RESUMO
The heterodimeric transcription factor hypoxia-inducible factor-1 (HIF-1) is activated under hypoxic conditions, resulting in the upregulation of its target genes plasminogen activator inhibitor-1 (PAI-1) and vascular endothelial growth factor (VEGF). PAI-1 and VEGF are also induced in response to vascular injury, which is characterized by the activation of platelets and the coagulation cascade as well as the generation of reactive oxygen species (ROS). However, it is not known whether HIF-1 is also stimulated by thrombotic factors. We investigated the role of thrombin, platelet-associated growth factors, and ROS derived from the p22(phox)-containing NADPH oxidase in the activation of HIF-1 and the induction of its target genes PAI-1 and VEGF in human vascular smooth muscle cells (VSMCs). Thrombin, platelet-derived growth factor-AB (PDGF-AB), and transforming growth factor-beta(1) (TGF-beta(1)) upregulated HIF-1alpha protein in cultured and native VSMCs. This response was accompanied by nuclear accumulation of HIF-1alpha as well as by increased HIF-1 DNA-binding and reporter gene activity. The thrombin-induced expression of HIF-1alpha, PAI-1, and VEGF was attenuated by antioxidant treatment as well as by transfection of p22(phox) antisense oligonucleotides. Inhibition of p38 mitogen-activated protein kinase and phosphatidylinositol-3-kinase significantly decreased thrombin-induced HIF-1alpha, PAI-1, and VEGF expression. These findings demonstrate that the HIF-1 signaling pathway can be stimulated by thrombin and platelet-associated growth factors and that a redox-sensitive cascade activated by ROS derived from the p22(phox)-containing NADPH oxidase is crucially involved in this response.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Membrana Transportadoras , Músculo Liso Vascular/metabolismo , NADPH Desidrogenase/fisiologia , NADPH Oxidases/fisiologia , Proteínas Nucleares/metabolismo , Fosfoproteínas/fisiologia , Transdução de Sinais , Trombina/farmacologia , Fatores de Transcrição , Antioxidantes/farmacologia , Células Cultivadas , Proteínas de Ligação a DNA/fisiologia , Fatores de Crescimento Endotelial/biossíntese , Fatores de Crescimento Endotelial/genética , Humanos , Fator 1 Induzível por Hipóxia , Subunidade alfa do Fator 1 Induzível por Hipóxia , Linfocinas/biossíntese , Linfocinas/genética , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/enzimologia , Proteínas Nucleares/fisiologia , Fosfatidilinositol 3-Quinases/fisiologia , Inibidor 1 de Ativador de Plasminogênio/biossíntese , Inibidor 1 de Ativador de Plasminogênio/genética , Fator de Crescimento Derivado de Plaquetas/farmacologia , RNA Mensageiro/biossíntese , Espécies Reativas de Oxigênio/fisiologia , Ativação Transcricional , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio Vascular , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
We present a strategy to characterize specific antigen/autoantibody systems using polyclonal sera from patients as a probe and crude antigenic preparations such as mitoplasts. Sera from patients with primary biliary cirrhosis (PBC) are characterized by at least one of two specific subtypes of antimitochondrial antibodies (AMA), anti-p48 or anti-p62. Immunoblotting of such sera with mitoplast preparations derived from human, rat and rabbit livers revealed three proteins of approximately 27, 48 and 68 kDa as target antigens. On the basis of the molecular weight of these antigens we were able to purify them by elution from preparative SDS gels. Immunization of NZB mice with the high molecular weight component (the 68 kDa antigen from human liver mitoplasts) elicited a monoclonal antibody. The 68 kDa protein was then isolated by affinity chromatography and may well represent the prime target antigen of anti-p62 antimitochondrial antibodies. This experimental approach could be applied to protein target antigens of other autoantibodies.
Assuntos
Anticorpos Monoclonais , Autoanticorpos , Autoantígenos/análise , Doenças Autoimunes/diagnóstico , Mitocôndrias Hepáticas/imunologia , Animais , Autoantígenos/imunologia , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Feminino , Humanos , Hibridomas/análise , Soros Imunes , Camundongos , Camundongos Endogâmicos NZB , Coelhos , Radioimunoensaio/métodos , RatosRESUMO
BACKGROUND: This study was designed to assess the diagnostic value of the oral calcium tolerance test with measurement of intact parathyroid hormone by the immunoradiometric assay (IRMA PTH) in the diagnosis of primary hyperparathyroidism in patients with symptoms who have minimal, intermittent, or no elevation of the levels of total calcium and/or intact PTH. METHODS: After baseline levels of IRMA PTH and total calcium were measured, an oral calcium load of 1000 mg elemental calcium was administered to 10 patients with hyperparathyroidism and 18 normal control subjects. Total calcium and IRMA PTH levels were measured at 30, 60, and 120 minutes after the oral calcium load was administered. RESULTS: The mean suppression of the baseline level of IRMA PTH in the patients with hyperparathyroidism was 83.7% +/- 6.5% (mean +/- 1 SEM), but the levels of the normal control subjects fell significantly (p < 0.05) lower to 58.8% +/- 3.7% (mean +/- 1 SEM). CONCLUSIONS: This study suggests that the oral calcium tolerance test may be a valuable adjunct in confirming the diagnosis of primary hyperparathyroidism in patients with symptoms who have minimal, intermittent, or no elevation of the levels of total calcium and/or IRMA PTH:
Assuntos
Cálcio , Hiperparatireoidismo/diagnóstico , Hormônio Paratireóideo/sangue , Administração Oral , Adulto , Idoso , Idoso de 80 Anos ou mais , Cálcio/sangue , Feminino , Humanos , Hiperparatireoidismo/sangue , Hiperparatireoidismo/cirurgia , Ensaio Imunorradiométrico , Masculino , Pessoa de Meia-Idade , Período Pós-Operatório , Valores de Referência , Fatores de TempoRESUMO
The chromatographic behavior of six calixarene-bonded stationary phases is reported. Varying analyte selectivities (i.e., for phenols, substituted aromatics, polycyclic aromatic hydrocarbons, barbituric acid derivatives, xanthines) exist as a function of the ring-size of the calix[n]arenes (n=4, 6, 8) and the substitution at the "upper rim" with para-tert.-butyl groups. Although eluents with unusually high proportions of water were used, a comparison with conventional reversed-phase (RP) columns shows a predominantly reversed-phase character with remarkable selectivities of these phases. The influences of several organic solvents on retention variations of solutes are compared for RP-C18, phenyl and calixarene phases.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Substâncias Macromoleculares , Calixarenos , Fenitoína/isolamento & purificação , Compostos Policíclicos/isolamento & purificação , Primidona/isolamento & purificaçãoRESUMO
The adductor canal was simulated using 2.6 cm muscular tissue and 2 fasciae to analyse the limits of colour-coded Doppler sonography (angiodynography) in this region. Defects in the spectral signal cause a significant underestimation of mean, peak systolic and peak diastolic (backflow) velocities and of calculated blood flow. Furthermore the pulsatility index is overestimated and the colour-coded visualisation of the arteries is almost lost. For the most part, these changes can be compensated by administration of a sonographic contrast agent (SH U 454). A minimum of 9 mg microbubbles/ml blood is required. Nevertheless, the adjustment of system controls (e.g. transducer power) becomes more difficult and an ideal setting impossible.
Assuntos
Meios de Contraste , Artéria Femoral/diagnóstico por imagem , Modelos Estruturais , Polissacarídeos , Humanos , Perna (Membro)/diagnóstico por imagem , Músculos/diagnóstico por imagem , UltrassonografiaRESUMO
A new practically orientated phantom for evaluating Doppler Duplex equipment was developed and used to determine the possibilities and limits of color coded Doppler Sonography (Angiodynography) in the diagnosis of arterial occlusive disease of the lower extremity. The problems that may occur in quantifying flow in these arteries are analyzed. The influence of superimposed slices of various soft-tissues on flow quantification, on the spectral waves and on color coded visualisation of the arteries and the possibility to compensate those changes by the use of sonographic contrast agents are discussed.
Assuntos
Arteriopatias Oclusivas/diagnóstico , Perna (Membro)/irrigação sanguínea , Ultrassonografia , Cor , Estudos de Avaliação como Assunto , Humanos , Modelos EstruturaisAssuntos
Equipamentos Odontológicos de Alta Rotação , Instrumentos Odontológicos , Desinfecção/instrumentação , Esterilização/instrumentação , Desinfecção/métodos , Desenho de Equipamento , Estudos de Avaliação como Assunto , Infecções por HIV/prevenção & controle , Hepatite Viral Humana/prevenção & controle , Humanos , Lubrificação , Esterilização/métodosRESUMO
Nicotinic acetylcholine receptors play important roles in numerous cognitive processes as well as in several debilitating central nervous system (CNS) disorders. In order to fully elucidate the diverse roles of nicotinic acetylcholine receptors in CNS function and dysfunction, a detailed knowledge of their cellular and subcellular localizations is essential. To date, methods to precisely localize nicotinic acetylcholine receptors in the CNS have predominantly relied on the use of anti-receptor subunit antibodies. Although data obtained by immunohistology and immunoblotting are generally in accordance with ligand binding studies, some discrepancies remain, in particular with electrophysiological findings. In this context, nicotinic acetylcholine receptor subunit-deficient mice should be ideal tools for testing the specificity of subunit-directed antibodies. Here, we used standard protocols for immunohistochemistry and western blotting to examine the antibodies raised against the alpha3-, alpha4-, alpha7-, beta2-, and beta4-nicotinic acetylcholine receptor subunits on brain tissues of the respective knock-out mice. Unexpectedly, for each of the antibodies tested, immunoreactivity was the same in wild-type and knock-out mice. These data imply that, under commonly used conditions, these antibodies are not suited for immunolocalization. Thus, particular caution should be exerted with regards to the experimental approach used to visualize nicotinic acetylcholine receptors in the brain.
Assuntos
Anticorpos/metabolismo , Especificidade de Anticorpos/imunologia , Imuno-Histoquímica/métodos , Neuroquímica/métodos , Subunidades Proteicas/imunologia , Receptores Nicotínicos/imunologia , Acetilcolina/metabolismo , Animais , Animais Recém-Nascidos , Anticorpos/química , Western Blotting , Bungarotoxinas/metabolismo , Córtex Cerebral/anatomia & histologia , Córtex Cerebral/imunologia , Córtex Cerebral/metabolismo , Eletroforese em Gel Bidimensional , Feminino , Hipocampo/anatomia & histologia , Hipocampo/imunologia , Hipocampo/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/imunologia , Neurônios/metabolismo , Subunidades Proteicas/análise , Subunidades Proteicas/genética , Receptores Nicotínicos/análise , Receptores Nicotínicos/genética , Transmissão Sináptica/imunologiaRESUMO
According to the Cholodny-Went hypothesis, gravitropic differential growth is brought about by the redistribution of auxin (indolyl-3-acetic acid, IAA). We reinvestigated the relevance of different auxins and studied the role of ethylene in hypocotyls of sunflower and shoots and roots of rye and maize seedlings. Incubation of coleoptiles and of sunflower hypocotyls in solutions of IAA and dichlorophenoxyacetic acid as well as naphthylacetic acid resulted in a two- to threefold length increase compared to water controls. In spite of this pronounced general effect on elongation growth, gravi-curvature was similar to water controls. In contrast to this, inhibition of ethylene synthesis by aminoethoxyvinylglycine prevented differential growth of both hypocotyls and coleoptiles and of roots of maize. In horizontally stimulated maize roots growing on surfaces, inhibition of ethylene perception by methylcyclopropene inhibited roots to adapt growth to the surface, resulting in a lasting vertical orientation of the root tips. This effect is accompanied by up- and down-regulation of a number of proteins as detected by two-dimensional matrix-assisted laser desorption-ionization time-of-flight mass spectrometry. Together the data query the regulatory relevance of IAA redistribution for gravitropic differential growth. They corroborate the crucial regulatory role of ethylene for gravitropic differential growth, both in roots and coleoptiles of maize as well as in hypocotyls.
Assuntos
Etilenos/metabolismo , Gravitropismo , Helianthus/crescimento & desenvolvimento , Secale/crescimento & desenvolvimento , Zea mays/crescimento & desenvolvimento , Ácido 2,4-Diclorofenoxiacético/farmacologia , Cotilédone/crescimento & desenvolvimento , Cotilédone/metabolismo , Ciclopropanos/farmacologia , Enzimas/metabolismo , Glicina/análogos & derivados , Glicina/farmacologia , Gravitropismo/efeitos dos fármacos , Helianthus/efeitos dos fármacos , Helianthus/metabolismo , Herbicidas/farmacologia , Hipocótilo/crescimento & desenvolvimento , Hipocótilo/metabolismo , Ácidos Indolacéticos/farmacologia , Modelos Biológicos , Ácidos Naftalenoacéticos/farmacologia , Mapeamento de Peptídeos , Reguladores de Crescimento de Plantas/farmacologia , Proteínas de Plantas/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Secale/efeitos dos fármacos , Secale/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Fatores de Tempo , Zea mays/efeitos dos fármacos , Zea mays/metabolismoRESUMO
An account is given on the long-term control of 27 unselected diabetics treated with Leo insulins in private internal medical practice. Both primary adjustments in recently manifested juvenile diabetes and change-over from other insulins or oral antidiabetics were carried out as outpatient treatment. Thorough training of the patient, which is a prerequisite to successful cooperation between the diabetic and his physician, is just as important as regular metabolic check-ups. The therapeutic results was considerably improved using Leo insulins. While 26 out of 27 patients were inadequately compensated before the beginning of therapy, the subsequent observation revealed very good adjustment in 11, good adjustment in 3 and satisfactory adjustment in 4 patients.
Assuntos
Diabetes Mellitus Tipo 1/tratamento farmacológico , Diabetes Mellitus/tratamento farmacológico , Insulina/uso terapêutico , Adolescente , Adulto , Idoso , Nefropatias Diabéticas/tratamento farmacológico , Neuropatias Diabéticas/tratamento farmacológico , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , AutoadministraçãoRESUMO
Statistical analysis of a group of 25 healthy individuals, examined via angiodynography, shows a large range of scatter for all measured parameters in pelvic and lower extremity arteries. Blood flow and velocity values, systolic acceleration and vessel diameter decrease whereas the pulsatility index rises from the centre towards the periphery. Problem areas for morphological and quantitative evaluation are the abdominal aorta, pelvic vessels, superficial femoral artery in the distal adductor canal and the fibular artery. Nearly all vessels showed tri- or multiphasic spectral patterns. Biphasic waveform, however, may be normal in lower extremity arteries. The data yielded by colour-coded Doppler sonography were comparable to results obtained with other procedures.
Assuntos
Perna (Membro)/irrigação sanguínea , Pelve/irrigação sanguínea , Ultrassonografia Doppler em Cores , Adolescente , Adulto , Artérias/diagnóstico por imagem , Velocidade do Fluxo Sanguíneo/fisiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Valores de ReferênciaRESUMO
Plasminogen activator inhibitor-1 (PAI-1) is the primary physiological inhibitor of both tissue-type and urokinase-type plasminogen activators. The balance between plasminogen activators and PAI-1 plays an important role in several physiological and pathophysiological processes such as atherosclerosis or thrombosis. Because these conditions are associated with hypoxia, it was the aim of the present study to investigate the influence of low O(2) tension on the expression of PAI-1 mRNA and protein using primary cultured rat hepatocytes as a model system. We found that PAI-1 mRNA and protein were induced by mild hypoxia (8% O(2)). The hypoxia-dependent PAI-1 mRNA induction was transcriptionally regulated because it was inhibited by actinomycin D (ActD). Luciferase (LUC) reporter gene constructs driven by about 800 bp of the 5'-flanking region of the rat PAI-1 gene were transiently transfected into primary rat hepatocytes; mild hypoxia caused a 3-fold induction, which was mediated by the PAI-1 promoter region -175/-158 containing 2 putative hypoxia response elements (HRE) binding the hypoxia-inducible factor (HIF-1). Mutation of the HRE-1 (-175/-168) or HRE-2 (-165/-158) also abolished the induction by mild hypoxia. Cotransfection of a HIF-1alpha vector and the PAI-1-LUC constructs, as well as gel shift assays, showed that the HRE-2 of the PAI-1 promoter was most critical for induction by hypoxia and HIF-1 binding. Thus, PAI-1 induction by mild hypoxia via a HIF-1 binding HRE in the rat PAI-1 promoter appears to be the mechanism causing the increase in PAI-1 in many clinical conditions associated with O(2) deficiency.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Fígado/metabolismo , Proteínas Nucleares/metabolismo , Inibidor 1 de Ativador de Plasminogênio/biossíntese , Elementos de Resposta , Fatores de Transcrição , Animais , Hipóxia Celular , Regulação da Expressão Gênica , Fator 1 Induzível por Hipóxia , Subunidade alfa do Fator 1 Induzível por Hipóxia , Fígado/patologia , Masculino , Oxigênio/metabolismo , Inibidor 1 de Ativador de Plasminogênio/genética , Ratos , Ratos WistarRESUMO
Right-heart catheterization and ergometry with arterial and mixed venous blood gas analysis were performed in 27 patients with a wide range of chronic obstructive pulmonary diseases. The purpose of the examination was to evaluate the risk in patients for lung surgery or to detect additional heart diseases. Patients who developed exertional hypoxia (group 1) were compared with others who did not (group 2). In all patients the steady-state maximal workload was determined by ventilatory dysfunction. Both groups had normal values for mixed venous pO2 and normal increase of the circulatory parameters during exercise. The patients with exertional hypoxia differed from the others in that they showed no decrease of venous admixture and alveolar-arterial oxygen gradient. In addition, these patients had increasing pCO2 values at rest compared with exercise, indicating alveolar hypoventilation and ventilation-perfusion mismatching. Because of the good correlation of the absolute values of FEV1 (forced expiratory volume in 1 s) with pulmonary artery pressures, parameters of gas exchange and working capacity, this lung function parameter seems to have a central role in predicting the functional state of patients with chronic obstructive disease. Ergometry and blood gas analysis should be performed in addition because these values cannot be predicted with the calculated postoperative FEV1.
Assuntos
Hemodinâmica , Pneumopatias Obstrutivas/fisiopatologia , Troca Gasosa Pulmonar , Idoso , Pressão Sanguínea , Débito Cardíaco , Volume Expiratório Forçado , Humanos , Hipertensão Pulmonar/etiologia , Hipóxia/etiologia , Pneumopatias Obstrutivas/complicações , Pessoa de Meia-Idade , Oxigênio/sangue , Esforço FísicoRESUMO
Apart from the proximal anastomosis, aortoiliac/-femoral bypass is accessible to angiodynography. The spectral waves can mostly be derived and analyzed very well. Femoropopliteal grafts can be demonstrated over the whole length, including the anastomoses if they are not located in the adductor (Hunter's) canal. Pathological findings such as stenosis or aneurysms can be easily detected. Because of a large range of scatter, blood velocity or flow are no suitable parameter to determine functioning and prognosis of the bypass. A triphasic spectral waveform in the graft, the failing of aneurysms, stenosis or turbulent blood flow in the anastomosis, a high "Pulsatility Index" and a short systolic blood flow acceleration distal to the bypass, proved to be more important parameters, that can be easily determined via colour coded Doppler sonography.
Assuntos
Prótese Vascular , Oclusão de Enxerto Vascular/diagnóstico por imagem , Isquemia/diagnóstico por imagem , Isquemia/cirurgia , Perna (Membro)/irrigação sanguínea , Complicações Pós-Operatórias/diagnóstico por imagem , Adulto , Idoso , Aorta Abdominal/diagnóstico por imagem , Aorta Abdominal/cirurgia , Velocidade do Fluxo Sanguíneo/fisiologia , Volume Sanguíneo/fisiologia , Feminino , Artéria Femoral/diagnóstico por imagem , Artéria Femoral/cirurgia , Humanos , Artéria Ilíaca/diagnóstico por imagem , Artéria Ilíaca/cirurgia , Masculino , Pessoa de Meia-Idade , Desenho de Prótese , Fluxo Pulsátil , UltrassonografiaRESUMO
Plasminogen activator inhibitor-1 (PAI-1) expression is induced by hypoxia (8% O(2)) via the PAI-1 promoter region -175/-159 containing a hypoxia response element (HRE-2) binding the hypoxia-inducible factor-1 (HIF-1) and an adjacent response element (HRE-1) binding a so far unknown factor. The aim of the present study was to identify this factor and to investigate its role in the regulation of PAI-1 expression. It was found by supershift assays that the upstream stimulatory factor-2a (USF-2a) bound mainly to the HRE-1 of the PAI-1 promoter and to a lesser extent to HRE-2. Overexpression of USF-2a inhibited PAI-1 messenger RNA and protein expression and activated L-type pyruvate kinase expression in primary rat hepatocytes under normoxia and hypoxia. Luciferase (Luc) gene constructs driven by 766 and 276 base pairs of the 5'-flanking region of the PAI-1 gene were transfected into primary hepatocytes together with expression vectors encoding wild-type USF-2a and a USF-2a mutant lacking DNA binding and dimerization activity (DeltaHU2a). Cotransfection of the wild-type USF-2a vector reduced Luc activity by about 8-fold, whereas cotransfection of DeltaHU2a did not influence Luc activity. Mutation of the HRE-1 (-175/-168) in the PAI-1 promoter Luc constructs decreased USF-dependent inhibition of Luc activity. Mutation of the HRE-2 (-165/-158) was less effective. Cotransfection of a HIF-1alpha vector could compete for the binding of USF at HRE-2. These results indicated that the balance between 2 transcriptional factors, HIF-1 and USF-2a, which can bind adjacent HRE sites, appears to be involved in the regulation of PAI-1 expression in many clinical conditions.
Assuntos
Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Proteínas Nucleares/genética , Inibidor 1 de Ativador de Plasminogênio/genética , Fatores de Transcrição/genética , Animais , Hipóxia Celular , Fator 1 Induzível por Hipóxia , Subunidade alfa do Fator 1 Induzível por Hipóxia , Fígado , Masculino , Regiões Promotoras Genéticas , Ratos , Fatores Estimuladores UpstreamRESUMO
We report here the preparative scale isolation of the four subunits of the nicotinic acetylcholine receptor (nAChR) applying short inverse pore gradient SDS gels on an elution-PAGE apparatus. The nAChR subunits are of similar molecular weights (alpha, 50.2 kDa; beta, 53.7 kDa; gamma, 56.3 kDa; delta, 57.6 kDa) and isoelectric point (approx 5.5) and share the typical properties of amphiphatic membrane proteins that are difficult to separate by chromatographic procedures. Preparative PAGE, which has proved to be the method of choice for nAChR-subunit fractionation, however, is time-consuming and achieves only moderate resolutions yielding dilute fractions. We present here the fractionation of a membrane preparation of Torpedo electric organ (2 mg of total protein) on short gels (2 cm) with linear or concave inverse pore gradients. All of the four subunits are isolated in homogeneous fractions of approx 70 micrograms/ml within only 4 h. Compared to the standard method, this means a 50% reduction in separation times with threefold higher concentrated protein fractions. We also give the theoretical explanation and experimental validation of the improved features resulting from short inverse-gradient polyacrylamide gels.
Assuntos
Eletroforese em Gel de Poliacrilamida/métodos , Proteínas de Membrana/química , Proteínas de Membrana/isolamento & purificação , Receptores Nicotínicos/química , Receptores Nicotínicos/isolamento & purificação , Animais , Estudos de Avaliação como Assunto , Ponto Isoelétrico , Modelos Teóricos , Peso Molecular , Conformação Proteica , TorpedoRESUMO
Liver glucokinase (GK) is localized predominantly in the perivenous zone. GK mRNA was induced by insulin maximally under venous O2 partial pressure (pO2) and only half-maximally under arterial pO2. CoCl2 and desferrioxamine mimicked venous pO2 and enhanced the insulin-dependent induction of GK mRNA under arterial pO2. H2O2 mimicked arterial pO2 and reduced insulin-induced GK mRNA under venous pO2 to the lower arterial levels. Thus the zonal O2 gradient in liver seems to have a key role in the heterogenous expression of the GK gene.
Assuntos
Monitorização Transcutânea dos Gases Sanguíneos , Glucoquinase/metabolismo , Peróxido de Hidrogênio/farmacologia , Insulina/farmacologia , Fígado/enzimologia , Animais , Antimutagênicos/farmacologia , Cobalto , Desferroxamina/farmacologia , Indução Enzimática , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Masculino , RNA Mensageiro/metabolismo , Ratos , Ratos WistarRESUMO
Using 2,8,5'-[3H]ATP as a direct photoaffinity label for membrane-bound nicotinic acetylcholine receptor (nAChR) from Torpedo marmorata, we have identified a binding site for ATP in the extracellular region of the beta-subunit of the receptor. Photolabeling was completely inhibited in the presence of saturating concentrations of nonradioactive ATP, whereas neither the purinoreceptor antagonists suramin, theophyllin, and caffeine nor the nAChR antagonists alpha-bungarotoxin and d-tubocurarine affected the labeling reaction. Competitive and noncompetitive nicotinic agonists and Ca2+ increased the yield of the photoreaction by up to 50%, suggesting that the respective binding sites are allosterically linked with the ATP site. The dissociation constant KD of binding of ATP to the identified site on the nAChR was of the order of 10(-4) M. Sites of labeling were found in the sequence regions Leu11-Pro17 and Asp152-His163 of the nAChR beta-subunit. These regions may represent parts of a single binding site for ATP, which is discontinuously distributed within the primary structure of the N-terminal extracellular domain. The existence of an extracellular binding site for ATP confirms, on the molecular level, that this nucleotide can directly act on nicotinic receptors, as has been suggested from previous electrophysiological and biochemical studies.