RESUMO
Treatment of the pancreatic beta-cell line betaTC6-F7 with an imidazoline compound, RX-871024, KCl, or tolbutamide resulted in increased threonine phosphorylation of a 220-kDa protein (p220) concurrent with enhanced insulin secretion, which can be partially antagonized by diazoxide, an ATP-sensitive potassium (K(ATP)) channel activator. Although phosphorylation of p220 was regulated by cytoplasmic free calcium concentration ([Ca(2+)](i)), membrane depolarization alone was not sufficient to induce phosphorylation. Phosphorylation of p220 also was not directly mediated by protein kinase A, protein kinase C, or insulin exocytosis. Analysis of subcellular fractions indicated that p220 is a hydrophilic protein localized exclusively in the cytosol. Subsequently, p220 was purified to homogeneity, sequenced, and identified as nonmuscle myosin heavy chain-A (MHC-A). Stimulation of threonine phosphorylation of nonmuscle MHC-A by KCl treatment also resulted in increased phosphorylation of a 40-kDa protein, which was coimmunoprecipitated by antibody to MHC-A. Our results suggest that both nonmuscle MHC-A and the 40-kDa protein may play roles in regulating signal transduction, leading to insulin secretion.
Assuntos
Hipoglicemiantes/farmacologia , Imidazóis/farmacologia , Indóis/farmacologia , Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Treonina/metabolismo , Tolbutamida/farmacologia , Sequência de Aminoácidos , Animais , Anti-Hipertensivos/farmacologia , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/fisiologia , Linhagem Celular , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Citosol/química , Citosol/enzimologia , Diazóxido/farmacologia , Exocitose/fisiologia , Humanos , Secreção de Insulina , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/enzimologia , Isomerismo , Potenciais da Membrana/efeitos dos fármacos , Proteínas de Membrana/análise , Proteínas de Membrana/metabolismo , Cadeias Pesadas de Miosina/análise , Cadeias Pesadas de Miosina/química , Nifedipino/farmacologia , Fosforilação , Cloreto de Potássio/farmacologia , Proteína Quinase C/metabolismo , Frações Subcelulares/químicaRESUMO
Phosphorylation of eukaryotic translation initiation factor-2alpha (eIF-2alpha) is one of the key steps where protein synthesis is regulated in response to changes in environmental conditions. The phosphorylation is carried out in part by three distinct eIF-2alpha kinases including mammalian double-stranded RNA-dependent eIF-2alpha kinase (PKR) and heme-regulated inhibitor kinase (HRI), and yeast GCN2. We report the identification and characterization of a related kinase, PEK, which shares common features with other eIF-2alpha kinases including phosphorylation of eIF-2alpha in vitro. We show that human PEK is regulated by different mechanisms than PKR or HRI. In contrast to PKR or HRI, which are dependent on autophosphorylation for their kinase activity, a point mutation that replaced the conserved Lys-614 with an alanine completely abolished the eIF-2alpha kinase activity, whereas the mutant PEK was still autophosphorylated when expressed in Sf-9 cells. Northern blot analysis indicates that PEK mRNA was predominantly expressed in pancreas, though low expression was also present in several tissues. Consistent with the high levels of mRNA in pancreas, the PEK protein was only detected in human pancreatic islets, and the kinase co-localized with somatostatin, a pancreatic delta cell-specific hormone. Thus PEK is believed to play an important role in regulating protein synthesis in the pancreatic islet, especially in islet delta cells.
Assuntos
Ilhotas Pancreáticas/enzimologia , Somatostatina/metabolismo , eIF-2 Quinase/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Caenorhabditis elegans/genética , DNA Complementar , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fosforilação , Mutação Puntual , Ratos , Homologia de Sequência de Aminoácidos , eIF-2 Quinase/genéticaRESUMO
In response to various environmental stresses, eukaryotic cells down-regulate protein synthesis by phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2alpha). In mammals, the phosphorylation was shown to be carried out by eIF-2alpha kinases PKR and HRI. We report the identification and characterization of a cDNA from rat pancreatic islet cells that encodes a new related kinase, which we term pancreatic eIF-2alpha kinase, or PEK. In addition to a catalytic domain with sequence and structural features conserved among eIF-2alpha kinases, PEK contains a distinctive amino-terminal region 550 residues in length. Using recombinant PEK produced in Escherichia coli or Sf-9 insect cells, we demonstrate that PEK is autophosphorylated on both serine and threonine residues and that the recombinant enzyme can specifically phosphorylate eIF-2alpha on serine-51. Northern blot analyses indicate that PEK mRNA is expressed in all tissues examined, with highest levels in pancreas cells. Consistent with our mRNA assays, PEK activity was predominantly detected in pancreas and pancreatic islet cells. The regulatory role of PEK in protein synthesis was demonstrated both in vitro and in vivo. The addition of recombinant PEK to reticulocyte lysates caused a dose-dependent inhibition of translation. In the Saccharomyces model system, PEK functionally substituted for the endogenous yeast eIF-2alpha kinase, GCN2, by a process requiring the serine-51 phosphorylation site in eIF-2alpha. We also identified PEK homologs from both Caenorhabditis elegans and the puffer fish Fugu rubripes, suggesting that this eIF-2alpha kinase plays an important role in translational control from nematodes to mammals.
Assuntos
Ilhotas Pancreáticas/enzimologia , Biossíntese de Proteínas/genética , eIF-2 Quinase/genética , Sequência de Aminoácidos , Animais , Clonagem Molecular , Dados de Sequência Molecular , Fosfoproteínas/análise , Fosforilação , Fosfosserina/análise , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/genética , Reticulócitos/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , eIF-2 Quinase/químicaRESUMO
In the retinas of diabetic animals, protein kinase C (PKC) activity is elevated, and Na+-K+-ATPase and calcium ATPase activities are subnormal. These abnormalities are also present in another model of diabetic retinopathy, experimental galactosemia. We have investigated the relationship between hyperglycemia-induced abnormalities of PKC and ATPases using a selective inhibitor of beta isoform of PKC (LY333531). Diabetes or experimental galactosemia of 2 months' duration resulted in > 50% elevation of PKC activity in the retina, and administration of LY333531 prevented the elevation. In retinas of the same rats, the LY333531 prevented hyperglycemia-induced decreases of both Na+-K+-ATPase and calcium ATPase activities. Retinal microvessels, the main site of lesions in diabetic retinopathy, likewise showed elevated activity of PKC and inhibition of ATPases in diabetes and in experimental galactosemia, and administration of LY333531 to diabetic animals prevented these abnormalities. PKC activity in sciatic nerves, in contrast, became subnormal in diabetes and experimental galactosemia, and LY333531 had no effect on PKC activity in the sciatic nerve. PKC activity in the cerebral cortex was not affected by diabetes or experimental galactosemia. The results suggest that diabetes-induced reductions in Na+-K+-ATPase and calcium ATPase in the retina are mediated in large part by PKC-beta. The availability of an agent that can normalize the hyperglycemia-induced increase in PKC activity in the retina should facilitate investigation of the role of PKC in the development of diabetic retinopathy.
Assuntos
Diabetes Mellitus Experimental/enzimologia , Galactosemias/enzimologia , Proteína Quinase C/metabolismo , Retina/enzimologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/enzimologia , Estudos de Coortes , Inibidores Enzimáticos/sangue , Inibidores Enzimáticos/farmacologia , Galactosemias/induzido quimicamente , Indóis/sangue , Indóis/farmacologia , Maleimidas/sangue , Maleimidas/farmacologia , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/efeitos dos fármacos , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Retina/efeitos dos fármacos , Nervo Isquiático/efeitos dos fármacos , Nervo Isquiático/enzimologia , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , ATPase Trocadora de Sódio-Potássio/efeitos dos fármacosRESUMO
C-peptide, a cleavage product from the processing of proinsulin to insulin, has been considered to possess little if any biological activity other than its participation in insulin synthesis. Injection of human C-peptide prevented or attenuated vascular and neural (electrophysiological) dysfunction and impaired Na+- and K+-dependent adenosine triphosphate activity in tissues of diabetic rats. Nonpolar amino acids in the midportion of the peptide were required for these biological effects. Synthetic reverse sequence (retro) and all-D-amino acid (enantio) C-peptides were equipotent to native C-peptide, which indicates that the effects of C-peptide on diabetic vascular and neural dysfunction were mediated by nonchiral interactions instead of stereospecific receptors or binding sites.
Assuntos
Peptídeo C/química , Peptídeo C/uso terapêutico , Angiopatias Diabéticas/prevenção & controle , Neuropatias Diabéticas/prevenção & controle , Sequência de Aminoácidos , Animais , Circulação Sanguínea/efeitos dos fármacos , Glicemia/metabolismo , Peptídeo C/farmacologia , Permeabilidade Capilar/efeitos dos fármacos , Dicroísmo Circular , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/fisiopatologia , Humanos , Masculino , Dados de Sequência Molecular , Condução Nervosa/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Estrutura Secundária de Proteína , Ratos , Ratos Sprague-Dawley , ATPase Trocadora de Sódio-Potássio/metabolismo , EstereoisomerismoAssuntos
Cloranfenicol O-Acetiltransferase/genética , Cromatografia/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Genes Reporter/genética , Animais , Bovinos , Células Cultivadas , Cloranfenicol O-Acetiltransferase/análise , Cromatografia/economia , Ensaio de Imunoadsorção Enzimática/economia , Humanos , Kit de Reagentes para Diagnóstico , Retina/citologia , Retina/enzimologia , Sensibilidade e Especificidade , TransfecçãoRESUMO
Protein kinase C (PKC) is a family of closely related serine and threonine kinases. Overactivation of some PKC isozymes has been postulated to occur in several diseases states, including diabetic complications. Selective inhibition of overactivated PKC isozymes may offer a unique therapeutic approach to disease states such as diabetic retinopathy. A novel series of 14-membered macrocycles containing a N-N'-bridged bisindolylmaleimide moiety is described. A panel of eight cloned human PKC isozymes (alpha, beta I, beta II, gamma, delta, epsilon, sigma, eta) was used to identify the series and optimize the structure and associated activity relationship. The dimethylamine analogue LY333531 (1), (S)-13-[(dimethylamino)methyl]-10,11,14,15-tetrahydro-4,9:16, 21-dimetheno-1H, 13H-dibenzo[e,k]pyrrolo[3,4-h][1,4,13]oxadiazacyclohexadecene++ +-1,3(2H)-dione, inhibits the PKC beta I (IC50 = 4.7 nM) and PKC beta II (IC50 = 5.9 nM) isozymes and was 76- and 61-fold selective for inhibition of PKC beta I and PKC beta II in comparison to PKC alpha, respectively. The additional analogues described in the series are also selective inhibitors of PKC beta. LY333531 (1) exhibits ATP dependent competitive inhibition of PKC beta I and is selective for PKC in comparison to other ATP dependent kinases (protein kinase A, calcium calmodulin, caesin kinase, src tyrosine kinase). The cellular activity of the series was assessed using bovine retinal capillary endothelial cells. Retinal endothelial cell dysfunction has been implicated in the development of diabetic retinopathy. Plasminogen activator activity stimulated by a phorbol ester (4 beta-phorbol 12,13-dibutyrate) in endothelial cells was inhibited by the compounds in the series with ED50 values ranging from 7.5 to 0.21 microM. A comparison of the PKC isozyme and related ATP dependent kinase inhibition profiles is provided for the series and compared to the profile for staurosporine, a nonselective PKC inhibitor. The cellular activity of the series is compared with that of the kinase inhibitor staurosporine.
Assuntos
Indóis/farmacologia , Isoenzimas/antagonistas & inibidores , Maleimidas/farmacologia , Proteína Quinase C/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Bovinos , Células Cultivadas , Humanos , Indóis/síntese química , Isoenzimas/metabolismo , Maleimidas/síntese química , Dados de Sequência Molecular , Estrutura Molecular , Ativadores de Plasminogênio/farmacologia , Proteína Quinase C/metabolismo , Proteína Quinase C betaRESUMO
The vascular complications of diabetes mellitus have been correlated with enhanced activation of protein kinase C (PKC). LY333531, a specific inhibitor of the beta isoform of PKC, was synthesized and was shown to be a competitive reversible inhibitor of PKC beta 1 and beta 2, with a half-maximal inhibitory constant of approximately 5 nM; this value was one-fiftieth of that for other PKC isoenzymes and one-thousandth of that for non-PKC kinases. When administered orally, LY333531 ameliorated the glomerular filtration rate, albumin excretion rate, and retinal circulation in diabetic rats in a dose-responsive manner, in parallel with its inhibition of PKC activities.
Assuntos
Diabetes Mellitus Experimental/complicações , Angiopatias Diabéticas/prevenção & controle , Inibidores Enzimáticos/farmacologia , Indóis/farmacologia , Isoenzimas/antagonistas & inibidores , Maleimidas/farmacologia , Proteína Quinase C/antagonistas & inibidores , Administração Oral , Albuminúria/prevenção & controle , Animais , Diabetes Mellitus Experimental/enzimologia , Diabetes Mellitus Experimental/fisiopatologia , Angiopatias Diabéticas/enzimologia , Angiopatias Diabéticas/etiologia , Diglicerídeos/metabolismo , Relação Dose-Resposta a Droga , Ativação Enzimática , Inibidores Enzimáticos/química , Taxa de Filtração Glomerular/efeitos dos fármacos , Humanos , Indóis/administração & dosagem , Indóis/química , Isoenzimas/metabolismo , Glomérulos Renais/metabolismo , Masculino , Maleimidas/administração & dosagem , Maleimidas/química , Músculo Liso Vascular/enzimologia , Fosforilação/efeitos dos fármacos , Proteína Quinase C/metabolismo , Proteína Quinase C beta , Ratos , Ratos Sprague-Dawley , Fluxo Sanguíneo Regional/efeitos dos fármacos , Fluxo Plasmático Renal/efeitos dos fármacos , Retina/metabolismo , Vasos Retinianos/fisiopatologia , Especificidade por SubstratoRESUMO
Previous studies have shown that high glucose levels and diabetes induce an elevation in protein kinase C (PKC) activity in vascular cells and tissues susceptible to diabetic complications. In addition, PKC activation has been shown to modulate vascular cell growth, permeability, and gene expression, processes thought to be involved in the development of vascular complications. Using two in vivo model systems, we have identified a novel inhibitor of diabetic vascular dysfunction, LY290181. LY290181 prevented glucose-induced increases in blood flow and permeability in rat granulation tissue and corresponding vascular changes in the retina, sciatic nerve, and aorta of diabetic rats. Tested for its ability to inhibit PKC-regulated processes, LY290181 inhibited phorbol ester-stimulated plasminogen activator activity in a dose-dependent manner in bovine retinal endothelial cells and in human dermal fibroblasts. In addition, LY290181 inhibited phorbol ester-stimulated activation of the porcine urokinase plasminogen activator (uPA) promoter (-4600/+398) linked to the chloramphenicol acetyltransferase (CAT) reporter gene (p4660CAT). More detailed analysis of the uPA promoter revealed that LY290181 inhibited phorbol ester-stimulated activation of the uPA phorbol response element (-2458/-2349) located upstream of the thymidine kinase promoter (puPATKCAT). LY290181 appears to inhibit uPA promoter activation by blocking phorbol ester-stimulated binding of nuclear proteins to the uPA PEA3/12-0-tetradecanoylphorbol 13-acetate responsive element (TRE). These results suggest that LY290181 may inhibit diabetes-induced vascular dysfunction by inhibiting transcription factor binding to specific PKC-regulated genes involved in vascular function.
Assuntos
Diabetes Mellitus Experimental/fisiopatologia , Angiopatias Diabéticas/fisiopatologia , Endotélio Vascular/metabolismo , Inibidores Enzimáticos/farmacologia , Naftalenos/farmacologia , Proteína Quinase C/metabolismo , Piranos/farmacologia , Fatores de Transcrição/antagonistas & inibidores , Ativação Transcricional , Ativador de Plasminogênio Tipo Uroquinase/genética , Animais , Sequência de Bases , Capilares , Bovinos , Células Cultivadas , Cloranfenicol O-Acetiltransferase/biossíntese , Angiopatias Diabéticas/prevenção & controle , Endotélio Vascular/efeitos dos fármacos , Inibidores Enzimáticos/uso terapêutico , Fibroblastos , Humanos , Masculino , Dados de Sequência Molecular , Naftalenos/uso terapêutico , Oligodesoxirribonucleotídeos , Dibutirato de 12,13-Forbol/farmacologia , Regiões Promotoras Genéticas , Proteína Quinase C/antagonistas & inibidores , Piranos/uso terapêutico , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/biossíntese , Vasos Retinianos , Pele/efeitos dos fármacos , Pele/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Ativação Transcricional/efeitos dos fármacosRESUMO
Livers from normal cats and dogs, cats with mucopolysaccharidoses (MPS) I and VI, and dogs with MPS VII were analyzed biochemically and morphometrically to determine the lysosomal storage of glycosaminoglycans (GAG) in these animal models of human genetic disease. Analyses were performed on liver samples from seven normal cats ranging in age from 13 weeks to 15 months; six MPS I-affected cats ranging in age from 10 weeks to 26 months; four MPS VI-affected cats ranging in age from 9 months to 32 months; four normal dogs ranging in age from 1 month to 47 months; and three MPS VII-affected dogs, 5 days, 11 days, and 14 months of age. All of the animals were from the breeding colony at the University of Pennsylvania School of Veterinary Medicine and were maintained in accordance with national standards for the care and use of laboratory animals. Each GAG subclass was quantitated, and total GAG concentration was determined. Liver from cats with MPS I had the highest total GAG concentration (5.7 times that of the control), followed by liver from dogs with MPS VII (1.8 times) and cats with MPS VI (1.5 times). These data were very closely correlated (R2 = 0.982) with the results of the morphometric analyses of hepatocyte and Kupffer cell vacuolation associated with lysosomal storage and support the validity of both methods. This is particularly important for the quantification of total and individual GAG concentrations in tissue preparations. The values obtained should prove useful in future assessments of therapeutic regimes, such as enzyme replacement, bone marrow transplantation, and gene therapy, for these genetic diseases.
Assuntos
Doenças do Gato/patologia , Doenças do Cão/patologia , Glicosaminoglicanos/metabolismo , Fígado/patologia , Mucopolissacaridoses/veterinária , Animais , Doenças do Gato/metabolismo , Gatos , Densitometria , Doenças do Cão/metabolismo , Cães , Eletroforese em Acetato de Celulose , Glicosaminoglicanos/análise , Células de Kupffer/patologia , Células de Kupffer/ultraestrutura , Fígado/química , Fígado/metabolismo , Fígado/ultraestrutura , Microscopia Eletrônica , Mucopolissacaridoses/metabolismo , Mucopolissacaridoses/patologia , Mucopolissacaridose I/metabolismo , Mucopolissacaridose I/patologia , Mucopolissacaridose I/veterinária , Mucopolissacaridose VI/metabolismo , Mucopolissacaridose VI/patologia , Mucopolissacaridose VI/veterinária , Mucopolissacaridose VII/metabolismo , Mucopolissacaridose VII/patologia , Mucopolissacaridose VII/veterinária , Vacúolos/ultraestruturaRESUMO
Regional differences in retinal pigment epithelial (RPE) cell glycosaminoglycan (GAG) and collagen metabolism were studied using cells obtained from normal cats and those with deficient activity of arylsulfatase B (ASB), a lysosomal enzyme involved in GAG catabolism. Control and ASB-deficient RPE cultures initiated from superior equatorial (superior) and inferior equatorial (inferior) regions of the eye were radiolabeled for 72 hr with 35SO4, and GAGs from the media and cell layers were analyzed separately. In ASB-deficient RPE, there was an accumulation of dermatan/chondroitin sulfate in the cell layer of cultures initiated from the superior region of the eye but not in those initiated from the inferior region. This agrees with previous in situ and in vitro morphologic observations that accumulation of inclusions in ASB-deficient RPE was greater in the superior region of the eye than in the inferior region. By contrast, media from ASB-deficient cultures initiated from the inferior region of the eye contained much higher levels of radiolabeled dermatan/chondroitin sulfate than ASB-deficient cultures from the superior region or normal cultures. Increased GAG content in the media may result from increased secretion of proteoglycans, increased turnover of cell surface or extracellular matrix components, or extrusion of lysosomal contents. These results indicate that one or more of these mechanisms vary regionally throughout the eye in the RPE of ASB-deficient animals. Collagen production was determined in normal and ASB-deficient RPE cultures. In normal RPE, no differences in collagen synthesis were noted between the inferior and superior regions.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Condro-4-Sulfatase/deficiência , Colágeno/metabolismo , Glicosaminoglicanos/metabolismo , Epitélio Pigmentado Ocular/metabolismo , Animais , Anti-Infecciosos Locais/metabolismo , Gatos , Células Cultivadas , Sulfatos de Condroitina/metabolismo , Dermatan Sulfato/metabolismo , Heparitina Sulfato/metabolismo , Epitélio Pigmentado Ocular/enzimologiaRESUMO
A beta-glucuronidase mediated pathway for the degradation of glycosaminoglycans is present in the retinal pigment epithelium. The pathway has been defined using ocular tissues and cultured cells from mutant animals having a recessively inherited deficiency of the lysosomal enzyme. In situ, storage products accumulate in secondary lysosomes of the retinal pigment epithelium, the cytoplasm fills with inclusions and the cells hypertrophy; severity of the disease increases with aging. Deficient activity of beta-glucuronidase is present in primary and second passage cultures. Radiolabel studies with 35SO4 show a significant retention of cell layer label by mutant retinal pigment epithelial cells during a 72-hr pulse or 24-hr chase period. The labels is in newly synthesized chondroitin sulfate and heparan sulfate, which are natural substrates for the deficient enzyme. There is no difference from normal in the total radioactivity and electrophoretic profile of the glycosaminoglycans that are synthesized and released into the media. A retroviral vector was used to transfer normal rat beta-glucuronidase cDNA into the mutant cells. The vector treatment results in restoration of enzyme activity and correction of the degradative defect; 35SO4 labeling shows that chondroitin sulfate and heparan sulfate levels return to normal. The vector treatment studies indicate that a single gene defect determines the abnormal beta-glucuronidase mediated pathway in the mutant retinal pigment epithelium.
Assuntos
Glucuronidase/deficiência , Glicosaminoglicanos/metabolismo , Epitélio Pigmentado Ocular/enzimologia , Doenças Retinianas/etiologia , Transfecção , Animais , Células Cultivadas , DNA Viral , Cães , Regulação Enzimológica da Expressão Gênica , Glucuronidase/genética , Glucuronidase/fisiologia , Doenças Retinianas/terapia , Simplexvirus/genéticaRESUMO
Retroviral vectors were constructed containing a rat beta-glucuronidase cDNA driven by heterologous promoters. Vector-mediated gene transfer into human and canine beta-glucuronidase-deficient mucopolysaccharidosis type VII fibroblasts completely corrected the deficiency in beta-glucuronidase enzymatic activity. In primary cultures of canine mucopolysaccharidosis type VII retinal pigment epithelial cells, which contain large amounts of undegraded glycosaminoglycan substrates, vector correction restored normal processing of specific glycosaminoglycans in the lysosomal compartment. In canine mucopolysaccharidosis type VII bone marrow cells, beta-glucuronidase was expressed at high levels in transduced cells. Thus, the vector-encoded beta-glucuronidase was expressed at therapeutic levels in the appropriate organelle and corrected the metabolic defect in cells exhibiting the characteristic pathology of this lysosomal storage disorder.
Assuntos
Glucuronidase/deficiência , Lisossomos/enzimologia , Mucopolissacaridoses/genética , Vírus 40 dos Símios/genética , Simplexvirus/genética , Transfecção , Animais , Medula Óssea/enzimologia , Células Cultivadas , Fibroblastos/enzimologia , Vetores Genéticos , Glucuronidase/genética , Humanos , Mucopolissacaridoses/enzimologia , Ratos , Pele/enzimologiaRESUMO
The synthesis and turnover of glycosaminoglycans (GAGs) in different fractions of cultured feline retinal pigment epithelium (RPE) were characterized. In one method of fractionation, trypsin was used to separate the extracellular components (referred to as trypsin-soluble glycocalyx) from the intracellular components. As a second method, the basal extracellular matrix (basal ECM) was separated from the rest of the GAGs (cell-associated GAGs) by extracting the cell layer with NH4OH. The incorporation of 35SO4 into cetylpyridinium chloride-precipitable GAGs in the cell-associated and the intracellular fractions increased throughout the labeling period, while in the trypsin-soluble glycocalyx and the basal ECM incorporation approached a maximum. While heparan sulfate was the predominant GAG in all compartments, most was located extracellularly. The majority of dermatan sulfate was localized in the intracellular fraction. GAGs in the trypsin-soluble glycocalyx exhibited a rapid rate of turnover, while GAGs in the intracellular compartment and basal ECM turned over much more slowly. Ascorbic acid increased the incorporation of 35SO4 into ECM chondroitin sulfate/dermatan sulfate, but not heparan sulfate, on a per cell basis. Cycloheximide reduced incorporation of 35SO4-GAGs into both the cell-associated compartment and the basal ECM. In contrast, monensin caused a reduction in basal ECM GAGs while increasing the GAGs in the cell-associated compartment. The intracellular accumulation of GAGs and resultant pathology in alpha-L-iduronidase (alpha-L-id)-deficient RPE indicated that this pathway for the intracellular degradation of GAGs is important in normal RPE function. However, the turnover of GAGs in the trypsin-soluble glycocalyx was not affected by deficient alpha-L-id activity or by the subsequent intracellular accumulation of GAGs. Therefore, normal lysosomal activity in the RPE is not a prerequisite for maintaining the rate of extracellular GAG turnover within normal limits.
Assuntos
Matriz Extracelular/metabolismo , Glicosaminoglicanos/metabolismo , Membranas Intracelulares/metabolismo , Epitélio Pigmentado Ocular/metabolismo , Animais , Células Cultivadas , Meios de Cultura , Iduronidase/deficiência , Iduronidase/metabolismo , Lisossomos/fisiologia , Mucopolissacaridoses/enzimologia , Mucopolissacaridoses/patologia , Epitélio Pigmentado Ocular/citologia , Epitélio Pigmentado Ocular/enzimologia , Valores de ReferênciaRESUMO
It has been suggested that rhodopsin may have a direct role in the attachment of shed rod outer segments (ROS) to retinal pigmented epithelial (RPE) cells initiating the events which lead to engulfment. We isolated the soluble tryptic glycopeptide from rhodopsin (GP-T1) and used it as a probe to test this hypothesis. In phagocytic assays using both cultured chick and cat RPE cells, GP-T1 did not inhibit the phagocytosis of ROS. Additionally, mannose glycoconjugates were not effective inhibitors of phagocytosis. However, glycopeptides released by tryptic digestion of intact ROS did inhibit ROS uptake by the RPE cells. The results suggest that phagocytosis of ROS is not mediated through a simple carbohydrate recognition system and that rhodopsin is not the ligand recognized by RPE cells.
Assuntos
Fagocitose , Células Fotorreceptoras/metabolismo , Epitélio Pigmentado Ocular/fisiologia , Pigmentos da Retina/fisiologia , Rodopsina/fisiologia , Segmento Externo da Célula Bastonete/metabolismo , Albumina Sérica , Animais , Gatos , Células Cultivadas , Embrião de Galinha , Glicopeptídeos/isolamento & purificação , Glicopeptídeos/farmacologia , Ligantes , Mananas/farmacologia , Manose/farmacologia , Fagocitose/efeitos dos fármacos , Receptores de Superfície Celular/fisiologia , Soroalbumina Bovina/farmacologia , Tripsina/farmacologiaRESUMO
The synthesis and secretion of glycosaminoglycans (GAGs) in primary cultures of feline retinal pigment epithelium (RPE) was measured. After 14 days in culture, the cells were incubated for 3 days with media containing 20 microCi/ml 35SO4 and 10 microCi/ml [3H]-glucosamine. The GAGs were precipitated from the media and cell layer with cetylpyridinium chloride and ethanol, separated in 0.1 M Ba acetate by cellulose acetate electrophoresis, visualized by Alcian blue staining and fluorography, and quantitated by scanning densitometry and liquid scintillation counting. The predominant radioactively-labeled GAG secreted into the media by RPE cultures was chondroitin sulfate (CS), which accounted for 63% of the 35SO4, and 54% of the 3H incorporated. Radioactively-labeled heparan sulfate (HS), dermatan sulfate (DS), and hyaluronic acid (HA) were also identified in the media. Heparan sulfate accounted for 24% of the 35SO4 and 22% of the 3H, DS contained 13% of the 35SO4 and 7% of the 3H, and HA accounted for 17% of the 3H incorporated into the media GAGs. In contrast, fibroblast cultures secreted primarily HA, which accounted for 84% of the 3H in the media GAGs. The profile of GAGs retained by the cell layer was different from that of the secreted GAGs. The predominant radioactively-labeled GAG associated with the cell layer was HS. This GAG contained 54% of the 35SO4, and 52% of the 3H incorporated into cell layer GAGs. Dermatan sulfate contained 23% of the 35SO4, and 23% of the 3H, while CS accounted for 23% of the 35SO4, and 18% of the 3H incorporated into cell layer GAGs. The remaining 7% of the incorporated 3H was associated with HA. Total GAG profiles were determined for primary cultures of feline RPE by Alcian blue staining, and compared with those of freshly isolated cell samples. The GAG profiles were similar in cultured and freshly-isolated samples; however, the percentage of HS in the freshly-isolated samples was about twice as high as the percentage of HS in cultured samples, while the proportion of DS in cultured samples was higher than in freshly-isolated samples. This study demonstrates that the profile of radioactively-labeled GAGs secreted by primary cultures of feline RPE is distinct from that retained by the cell layer. In addition, the total GAG profile of cultured RPE is similar, but not identical, to that of freshly-isolated cells.
Assuntos
Glicosaminoglicanos/metabolismo , Epitélio Pigmentado Ocular/metabolismo , Animais , Gatos , Células Cultivadas , Sulfatos de Condroitina/metabolismo , Dermatan Sulfato/metabolismo , Glucosamina/metabolismo , Heparitina Sulfato/metabolismo , Ácido Hialurônico/metabolismo , Sulfatos/metabolismoRESUMO
Inositol triphosphate (IP3) and diacylglycerol (DG) are second messengers which control ionic events implicated in cell proliferation in a variety of tissues. In order to determine if these two second messengers control the proliferation of bovine retinal capillary pericytes (BRCP) or feline retinal pigment epithelial cells (FRPE) in culture, both intact BRCP or FRPE or BRCP or FRPE made permeable by saponin were used to study the effects of IP3 and DG on [3H]thymidine incorporation into DNA. [3H]Thymidine incorporation by BRCP made permeable to saponin showed specific IP3 dose-dependence; the apparent Km was 0.3 microM of IP3. Similar effects of A23187, a Ca2+ ionophore, or synthetic DG (1-oleoyl-2 acetyl-glycerol) were also observed. The combination of synthetic DG (0-,2-,4-, 8 micrograms ml-1) and 1 microM A23187 produced greater stimulation of [3H]thymidine incorporation by intact BRCP than was seen with DG or A23187 alone. In contrast to BRCP. [3H]thymidine incorporation by FRPE was not stimulated by IP3, A23187 or synthetic DG. The synergistic activation of IP3 and DG provided direct evidence to support the view that BRCP proliferation in vitro were regulated by the levels of the two second messengers.
Assuntos
Diglicerídeos/farmacologia , Glicerídeos/farmacologia , Fosfatos de Inositol/farmacologia , Mitose/efeitos dos fármacos , Vasos Retinianos/citologia , Fosfatos Açúcares/farmacologia , Animais , Calcimicina/farmacologia , Capilares/citologia , Capilares/efeitos dos fármacos , Capilares/metabolismo , Gatos , Bovinos , Células Cultivadas , DNA/biossíntese , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Inositol 1,4,5-Trifosfato , Epitélio Pigmentado Ocular/citologia , Vasos Retinianos/efeitos dos fármacos , Vasos Retinianos/metabolismo , Timidina/metabolismoRESUMO
The synthesis and secretion of glycosaminoglycans (GAGs) was characterized in subcultures of bovine retinal capillary pericytes. The GAGs were metabolically labeled with [3H]glucosamine and 35SO4 for 3 days, and then precipitated from the cell layer or media by cetylpyridinium chloride and ethanol, separated by cellulose acetate electrophoresis and further identified by their susceptibility to degradative procedures. The predominant radioactively labeled GAG associated with the pericyte-cell layer was heparan sulfate (HS). Radioactively labeled chondroitin sulfate (CS) and hyaluronic acid (HA) were also present in the pericyte-cell layer. No radioactively labeled dermatan sulfate (DS) was detected. The profile of radioactively labeled GAGs secreted by pericytes into the media differed considerably from that associated with the cell layer. Equal amounts of radioactivity were incorporated into HS and CS. Small quantities of radioactively labeled HA were also present in the media. Although no radioactively labeled DS was detected in the pericyte-cell layer, it was present in the media. The total pericyte-cell layer GAG profile was determined by scanning densitometry of the three bands resolved after cellulose acetate electrophoresis and Alcian Blue staining. The slowest band was identified as HS, and accounted for 17% of the total GAGs. The middle band was identified as DS, and accounted for 34% of the total GAGs. The fastest band was tentatively identified as either DS or chondroitinase AC-resistant CS, and constituted 49% of the total GAGs. The GAGs associated with the fibroblast-cell layer and secreted into the media by fibroblasts also were characterized and compared with those produced by pericytes. The major differences were in the secretion of large amounts of HA into the media by fibroblasts, and the presence of radioactively labeled DS in the cell layer of fibroblasts.
Assuntos
Glicosaminoglicanos/biossíntese , Vasos Retinianos/metabolismo , Animais , Capilares/metabolismo , Bovinos , Células Cultivadas , Sulfatos de Condroitina/biossíntese , Dermatan Sulfato/biossíntese , Heparitina Sulfato/biossíntese , Ácido Hialurônico/biossínteseRESUMO
Transport of ascorbate by primary cultures of cat retinal pigment epithelial cells (RPE) was studied. Confluent primary cultures were incubated with 10-500 microM L-[carboxyl-14C] ascorbic acid in balanced salt solution (BSS) at 37 degrees C for 1 to 40 min. The uptake of radioactive ascorbate followed saturation kinetics with a Km of 42 microM and Vmax of 117 pmol min-1 microgram-1 DNA. Cells incubated with 10 microM radioactive ascorbate for 40 min showed a ratio of intracellular to extracellular radioactive ascorbate of greater than 40. The transport of ascorbate was sodium- and energy-dependent. Replacement of 150 mM NaCl in BSS with 150 mM LiCl reduced ascorbate uptake significantly. Ouabain, 2,4-dinitrophenol, alpha-D-glucose, 3-O-methyl-D-glucose, and the ascorbate analogues, D-isoascorbate and dehydroascorbate, each inhibited ascorbate uptake into RPE cells. The efflux of radioactivity into the incubation media was slow when cells were preloaded with either 50- or 500 microM radioactive ascorbate, but increased when cells preloaded with 50 microM ascorbate were incubated in the presence of excess non-radioactive ascorbate. These studies demonstrated that a sodium-dependent carrier system is involved in transport of ascorbate in primary cultures of cat RPE.