RESUMO
Alzheimer disease (AD) is one of the most disabling disorders of the elderly and the number of people worldwide facing dementia is expected to dramatically increase in the near future. Thus, one of the major concerns of modern society is to identify putative biomarkers that serve as a valuable early diagnostic tool to identify a subset of patients with increased risk to develop AD. An ideal biomarker should be present in blood before dementia is clinically confirmed, have high sensitivity and specificity, and be reproducible. Proteomics platforms offer a powerful strategy to reach these goals and recently have been demonstrated to be promising approaches. However, the high variability of technologies and studied populations has led to contrasting results. To increase specificity, we analyzed both protein expression profiles and oxidative modifications (carbonylation) of plasma proteins in mild cognitive impairment (MCI) and AD subjects compared with age-matched controls. Most of the proteins found to have differential levels in MCI and AD confirmed results already obtained in other cohort studies. Interestingly, we applied for the first time in MCI a redox proteomics approach to specifically identify oxidized proteins. Among them, haptoglobin, one of the most abundantly secreted glycoproteins with chaperone function, was found to be either increasingly downregulated or increasingly oxidized in AD and MCI compared with controls. We also demonstrated that in vitro oxidation of haptoglobin affects the formation of amyloid-ß fibrils, thus suggesting that oxidized haptoglobin is not able to act as an extracellular chaperone to prevent or slow formation of amyloid-ß aggregates. Another chaperone protein, α2-macroglobulin, was found to be selectively oxidized in AD patients compared with controls. Our findings suggest that alterations in proteins acting as extracellular chaperones may contribute to exacerbating amyloid-ß toxicity in the peripheral system and may be considered a putative marker of disease progression.
Assuntos
Doença de Alzheimer/sangue , Haptoglobinas/metabolismo , Chaperonas Moleculares/metabolismo , alfa-Macroglobulinas/metabolismo , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/diagnóstico , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/biossíntese , Biomarcadores/sangue , Proteínas Sanguíneas/metabolismo , Progressão da Doença , Regulação para Baixo , Diagnóstico Precoce , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Masculino , Oxirredução , ProteômicaRESUMO
Among the pathological factors known to be associated with Alzheimer disease (AD), oxidative stress induced by the amyloid-ß peptide (Aß) has been demonstrated to play a key role in human brain and animal models of AD. Recently, we reported elevated levels of oxidative damage in the brain of a transgenic (Tg) AD mouse model with Swedish and Indiana familial AD mutations in human amyloid precursor protein (APP) [PDAPP mice, line J20], as evidenced by increased levels of protein carbonyls, 3-nitrotyrosine, and protein-bound 4-hydroxy-2-nonenal. This oxidative damage was dependent on the methionine 35 residue within the Aß peptide. Further insight into the molecular pathways affected in this Tg model of AD may be gained with discovery-based proteomics studies; therefore, two-dimensional gel-based expression proteomics was performed to compare differences in brain protein levels of J20 Tg mice with non-transgenic (NTg) littermate controls. Based on our studies, we identified six proteins that had significantly increased levels in J20 Tg relative to NTg mice: calcineurin subunit B type 1, ρ GDP-dissociation inhibitor 1, T-complex protein 1 subunit α A, α-enolase, peptidyl-prolyl cis-trans isomerase (Pin-1), and ATP synthase subunit α mitochondrial. Several of these proteins have previously been implicated in in vitro and in vivo models and subjects with AD. Additionally, using redox proteomics analyses we identified two oxidatively-modified proteins: phosphatidylethanolamine-binding protein 1 and Pin-1 with decreased levels of protein 3-nitrotyrosine in J20 Tg mice relative to NTg. Western blotting and immunoprecipitation analyses were used to validate proteomics results. Overall, these studies provide information about changes in the brain proteome as a result of Aß deposition and clues with which to further direct studies on elucidating AD pathogenesis.
Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/fisiologia , Precursor de Proteína beta-Amiloide/fisiologia , Proteoma/química , Proteômica , Sequência de Aminoácidos , Peptídeos beta-Amiloides/biossíntese , Peptídeos beta-Amiloides/química , Precursor de Proteína beta-Amiloide/biossíntese , Precursor de Proteína beta-Amiloide/química , Animais , Modelos Animais de Doenças , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dados de Sequência Molecular , Oxirredução , Proteoma/biossíntese , Proteômica/métodosRESUMO
Adriamycin (ADR) is a chemotherapeutic for the treatment of solid tumors. This quinone-containing anthracycline is well known to produce large amounts of reactive oxygen species (ROS) in vivo. A common complaint of patients undergoing long-term treatment with ADR is somnolence, often referred to as "chemobrain." While ADR itself does not cross the blood brain barrier (BBB), we recently showed that ADR administration causes a peripheral increase in tumor necrosis factor alpha (TNF-alpha), which migrates across the BBB and leads to inflammation and oxidative stress in brain, most likely contributing to the observed decline in cognition. In the current study, we measured levels of the antioxidant glutathione (GSH) in brains of mice injected intraparitoneally (i.p.) with ADR, as well as the levels and activities of several enzymes involved in brain GSH metabolism. We observed significantly decreased GSH levels, as well as altered GSH/GSSG ratio in brains of ADR treated mice relative to saline-treated controls. Also observed in brains of ADR treated mice were increased levels of glutathione peroxidase (GPx), glutathione-S-transferase (GST), and glutathione reductase (GR). We also observed increased activity of GPx, but a significant reduction in GST and GR activity in mice brain, 72 h post i.p. injection of ADR (20 mg/kg body weight). Furthermore, we used redox proteomics to identify specific proteins that are oxidized and/or have differential levels in mice brains as a result of a single i.p. injection of ADR. Visinin like protein 1 (VLP1), peptidyl prolyl isomerase 1 (Pin1), and syntaxin 1 (SYNT1) showed differential levels in ADR treated mice relative to saline-treated controls. Triose phosphate isomerase (TPI), enolase, and peroxiredoxin 1 (PRX-1) showed significantly increased specific carbonylation in ADR treated mice brain. These results further support the notion ADR induces oxidative stress in brain despite not crossing the BBB, and that antioxidant intervention may prevent ADR-induced cognitive dysfunction.
Assuntos
Antibióticos Antineoplásicos/efeitos adversos , Antioxidantes/metabolismo , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Doxorrubicina/efeitos adversos , Estresse Oxidativo , Animais , Encéfalo/enzimologia , Glutationa/metabolismo , Glutationa Peroxidase/metabolismo , Glutationa Redutase/metabolismo , Injeções Intraperitoneais , Masculino , Camundongos , Oxirredução , Carbonilação Proteica , ProteômicaRESUMO
2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is carcinogenic in multiple organs and numerous species. Bioactivation of PhIP is initiated by PhIP N(2)-hydroxylation catalysed by cytochrome P450s. Following N-hydroxylation, O-acetylation catalysed by N-acetyltransferase 2 (NAT2) is considered a further possible activation pathway. Genetic polymorphisms in NAT2 may modify cancer risk following exposure. Nucleotide excision repair-deficient Chinese hamster ovary (CHO) cells stably transfected with human cytochrome P4501A1 (CYP1A1) and a single copy of either NAT2*4 (rapid acetylator) or NAT2*5B (slow acetylator) alleles were used to test the effect of CYP1A1 and NAT2 polymorphism on PhIP genotoxicity. Cells transfected with NAT2*4 had significantly higher levels of N-hydroxy-PhIP O-acetyltransferase (p = 0.0150) activity than cells transfected with NAT2*5B. Following PhIP treatment, CHO cell lines transfected with CYP1A1, CYP1A1/NAT2*4 and CYP1A1/NAT2*5B each showed concentration-dependent cytotoxicity and hypoxanthine phosphoribosyl transferase (hprt) mutagenesis not observed in untransfected CHO cells. dG-C8-PhIP was the primary DNA adduct formed and levels were dose dependent in transfected CHO cells in the order: CYP1A1 < CYP1A1 and NAT2*5B < CYP1A1 and NAT2*4, although levels did not differ significantly (p > 0.05) following one-way analysis of variance. These results strongly support activation of PhIP by CYP1A1 with little effect of human NAT2 genetic polymorphism on mutagenesis and DNA damage.
Assuntos
Arilamina N-Acetiltransferase/metabolismo , Carcinógenos/farmacologia , Citocromo P-450 CYP1A1/metabolismo , Adutos de DNA/metabolismo , Imidazóis/farmacologia , Mutagênicos/farmacologia , Animais , Arilamina N-Acetiltransferase/genética , Células CHO , Cricetinae , Cricetulus , Citocromo P-450 CYP1A1/genética , Dano ao DNA , Humanos , Mutagênese , Polimorfismo Genético , TransfecçãoRESUMO
Acrolein, the most reactive of the alpha,beta-unsaturated aldehydes, is endogenously produced by lipid peroxidation, and has been found increased in the brain of patients with Alzheimer's disease. Although it is known that acrolein increases total protein carbonylation and impairs the function of selected proteins, no study has addressed which proteins are selectively carbonylated by this aldehyde. In this study we investigated the effect of increasing concentrations of acrolein (0, 0.005, 0.05, 0.5, 5, 50 microM) on protein carbonylation in gerbil synaptosomes. In addition, we applied proteomics to identify synaptosomal proteins that were selectively carbonylated by 0.5 microM acrolein. Acrolein increased total protein carbonylation in a dose-dependent manner. Proteomic analysis (two-dimensional electrophoresis followed by mass spectrometry) revealed that tropomyosin-3-gamma isoform 2, tropomyosin-5, beta-actin, mitochondrial Tu translation elongation factor (EF-Tu(mt)) and voltage-dependent anion channel (VDAC) were significantly carbonylated by acrolein. Consistent with the proteomics studies that have identified specifically oxidized proteins in Alzheimer's disease (AD) brain, the proteins identified in this study are involved in a wide variety of cellular functions including energy metabolism, neurotransmission, protein synthesis, and cytoskeletal integrity. Our results suggest that acrolein may significantly contribute to oxidative damage in AD brain.
Assuntos
Acroleína/farmacologia , Carbonilação Proteica/efeitos dos fármacos , Sinaptossomos/efeitos dos fármacos , Animais , Encéfalo/ultraestrutura , Relação Dose-Resposta a Droga , Eletroforese em Gel Bidimensional/métodos , Feminino , Gerbillinae , Masculino , Espectrometria de Massas/métodosRESUMO
The cannabinoid receptor 1 (CB1) cannabinoid receptor is an essential component of the cannabinergic system. It has been recognized as a therapeutic target for treating numerous diseases and is currently receiving considerable attention by the pharmaceutical community. Target-based drug design, utilizing three-dimensional information of receptor structure and ligand-binding motifs, requires significant amounts of purified protein. To facilitate the purification of CB1, we have expressed the receptor fused to various epitope tags using the baculovirus expression system. In addition, expression levels and ligand-binding profiles corresponding to the expressed fusion proteins have been compared. C-terminal histidine (His)-tagged CB1 gave a Bmax higher than most other systems previously reported in the literature, and was selected for subsequent metal affinity chromatography purification and mass spectroscopic (MS) analysis. Moreover, cells expressing C-terminal His-tagged CB1 were shown to inhibit forskolin-stimulated cyclic adenosine 3',5'-monophosphate (cAMP) production in a concentration-dependent manner in the presence of CP-55,940, confirming the expressed receptor's functional characteristics. A Western blot analysis of the purified receptor showed several forms of CB1, the most abundant being a 57 kDa monomeric protein. The purified CB1 preparations were subjected to protein digestion followed by MS. Fragments corresponding to >70% of the receptor were identified by this method, confirming the identity and purity of the expressed protein. The work presented here demonstrates that epitope-tagged CB1 can be expressed in sufficient amounts and purified to homogeneity for MS analysis. Moreover, these results will serve as a basis for future experiments aimed at characterizing the ligand-binding domains using covalently reacting receptor probes.
Assuntos
Espectrometria de Massas/métodos , Receptor CB1 de Canabinoide/isolamento & purificação , Sequência de Aminoácidos , Animais , Baculoviridae/genética , Sequência de Bases , Clonagem Molecular , Humanos , Modelos Biológicos , Dados de Sequência Molecular , Plasmídeos , Receptor CB1 de Canabinoide/química , Receptor CB1 de Canabinoide/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
Acrylonitrile (AN) is an industrial vinyl monomer that is acutely toxic. When administered to rats, AN covalently binds to tissue proteins in a dose-dependent but nonlinear manner [Benz, F. W., Nerland, D. E., Li, J., and Corbett, D. (1997) Fundam. Appl. Toxicol. 36, 149-156]. The nonlinearity in covalent binding stems from the fact that AN rapidly depletes liver glutathione after which the covalent binding to tissue proteins increases disproportionately. The identity of the tissue proteins to which AN covalently binds is unknown. The experiments described here were conducted to begin to answer this question. Male Sprague-Dawley rats were injected subcutaneously with 115 mg/kg (2.2 mmol/kg) [2,3-(14)C]AN. Two hours later, the livers were removed, homogenized, and fractionated into subcellular components, and the radioactively labeled proteins were separated on SDS-PAGE. One set of labeled proteins was found to be glutathione S-transferase (GST). Specific labeling of the mu over the alpha class was observed. Separation of the GST subunits by HPLC followed by scintillation counting showed that AN was selective for subunit rGSTM1. Mass spectral analysis of tryptic digests of the GST subunits indicated that the site of labeling was cysteine 86. The reason for the high reactivity of cysteine 86 in rGSTM1 was hypothesized to be due to its potential interaction with histidine 84, which is unique in this subunit.
Assuntos
Acrilonitrila/metabolismo , Carcinógenos/metabolismo , Glutationa Transferase/metabolismo , Fígado/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Relação Dose-Resposta a Droga , Glutationa Transferase/biossíntese , Glutationa Transferase/química , Marcação por Isótopo , Fígado/efeitos dos fármacos , Fígado/enzimologia , Masculino , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Ratos , Ratos Sprague-DawleyRESUMO
Administration of dehydroepiandrosterone (DHEA) to rodents produces many unique biological responses, some of which may be due to metabolism of DHEA to more biologically active products. In the current study, DHEA metabolism was studied using human and rat liver microsomal fractions. In both species, DHEA was extensively metabolized to multiple products; formation of these products was potently inhibited in both species by miconazole, demonstrating a principal role for cytochrome P450. In the rat, use of P450 form-selective inhibitors suggested the participation of P4501A and 3A forms in DHEA metabolism. Human liver samples displayed interindividual differences in that one of five subjects metabolized DHEA to a much greater extent than the others. This difference correlated with the level of P4503A activity present in the human liver samples. For one subject, troleandomycin inhibited hepatic microsomal metabolism of DHEA by 78%, compared to 81% inhibition by miconazole, suggesting the importance of P4503A in these reactions. Form-selective inhibitors of P4502D6 and P4502E1 had a modest inhibitory effect, suggesting that these forms may also contribute to metabolism of DHEA in humans. Metabolites identified by LC-MS in both species included 16alpha-hydroxy-DHEA, 7alpha-hydroxy-DHEA, and 7-oxo-DHEA. While 16alpha-hydroxy-DHEA appeared to be the major metabolite produced in rat, the major metabolite produced in humans was a mono-hydroxylated DHEA species, whose position of hydroxylation is unknown.
Assuntos
Hidrocarboneto de Aril Hidroxilases , Sistema Enzimático do Citocromo P-450/metabolismo , Desidroepiandrosterona/metabolismo , Microssomos Hepáticos/metabolismo , Adulto , Animais , Benzoflavonas/farmacologia , Cromatografia Líquida , Citocromo P-450 CYP3A , Inibidores das Enzimas do Citocromo P-450 , Desidroepiandrosterona/análogos & derivados , Desidroepiandrosterona/química , Humanos , Técnicas In Vitro , Masculino , Espectrometria de Massas , Miconazol/farmacologia , Microssomos Hepáticos/efeitos dos fármacos , Pessoa de Meia-Idade , Oxirredutases N-Desmetilantes/antagonistas & inibidores , Oxirredutases N-Desmetilantes/metabolismo , Ratos , Ratos Sprague-Dawley , Troleandomicina/farmacologiaRESUMO
Using two-dimensional electrophoresis, mass spectrometry, immunoblotting, and affinity pull-down assays, we found that myocardial protein kinase C epsilon (PKCepsilon) is physically associated with at least 36 known proteins that are organized into structural proteins, signaling molecules, and stress-responsive proteins. Furthermore, we found that the cardioprotection induced by activation of PKCepsilon is coupled with dynamic modulation and recruitment of PKCepsilon-associated proteins. The results suggest heretofore-unrecognized functions of PKCepsilon and provide an integrated framework for the understanding of PKCepsilon-dependent signaling architecture and cardioprotection.
Assuntos
Isoenzimas/análise , Miocárdio/química , Proteína Quinase C/análise , Proteoma/análise , Transdução de Sinais , Animais , Fármacos Cardiovasculares/análise , Eletroforese em Gel Bidimensional , Isoenzimas/genética , Isoenzimas/fisiologia , Camundongos , Camundongos Transgênicos , Miocárdio/enzimologia , Proteína Quinase C/genética , Proteína Quinase C/fisiologia , Proteína Quinase C-épsilon , Processamento de Proteína Pós-Traducional , Proteoma/fisiologiaRESUMO
GM2 synthase is a homodimer in which the subunits are joined by lumenal domain disulfide bond(s). To define the disulfide bond pattern of this enzyme, we analyzed a soluble form by chemical fragmentation, enzymatic digestion, and mass spectrometry and a full-length form by site-directed mutagenesis. All Cys residues of the lumenal domain of GM2 synthase are disulfide bonded with Cys(429) and Cys(476) forming a disulfide-bonded pair while Cys(80) and Cys(82) are disulfide bonded in combination with Cys(412) and Cys(529). Partial reduction to produce monomers converted Cys(80) and Cys(82) to free thiols while the Cys(429) to Cys(476) disulfide remained intact. CNBr cleavage at amino acid 330 produced a monomer-sized band under nonreducing conditions which was converted upon reduction to a 40-kDa fragment and a 24-kDa myc-positive fragment. Double mutation of Cys(80) and Cys(82) to Ser produced monomers but not dimers. In summary these results demonstrate that Cys(429) and Cys(476) form an intrasubunit disulfide while the intersubunit disulfides formed by both Cys(80) and Cys(82) with Cys(412) and Cys(529) are responsible for formation of the homodimer. This disulfide bond arrangement results in an antiparallel orientation of the catalytic domains of the GM2 synthase homodimer.
Assuntos
N-Acetilgalactosaminiltransferases/química , Sequência de Aminoácidos , Animais , Células CHO , Domínio Catalítico , Cricetinae , Dimerização , Dissulfetos , Glicosilação , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Polipeptídeo N-AcetilgalactosaminiltransferaseRESUMO
The apr locus of Pseudomonas aeruginosa encodes alkaline proteinase (APR), a member of the metzincin metalloendopeptidase superfamily, and an 11.4-kDa alkaline proteinase inhibitor (APRin). We describe here the expression in Escherichia coli and characterization of full-length and N-terminally truncated APRin proteins. Fluorescence and circular dichroism spectra indicated that the recombinant proteins were folded into native-like structures. Analytical ultracentrifugation showed that APRin was monomeric and formed a 1:1 complex with APR. Binding of wild-type APRin to APR occurred with association (k(on)) and dissociation (k(off)) rate constants of 0.29 +/- 0.06 x 10(6) m(-1) s(-1) and 1.15 +/- 0.08 x 10(-6) s(-1) to give an equilibrium dissociation constant (K(D)) of approximately 4 x 10(-12) m (25 degrees C, pH 7.0, ionic strength 2.4 m). The association rate decreased by approximately 2-fold in 20% glycerol and increased by approximately 3-fold in 0.1 m NaCl. The glycerol effect suggests a diffusion-limited reaction, and the small salt effect indicates that electrostatic interactions contribute little to binding. Deletion of residues 1-10, 1-6, or 6-10 abolished inhibition, and deletion of residues 1-2, 1-3, 1-4, and 1-5 resulted in a progressively decreased affinity of APRin for APR (K(D) = 0.12 micrometer the Delta(1-5) mutant). Substitution of APRin residues 6-10 with a (Gly)(5) or (Pro)(5) linker restored inhibitory activity of the Delta(6-10) mutant but with a 100- and 50-fold reduction in K(D). Log k(on) for the full-length and truncated inhibitors correlated with the solvent-accessible surface area of their N-terminal regions, suggesting that increased interactions and/or desolvation of these residues in the transition state for binding contribute to the enhanced association rate. Treatment of APRin with pseudolysin, also secreted by P. aeruginosa, resulted in removal of residues 1-5. APRin was neither an inhibitor nor a substrate of other metzincins, including collagenase or gelatinases A or B.
Assuntos
Fosfatase Alcalina/antagonistas & inibidores , Metaloendopeptidases/metabolismo , Pseudomonas/enzimologia , Pseudomonas/genética , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Sequência de Aminoácidos , Clonagem Molecular , Escherichia coli , Cinética , Mutagênese , Fragmentos de Peptídeos/química , Reação em Cadeia da Polimerase , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Serina Endopeptidases/químicaRESUMO
The use of a fish oil vehicle for cyclosporin A (CsA) can decrease the toxic effects of CsA but the mechanism is unclear. Here we examine the mechanism by which docosahexaenoic acid (DHA), a fish oil-derived polyunsaturated fatty acid, can alter the toxic effects of CsA on mouse organ function, endothelial macromolecular permeability, and membrane bilayer function. Mice given CsA and fish oil showed increased liver toxicity, kidney toxicity, incorporation of DHA, and evidence of oxidized fatty acids compared to control animals. We hypothesized that the toxic effects of CsA were primarily a result of membrane perturbation, which could be decreased if DHA were not oxidized. The presence of CsA (10 mol%) alone increased dipalmitoylphosphatidylcholine membrane permeability by seven fold over control (no CsA, no DHA). However, if non-oxidized DHA (15 mol%) and CsA were added to the membrane, the permeability returned to control levels. Interestingly, if the DHA was oxidized, the antagonistic effect of DHA on CsA was completely lost. While CsA alone increased endothelial permeability to albumin, the combination of non-oxidized DHA and CsA had no effect on endothelial macromolecular permeability. However the combination of oxidized DHA and CsA was no different than the effects of CsA only. CsA increased the fluorescence anisotropy of DPH in the liquid crystalline state of DPPC, while DHA decreased fluorescence anisotropy. However the combination of CsA and DHA was no different than DHA alone. We conclude that non-oxidized DHA can reverse the membrane perturbing effects of CsA, and the increases in endothelial macromolecular permeability, which may explain how fish oil is capable of decreasing the toxicity of CsA.
Assuntos
Ciclosporina/toxicidade , Ácidos Docosa-Hexaenoicos/farmacologia , Óleos de Peixe/farmacologia , Membranas Artificiais , Animais , Células Cultivadas , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Feminino , Fluidez de Membrana , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Zinc deficiency is associated with a reduced rate of bone formation that can be corrected by supplementation of the deficient diet with adequate amounts of zinc. This study was conducted to examine the effects of zinc on bone cell parameters associated with bone formation. Tibiae were removed from 19-d-old chicken embryos and incubated for 48 h in Dulbecco's modified Eagle's medium supplemented with antibiotics, bovine serum albumin, and HEPES. The addition of zinc (25-200 g/dL) to tibial cultures resulted in a concentration-dependent increase in alkaline phosphatase activity, an increase in the incorporation of proline into bone protein and an increase in the post-translational oxidation of proline to peptidyl hydroxyproline. These effects of zinc were all diminished by the addition of 2,6-pyridine dicarboxylic acid, a chelator of zinc. The addition of either cycloheximide (10(-5)M), dactinomycin (10(-8)M), or hydroxyurea (10(-3)M) to tibial cultures also attenuated the effects of zinc. The effect of zinc on bone cell DNA synthesis was measured by following the incorporation of 3H-thymidine into DNA and by fluorometric measurement of cellular DNA content. These methods revealed that the addition of zinc to cultured tibiae resulted in a concentration-dependent increase in tibial DNA content and synthesis rate. The magnitude of the zinc-induced DNA increase was similar to the magnitude of the zinc-induced increases in alkaline phosphatase activity, proline incorporation, and hydroxyproline synthesis. Normalization of these latter responses to tibial DNA content yield data indicating that the effect of zinc on bone formation results from a zinc-induced increase in bone cell proliferation.
Assuntos
Desenvolvimento Ósseo/efeitos dos fármacos , Zinco/farmacologia , Fosfatase Alcalina/metabolismo , Animais , Biomarcadores , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/enzimologia , Osso e Ossos/metabolismo , Embrião de Galinha , Replicação do DNA/efeitos dos fármacos , Hidroxiprolina/biossíntese , Hidroxiprolina/metabolismo , Prolina/metabolismo , Inibidores da Síntese de Proteínas/farmacologiaRESUMO
Carbonic anhydrase (CA) inhibitors lower the rate of aqueous humor (AH) secretion into the eye. Different CA isozymes might play different roles in the response. Here we have studied the effects of carbonic anhydrase inhibitors on cytoplasmic pH (pHi) regulation, using a dextran-bound CA inhibitor (DBI) to selectively inhibit membrane-associated CA in a cell line derived from rabbit NPE. pHi was measured using the fluorescent dye BCECF and the pHi responses to the cell permeable CA inhibitor acetazolamide (ACTZ) and DBI were compared. ACTZ markedly inhibited the rapid pHi changes elicited by bicarbonate/CO2 removal and readdition but DBI was ineffective in this respect, consistent with the inability of DBI to enter the cell and inhibit cytoplasmic CA isozymes. Added alone, ACTZ and DBI caused a similar reduction (0.2 pH units) of baseline pHi. We considered whether CA-IV might facilitate H+ extrusion via Na-H exchange. The Na-H exchanger inhibitor amiloride (1 mM) reduced pHi 0.52 +/- 0.10 pH units. In the presence of DBI, the magnitude of pHi reduction caused by amiloride was significantly (P < 0.05) reduced to 0.26 +/- 0.09 pH units. ACTZ similarly reduced the magnitude of the pHi reduction. DBI also reduced by approximately 40% the rate of pHi recovery in cells acidified by an ammonium chloride (20 mM) prepulse; a reduction in pHi recovery rate was also caused by ACTZ and amiloride. DBI failed to alter the pHi alkalinization response caused by elevating external potassium concentration, a response insensitive to amiloride but sensitive to ACTZ. These observations are consistent with a reduction in Na-H exchanger activity in the presence of DBI or ACTZ. We suggest that the CA-IV isozyme might catalyze rapid equilibration of H+ and HCO3- with CO2 in the unstirred layer outside the plasma membrane, preventing local accumulation of H+ which competes with sodium for the same external Na-H exchanger binding site. Inhibition of CA-IV could produce pHi changes that might alter the function of other ion transporters and channels in the NPE.
Assuntos
Acetazolamida/farmacologia , Inibidores da Anidrase Carbônica/farmacologia , Dextranos/farmacologia , Células Epiteliais/efeitos dos fármacos , Sulfonamidas/farmacologia , Amilorida/farmacologia , Cloreto de Amônio/farmacologia , Animais , Carbonatos/metabolismo , Linhagem Celular , Corpo Ciliar/citologia , Citoplasma/efeitos dos fármacos , Citoplasma/fisiologia , Células Epiteliais/fisiologia , Epitélio , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Potássio/farmacologia , CoelhosRESUMO
The addition of physiological concentrations of zinc (25-200 (microg/dL) to Dulbecco's Modified Eagle's Medium containing tibiae from 19-d chick embryos resulted in a concentration-dependent increase in tibial content of tartrate-resistant acid phosphatase (TRAP) and an increase in bone resorption, as measured by tibial calcium release. This increase in bone resorption was additive to the resorptive effect resulting from the addition of 10(-9)-10(-7) M parathyroid hormone (PTH), but was not additive to similar effects produced by the addition of 10(-9)-10(-7) M prostaglandin E2 (PGE2). An inhibitor of prostaglandin synthesis, flurbiprofen (10[-6] M), did not influence the effect of zinc on bone resorption. However, the addition of 2,6-pyridinedicarboxylic acid (10[-3] M, 2,6-PDCA), a chelator of zinc, did attenuate the effects of zinc, as did the addition of an inhibitor of DNA replication (hydroxyurea, 10[-3] M). Hydroxyurea also attenuated the bone resorptive response to PGE2, but had no influence on the effects of PTH. These results indicate that physiological concentrations of zinc alter bone resorptive rates in vitro by a mechanism that is dependent on DNA replication.
Assuntos
Reabsorção Óssea/metabolismo , Tíbia/metabolismo , Zinco/farmacologia , Fosfatase Ácida/metabolismo , Animais , Reabsorção Óssea/etiologia , Cálcio/metabolismo , Quelantes/farmacologia , Embrião de Galinha , Inibidores de Ciclo-Oxigenase/farmacologia , Replicação do DNA/efeitos dos fármacos , Dinoprostona , Flurbiprofeno/farmacologia , Hidroxiureia/farmacologia , Técnicas In Vitro , Isoenzimas/metabolismo , Inibidores da Síntese de Ácido Nucleico/farmacologia , Hormônio Paratireóideo , Ácidos Picolínicos/farmacologia , Fosfatase Ácida Resistente a Tartarato , Tíbia/efeitos dos fármacos , Tíbia/embriologiaRESUMO
PURPOSE: Cultured rabbit nonpigmented ciliary epithelium (NPE) transports ascorbic acid (ASC) inward through a sodium-dependent mechanism. This study was conducted to test whether Na-K transport is activated to export the additional sodium, which enters the cell in cotransport with ASC. METHODS: Studies were conducted using a cell line derived from rabbit NPE. ASC uptake was measured using [14C]ascorbic acid. The ouabain-sensitive potassium (86Rb) uptake rate was measured as an index of active Na-K transport. Cellular sodium was measured by atomic absorption spectrophotometry or SBFI fluorescence. RESULTS: In the presence of 200 microM ASC, ouabain-sensitive potassium (86Rb) uptake rate increased approximately 70%; lesser concentrations of ASC produced lesser increases. Phloridzin (100 microM) inhibited ASC uptake and inhibited the stimulatory effect of external ASC on 86Rb uptake. Dehydroascorbic acid (DHA) did not increase 86Rb uptake. Neither DHA nor ASC altered the Na,K-ATPase activity measured in isolated membrane material. External ASC appeared to stimulate active sodium transport through a mechanism involving an increase of cytoplasmic sodium. In the presence of 200 microM ASC, cellular sodium increased approximately 26%; studies with cells, sodium loaded by nigericin treatment, suggested that this sodium increase could account for the degree of 86Rb uptake stimulation observed in ASC-treated cells. However, the cellular sodium increase could not be explained simply on the basis of sodium entry through the ASC transporter. An additional sodium-entry pathway seemed to be activated in cells that accumulated ASC. Dimethylamiloride (DMA) abolished both the cellular sodium increase and the 86Rb uptake stimulation caused by ASC. DMA did not prevent ASC uptake. CONCLUSIONS: ASC significantly stimulated active Na-K transport in cultured NPE. The mechanism appeared to involve activation of a DMA-sensitive sodium entry pathway, which caused cytoplasmic sodium concentration to increase.
Assuntos
Ácido Ascórbico/farmacologia , Corpo Ciliar/metabolismo , Epitélio Pigmentado Ocular/metabolismo , Sódio/metabolismo , Amilorida/análogos & derivados , Amilorida/farmacologia , Animais , Ácido Ascórbico/metabolismo , Transporte Biológico Ativo/efeitos dos fármacos , Linhagem Celular , Células Cultivadas , Corpo Ciliar/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Epitélio/metabolismo , Ouabaína/farmacologia , Florizina/farmacologia , Epitélio Pigmentado Ocular/efeitos dos fármacos , Potássio/metabolismo , Coelhos , Radioisótopos de Rubídio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Espectrofotometria AtômicaRESUMO
Previously, we described a new metabolite derived from endogenous cholesterol in the presence of hamster liver microsomal protein and NADPH (Song et al., 1991, Biochem. Pharmacol. 41, 1439-1447). Through gas chromatography/mass spectral analysis of the metabolite and its methoxime-3-dimethyl-t-butylsilyl ether derivative, this metabolite has been definitively identified as 7-oxocholesterol. Isotope incorporation experiments using molecular 18O2 demonstrated that no oxygen atoms from molecular oxygen were incorporated into the product, 7-oxocholesterol, when 7 alpha-hydroxycholesterol was used as substrate. In contrast, one atom of 18O was incorporated into cholesterol from 18O2 during its metabolism to form 7 alpha-hydroxycholesterol. Formation of 7-oxocholesterol was dependent upon the presence of NADP+, 7 alpha-hydroxycholesterol, and hamster liver microsomes. This enzyme appears to be a membrane-bound protein and its activity was most abundant in liver microsomal fractions and to a lesser extent in mitochondrial fractions; little or no activity was observed in nuclei or cytosol. The enzyme activity was present in highest content in the livers of hamsters and was also observed in human and bovine liver microsomes, but not those of mouse, rabbit, or rat. The reaction was inhibited by 2'-AMP, but not by anti-NADPH:cytochrome-P450 oxidoreductase globulin, carbon monoxide, metyrapone, nor miconazole. In contrast to the previously characterized 3 beta-hydroxy-delta 5-C27-steroid oxidoreductase activity, NAD+ did not serve as an effective cofactor for 7-oxocholesterol formation. The ability of NADPH to partially serve as a cofactor in this reaction was shown to be due to a high NADPH-oxidase activity of hamster liver microsomes, thereby providing sufficient NADP+ to serve as the oxidizing pyridine nucleotide for the reaction. These results document the existence of a non-P450, NADP(+)-dependent 7 alpha-hydroxycholesterol dehydrogenase in liver microsomes which catalyzes this reaction. The product, 7-oxocholesterol, is produced enzymatically in the livers of hamsters and other mammals and may regulate bile acid metabolism or other processes due to its action as an oxysterol.
Assuntos
Hidroxiesteroide Desidrogenases/metabolismo , Cetocolesteróis/metabolismo , Microssomos Hepáticos/enzimologia , Animais , Bovinos , Cromatografia Líquida de Alta Pressão , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Cinética , Fígado/enzimologia , Masculino , Camundongos , NAD/metabolismo , NADP/metabolismo , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Coelhos , Ratos , Ratos Sprague-Dawley , Especificidade da Espécie , Frações Subcelulares/enzimologiaRESUMO
This paper describes a technique for quantitative analysis of 2-oxoglutarate (alpha-ketoglutarate) in biological samples using liquid chromatography with electrochemical detection (LC-EC). This method utilizes a simplified, efficient sample preparation designed to select for 2-oxoglutarate and similar compounds by derivatization with phenylhydrazine. The response was linear over the range from 62.5 to 1000 ng/ml. The least quantifiable concentration was 62.5 ng/ml and the least detectable concentration was 25 ng/ml. To test the ability of the assay to measure 2-oxoglutarate in biological samples, this method was used to quantitate the 2-oxoglutarate content in chick osteoblast cultures and to determine the ability of the assay to accurately measure a standard addition of 500 ng/ml 2-oxoglutarate when added to a sample of the forementioned groups. The 2-oxoglutarate content of these cells was 6.67 +/- 1.20 ng/micrograms DNA or 105 +/- 18 ng/cell layer (mean +/- 95% confidence interval) and the assay accurately measured the standard addition. This method was also used to quantitate 2-oxoglutarate content in whole embryonic chick calvariae containing 6.40 +/- 0.95 ng/mg dry bone weight or 37.5 +/- 5.5 ng/bone. This assay provides significantly lower detection limits than the currently available procedures and is suitable for determination of 2-oxoglutarate in biological samples where very low amounts of 2-oxoglutarate are found. This method is the first application of LC-EC for quantitating 2-oxoglutarate.
Assuntos
Cromatografia Líquida/métodos , Ácidos Cetoglutáricos/análise , Animais , Células Cultivadas , Embrião de Galinha , Eletroquímica , Osteoblastos/química , Crânio/química , Crânio/embriologiaRESUMO
Studies evaluated a novel series of biscarbonylamides of 2-amino-1,3,4-thiadiazole-5-sulfonamide (2-ATS) for topical use as ocular hypotensive carbonic anhydrase inhibitors (CAI). Transcorneal accession rate constants (k(in)) for ethyloxaloylazolamide (EtOxAz), ethylsuccinylazolamide (EtSuxAz) and ethyladipoylazolamide (EtAdipAz), and activity against carbonic anhydrase (CA) were determined in vitro by an enzymatic assay and High Performance Liquid Chromatography (HPLC). The ocular hypotensive effect was measured by pneumatonometry on conscious normotensive New Zealand White (NZW) rabbits, using masked, randomly assigned paired-eye design for treatment vs. control. At various time points following treatment, aqueous humor, ciliary processes and corneal buttons were collected and assayed for drug concentrations using enzymatic assay and HPLC. Transcorneal accession rates for the novel compounds were 1.5 to 18 times that of the parent compound, acetazolamide (Actz). The activity factor for EtOxAz was 72.8 x 10(3) hr-1 of 23 times that of Actz. The activity factors for EtSuxAz and EtAdipAz were 6.8 and 1.1 x 10(3) hr-1, respectively. Subcutaneous administration of EtOxAz. EtSuxAz, and EtAdipAz, in 225 mumol kg-1 concentrations, induced a significant decrease in the intraocular pressure (IOP) at 1 hr post injection of 4, 5.8 and 6 mmHg for EtOxAz, EtSuxAz and EtAdipAz, respectively (P < 0.05 for each). Topical application of 75 mM EtOxAz lowered the IOP by 3.0 mmHg (P < 0.05). This effect was maximal after 60 min and persisted for at least 5 hr. EtSuxAz and EtAdipAz did not alter the IOP significantly when given topically. Subcutaneous administration of the three compounds was associated with acidosis (pH as low as 7.21). Topical application did not cause any changes in the acid-base balance. There was a direct correlation between the amount of drug delivered to the ciliary process and the magnitude of ocular hypotensive effect. Following topical application EtOxAz reached the ciliary epithelium in concentrations sufficient to inhibit more than 99.95% of the ciliary carbonic anhydrase (> 8 microM), while plasma drug concentrations were below the limit of detection by the assay (< 0.2 microM). Within the first hour after topical application, half of the EtOxAz was eliminated from the anterior uvea. In summary, EtOxAz is a topically effective CAI. Structural modifications of thiadiazole sulfonamides, with the increase of both water and lipid solubilities, improved the transcorneal accession while preserving sufficient CA inhibitory activity, resulting in a significant IOP decrease following topical application of EtOxAz.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Equilíbrio Ácido-Base/efeitos dos fármacos , Inibidores da Anidrase Carbônica/farmacocinética , Pressão Intraocular/efeitos dos fármacos , Tiadiazóis/farmacocinética , Administração Tópica , Animais , Inibidores da Anidrase Carbônica/farmacologia , Bovinos , Córnea/metabolismo , Olho/metabolismo , Técnicas In Vitro , Injeções Subcutâneas , Coelhos , Tiadiazóis/farmacologia , Fatores de Tempo , Distribuição TecidualRESUMO
A novel homologous series of bis(carbonyl)amidothiadiazole sulfonamides has been synthesized for structure-activity relationship studies, and initial characterization has been performed. The goal was synthesis of thiadiazole derivatives with appropriate lipid and water solubilities for utility as topically (corneal application) active carbonic anhydrase (CA) inhibitors. This series has solubility properties and pKa which bracket those of acetazolamide--the prototypical CA inhibitor. All of these compounds are active as in vitro CA inhibitors, and are 10-25% as potent as acetazolamide as in vitro enzyme inhibitors. Two of these compounds act as ocular hypotensive agents after topical application of a single dose to the corneas of normotensive New Zealand albino rabbits. The efficacy of the lead compound of this series (in this one model) is approximately equivalent to that of topical CA inhibitors that are presently in clinical trial. None of these novel compounds reacts to an appreciable extent with free sulfhydryl groups (a predictor of toxicity). This family of compounds will be useful for future studies of ocular pharmacokinetics, as well as ocular and systemic effects of topical administration of CA inhibitors. These and future studies may lead to development of thiadiazole sulfonamides useful in the management of glaucoma.