RESUMO
We have used in situ hybridization and immunocytochemistry to characterize the cellular distribution of cystic fibrosis (CF) gene expression in human bronchus. The cystic fibrosis transmembrane conductance regular (CFTR) was primarily localized to cells of submucosal glands in bronchial tissues from non-CF individuals notably in the serous component of the secretory tubules as well as a subpopulation of cells in ducts. Normal distribution of CFTR mRNA was found in CF tissues while expression of CFTR protein was genotype specific, with delta F508 homozygotes demonstrating no detectable protein and compound heterozygotes expressing decreased levels of normally distributed protein. Our data suggest mechanisms whereby defects in CFTR expression could lead to abnormal production of mucus in human lung.
Assuntos
Brônquios/química , Fibrose Cística/metabolismo , Glândulas Exócrinas/química , Proteínas de Membrana/análise , Proteínas de Membrana/genética , Regulador de Condutância Transmembrana em Fibrose Cística , Células Epiteliais , Epitélio/química , Humanos , Hibridização In Situ , Modelos Biológicos , Mucosa/química , Pleura/química , Sondas RNA , RNA Antissenso , RNA Mensageiro/análiseRESUMO
Although the number of pathologies known to arise from the inappropriate folding of proteins continues to grow, mechanisms underlying the recognition and ultimate disposition of misfolded polypeptides remain obscure. For example, how and where such substrates are identified and processed is unknown. We report here the identification of a specific subcellular structure in which, under basal conditions, the 20S proteasome, the PA700 and PA28 (700- and 180-kD proteasome activator complexes, respectively), ubiquitin, Hsp70 and Hsp90 (70- and 90-kD heat shock protein, respectively) concentrate in HEK 293 and HeLa cells. The structure is perinuclear, surrounded by endoplasmic reticulum, adjacent to the Golgi, and colocalizes with gamma-tubulin, an established centrosomal marker. Density gradient fractions containing purified centrosomes are enriched in proteasomal components and cell stress chaperones. The centrosome-associated structure enlarges in response to inhibition of proteasome activity and the level of misfolded proteins. For example, folding mutants of CFTR form large inclusions which arise from the centrosome upon inhibition of proteasome activity. At high levels of misfolded protein, the structure not only expands but also extensively recruits the cytosolic pools of ubiquitin, Hsp70, PA700, PA28, and the 20S proteasome. Thus, the centrosome may act as a scaffold, which concentrates and recruits the systems which act as censors and modulators of the balance between folding, aggregation, and degradation.
Assuntos
Adenosina Trifosfatases/metabolismo , Centrossomo/química , Centrossomo/enzimologia , Cisteína Endopeptidases/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Complexos Multienzimáticos/metabolismo , Adenosina Trifosfatases/análise , Animais , Galinhas , Cisteína Endopeptidases/análise , Regulador de Condutância Transmembrana em Fibrose Cística/análise , Regulador de Condutância Transmembrana em Fibrose Cística/química , Feto/química , Feto/metabolismo , Regulação Enzimológica da Expressão Gênica , Proteínas de Choque Térmico HSP70/análise , Proteínas de Choque Térmico HSP70/metabolismo , Células HeLa , Humanos , Rim/citologia , Complexos Multienzimáticos/análise , Mutagênese/fisiologia , Complexo de Endopeptidases do Proteassoma , Dobramento de Proteína , Tubulina (Proteína)/análise , Tubulina (Proteína)/metabolismoRESUMO
Cystic fibrosis (CF) is characterized by an abnormality in cAMP-regulated chloride transport that results from a primary defect in the protein product of the CF gene, the CF transmembrane conductance regulator (CFTR). In this report, antibodies against CFTR peptides were used to localize the CFTR protein in human pancreas. An affinity purified antibody (alpha-1468) raised against a synthetic CFTR peptide identified a 155-170-kD protein on immunoblot. Cytochemical studies with alpha-1468 localized CFTR to small branching, tubular structures. The same structures were recognized by two other antibodies raised against different regions of the CFTR molecule. To identify the cells being stained, double-label immunofluorescence studies were performed using alpha-1468 and a monoclonal antibody which stains pancreatic centroacinar and intralobular duct cells. Both antibodies localized to the same population of cells, with alpha-1468 being confined to the apical domain of these cells. No conclusive staining of acinar cells was evident. These findings suggest that proximal duct epithelial cells play a key role in the early events leading to pancreatic insufficiency in CF, and imply that apical chloride transport by these cells is essential for normal pancreatic secretory function.
Assuntos
Fibrose Cística/metabolismo , Proteínas de Membrana/metabolismo , Ductos Pancreáticos/metabolismo , Sequência de Aminoácidos , Cloretos/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística , Humanos , Immunoblotting , Imuno-Histoquímica , Dados de Sequência MolecularRESUMO
Defective trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR) is the most common cause of cystic fibrosis. In chloride-secreting epithelia, it is well established that CFTR localizes to intracellular organelles and to apical membranes. However, it is controversial whether secretagogues regulate the trafficking of CFTR. To investigate whether acute hormonal stimulation of chloride secretion is coupled to the trafficking of CFTR, we used the intact shark rectal gland, a model tissue in which salt secretion is dynamically regulated and both chloride secretion and cellular CFTR immunofluorescence can be quantified in parallel. In rectal glands perfused under basal conditions without secretagogues, Cl- secretion was 151+/-65 microeq/h/g. Vasoactive intestinal peptide (VIP), forskolin, and genistein led to 10-, 6-, and 4-fold increases in Cl- secretion. In basal glands, quantitative confocal microscopy revealed CFTR immunofluorescence extending from the apical membrane deeply into the cell (7.28+/-0.35 micron). During stimulation with secretagogues, apical extension of CFTR immunofluorescence into the cell was reduced significantly to 3.24+/-0.08 micron by VIP, 4.08+/-0.13 by forskolin, and 3.19+/-0.1 by genistein (P < 0.001). Moreover, the peak intensity of CFTR fluorescence shifted towards the apical membrane (peak fluorescence 2.5+/-0.13 micron basal vs. 1.51+/-0.06, 1.77+/-0.1, and 1.38+/-0.05 for VIP, forskolin, and genistein; all P < 0.001). The increase in both Cl- secretion and apical CFTR trafficking reversed to basal values after removal of VIP. These data provide the first quantitative morphological evidence for acute hormonal regulation of CFTR trafficking in an intact epithelial tissue.
Assuntos
Antimetabólitos/farmacologia , Colforsina/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Genisteína/farmacologia , Glândula de Sal/efeitos dos fármacos , Peptídeo Intestinal Vasoativo/farmacologia , Animais , Anticorpos/metabolismo , Cloretos/metabolismo , Cação (Peixe) , Feminino , Humanos , Masculino , Microscopia de Fluorescência/métodos , Perfusão , Glândula de Sal/metabolismo , Xenopus laevisRESUMO
The most prevalent mutation (delta F508) in cystic fibrosis patients inhibits maturation and transfer to the plasma membrane of the mutant cystic fibrosis transmembrane conductance regulator (CFTR). We have analyzed the properties of a delta F508 CFTR mouse model, which we described recently. We show that the mRNA levels of mutant CFTR are normal in all tissues examined. Therefore the reduced mRNA levels reported in two similar models may be related to their intronic transcription units. Maturation of mutant CFTR was greatly reduced in freshly excised oviduct, compared with normal. Accumulation of mutant CFTR antigen in the apical region of jejunum crypt enterocytes was not observed, in contrast to normal mice. In cultured gallbladder epithelial cells from delta F508 mice, CFTR chloride channel activity could be detected at only two percent of the normal frequency. However, in mutant cells that were grown at reduced temperature the channel frequency increased to over sixteen percent of the normal level at that temperature. The biophysical characteristics of the mutant channel were not significantly different from normal. In homozygous delta F508 mice we did not observe a significant effect of genetic background on the level of residual chloride channel activity, as determined by the size of the forskolin response in Ussing chamber experiments. Our data show that like its human homologue, mouse delta F508-CFTR is a temperature sensitive processing mutant. The delta F508 mouse is therefore a valid in vivo model of human delta F508-CFTR. It may help us to elucidate the processing pathways of complex membrane proteins. Moreover, it may facilitate the discovery of new approaches towards therapy of cystic fibrosis.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/genética , Animais , Western Blotting , Células Cultivadas , Regulador de Condutância Transmembrana em Fibrose Cística/imunologia , Modelos Animais de Doenças , Tubas Uterinas/metabolismo , Feminino , Vesícula Biliar/citologia , Vesícula Biliar/metabolismo , Imuno-Histoquímica , Jejuno/metabolismo , Camundongos , Camundongos Knockout , Técnicas de Patch-Clamp , Mutação Puntual , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , TemperaturaRESUMO
Malignant ascites is a manifestation of advanced malignant disease that is associated with significant morbidity. Mainstays of treatment include diuretics and recurrent large volume paracentesis. Although lymphatic obstruction has been considered the major pathophysiologic mechanism behind its formation, recent evidence suggests that immune modulators, vascular permeability factors, and metalloproteinases are contributing significantly to the process. These new observations offer the opportunity for development of new, more targeted therapies for the treatment of malignant ascites. This article uses a clinical case to highlight the problem, then reviews these new concepts in the pathophysiology of malignant ascites formation. The diagnosis and management of this challenging medical problem are subsequently discussed, with emphasis on how these new pathophysiologic insights are being applied to the development of novel therapies that may soon change how we manage this troubling clinical condition.
Assuntos
Ascite/etiologia , Neoplasias de Cabeça e Pescoço/complicações , Melanoma/complicações , Couro Cabeludo , Neoplasias Cutâneas/complicações , Ascite/diagnóstico , Ascite/fisiopatologia , Ascite/terapia , Diuréticos/uso terapêutico , Humanos , Imunoterapia , Masculino , Pessoa de Meia-Idade , ParacenteseRESUMO
To detect potential substrate proteins for Ca2+/calmodulin-dependent protein kinase II outside the central nervous system, antibodies were made to a synthetic peptide corresponding to a sequence within synapsin I which is phosphorylated by this enzyme. In neural tissues, this antibody (212) identified an 86/80 kDa doublet corresponding to synapsin I. In rat liver, intestinal enterocytes and the clone 9 cell line this antibody identified two proteins of 170 and 85 kDa. These proteins were present in the particulate fraction of liver postnuclear supernatant, and were released into the soluble fraction when extracted with 100 mM NaCl. In liver, enterocytes, and clone 9 cells, these antigens were localized by immunocytochemical techniques to small intracellular vesicles. The endocytic compartment of clone 9 cells was labeled by continuous uptake of horseradish peroxidase; antibody 212-labeled vesicles exhibited overlap with the compartment. To confirm the identity of this compartment as endosomal, rat liver endosomes were labeled in vivo by intravenous injection of horseradish peroxidase. Horseradish peroxidase-containing endosomes of approximately 80 nm were recognized by antibody 212. Occasionally, larger endosomes (approximately 300-500 nm) were also labeled. In clone 9 cells, partial overlap was observed between the 212 antigen and a transferrin receptor-positive, brefeldin A-sensitive compartment. In clone 9 cells double-labeled with anti-tubulin and antibody 212, then imaged using confocal microscopy, these vesicles appeared to be associated with microtubules. This antigen has properties similar to that of CLIP-170, a membrane-associated endosomal phosphoprotein. These findings demonstrate that a 170/85 kDa antigen containing an epitope for the Ca2+/calmodulin-dependent protein kinase II phosphorylation sequence is associated with an endocytic compartment.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/imunologia , Compartimento Celular/imunologia , Endocitose/imunologia , Epitopos/análise , Sinapsinas/imunologia , Animais , Anticorpos Monoclonais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Linhagem Celular , Fígado/citologia , Fígado/imunologia , Proteínas de Membrana/imunologia , Peso Molecular , Neurônios , Fosfoproteínas/química , Fosforilação , Ratos , Especificidade por SubstratoRESUMO
This study reports on the use of a new sensitive assay of cAMP-dependent protein kinase activity to examine the effect of cholecystokinin (CCK) on the cAMP second messenger cascade in rat pancreatic acini. Treatment of acini with both low (pM) and high (nM) concentrations of CCK was associated with an increase in cAMP-dependent protein kinase activity. The increases in kinase activity were detected in the absence of phosphodiesterase inhibition, a condition required to detect a measurable increase in cellular cAMP in these cells. Furthermore, the cAMP cascade was dissociated from the secretory effects of CCK, since the CCK analogue, OPE, mediates enzyme secretion but does not increase cellular cAMP levels or kinase activity.
Assuntos
Colecistocinina/farmacologia , Pâncreas/efeitos dos fármacos , Proteínas Quinases/metabolismo , 1-Metil-3-Isobutilxantina/farmacologia , Animais , AMP Cíclico/fisiologia , Ativação Enzimática/efeitos dos fármacos , Técnicas In Vitro , Masculino , Pâncreas/enzimologia , Pâncreas/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
The DeltaF508 gene mutation prevents delivery of the cystic fibrosis transmembrane conductance regulator (CFTR) to the plasma membrane. The current study examines the biochemical basis for the upregulation of DeltaF508 CFTR expression by sodium butyrate and low temperature. Surface CFTR protein expression was determined by quantitative immunoblot following surface biotinylation and streptavidin extraction. CF gene expression was measured by Northern analysis and CFTR function by forskolin-stimulated (125)I efflux. Butyrate increased DeltaF508 mRNA levels and protein expression but did not increase the biochemical or functional expression of DeltaF508 CFTR at the cell surface. Low temperature increased the biochemical and functional expression of DeltaF508 CFTR at the cell surface but did not increase CFTR mRNA levels. Combining treatments led to a synergistic increase in both DeltaF508 mRNA and surface protein levels that results from the stabilization of CFTR mRNA and protein by low temperature. These findings indicate that surface expression of DeltaF508 CFTR can be markedly enhanced by carefully selected combination agents.
Assuntos
Butiratos/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Animais , Linhagem Celular , Membrana Celular/metabolismo , Colforsina/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Feminino , Iodetos/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Mutação , RNA Mensageiro/metabolismo , Temperatura , Transfecção , Regulação para Cima/efeitos dos fármacosRESUMO
Although the biosynthetic arrest of the DeltaF508 mutant of cystic fibrosis transmembrane conductance regulator (CFTR) can be partially reversed by physical and chemical means, recent evidence suggests that the functional stability of the mutant protein after reaching the cell surface is compromised. To understand the molecular basis for this observation, the current study directly measured the half-life of Delta F508 and wild-type CFTR at the cell surface of transfected LLC-PK(1) cells. Plasma membrane CFTR expression over time was characterized biochemically and functionally in these polarized epithelial cells. Surface biotinylation, streptavidin extraction, and quantitative immunoblot analysis determined the biochemical half-life of plasma membrane DeltaF508 CFTR to be approximately 4 h, whereas the plasma membrane half-life of wild-type CFTR exceeded 48 h. This difference in biochemical stability correlated with CFTR-mediated transport function. These findings indicate that the Delta F508 mutation decreases the biochemical stability of CFTR at the cell surface. We conclude that the Delta F508 mutation triggers more rapid internalization of CFTR and/or its preferential sorting to a pathway of rapid degradation.
Assuntos
Membrana Celular/genética , Polaridade Celular/fisiologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Células Epiteliais/metabolismo , Animais , Biotinilação , Butiratos/farmacologia , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Fibrose Cística/genética , Fibrose Cística/metabolismo , Fibrose Cística/fisiopatologia , Regulador de Condutância Transmembrana em Fibrose Cística/química , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Endocitose/fisiologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/ultraestrutura , Meia-Vida , Células LLC-PK1/efeitos dos fármacos , Células LLC-PK1/metabolismo , Células LLC-PK1/ultraestrutura , Mutação/fisiologia , Transporte Proteico/genética , ATPase Trocadora de Sódio-Potássio/metabolismo , Suínos , TransfecçãoRESUMO
An antiserum raised against purified rat parotid secretory granule membrane proteins has been used to identify organelle-specific protein phosphorylation events following stimulation of intact cells from the rat parotid gland. After lobules were prelabeled with [32P]orthophosphate and exposed to secretagogues, phosphoproteins were immunoprecipitated with the granule membrane protein antiserum, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and visualized by autoradiography. Parallel studies of stimulated amylase release were performed. Isoproterenol treatment of parotid lobules resulted in an increase in the phosphate content of immunoprecipitable 60- and 72-kDa proteins that correlated with amylase release in a time-dependent manner. Forskolin addition mimicked these effects, but only the isoproterenol effects were reversed by propranolol treatment. To confirm the specificity of the antiserum to the secretory granule membrane fraction, subcellular isolation techniques were employed following in situ phosphorylation. The 60- and 72-kDa phosphoproteins were immunoprecipitated from both a particulate fraction and a purified secretory granule fraction. Furthermore, the extraction properties of both species suggest that they are integral membrane proteins. These findings support the possibility that stimulus-regulated secretion may involve phosphorylation of integral membrane proteins of the exocrine secretory granule.
Assuntos
Grânulos Citoplasmáticos/metabolismo , Proteínas de Membrana/metabolismo , Glândula Parótida/fisiologia , Amilases/metabolismo , Animais , Autorradiografia , Colforsina/farmacologia , Grânulos Citoplasmáticos/efeitos dos fármacos , Grânulos Citoplasmáticos/ultraestrutura , Eletroforese em Gel de Poliacrilamida , Cinética , Masculino , Proteínas de Membrana/isolamento & purificação , Microscopia Eletrônica , Peso Molecular , Glândula Parótida/efeitos dos fármacos , Glândula Parótida/metabolismo , Fosfatos/metabolismo , Fosfoproteínas/isolamento & purificação , Radioisótopos de Fósforo , Fosforilação , Ratos , Ratos EndogâmicosRESUMO
We describe the case of a patient who presented with abdominal pain, weight loss, and night sweats of 8 wk duration due to the impalement of a toothpick in the transverse colon. The toothpick, which was inadvertently swallowed, was successfully removed during colonoscopy. Therapeutic colonoscopy has replaced or lessened to a significant degree the need for traditional open surgical procedures to extract foreign bodies from the colon. Unexplained abdominal pain and constitutional symptoms should alert the clinician to the possibility of a foreign body.
Assuntos
Colo , Colonoscopia , Corpos Estranhos/terapia , Adulto , Corpos Estranhos/diagnóstico , Humanos , MasculinoRESUMO
Plasma membrane chloride transport by the cystic fibrosis transmembrane conductance regulator (CFTR) may be regulated by cellular processes that affect the cycling of CFTR with the plasma membrane. Testing this hypothesis requires cytochemical evidence for the presence of a subcellular compartment of CFTR. In this study, the subcellular distribution of CFTR in a normal epithelial cell population was characterized using immunofluorescence and immunoelectron microscopy. Two anti-CFTR antibodies, raised against different epitopes of the CFTR molecule, specifically labeled the apical pole of striated duct epithelial cells in tissue sections of rat submandibular gland. By use of electron microscopy, the CFTR immunoreactivity was associated with the apical plasma membrane and the membranes of many subapical vesicles. In this preparation, some of the CFTR-labeled vesicles were also labeled with antibodies against transferrin receptor and rab4, two markers of early endosomes and receptor-mediated endocytosis. These observations provide direct cytochemical evidence for the existence of peripherally located CFTR-expressing endosomes and support the hypothesis that membrane recycling may contribute to CFTR function.
Assuntos
Proteínas de Membrana/metabolismo , Frações Subcelulares/metabolismo , Glândula Submandibular/metabolismo , Animais , Membrana Celular/metabolismo , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística , Endocitose , Epitélio/metabolismo , Imunofluorescência , Imuno-Histoquímica/métodos , Masculino , Microscopia de Fluorescência , Organelas/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Superfície Celular/metabolismo , Glândula Submandibular/citologia , Glândula Submandibular/ultraestrutura , Distribuição TecidualRESUMO
An anti-peptide antibody raised to the C-terminal sequence of the cystic fibrosis transmembrane conductance regulator (CFTR) was used to examine CFTR immunoreactivity in the T84 colonocyte cell line. Immunoblots of T84 cell lysates detected CFTR as a 170-kDa protein that appeared as a broad band or doublet in SDS/PAGE. This protein comigrated with the predominant immunoblot signal detected in human pancreas and colon. An equivalent protein was detected as a prominent substrate for protein kinase A and for protein kinase C in T84 cell immunoprecipitates with this antibody. The immunoprecipitated protein resembled the protein detected by immunoblot in that both proteins showed the same change in electrophoretic mobility after digestion by N-Glycanase. The precipitated protein was indentified as CFTR by two criteria. First, the same protein was immunoprecipitated with an antibody to a different CFTR peptide, [Lys102]CFTR-(102-116). Second, two-dimensional phosphopeptide mapping was used to compare the immunoprecipitated protein with a bacterially expressed protein known to contain most of the predicted protein kinase A phosphorylation sites in CFTR. Because the six most prominent peptides in each map were equivalent, these maps confirm that the precipitated protein is CFTR. By using these antibodies for immunofluorescence and immunoperoxidase staining, CFTR was localized to the apical region of T84 cells grown in tumors and in monolayers. Thus, T84 cells express CFTR at sufficient levels to permit identification and immunochemical studies of this protein in its endogenously occurring form.
Assuntos
Proteínas de Membrana/análise , Sequência de Aminoácidos , Linhagem Celular , Colo , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística , Imunofluorescência , Humanos , Immunoblotting , Técnicas Imunoenzimáticas , Imuno-Histoquímica , Proteínas de Membrana/imunologia , Dados de Sequência Molecular , Mapeamento de Peptídeos , Peptídeos/síntese química , Peptídeos/imunologia , Fosfopeptídeos/análiseRESUMO
BACKGROUND/AIMS: In the intestine, the cystic fibrosis transmembrane conductance regulator (CFTR) has been localized to the apical pole of crypt epithelial cells. Recent data indicate that some villus cells may also express CFTR, although the identity of these cells has not been established. The aim of the current study was to characterize the distribution, morphology, and surface marker expression of CFTR-expressing villus cells. METHODS: Immunofluorescence and immunoelectron microscopy was performed using anti-CFTR and enzyme marker antibodies. RESULTS: In the rat and human proximal small intestine, a subpopulation of scattered villus and superficial crypt epithelial cells label brightly with anti-CFTR antibodies. The fluorescent signal is detected throughout the cells with its greatest concentration apically. At the ultrastructural level, labeling involves the brush border and a prominent subapical vesicular compartment. The cells resemble adjacent villus enterocytes in their abundance of mitochondria and expression of basolateral Na(+)-K(+)-adenosine triphosphatase yet differ in their absence of brush-border sucrase and lactase expression. CONCLUSIONS: A previously uncharacterized subpopulation of villus cells with high levels of intracellular CFTR expression exists in the proximal small intestine. Morphological and cytochemical studies suggest that this subset of villus cells has a unique transport function.
Assuntos
Fibrose Cística/metabolismo , Mucosa Intestinal/metabolismo , Proteínas de Membrana/metabolismo , Animais , Regulador de Condutância Transmembrana em Fibrose Cística , Células Epiteliais , Epitélio/enzimologia , Epitélio/metabolismo , Imunofluorescência , Humanos , Imuno-Histoquímica , Mucosa Intestinal/citologia , Mucosa Intestinal/enzimologia , Lactase , Microscopia Imunoeletrônica , Ratos , Sacarase/metabolismo , beta-Galactosidase/metabolismoRESUMO
The cellular mechanisms of HCO(-)(3) secretion in the human pancreas are unclear. Expression of a Na(+)-HCO(-)(3) cotransporter (NBC) mRNA has been observed recently, but the distribution and physiological role of the NBC protein are not known. Here we examined the expression and localization of NBC in human pancreas by Northern blot, immunoblot, and immunofluorescence microscopy. Rat kidney NBC probes detected a single 9.5-kb band by Northern blot. On immunoblots, two polyclonal antisera directed against different epitopes of rat kidney NBC identified a single approximately 130-kDa protein. In cryosections of normal human pancreas, both antisera labeled basolateral membranes of large, morphologically identifiable ducts and produced a distinct labeling pattern in the remainder of the parenchyma. In double-labeling experiments, NBC immunoreactivity in the parenchyma colocalized with the Na(+)-K(+) pump, a basolateral marker. In contrast, NBC and cystic fibrosis transmembrane conductance regulator, an apical membrane marker, were detected within the same histological structures but at different subcellular localizations. The NBC antisera did not label acinar or islet cells. Our observations suggest that secretion of HCO(-)(3) by human pancreatic duct cells involves the basolateral uptake of Na(+) and HCO(-)(3) via NBC, an electrogenic Na(+)-HCO(-)(3) cotransporter.
Assuntos
Proteínas de Transporte/metabolismo , Pâncreas/metabolismo , Idoso , Anticorpos Monoclonais , Northern Blotting , Proteínas de Transporte/genética , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Immunoblotting , RNA Mensageiro/metabolismo , Valores de Referência , Simportadores de Sódio-Bicarbonato , Distribuição TecidualRESUMO
Using an 125I- efflux assay, we have studied the expression of various types of chloride channels in isolated neonatal rat cardiomyocytes. Three different classes of anion conductances were distinguished: (1) a Ca(2+)-sensitive Cl- conductance, triggered upon stimulation of the cells with endothelin-1 or Ca(2+)-ionophore; (2) a cAMP/protein kinase A-operated Cl- conductance, activated by addition of forskolin. This anion channel could be identified as the Cystic Fibrosis Transmembrane conductance Regulator (CFTR-CI- channel) by Western blotting as well as by its enhanced activity in cultures pretreated with the tyrosine kinase inhibitor genistein; (3) a distinct class of cell volume-regulated Cl- channels, potentiated in the presence of endothelin-1 or the phosphotyrosine phosphatase inhibitor pervanadate. The potential role of each class of Cl- channels in the generation and/or modulation of action potentials as well as in maintaining cell volume is discussed.
Assuntos
Canais de Cloreto/biossíntese , Expressão Gênica , Coração/efeitos dos fármacos , Iodetos/metabolismo , Miocárdio/metabolismo , 1-Metil-3-Isobutilxantina/farmacologia , Animais , Animais Recém-Nascidos , Calcimicina/farmacologia , Cálcio/metabolismo , Células Cultivadas , Canais de Cloreto/fisiologia , Colforsina/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/biossíntese , Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Endotelinas/farmacologia , Inibidores Enzimáticos/farmacologia , Expressão Gênica/efeitos dos fármacos , Genisteína , Coração/fisiologia , Soluções Hipertônicas , Soluções Hipotônicas , Radioisótopos do Iodo , Isoflavonas/farmacologia , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Ratos , Ratos Wistar , Transdução de Sinais , Vanadatos/farmacologiaRESUMO
Cystic fibrosis results from defective Cl- channel activity mediated by the cystic fibrosis transmembrane conductance regulator (CFTR) gene product. In the gastrointestinal tract this is manifested in abnormal salivary secretion and pancreatic insufficiency. This is generally attributed to defective Cl- transport by the ductal system of the glands. We provide the first immunocytochemical and functional evidence for expression of CFTR protein and Cl- current in rat and mouse submandibular gland (SMG) and pancreatic acinar cells, a site proximal to the ductal system of these secretory glands. Monoclonal and polyclonal antibodies recognizing COOH-terminal epitopes of CFTR show that duct and acinar cells from the two glands express CFTR in the luminal membrane. Specificity of the polyclonal antibody was verified by absence of staining in duct and acinar cells of the SMG of cf-/cf- and delta F/delta F mice. Identification of CFTR in acinar cells was aided by demonstrating coexpression of CFTR and type 3 inositol 1,4,5-trisphosphate receptors in the luminal pole of acini and absence of type 3 inositol 1,4,5-trisphosphate receptors in ducts. Electrophysiological characterization in single SMG duct and acinar cells shows the presence of a protein kinase A-activated, voltage- and time-independent, ohmic Cl- current and absence of repolarization-dependent tail currents, all of which are kinetic properties of the CFTR-dependent Cl- channel. In addition, the channel was activated by the nonhydrolyzable ATP analog 5'-adenylylimidodiphosphate and the benzimidazalone NS-004. Channels activated by all activators were inhibited by glibenclamide and a known inhibitory antiserum [anti-CFTR-(505-511)]. Combined immunologic, functional, and pharmacological evidence allows us to conclude that acinar cells of the SMG and pancreas express functional CFTR-dependent Cl- channels. Because this site is proximal to the duct, modification of activity of this channel in acinar cells is likely to contribute to abnormal salivary secretion and pancreatic insufficiency typical of cystic fibrosis.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Pâncreas/metabolismo , Ductos Pancreáticos/metabolismo , Glândula Submandibular/metabolismo , Animais , Cloretos/fisiologia , Condutividade Elétrica , Imuno-Histoquímica , Camundongos/genética , Pâncreas/citologia , Ductos Pancreáticos/citologia , Reação em Cadeia da Polimerase , Ratos , Transcrição GênicaRESUMO
A central function of cystic fibrosis transmembrane conductance regulator (CFTR)-expressing tissues is the secretion of fluid containing 100-140 mM HCO3-. High levels of HCO3- maintain secreted proteins such as mucins (all tissues) and digestive enzymes (pancreas) in a soluble and/or inactive state. HCO3- secretion is impaired in CF in all CFTR-expressing, HCO3--secreting tissues examined. The mechanism responsible for this critical problem in CF is unknown. Since a major component of HCO3- secretion in CFTR-expressing cells is mediated by the action of a Cl-/HCO3- exchanger (AE), in the present work we examined the regulation of AE activity by CFTR. In NIH 3T3 cells stably transfected with wild type CFTR and in HEK 293 cells expressing WT and several mutant CFTR, activation of CFTR by cAMP stimulated AE activity. Pharmacological and mutagenesis studies indicated that expression of CFTR in the plasma membrane, but not the Cl- conductive function of CFTR was required for activation of AE. Furthermore, mutations in NBD2 altered regulation of AE activity by CFTR independent of their effect on Cl- channel activity. At very high expression levels CFTR modified the sensitivity of AE to 4,4'-diisothiocyanatostilbene-2, 2'-disulfonate. The novel finding of regulation of Cl-/HCO3- exchange by CFTR reported here may have important physiological implications and explain, at least in part, the impaired HCO3- secretion in CF.
Assuntos
Bicarbonatos/metabolismo , Cloretos/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células 3T3 , Animais , Sequência de Bases , Linhagem Celular , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Primers do DNA , Humanos , Transporte de Íons , Potenciais da Membrana , Camundongos , Mutagênese Sítio-Dirigida , TransfecçãoRESUMO
BACKGROUND & AIMS: The progenitor cells responsible for transforming growth factor (TGF)-alpha-induced pancreatic ductal metaplasia and neoplasia remain uncharacterized. During pancreatic development, differentiated cell types arise from ductal progenitor cells expressing the Pdx1 homeodomain transcription factor. The aims of this study were, first, to evaluate the role of Pdx1-expressing stem cells in MT-TGFalpha transgenic mice, and second, to further characterize cell proliferation and differentiation in this model. METHODS: To assess Pdx1 gene expression in normal and metaplastic epithelium, we performed in vivo reporter gene analysis using heterozygous Pdx1(lacZ/+) and bigenic Pdx1(lacZ/+)/MT-TGFalpha mice. RESULTS: Pdx1(lacZ/+)/MT-TGFalpha bigenics showed up-regulated Pdx1 expression in premalignant metaplastic ductal epithelium. In addition to Pdx1 gene activation, TGF-alpha-induced metaplastic epithelium demonstrated a pluripotent differentiation capacity, as evidenced by focal expression of Pax6 and initiation of islet cell neogenesis. The majority of Pdx1-positive epithelial cells showed no expression of insulin, similar to the pattern observed during embryonic development. CONCLUSIONS: Overexpression of TGF-alpha induces expansion of a Pdx1-expressing epithelium characterized by focal expression of Pax6 and initiation of islet neogenesis. These findings suggest that premalignant events induced by TGF-alpha in mouse pancreas may recapitulate a developmental program active during embryogenesis.