction monitoring mode of a triple-quadrupole tandem mass spectrometer. We found a good linear response for each NAD(+)-related compound. The limits of quantification for NAD(+) and related compounds range from 0.1 to 1 pmol. The extraction efficiency of NAD(+) and related compounds from mouse erythrocytes is between 84 and 114%. The coefficients of variation for the analyses are all less than 6%. Using our method, we measured, in a single analysis, the amounts of NMN, NAMN, NAD(+), and 5'AMP present in mouse erythrocytes. Additionally, this is the first report of a direct determination of the amounts of NMN and NAMN present in any type of cell. These results indicate that our method sensitively, specifically, and simultaneously measures cellular NAD(+) and related compounds.
Assuntos
NAD/análise , Espectrometria de Massas por Ionização por Electrospray/métodos , Adenosina Difosfato Ribose/análise , Monofosfato de Adenosina/análise , Animais , Cromatografia Líquida/métodos , Eritrócitos/química , Células HL-60 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , NAD/análogos & derivados , Niacina/análise , Niacinamida/análise , Mononucleotídeo de Nicotinamida/análogos & derivados , Mononucleotídeo de Nicotinamida/análise , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: Morphine can desensitize mu-opioid receptor (MOR), but it does not cause internalization of the receptor after binding. Acute desensitization of MOR impairs the efficiency of signaling, whereas the receptor internalization restores the cell responsiveness to the agonists. Thereby, the property of morphine may limit the analgesic effects of this opiate drug. It has been shown that [D-Ala2,MePhe4,Gly-ol5]enkephalin (DAMGO), a potent MOR agonist inducing the internalization, facilitates morphine to internalize MOR, suggesting that MOR agonists with low relative activity versus endocytosis (RAVE) values such as DAMGO can potentiate analgesic effects of morphine through stimulating MOR internalization. The authors examined whether the acute analgesic effect of morphine can be potentiated by low relative activity versus endocytosis agonists DAMGO and fentanyl. METHODS: Rats injected intrathecally with opioids were subjected to a hot plate test for antinociceptive effect. Immunostained spinal dorsal horn was analyzed by confocal microscopy. RESULTS: Fentanyl induced MOR internalization to a lesser extent than DAMGO at equianalgesic doses. Coadministration of fentanyl promoted morphine-induced MOR internalization. The analgesic effect of morphine was greatly potentiated together with decrease in the relative activity versus endocytosis value when MOR internalization was induced by coadministration of a subanalgesic dose of DAMGO or fentanyl. In contrast, the combination of DAMGO and fentanyl increased neither the analgesic effect nor the internalization of MOR. CONCLUSIONS: The results suggest that the coadministration of morphine with MOR-internalizing agonist is clinically applicable to develop successful pain-management regimens to achieve satisfactory analgesia using less morphine.
Assuntos
Analgésicos Opioides/farmacologia , Endocitose/efeitos dos fármacos , Morfina/farmacologia , Receptores Opioides mu/metabolismo , Animais , Ala(2)-MePhe(4)-Gly(5)-Encefalina/farmacologia , Fentanila/farmacologia , Masculino , Morfina/farmacocinética , Células do Corno Posterior/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
NAD synthetase catalyzes the final step in the biosynthesis of NAD. In the present study, we obtained cDNAs for two types of human NAD synthetase (referred as NADsyn1 and NADsyn2). Structural analysis revealed in both NADsyn1 and NADsyn2 a domain required for NAD synthesis from ammonia and in only NADsyn1 an additional carbon-nitrogen hydrolase domain shared with enzymes of the nitrilase family that cleave nitriles as well as amides to produce the corresponding acids and ammonia. Consistent with the domain structures, biochemical assays indicated (i) that both NADsyn1 and NADsyn2 have NAD synthetase activity, (ii) that NADsyn1 uses glutamine as well as ammonia as an amide donor, whereas NADsyn2 catalyzes only ammonia-dependent NAD synthesis, and (iii) that mutant NADsyn1 in which Cys-175 corresponding to the catalytic cysteine residue in nitrilases was replaced with Ser does not use glutamine. Kinetic studies suggested that glutamine and ammonia serve as physiological amide donors for NADsyn1 and NADsyn2, respectively. Both synthetases exerted catalytic activity in a multimeric form. In the mouse, NADsyn1 was seen to be abundantly expressed in the small intestine, liver, kidney, and testis but very weakly in the skeletal muscle and heart. In contrast, expression of NADsyn2 was observed in all tissues tested. Therefore, we conclude that humans have two types of NAD synthetase exhibiting different amide donor specificity and tissue distributions. The ammonia-dependent synthetase has not been found in eucaryotes until this study. Our results also indicate that the carbon-nitrogen hydrolase domain is the functional domain of NAD synthetase to make use of glutamine as an amide donor in NAD synthesis. Thus, glutamine-dependent NAD synthetase may be classified as a possible glutamine amidase in the nitrilase family. Our molecular identification of NAD synthetases may prove useful to learn more of mechanisms regulating cellular NAD metabolism.