RESUMO
Fibrinogen (Fg) is a high molecular weight plasma adhesion protein and a biomarker of inflammation. Many cardiovascular and cerebrovascular disorders are accompanied by increased blood content of Fg. Increased levels of Fg result in changes in blood rheological properties such as increases in plasma viscosity, erythrocyte aggregation, platelet thrombogenesis, alterations in vascular reactivity and compromises in endothelial layer integrity. These alterations exacerbate the complications in peripheral blood circulation during cardiovascular diseases such as hypertension, diabetes and stroke. In addition to affecting blood viscosity by altering plasma viscosity and erythrocyte aggregation, growing experimental evidence suggests that Fg alters vascular reactivity and impairs endothelial cell layer integrity by binding to its endothelial cell membrane receptors and activating signalling mechanisms. The purpose of this review is to discuss experimental data, which demonstrate the effects of Fg causing vascular dysfunction and to offer possible mechanisms for these effects, which could exacerbate microcirculatory complications during cardiovascular diseases accompanied by increased Fg content.
Assuntos
Doenças Cardiovasculares/metabolismo , Doenças Cardiovasculares/fisiopatologia , Fibrinogênio/metabolismo , Microcirculação/fisiologia , Animais , Viscosidade Sanguínea/fisiologia , Agregação Eritrocítica/fisiologia , HumanosRESUMO
BACKGROUND: The plasma membrane Ca(2+)-ATPase (PMCA) plays an essential role in maintaining low intracellular Ca(2+) ([Ca(2+)](i)) in resting platelets. Earlier studies demonstrated that platelet activation by thrombin results in tyrosine phosphorylation of PMCA, which inhibits pump activity. OBJECTIVES: The objective was to determine the functional consequences of PMCA tyrosine phosphorylation. METHODS: A decapeptide including the tyrosine phosphorylation site of PMCA and a scrambled version were synthesized and introduced into human platelets using saponin. Fura-2 calcium monitoring and aggregometry were used to characterize the effects of inhibition of tyrosine phosphorylation. RESULTS: Western blot analysis of immunoprecipitates showed that introduction of the inhibitory peptide decreased tyrosine phosphorylation of PMCA by nearly 60% in saponin-permeabilized, thrombin-treated platelets as compared with the scrambled control peptide. Concomitant with inhibition of PMCA tyrosine phosphorylation was a significant decrease in [Ca(2+)](i) during thrombin-mediated platelet activation. The functional consequence of reduced PMCA tyrosine phosphorylation and decreased [Ca(2+)](i) was a significant delay in the onset of thrombin-mediated platelet aggregation. CONCLUSIONS: The results demonstrate that PMCA tyrosine phosphorylation regulates [Ca(2+)](i) during platelet activation, which affects downstream events in the activation process. Moreover, PMCA tyrosine phosphorylation and resultant inhibition of PMCA activity produces a positive feedback loop mechanism by enhancing the increase in [Ca(2+)](i) accompanying platelet activation.
Assuntos
Plaquetas/citologia , ATPases Transportadoras de Cálcio/metabolismo , Tirosina/metabolismo , Sequência de Aminoácidos , Plaquetas/enzimologia , ATPases Transportadoras de Cálcio/química , Membrana Celular/enzimologia , Humanos , Cinética , Fosforilação , Agregação PlaquetáriaRESUMO
Calcium transport across a monolayer of Madin-Darby canine kidney (MDCK) cells was measured in response to stimulation of the basal surface with calcium-sensing receptor (CaR) agonists. Stimulation of the CaR resulted in a time- and concentration-dependent inhibition of calcium transport but did not change transepithelial voltage or resistance. Inhibition of transport was not altered by pretreatment of cells with pertussis toxin but was blocked by the phospholipase C (PLC) inhibitor U-73122. To determine a potential mechanism by which the CaR could inhibit calcium transport, we measured activity of the plasma membrane calcium ATPase (PMCA). Stimulation of the CaR on the basal surface resulted in an inhibition of the PMCA in a concentration- and PLC-dependent manner. Thus stimulation of the CaR inhibits both calcium transport and PMCA activity through a PLC-dependent pathway. These studies provide the first direct evidence that calcium can inhibit its own transcellular absorption in a model of the distal tubule. In addition, they provide a potential mechanism for the CaR to inhibit calcium transport, inhibition of PMCA.
Assuntos
ATPases Transportadoras de Cálcio/antagonistas & inibidores , Cálcio/metabolismo , Rim/metabolismo , Receptores de Superfície Celular/metabolismo , Absorção , Algoritmos , Animais , Bovinos , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Eletrofisiologia , Proteínas de Ligação ao GTP/metabolismo , Rim/enzimologia , Toxina Pertussis , Receptores de Detecção de Cálcio , Fosfolipases Tipo C/metabolismo , Fatores de Virulência de Bordetella/toxicidadeRESUMO
The plasma membrane Ca(2+)-ATPase (PMCA) plays an essential role in maintaining low cytosolic Ca(2+) in resting platelets. During platelet activation PMCA is phosphorylated transiently on tyrosine residues resulting in inhibition of the pump that enhances elevation of Ca(2+). Tyrosine phosphorylation of many proteins during platelet activation results in their association with the cytoskeleton. Consequently, in the present study we asked if PMCA interacts with the platelet cytoskeleton. We observed that very little PMCA is associated with the cytoskeleton in resting platelets but that approximately 80% of total PMCA (PMCA1b + PMCA4b) is redistributed to the cytoskeleton upon activation with thrombin. Tyrosine phosphorylation of PMCA during activation was not associated with the redistribution because tyrosine-phosphorylated PMCA was not translocated specifically to the cytoskeleton. Because PMCA b-splice isoforms have C-terminal PSD-95/Dlg/ZO-1 homology domain (PDZ)-binding domains, a C-terminal peptide was used to disrupt potential PDZ domain interactions. Activation of saponin-permeabilized platelets in the presence of the peptide led to a significant decrease of PMCA in the cytoskeleton. PMCA associated with the cytoskeleton retained Ca(2+)-ATPase activity. These results suggest that during activation active PMCA is recruited to the cytoskeleton by interaction with PDZ domains and that this association provides a microenvironment with a reduced Ca(2+) concentration.
Assuntos
Plaquetas/enzimologia , ATPases Transportadoras de Cálcio/metabolismo , Citoesqueleto/enzimologia , Sequência de Aminoácidos , ATPases Transportadoras de Cálcio/química , Membrana Celular/enzimologia , Humanos , Fosforilação , Ativação Plaquetária , Tirosina/metabolismoRESUMO
We have compared the primary sequence and enzymatic properties of the sarcoplasmic reticulum Ca(2+)-ATPases from a cold-tolerant frog Rana sylvatica with those of a closely related cold-intolerant frog, Rana clamitans. Sarcoplasmic reticulum isolated from leg muscles of both species contains a major protein ( approximately 100 kDa) that reacts with a monoclonal antibody against sarco(endo)plasmic reticulum Ca(2+)-ATPase type 1 (SERCA1). The apparent molecular mass of R. sylvatica SERCA1 is 115 kDa, whereas that of R. clamitans is 105 kDa. However, the deduced amino acid sequences obtained from cDNAs do not indicate a difference in molecular weight, thus suggesting post-translational protein modification of R. sylvatica SERCA1. Comparison of the temperature dependence of both ATP hydrolysis and Ca(2+) transport indicates that R. sylvatica SERCA1 exhibits significantly lower activation energy below 20 degrees C and an approximately 2-fold greater Ca(2+)-ATPase activity near 0 degrees C. Furthermore, R. sylvatica SERCA1 exhibits simple Michaelis-Menten kinetics with ATP and Ca(2+) as opposed to the two-site ATP kinetics and positive cooperativity with Ca(2+) observed for R. clamitans and mammalian SERCA1s. Cooperativity has been linked to protein-protein interaction in SERCA1, and this property may be altered in R. sylvatica SERCA1. Primary sequence comparison shows that R. sylvatica SERCA1 exhibits seven unique amino acid substitutions, three of which are in the ATP binding domain. We also report for the first time the presence of alternative splicing in the frog, resulting in isoforms SERCA1a and SERCA1b. Thus, it appears that the low temperature muscle contractility of R. sylvatica can be explained partially by significant functional and structural differences in SERCA1.
Assuntos
ATPases Transportadoras de Cálcio/metabolismo , Músculo Esquelético/enzimologia , Adaptação Fisiológica , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Cálcio/metabolismo , ATPases Transportadoras de Cálcio/química , ATPases Transportadoras de Cálcio/genética , Temperatura Baixa , Primers do DNA , DNA Complementar , Hidrólise , Cinética , Dados de Sequência Molecular , Músculo Esquelético/fisiologia , Ranidae , Reação em Cadeia da Polimerase Via Transcriptase Reversa , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático , Homologia de Sequência de AminoácidosRESUMO
The seminal work of McGrath and Solter (1) and independently of Surani et al. (2) in 1984 established the fundamental principle of nuclear nonequivalency; that is, chromosomes of both paternal and maternal origin are required for development to term in mammals. This was achieved through the creation of diploid reconstituted zygotes, which contained either two maternal or two paternal pronuclei. Embryos containing pronuclei exclusively of maternal or paternal origin display characteristic developmental abnormalities and fail to develop to term. This failure is partially explained by the observation that paternally and maternally derived genomes have complementary roles during embryogenesis, contributing differentially to embryonic and extraembryonic lineages (2-5). These reconstitutions were accomplished by nuclear transplantation and karyoplast fusion using HVJ or Sendai virus-assisted fusion (1). These experiments laid the foundation for the discovery and exploration of this unique form of non-Mendelian mammalian gene regulation whereby expression of genes and hence phenotype were dictated by the parent from whom they where inherited. This parent-of-origin phenomenon is known as genomic imprinting.
Assuntos
Núcleo Celular/fisiologia , Técnicas de Cultura Embrionária/métodos , Embrião de Mamíferos/fisiologia , Impressão Genômica/genética , Animais , Núcleo Celular/metabolismo , Embrião de Mamíferos/metabolismo , Feminino , Humanos , Masculino , Gravidez , Pseudogravidez , Zigoto/fisiologiaRESUMO
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
Intracellular Ca(2+) is increased in the platelets of hypertensive individuals. Previously, we demonstrated that platelet plasma membrane Ca(2+)-ATPase (PMCA) activity inversely correlates with diastolic blood pressure and that inhibition of this Ca(2+) pump could explain the elevation of cytosolic Ca(2+) in hypertension. More recently, we discovered that PMCA is phosphorylated on tyrosine residues during thrombin-stimulated platelet aggregation and that this phosphorylation causes inhibition of PMCA activity. In the present work, we tested the hypothesis that tyrosine phosphorylation of PMCA in hypertensive patients could account for the observed inhibition of the Ca(2+) pump. Platelets were obtained from untreated hypertensive and normotensive volunteers. PMCA was immunoprecipitated from solubilized platelets, and tyrosine phosphorylation was quantified by chemiluminescence of immunoblots treated with anti-phosphotyrosine. PMCA content was measured on the same immunoblots by stripping and reprobing with anti-PMCA. Phosphorylation was reported as normalized phosphotyrosine chemiluminescence per nanogram PMCA (mean+/-SE). The average PMCA tyrosine phosphorylation for 15 normotensive subjects was 0.53+/-0. 09, whereas the average for 8 hypertensive individuals was 1.82+/-0. 25 (P<0.0005, Mann-Whitney U test). Age, gender, and systolic blood pressure did not correlate with PMCA phosphorylation. These results suggest that PMCA in platelets of hypertensive individuals is inhibited because of tyrosine phosphorylation, resulting in increased platelet intracellular Ca(2+), hyperactive platelets, and increased risk of heart attack and stroke.
Assuntos
Plaquetas/enzimologia , ATPases Transportadoras de Cálcio/sangue , ATPases Transportadoras de Cálcio/química , Hipertensão/enzimologia , Adulto , ATPases Transportadoras de Cálcio/antagonistas & inibidores , Estudos de Casos e Controles , Membrana Celular/enzimologia , Feminino , Humanos , Hipertensão/complicações , Técnicas In Vitro , Masculino , Pessoa de Meia-Idade , Fosforilação , Fatores de Risco , Trombose/etiologia , Tirosina/químicaRESUMO
PURPOSE: Although thrombin is best known for its role in blood coagulation, it has been reported to change the activity of ion motive ATPases in some tissues. In the present study, experiments were conducted to determine the influence of thrombin on active sodium-potassium transport in porcine lenses. METHODS: Ouabain-sensitive potassium (86Rb) uptake by intact porcine lenses was used as an index of Na,K-ATPase-mediated active sodium-potassium transport. Na,K-ATPase activity was measured by determining ouabain-sensitive ATP hydrolysis in isolated membrane material. RESULTS: In the presence of thrombin (1 unit/ml) the rate of ouabain-sensitive potassium (86Rb) uptake was reduced by 40% to 60%, but ouabain-insensitive potassium (86Rb) uptake was unchanged. The inhibitory effect of thrombin on ouabain-sensitive potassium (86Rb) uptake was suppressed in the presence of hirudin (an antagonist for thrombin receptors) but persisted in the presence of amphotericin B (a pseudo ionophore that effectively clamps plasma membrane sodium permeability at a high value). Enzyme measurements showed ouabain-sensitive ATP hydrolysis (Na,K-ATPase activity) was significantly inhibited in membrane material isolated from the capsule-epithelium of lenses, which had been pretreated with thrombin for 30 minutes. However, thrombin failed to exert a direct inhibitory effect on Na,K-ATPase activity when added directly to membrane fragments isolated from the epithelium of control (nonincubated) lenses. Both genistein and herbimycin (tyrosine kinase inhibitors) suppressed the effect of thrombin on the 86Rb uptake response. Results from Western blot studies suggested that tyrosine kinases are activated in the epithelium of lenses exposed to thrombin. CONCLUSIONS: The results suggest the inhibitory effect of thrombin on lens active sodium-potassium transport could involve the activation of a receptor-second-messenger mechanism in intact lens cells. The response appears to involve a tyrosine kinase-mediated step. The functional significance of the thrombin-mediated change of lens active sodium-potassium transport is unclear since appreciable amounts of thrombin may only be presented to the lens during instances of blood-aqueous-barrier breakdown. It is possible that lens receptors are functionally activated by other proteases, possibly cathepsins, which may enter aqueous humor from the ciliary body.
Assuntos
Cristalino/efeitos dos fármacos , Potássio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Sódio/metabolismo , Trombina/farmacologia , Animais , Benzoquinonas , Western Blotting , Inibidores Enzimáticos/farmacologia , Genisteína/farmacologia , Transporte de Íons/efeitos dos fármacos , Lactamas Macrocíclicas , Cristalino/metabolismo , Ouabaína/farmacologia , Quinonas/farmacologia , Rifabutina/análogos & derivados , Radioisótopos de Rubídio/metabolismo , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , Suínos , Trombina/antagonistas & inibidoresRESUMO
Membrane oxidation may contribute to cataractogenesis. In our pursuit to understand the etiology of cataracts, we assessed the effect of membrane oxidation products on the activity of the lens epithelium calcium pump. Microsome preparations from bovine lens epithelium were oxidized to varying degrees with a ferrous and ferric ascorbate system to generate hydrogen peroxide and superoxide. Ca2+ -ATPase activity was measured using a colorometric assay. Lipid oxidation was quantified by infrared spectroscopy. Ca2+ -ATPase activity decreased as a function of ascorbate concentration between 0 and 200 microM. The level of Ca2+ -ATPase inhibition was correlated to both the level of lipid oxidation and the degree of lipid hydrocarbon chain order. At 25 degrees C when lipids are more ordered, the Ca2+ -ATPase activity was similar to that observed in the oxidized system measured at 37 degrees C. Glutathione, mercaptoethanol, and iodoacetate were able to reverse the oxidative inhibition of the calcium pump, suggesting that the ascorbate/iron oxidant directly oxidized the protein sulfhydryl moieties. To further probe the mechanism of Ca2+ ATPase inhibition, hydrogen peroxide was used to oxidize muscle sarcoplasmic reticulum Ca2+ -ATPase reconstituted in its native lipid vesicles, egg phosphatidylcholine, and dihydrosphingomyelin, with saturated hydrocarbon chains. In these systems, oxidation inhibited the Ca2+ -ATPase pump by 60-80%. There was no statistical difference between the level of oxidative inhibition and the percentage of dihydrosphingomyelin. Because dihydrosphingomyelin cannot be oxidized, whereas egg phosphatidylcholine (PC) can, and because the percentage of inhibition was the same for reconstituted systems using either lipid, the mechanism of inhibition is likely not via a secondary process involving oxidation-induced lipid structural changes or products of lipid oxidation.
Assuntos
ATPases Transportadoras de Cálcio/metabolismo , Cristalino/metabolismo , Lipídeos de Membrana/metabolismo , Microssomos/metabolismo , Animais , Bovinos , Membrana Celular/metabolismo , Epitélio , Glutationa , Iodoacetatos , Cinética , Mercaptoetanol , Oxirredução , CoelhosRESUMO
PURPOSE: To determine whether dopamine receptor stimulation regulates Na,K-ATPase-mediated ion transport in cultured nonpigmented ciliary epithelium (NPE). METHODS: Using a rabbit NPE cell line, active Na-K transport activity was determined by measuring ouabain-sensitive potassium (86Rb) uptake in cell monolayers. Western blot analysis of membrane material obtained from cell homogenates was conducted to examine tyrosine phosphorylation of membrane proteins. RESULTS: Ouabain-sensitive potassium (86Rb) uptake was inhibited in the presence of either dopamine or the D1-selective agonist SKF82958. The response was suppressed by SCH23390, a D1 antagonist, but not by sulpiride, a D2-selective antagonist. Quinpirole, a D2-selective agonist, did not cause inhibition of ouabain-sensitive potassium (86Rb) uptake. Cyclic adenosine monophosphate (cAMP) was detectably increased in SKF82958-treated cells, although the concentration of SKF required to elevate cell cAMP was higher than the concentration needed to inhibit ouabain-sensitive potassium (86Rb) uptake. The protein kinase A inhibitor H89 prevented the 86Rb uptake response to SKF82958. Genistein, an inhibitor of tyrosine kinases, also prevented the 86Rb uptake response to SKF82958. Membrane material isolated from cells exposed to SKF82958 showed an increase in the density of several phosphotyrosine bands. These changes in phosphotyrosine immunoblot density were not observed in material isolated from cells that received either genistein or SCH23390 before SKF82958 treatment. CONCLUSIONS: The results of this study suggest D1 agonists cause a reduction of Na,K-ATPase-mediated ion transport by a mechanism that could involve a tyrosine kinase step.
Assuntos
Corpo Ciliar/efeitos dos fármacos , Agonistas de Dopamina/farmacologia , Inibidores Enzimáticos/farmacologia , Genisteína/farmacologia , Epitélio Pigmentado Ocular/efeitos dos fármacos , Potássio/metabolismo , Sódio/metabolismo , Animais , Transporte Biológico Ativo/efeitos dos fármacos , Western Blotting , Linhagem Celular , Células Cultivadas , Corpo Ciliar/metabolismo , AMP Cíclico/metabolismo , Transporte de Íons/efeitos dos fármacos , Epitélio Pigmentado Ocular/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Coelhos , Receptores de Dopamina D1/metabolismo , Radioisótopos de Rubídio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
A fundamental keystone of developmental biology has been the growing use of reporter genes in model transgenic systems. Their use has greatly facilitated investigations of cell lineage and cell fate in addition to aiding experiments aimed at determining patterns of gene expression, gene interaction and gene regulation. Through construction of transgenic mice, ubiquitously expressing human placental alkaline phosphatase (PLAP), we demonstrate the suitability of PLAP as a reporter gene for use in conjunction with, or as an alternative to, beta-galactosidase (lacZ). Our findings demonstrate that over-expression of PLAP has no adverse effects on mouse development or viability, despite a widespread pattern of expression. This technology provides a simple yet effective mechanism based on eukaryotic reporter gene technology to facilitate the identification of transgenic cells within complex in vivo systems.
Assuntos
Genes Reporter , Camundongos Transgênicos , Biologia Molecular/métodos , Proteínas/metabolismo , beta-Galactosidase/metabolismo , Animais , Vírus do Sarcoma Aviário/metabolismo , Células Cultivadas , Quimera , Embrião de Mamíferos/anatomia & histologia , Embrião de Mamíferos/metabolismo , Humanos , Camundongos , Músculo Esquelético/anatomia & histologia , Músculo Esquelético/fisiologia , PlasmídeosRESUMO
We have used an insulin-like growth factor (IGF)-II transgenic mouse model in which mouse IGF-II is widely overexpressed, resulting in increased fetal size and selective organ overgrowth, to investigate the effects on the development of the endocrine pancreas. Fetuses examined on day 19.5-20 of gestation had significantly elevated circulating levels of IGF-II, compared with control mice. The pancreatic islets in transgenic animals were of irregular shape and had a mean area five times greater than in controls, whereas the mean number of islets per tissue section was not altered. The size of individual endocrine cells was not altered. Although the islets in animals expressing the IGF-II transgene were considerably larger, immunohistochemistry for insulin and glucagon showed that the relative proportion of beta-cells was significantly less, and that of alpha-cells was higher. Normal islet morphology was disrupted, with alpha-cells appearing in small groups within the islets, as well as on the periphery, whereas beta-cells were often seen at the edge of the islets. Twice as many islet cells (21.9% vs. 11.4%) were involved in cell replication, detected by the presence of immunoreactive proliferating cell nuclear antigen, in pancreata from transgenic mice vs. controls, whereas the number of cells undergoing apoptosis was significantly reduced. Abundant IGF-II messenger RNAwas found within the islets of transgenic animals by in situ hybridization, and the relative area of islets demonstrating immunoreactive IGF-II was significantly greater. Immunoreactive IGF-I was much less abundant and was further reduced in islets of transgenic animals. The area of islets immunopositive for IGF binding protein-2 was unaltered. Despite the presence of islet hyperplasia, circulating insulin levels and serum glucose levels were not significantly different between transgenic and control mice. These results show that an overexpression of IGF-II in fetal life has a profound effect on islet morphology and causes islet hyperplasia while reducing the attrition of islet cells by apoptosis.
Assuntos
Expressão Gênica , Fator de Crescimento Insulin-Like II/genética , Ilhotas Pancreáticas/patologia , Animais , Apoptose , Hiperplasia , Hibridização In Situ , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/análise , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/análise , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/análise , Fator de Crescimento Insulin-Like I/análise , Ilhotas Pancreáticas/química , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Antígeno Nuclear de Célula em Proliferação/análise , RNA Mensageiro/análiseRESUMO
The presence of hypochlorite-modified lipoproteins in atherosclerotic lesions suggests that HOCl, a naturally occurring oxidant formed by the myeloperoxidase-catalyzed reaction of H2O2 and Cl-, is a candidate for generation of modified lipoproteins in vivo. We have previously demonstrated that Cu(2+)-oxidized LDL inhibits platelet plasma membrane Ca(2+)-ATPase (PMCA) in isolated membranes and causes an increase in cytosolic Ca2+ in resting whole platelets. However, Cu(2+)-oxidized LDL may not be identical in structure and function to the physiologically modified lipoprotein. Since platelet function may be affected by native and modified lipoproteins, the effect of HOCl-modified LDL and HDL3 on platelet PMCA and on the free intracellular Ca2+ concentration ([Ca2+]i) of whole platelets has been investigated. We demonstrate that in contrast to Cu(2+)-oxidized LDL, HOCl-modified LDL and HDL3 stimulate platelet PMCA activity in isolated membranes and that this effect results in a decrease of [Ca2+]i in vivo. Thus, HOCl-oxidation produces modified lipoproteins with the potential for altering platelet function and with properties different from those of the Cu(2+)-oxidized counterparts.
Assuntos
Plaquetas/metabolismo , ATPases Transportadoras de Cálcio/metabolismo , Cálcio/metabolismo , Membrana Celular/metabolismo , Lipoproteínas HDL/farmacologia , Plaquetas/efeitos dos fármacos , ATPases Transportadoras de Cálcio/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Fura-2/análise , Humanos , Ácido Hipocloroso/química , Lipoproteínas HDL/químicaRESUMO
Thrombin activation of human platelets is accompanied by tyrosine phosphorylation of many cellular proteins, as well as changes in the cytosolic Ca2+ concentration. Consequently, we assessed tyrosine phosphorylation of the platelet inositol 1,4,5-trisphosphate receptor (IP(3)R) to determine if phosphorylation modulates Ca2+ release from internal stores. Stimulation of platelets with thrombin resulted in tyrosine phosphorylation of the type 1 IP(3)R, which peaked at 3 min followed by dephosphorylation to the basal level by 10 min. Thrombin also increased the extent of IP(3)R-mediated Ca2+ release 1.6-fold in crude platelet membranes isolated from thrombin-stimulated (3 min) platelets. Since we have previously shown that the plasma membrane Ca2+-ATPase (PMCA) is also phosphorylated on tyrosine residues during thrombin-stimulated platelet activation, resulting in inhibition of the pump (Dean et al., J Biol Chem 1997; 272: 15113-9), the present results suggest that early in thrombin-stimulated platelet activation, tyrosine phosphorylation of PMCA and IP(3)R results in transient enhancement in platelet cytosolic Ca2+, but that this enhancement is attenuated by subsequent dephosphorylation.
RESUMO
While studying the bile acid synthetic pathway of hamsters, we discovered an NADP+-dependent liver microsomal 7alpha-hydroxycholesterol dehydrogenase (7alpha-HCD) activity that was not observed in rat liver microsomal fractions. The hamster liver microsomal 7alpha-HCD was purified to homogeneity using 2', 5'-ADP and cholic acid-agarose affinity chromatography. 7alpha-HCD displayed a molecular weight of approximately 34,000 on SDS-polyacrylamide gel electrophoresis; it is an intrinsic membrane protein of the hamster liver endoplasmic reticulum and exists as a multimeric aggregate in pure form. Partial N-terminal amino acid sequence analysis showed that 7alpha-HCD had high sequence similarity to human 11beta-hydroxysteroid dehydrogenase (11beta-HSD; 24/30 amino acid identity). The Km values for corticosterone and 7alpha-hydroxycholesterol were 1.2 and 1.9 microM, respectively, for purified 7alpha-HCD; both reactions displayed identical Vmax values (approximately 170 nmol/min/mg of protein). The IC50 of carbenoxolone, a competitive inhibitor of 11beta-HSD, was 75 nM for 7alpha-hydroxycholesterol dehydrogenation and 210 nM for corticosterone dehydrogenation. The tissue-specific expression in hamster was as follows: adrenal >/= liver > kidney > testis >> brain > lung. Microsomal 7alpha-HCD is uniquely expressed in hamster liver and to some extent in human liver but not in rat liver. Western blot analysis with two antibodies elicited against an N-terminal peptide of the human 11beta-HSD and purified hamster liver 7alpha-HCD, respectively, suggested the presence of multiple forms of 7alpha-HCD in hamster liver, most likely due to the existence of a family of 11beta-HSD proteins. Since 7-oxocholesterol is a potent inhibitor of cholesterol 7alpha-hydroxylase, alternative mechanisms for regulation of bile acid synthesis may exist in human and hamster liver due to production of this metabolite and its potential as an oxysterol.
Assuntos
Hidroxiesteroide Desidrogenases/química , Hidroxiesteroide Desidrogenases/isolamento & purificação , Microssomos Hepáticos/enzimologia , 11-beta-Hidroxiesteroide Desidrogenases , Sequência de Aminoácidos , Animais , Carbenoxolona/farmacologia , Catálise , Cricetinae , Inibidores Enzimáticos/farmacologia , Humanos , Técnicas In Vitro , Cinética , Masculino , Mesocricetus , Dados de Sequência Molecular , Coelhos , Ratos , Ratos Sprague-DawleyRESUMO
In human and mouse, most imprinted genes are arranged in chromosomal clusters. Their linked organization suggests co-ordinated mechanisms controlling imprinting and gene expression. The identification of local and regional elements responsible for the epigenetic control of imprinted gene expression will be important in understanding the molecular basis of diseases associated with imprinting such as Beckwith-Wiedemann syndrome. We have established a complete contig of clones along the murine imprinting cluster on distal chromosome 7 syntenic with the human imprinting region at 11p15.5 associated with Beckwith-Wiedemann syndrome. The cluster comprises approximately 1 Mb of DNA, contains at least eight imprinted genes and is demarcated by the two maternally expressed genes Tssc3 (Ipl) and H19 which are directly flanked by the non-imprinted genes Nap1l4 (Nap2) and Rpl23l (L23mrp), respectively. We also localized Kcnq1 (Kvlqt1) and Cd81 (Tapa-1) between Cdkn1c (p57(Kip2)) and Mash2. The mouse Kcnq1 gene is maternally expressed in most fetal but biallelically transcribed in most neonatal tissues, suggesting relaxation of imprinting during development. Our findings indicate conserved control mechanisms between mouse and human, but also reveal some structural and functional differences. Our study opens the way for a systematic analysis of the cluster by genetic manipulation in the mouse which will lead to animal models of Beckwith-Wiedemann syndrome and childhood tumours.
Assuntos
Síndrome de Beckwith-Wiedemann/genética , Cromossomos Humanos Par 11/genética , Impressão Genômica/genética , Família Multigênica/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Homologia de Sequência do Ácido Nucleico , Sequência de Aminoácidos , Animais , Mapeamento de Sequências Contíguas , Proteínas de Ligação a DNA , Feminino , Marcadores Genéticos , Humanos , Canais de Potássio KCNQ , Canal de Potássio KCNQ1 , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Proteínas Nucleares/genética , Mapeamento Físico do Cromossomo , Canais de Potássio/genéticaRESUMO
PURPOSE: In a previous study, an increase in Na,K-ATPase alpha 2 expression was detected in the epithelium of porcine lenses exposed to amphotericin B, an ionophore that also increases lens sodium and stimulates active sodium transport. The purpose of the present study was to determine whether an increase of Na,K-ATPase alpha 2 synthesis is a response to an episode of rapid Na-K transport or whether the increase in lens sodium alone can initiate the response. METHODS: Western blot analyses were conducted to probe for Na,K-ATPase alpha polypeptides in membrane material isolated from porcine lens epithelium. Ouabain-sensitive adenosine triphosphate hydrolysis was used as an index of Na,K-ATPase activity, and lens ion content was determined by atomic absorption spectrophotometry. 86-Rubidium (86Rb) uptake was measured as an indicator for active potassium transport. RESULTS: 86Rb uptake was markedly diminished in lenses exposed to dihydro-ouabain (DHO), signifying inhibition of active sodium-potassium transport. Consistent with this, the sodium content of DHO-treated lenses increased. By western blot analysis, a marked increase of Na,K-ATPase alpha 2 polypeptide could be detected in the epithelium of DHO-treated lenses. To rule out the possibility that apparent stimulation of Na,K-ATPase alpha 2 synthesis stemmed from binding of DHO to Na,K-ATPase sites, experiments were conducted to confirm an increase of Na,K-ATPase alpha 2 polypeptide in the epithelium of lenses exposed to low-potassium medium to inhibit active sodium-potassium transport. Consistent with the apparent increase of Na,K-ATPase polypeptide, Na,K-ATPase activity was detectably increased in epithelial material isolated from lenses pretreated with DHO or low-potassium medium. CONCLUSIONS: An increase in Na,K-ATPase alpha 2 polypeptide can occur in the epithelium of lenses subjected to an episode of sodium pump inhibition. This suggests the response could be triggered by an increase in cell sodium and does not necessarily require a period of stimulated active sodium-potassium transport.
Assuntos
Cristalino/enzimologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Regulação para Cima , Animais , Transporte Biológico/efeitos dos fármacos , Western Blotting , Inibidores Enzimáticos/farmacologia , Epitélio/efeitos dos fármacos , Epitélio/enzimologia , Cristalino/efeitos dos fármacos , Ouabaína/análogos & derivados , Ouabaína/farmacologia , Potássio/metabolismo , Radioisótopos de Rubídio/metabolismo , Sódio/metabolismo , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , Espectrofotometria Atômica , SuínosRESUMO
A baculovirus expression system was used to determine the contribution of carboxyl methylation of specific G protein gamma subunits to the interaction between alpha and beta gamma subunits. beta gamma subunits were carboxyl methylated by a membrane bound methyltransferase in Sf9 cells, and periodate-oxidized adenosine inhibited this methylation by 90%. Carboxyl methylation of beta(1) gamma(2), beta(2) gamma(3), and beta(2) gamma(7) enhanced pertussis toxin-catalyzed ADP-ribosylation of alpha(i2) and alpha(i3) by about 2-fold. On the other hand, methylation did not enhance membrane attachment of beta gamma subunits. These results suggest that methylation of isoprenylated gamma subunits is required for optimal G protein-mediated signal transduction, but not membrane attachment.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Adenosina Difosfato Ribose/metabolismo , Animais , Catálise , Linhagem Celular , Membrana Celular/metabolismo , Proteínas de Ligação ao GTP/genética , Metilação , Metiltransferases/antagonistas & inibidores , Metiltransferases/metabolismo , Toxina Pertussis , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Spodoptera , Fatores de Virulência de Bordetella/farmacologiaRESUMO
As a consequence of its central role in the regulation of calcium metabolism in the platelet, the plasma membrane Ca2+-ATPase (PMCA) was assessed for cAMP-dependent and tyrosine phosphorylation. Addition of forskolin or prostaglandin E1, agents known to elevate platelet cAMP and calcium efflux, to platelets pre-labeled with [32P]PO4 resulted in the direct phosphorylation of platelet PMCA. Similarly, addition of the catalytic subunit of protein kinase A to platelet plasma membranes resulted in a 1.4-fold stimulation of activity. Thus, the previously reported inhibition of platelet activation by elevated intracellular cAMP may be accomplished in part by stimulation of PMCA, likely resulting in a decrease in intracellular calcium. Treatment with thrombin evoked tyrosine phosphorylation of platelet PMCA, while PMCA from resting platelets exhibited little tyrosine phosphorylation. Phosphorylation of platelet plasma membranes by pp60(src) resulted in 75% inhibition of PMCA activity within 15 min. Similarly, membranes isolated from thrombin-treated platelets exhibited 40% lower PMCA activity than those from resting platelets. Phosphorylation of erythrocyte ghosts and purified PMCA by pp60(src) also resulted in up to 75% inhibition of Ca2+-ATPase activity, and inhibition was correlated with tyrosine phosphorylation. Sequencing of a peptide obtained after 32P labeling of purified erythrocyte PMCA in vitro showed that tyrosine 1176 of PMCA4b is phosphorylated by pp60(src). These results indicate that tyrosine phosphorylation of platelet PMCA may serve as positive feedback to inhibit PMCA and increase intracellular calcium during platelet activation.