RESUMO
Iodide is captured by thyrocytes through the Na(+)/I(-) symporter (NIS) before being released into the follicular lumen, where it is oxidized and incorporated into thyroglobulin for the production of thyroid hormones. Several reports point to pendrin as a candidate protein for iodide export from thyroid cells into the follicular lumen. Here, we show that a recently discovered Ca(2+)-activated anion channel, TMEM16A or anoctamin-1 (ANO1), also exports iodide from rat thyroid cell lines and from HEK 293T cells expressing human NIS and ANO1. The Ano1 mRNA is expressed in PCCl3 and FRTL-5 rat thyroid cell lines, and this expression is stimulated by thyrotropin (TSH) in rat in vivo, leading to the accumulation of the ANO1 protein at the apical membrane of thyroid follicles. Moreover, ANO1 properties, i.e., activation by intracellular calcium (i.e., by ionomycin or by ATP), low but positive affinity for pertechnetate, and nonrequirement for chloride, better fit with the iodide release characteristics of PCCl3 and FRTL-5 rat thyroid cell lines than the dissimilar properties of pendrin. Most importantly, iodide release by PCCl3 and FRTL-5 cells is efficiently blocked by T16Ainh-A01, an ANO1-specific inhibitor, and upon ANO1 knockdown by RNA interference. Finally, we show that the T16Ainh-A01 inhibitor efficiently blocks ATP-induced iodide efflux from in vitro-cultured human thyrocytes. In conclusion, our data strongly suggest that ANO1 is responsible for most of the iodide efflux across the apical membrane of thyroid cells.
Assuntos
Polaridade Celular , Canais de Cloreto/metabolismo , Iodetos/metabolismo , Proteínas de Neoplasias/metabolismo , Glândula Tireoide/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Anoctamina-1 , Transporte Biológico , Cálcio/metabolismo , Canais de Cloreto/antagonistas & inibidores , Canais de Cloreto/genética , Células HEK293 , Humanos , Moduladores de Transporte de Membrana/farmacologia , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Interferência de RNA , Ratos , Glândula Tireoide/citologia , Glândula Tireoide/efeitos dos fármacos , Tireotropina/metabolismo , Fatores de Tempo , TransfecçãoRESUMO
The use of substances that could activate the defective chloride channels of the mutant cystic fibrosis transmembrane conductance regulator (CFTR) has been suggested as possible therapy for cystic fibrosis. Using epithelia formed by cells stably transfected with wildtype or mutant (G551D, G1349D) CFTR, we estimated the apparent dissociation constant, K(D), of a series of CFTR activators by measuring the increase in the apical membrane current. Modification of apparent K(D) of CFTR activators by mutations of the nucleotide-binding domains (NBDs) suggests that the binding site might be in these regions. The human NBD structure was predicted by homology with murine NBD1. An NBD1-NBD2 complex was constructed by overlying monomers to a bacterial ABC transporter NBD dimer in the "head-to-tail" conformation. Binding sites for CFTR activators were predicted by molecular docking. Comparison of theoretical binding free energy estimated in the model to free energy estimated from the apparent dissociation constants, K(D), resulted in a remarkably good correlation coefficient for one of the putative binding sites, located in the interface between NBD1 and NBD2.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/agonistas , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Transportadores de Cassetes de Ligação de ATP/agonistas , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/fisiologia , Nucleotídeos de Adenina/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/genética , Linhagem Celular , Permeabilidade da Membrana Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/genética , Permeabilidade da Membrana Celular/fisiologia , Fibrose Cística/tratamento farmacológico , Regulador de Condutância Transmembrana em Fibrose Cística/química , Dimerização , Condutividade Elétrica , Eletrofisiologia , Genisteína/química , Genisteína/farmacologia , Humanos , Camundongos , Dados de Sequência Molecular , Mutação/genética , Estrutura Terciária de Proteína , Ratos , Alinhamento de Sequência , TermodinâmicaRESUMO
A number of genetic diseases, including cystic fibrosis, have been identified as disorders of protein trafficking associated with retention of mutant protein within the endoplasmic reticulum. In the presence of the benzo(c)quinolizinium drugs, MPB-07 and its congener MPB-91, we show the activation of cystic fibrosis transmembrane conductance regulator (CFTR) delF508 channels in IB3-1 human cells, which express endogenous levels of delF508-CFTR. These drugs were without effect on the Ca(2+)-activated Cl- transport, whereas the swelling-activated Cl- transport was found altered in MPB-treated cells. Immunoprecipitation and in vitro phosphorylation shows a 20% increase of the band C form of delF508 after MPB treatment. We then investigated the effect of these drugs on the extent of mislocalisation of delF508-CFTR in native airway cells from cystic fibrosis patients. We first showed that delF508 CFTR was characteristically restricted to an endoplasmic reticulum location in approximately 80% of untreated cells from CF patients homozygous for the delF508-CFTR mutation. By contrast, 60-70% of cells from non-CF patients showed wild-type CFTR in an apical location. MPB-07 treatment caused dramatic relocation of delF508-CFTR to the apical region such that the majority of delF508/delF508 CF cells showed a similar CFTR location to that of wild-type. MPB-07 had no apparent effect on the distribution of wild-type CFTR, the apical membrane protein CD59 or the ER membrane Ca(2+),Mg-ATPase. We also showed a similar pharmacological effect in nasal cells freshly isolated from a delF508/G551D CF patient. The results demonstrate selective redirection of a mutant membrane protein using cell-permeant small molecules of the benzo(c)quinolizinium family and provide a major advance towards development of a targetted drug treatment for cystic fibrosis and other disorders of protein trafficking.
Assuntos
Cloretos/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/metabolismo , Quinolizinas/farmacologia , Mucosa Respiratória/efeitos dos fármacos , Cálcio/metabolismo , Polaridade Celular , Células Cultivadas , AMP Cíclico/agonistas , AMP Cíclico/metabolismo , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Inibidores Enzimáticos/farmacologia , Humanos , Imuno-Histoquímica , Iodetos/metabolismo , Quinolizinas/química , Mucosa Respiratória/citologia , Mucosa Respiratória/metabolismoRESUMO
We have designed and synthesized benzo[c]quinolizinium derivatives and evaluated their effects on the activity of G551D cystic fibrosis transmembrane conductance regulator (CFTR) expressed in Chinese hamster ovary and Fisher rat thyroid cells. We demonstrated, using iodide efflux, whole cell patch clamp, and short-circuit recordings, that 5-butyl-6-hydroxy-10-chlorobenzo[c]quinolizinium chloride (MPB-91) restored the activity of G551D CFTR (EC(50) = 85 microM) and activated CFTR in Calu-3 cells (EC(50) = 47 microM). MPB-91 has no effect on the ATPase activity of wild-type and G551D NBD1/R/GST fusion proteins or on the ATPase, GTPase, and adenylate kinase activities of purified NBD2. The activation of CFTR by MPB-91 is independent of phosphorylation because 1) kinase inhibitors have no effect and 2) the compound still activated CFTR having 10 mutated protein kinase A sites (10SA-CFTR). The new pharmacological agent MPB-91 may be an important candidate drug to ameliorate the ion transport defect associated with CF and to point out a new pathway to modulate CFTR activity.
Assuntos
Adenosina Trifosfatases/fisiologia , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Ativadores de Enzimas/farmacologia , Quinolizinas/farmacologia , Adenosina Trifosfatases/metabolismo , Animais , Células CHO , Canais de Cloreto/efeitos dos fármacos , Canais de Cloreto/metabolismo , Cricetinae , Eletrofisiologia , Iodetos/metabolismo , Técnicas de Patch-Clamp , Fosforilação , Ratos , Ratos Endogâmicos F344 , Glândula Tireoide/citologia , Glândula Tireoide/efeitos dos fármacos , Glândula Tireoide/metabolismoRESUMO
The green fluorescent protein YFP-H148Q is sensitive to halides by a mechanism involving halide binding and a shift in pK(a). However, a limitation of YFP-H148Q is its low halide sensitivity, with K(d)>100 mM for Cl(-). Indicators with improved sensitivities are needed for cell transport studies, particularly in drug discovery by high-throughput screening, and for measurement of Cl(-) concentration in subcellular organelles. YFP-H148Q libraries were generated in which pairs of residues in the vicinity of the halide binding site were randomly mutated. An automated procedure was developed to screen bacterial colonies for improved halide sensitivity. Analysis of 1536 clones revealed improved anion sensitivities with K(d) down to 2 mM for I(-) (I152L), 40 mM for Cl(-) (V163S), and 10 mM for NO(3)(-) (I152L). The anion-sensitive mechanism of these indicators was established and their utility in cells was demonstrated using transfected cells expressing the cystic fibrosis transmembrane conductance regulator chloride channel.
Assuntos
Cloretos/metabolismo , Iodetos/metabolismo , Proteínas Luminescentes/metabolismo , Células 3T3 , Animais , Escherichia coli/genética , Proteínas de Fluorescência Verde , Cinética , Proteínas Luminescentes/genética , Camundongos , Mutação , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transformação BacterianaRESUMO
The flavonoid genistein and the benzo[c]quinolizinium MPB-07 have been shown to activate the cystic fibrosis transmembrane conductance regulator (CFTR), the protein that is defective in cystic fibrosis. Lead-based combinatorial and parallel synthesis yielded 223 flavonoid, quinolizinium, and related heterocyclic compounds. The compounds were screened for their ability to activate CFTR at 50 microm concentration by measurement of the kinetics of iodide influx in Fisher rat thyroid cells expressing wild-type or G551D CFTR together with the green fluorescent protein-based halide indicator YFP-H148Q. Duplicate screenings revealed that 204 compounds did not significantly affect CFTR function. Compounds of the 7,8-benzoflavone class, which are structurally intermediate between flavones and benzo[c]quinoliziniums, were effective CFTR activators with the most potent being 2-(4-pyridinium)benzo[h]4H-chromen-4-one bisulfate (UCcf-029). Compounds of the novel structural class of fused pyrazolo heterocycles were also strong CFTR activators with the most potent being 3-(3-butynyl)-5-methoxy-1-phenylpyrazole-4-carbaldehyde (UCcf-180). A CFTR inhibitor was also identified. The active compounds did not induce iodide influx in null cells deficient in CFTR. Short-circuit current measurements showed that the CFTR activators identified by screening induced strong anion currents in the transfected cell monolayers grown on porous supports. Compared with genistein, the most active compounds had up to 10 times greater potency in activating wild-type and/or G551D-CFTR. The activators had low cellular toxicity and did not elevate cellular cAMP concentration or inhibit phosphatase activity, suggesting that CFTR activation may involve a direct interaction. These results establish an efficient screening procedure to identify CFTR activators and inhibitors and have identified 7,8-benzoflavones and pyrazolo derivatives as novel classes of CFTR activators.
Assuntos
Técnicas de Química Combinatória , Regulador de Condutância Transmembrana em Fibrose Cística/agonistas , Flavonoides/química , Quinazolinas/química , Animais , Células Cultivadas , Ratos , Ratos Endogâmicos F344RESUMO
The pharmacological activation of the cystic fibrosis gene protein cystic fibrosis transmembrane conductance regulator (CFTR) was studied in human airway epithelial Calu-3 cells, which express a high level of CFTR protein as assessed by Western blot and in vitro phosphorylation. Immunolocalization shows that CFTR is located in the apical membrane. We performed iodide efflux, whole cell patch-clamp, and short-circuit recordings to demonstrate that the novel synthesized xanthine derivative 3, 7-dimethyl-1-isobutylxanthine (X-33) is an activator of the CFTR channel in Calu-3 cells. Whole cell current activated by X-33 or IBMX is linear, inhibited by glibenclamide and diphenylamine-2-carboxylate but not by DIDS or TS-TM calix[4]arene. Intracellular cAMP was not affected by X-33. An outwardly rectifying Cl(-) current was recorded in the absence of cAMP and X-33 stimulation, inhibited by DIDS and TS-TM calix[4]arene. With the use of short-circuit recordings, X-33 and IBMX were able to stimulate a large concentration-dependent CFTR transport that was blocked by glibenclamide but not by DIDS. Our results show that manipulating the chemical structure of xanthine derivatives offers an opportunity to identify further specific activators of CFTR in airway cells.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Mucosa Respiratória/citologia , Mucosa Respiratória/efeitos dos fármacos , Teofilina/análogos & derivados , Xantinas/farmacologia , 1-Metil-3-Isobutilxantina/farmacologia , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/farmacologia , Animais , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Células CHO , Cafeína/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Cloretos/metabolismo , Colforsina/farmacologia , Cricetinae , AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Glibureto/farmacologia , Hipoglicemiantes/farmacologia , Técnicas In Vitro , Iodetos/farmacocinética , Radioisótopos do Iodo , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Proteínas de Membrana/metabolismo , Técnicas de Patch-Clamp , Inibidores de Fosfodiesterase/farmacologia , Fosforilação , Mucosa Respiratória/fisiologia , Teofilina/farmacologia , Xantinas/síntese química , ortoaminobenzoatos/farmacologiaRESUMO
Human bronchial epithelial cells were treated in vitro with interferon-gamma or tumor necrosis factor-alpha to assess their effect on transepithelial ion transport. Short-circuit current measurements revealed that Na(+) absorption was markedly inhibited by interferon-gamma (10-1,000 U/ml). The cystic fibrosis transmembrane conductance regulator was also downregulated by interferon-gamma as evident at the protein level and by the decrease in the cAMP-dependent current. On the other hand, interferon-gamma caused an increase of the current elicited by apical UTP application, which is due to the activity of Ca(2+)-dependent Cl(-) channels. Tumor necrosis factor-alpha caused few changes in ion transport. Transepithelial fluid transport was measured in normal and cystic fibrosis cells. At rest, both types of cells showed an amiloride-sensitive fluid absorption that was inhibited by interferon-gamma but not by tumor necrosis factor-alpha. Our results show that interferon-gamma alters the transepithelial ion transport of cultured bronchial cells. This effect may change the ion composition and/or volume of periciliary fluid.
Assuntos
Brônquios/metabolismo , Interferon gama/farmacologia , Transporte Biológico/efeitos dos fármacos , Líquidos Corporais/metabolismo , Brônquios/citologia , Brônquios/efeitos dos fármacos , Cálcio/metabolismo , Membrana Celular/efeitos dos fármacos , Células Cultivadas , Canais de Cloreto/efeitos dos fármacos , Canais de Cloreto/fisiologia , AMP Cíclico/fisiologia , Fibrose Cística/metabolismo , Fibrose Cística/patologia , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Regulação para Baixo , Condutividade Elétrica , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , Íons , Valores de Referência , Canais de Sódio/efeitos dos fármacos , Canais de Sódio/fisiologia , Fator de Necrose Tumoral alfa/farmacologia , Uridina Trifosfato/farmacologiaRESUMO
We describe the identification and characterization of a new gene deleted in the AMME contiguous gene syndrome. This gene is predominantly expressed in heart, skeletal muscle, spinal cord, and brain. Screening of placenta and NT2 cDNA libraries enabled us to obtain the 1.5-kb full-length transcript, which shows a 426-bp open reading frame. Since the resulting 142-amino-acid peptide has a single putative transmembrane domain and a weak but suggestive homology with KCNE1 (minK), a protein associated with the KCNQ1 potassium channel (KVLQT1), we named this new gene KCNE1-like (KCNE1L). To obtain greater insight into this new member of an apparently distinct protein family, we have identified and characterized the homologous mouse gene (Kcne1l), which encodes a peptide of 143 amino acids with 91% homology and 80% identity. The expression pattern of mouse Kcne1l in the developing embryo revealed strong signal in ganglia, in the migrating neural crest cells of cranial nerves, in the somites, and in the myoepicardial layer of the heart. The specific distribution in adult tissues, the putative channel function, and the expression pp6tern in the developing mouse embryo suggest that KCNE1L could be involved in the development of the cardiac abnormalities as well as of some neurological signs observed in patients with AMME contiguous gene syndrome.
Assuntos
Deleção de Genes , Deficiência Intelectual/genética , Nefrite Hereditária/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Canais de Potássio/genética , Cromossomo X/genética , Animais , Mapeamento Cromossômico , Células Clonais , Bases de Dados Factuais , Condutividade Elétrica , Eletrocardiografia , Expressão Gênica , Cardiopatias Congênitas/genética , Humanos , Hibridização In Situ , Masculino , Proteínas de Membrana/genética , Camundongos , Dados de Sequência Molecular , Canais de Potássio/química , Homologia de Sequência do Ácido Nucleico , SíndromeRESUMO
Chloride channels play an important role in the physiology and pathophysiology of epithelia, but their pharmacology is still poorly developed. We have chemically synthesized a series of substituted benzo[c]quinolizinium (MPB) compounds. Among them, 6-hydroxy-7-chlorobenzo[c]quinolizinium (MPB-27) and 6-hydroxy-10-chlorobenzo[c]quinolizinium (MPB-07), which we show to be potent and selective activators of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. We examined the effect of MPB compounds on the activity of CFTR channels in a variety of established epithelial and nonepithelial cell systems. Using the iodide efflux technique, we show that MPB compounds activate CFTR chloride channels in Chinese hamster ovary (CHO) cells stably expressing CFTR but not in CHO cells lacking CFTR. Single and whole cell patch clamp recordings from CHO cells confirm that CFTR is the only channel activated by the drugs. Ussing chamber experiments reveal that the apical addition of MPB to human nasal epithelial cells produces a large increase of the short circuit current. This current can be totally inhibited by glibenclamide. Whole cell experiments performed on native respiratory cells isolated from wild type and CF null mice also show that MPB compounds specifically activate CFTR channels. The activation of CFTR by MPB compounds was glibenclamide-sensitive and 4, 4'-diisothiocyanostilbene-2,2'-disulfonic acid-insensitive. In the human tracheal gland cell line MM39, MPB drugs activate CFTR channels and stimulate the secretion of the antibacterial secretory leukoproteinase inhibitor. In submandibular acinar cells, MPB compounds slightly stimulate CFTR-mediated submandibular mucin secretion without changing intracellular cAMP and ATP levels. Similarly, in CHO cells MPB compounds have no effect on the intracellular levels of cAMP and ATP or on the activity of various protein phosphatases (PP1, PP2A, PP2C, or alkaline phosphatase). Our results provide evidence that substituted benzo[c]quinolizinium compounds are a novel family of activators of CFTR and of CFTR-mediated protein secretion and therefore represent a new tool to study CFTR-mediated chloride and secretory functions in epithelial tissues.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/efeitos dos fármacos , Quinolizinas/farmacologia , Animais , Células CHO , Cílios/efeitos dos fármacos , Cílios/fisiologia , Colforsina/farmacologia , Cricetinae , Regulador de Condutância Transmembrana em Fibrose Cística/deficiência , Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Desenho de Fármacos , Feminino , Glibureto/farmacologia , Humanos , Masculino , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Estrutura Molecular , Mucosa Nasal/efeitos dos fármacos , Mucosa Nasal/fisiologia , Técnicas de Patch-Clamp , Quinolinas/síntese química , Quinolinas/química , Quinolinas/farmacologia , Quinolizinas/síntese química , Quinolizinas/química , Proteínas Recombinantes/efeitos dos fármacos , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , TransfecçãoRESUMO
OBJECTIVE: To test in vitro and in vivo the hypothesis that sodium nedocromil could modulate the expression of surface molecules on airway epithelial cells. METHODS: Human bronchial epithelial cells, obtained from surgically resected bronchi, were cultured and stimulated with recombinant IFN-gamma in the presence of sodium nedocromil. The intensity of the expression of surface molecules HLA-DR and ICAM-1 molecules on bronchial epithelial cells in vitro, was quantified by specific antibody staining and flow-cytometry analysis. Furthermore, we studied the effect of the drug on airway inflammation in vivo and on allergic rhinitis patients sensitized to house dust mites. Nasal epithelial cells were collected by brushing, at baseline and 2 to 3 weeks after treatment with sodium nedocromil. The expression of HLA-DR and ICAM-1 molecules was measured by flow-cytometry, and the proportions of neutrophils and eosinophils "contaminating" the epithelial cells evaluated by light microscopy examination of nasal brushings. RESULTS: The enhanced HLA-DR and ICAM-1 expression, induced by IFN-gamma, was effectively downregulated, in a dose-dependent manner, by sodium nedocromil. At all the concentrations tested (10(-9) to 10(-4) M), the inhibitory activity of the drug was stronger on HLA-DR than on ICAM-1 expression (P<.05, all comparisons). As compared with healthy subjects, patients with allergic rhinitis had a higher expression of HLA-DR (P<.05) but not of ICAM-1 molecules (P>.05) on nasal epithelial cells, and higher proportions of nasal eosinophils (P<.05). Treatment with sodium nedocromil downregulated the expression of HLA-DR (P<.05), but not of ICAM-1 (P>.05), and induced a mild, but not statistically significant, decrease of nasal eosinophilia (P>.05). CONCLUSION: These data demonstrate that the antiinflammatory activity of sodium nedocromil may include modulation of surface molecule expression on airway epithelial cells.
Assuntos
Brônquios/citologia , Células Epiteliais/metabolismo , Antígenos HLA-DR/biossíntese , Molécula 1 de Adesão Intercelular/biossíntese , Nedocromil/farmacologia , Adolescente , Antialérgicos/uso terapêutico , Comunicação Celular/efeitos dos fármacos , Células Cultivadas , Criança , Regulação para Baixo/efeitos dos fármacos , Feminino , Humanos , Hipersensibilidade Imediata/tratamento farmacológico , Hipersensibilidade Imediata/patologia , Interferon gama/farmacologia , Masculino , Pessoa de Meia-Idade , Mucosa Nasal/imunologia , Nedocromil/uso terapêuticoRESUMO
The HIV-1 Nef protein plays an important role in the development of the pathology associated with AIDS. Despite various studies that have dealt with different aspects of Nef function, the complete mechanism by which it alters the physiology of infected cells remains to be established. Nef can associate with cell membranes, therefore supporting the hypothesis that it might interact with membrane proteins as ionic channels and modify their electrical properties. By using the patch-clamp technique, we found that Nef expression determines a 25-mV depolarization of lymphoblastoid CEM cells. Both charybdotoxin (CTX) and the membrane-permeable Ca2+ chelator BAPTA/AM depolarized the membrane of native cells without modifying that of Nef-transfected cells. These data suggested that the resting potential in native CEM cells is settled by a CTX- and Ca2+-sensitive K+ channel (KCa,CTX), whose activity is absent in Nef-expressing cells. This was confirmed by direct measurements of whole-cell KCa,CTX currents. Single-channel recordings on excised patches showed that a KCa,CTX channel of 35 pS with a half-activation near 400 nM Ca2+ was present in both native and Nef-transfected cells. The measurements of free intracellular Ca2+ were not different in the two cell lines, but Nef-transfected cells displayed an increased Ca2+ content in ionomycin-sensitive stores. Taken together, these results indicate that Nef expression alters the resting membrane potential of the T lymphocyte cell line by inhibiting a KCa,CTX channel, possibly by intervening in the regulation of intracellular Ca2+ homeostasis.
Assuntos
Cálcio/fisiologia , Produtos do Gene nef/biossíntese , HIV-1/fisiologia , Bloqueadores dos Canais de Potássio , Linfócitos T/fisiologia , Linfócitos T/virologia , Fosfatase Alcalina/antagonistas & inibidores , Cálcio/metabolismo , Linhagem Celular Transformada , Charibdotoxina/farmacologia , Inibidores Enzimáticos/farmacologia , Produtos do Gene nef/genética , Genisteína/farmacologia , HIV-1/genética , Humanos , Concentração de Íons de Hidrogênio , Líquido Intracelular/fisiologia , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/genética , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Técnicas de Patch-Clamp , Canais de Potássio/genética , Canais de Potássio/fisiologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Linfócitos T/enzimologia , Transfecção , Células Tumorais Cultivadas , Vanadatos/farmacologia , Produtos do Gene nef do Vírus da Imunodeficiência HumanaRESUMO
The activity of volume-sensitive Cl- channels was studied in human tracheal epithelial cells (9HTEo-) by taurine efflux experiments. The efflux elicited by a hypotonic shock was partially inhibited by adenosine receptor antagonists, by alpha,beta-methyleneadenosine 5'-diphosphate (alphabetaMeADP), an inhibitor of the 5'-ectonucleotidase, and by adenosine deaminase. On the other hand, dipyridamole, a nucleoside transporter inhibitor, increased the swelling-induced taurine efflux. Extracellular ATP and adenosine increased taurine efflux by potentiating the effect of hypotonic shock. alphabetaMeADP strongly inhibited the effect of extracellular ATP but not that of adenosine. These results suggest that anion channel activation involves the release of intracellular ATP, which is then degraded to adenosine by specific ectoenzymes. Adenosine then binds to purinergic receptors, causing the activation of the channels. To directly demonstrate ATP efflux, cells were loaded with [3H]AMP, and the release of radiolabeled molecules was analyzed by high performance liquid chromatography. During hypotonic shock, cell supernatants showed the presence of ATP, ADP, and adenosine. alphabetaMeADP inhibited adenosine formation and caused the appearance of AMP. Under hypotonic conditions, elevation of intracellular Ca2+ by ionomycin caused an increase of ATP and adenosine in the extracellular solution. Our results demonstrate that volume-sensitive anion channels are regulated with an autocrine mechanism involving swelling-induced ATP release and then hydrolysis to adenosine.
Assuntos
Adenosina/farmacologia , Canais de Cloreto/efeitos dos fármacos , Traqueia/efeitos dos fármacos , Nucleotídeos de Adenina/metabolismo , Linhagem Celular Transformada , Canais de Cloreto/metabolismo , Cromatografia Líquida de Alta Pressão , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , Pressão Osmótica , Traqueia/citologia , Traqueia/metabolismo , Fosfolipases Tipo C/metabolismoRESUMO
The bovine CaCC protein is a putative Ca2+-dependent Cl- channel of airway epithelial cells. Therefore, CaCC proteins could contribute to transepithelial Cl- transport and accordingly modify the phenotype of cystic fibrosis (CF) patients. We have identified a murine EST containing a full-length cDNA coding for a 902-amino-acid protein highly homologous to bovine CaCC. The murine gene (mCaCC) maps to chromosome 3 at the H2-H3 band and is expressed, as indicated by Northern blot analysis, in mouse skin and kidney but not in brain, heart, lung or testis. RT-PCR indicates a low expression in tracheal epithelial cells. Heterologous expression of mCaCC in Xenopus oocytes elicits membrane currents that are anion-selective and inhibited by DIDS and by niflumic acid, a blocker of the endogenous chloride current in oocytes. The identification of genes belonging to the CaCC family will help to evaluate their role as ion channels or channel regulators and their actual contribution to epithelial chloride transport.
Assuntos
Cálcio/fisiologia , Canais de Cloreto/genética , Genes/genética , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/farmacologia , Sequência de Aminoácidos , Animais , Northern Blotting , Bovinos , Canais de Cloreto/administração & dosagem , Mapeamento Cromossômico , Etiquetas de Sequências Expressas , Feminino , Expressão Gênica , Hibridização in Situ Fluorescente , Ionomicina/farmacologia , Ionóforos/farmacologia , Masculino , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Microinjeções , Dados de Sequência Molecular , Ácido Niflúmico/farmacologia , Oócitos/citologia , Oócitos/efeitos dos fármacos , Oócitos/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Cloreto de Sódio/farmacologia , Distribuição Tecidual , XenopusRESUMO
Chromones (sodium cromoglycate and sodium nedocromil) block cell swelling-activated Cl- channels in NIH-3T3 fibroblasts and endothelial cells. This has led to hypothesize that cell volume regulation might be involved in asthma pathogenesis. Using whole-cell patch-clamp experiments, we studied the effect of chromones on volume-sensitive Cl- currents in transformed human tracheal epithelial cells (9HTEo-) and in primary cultures of human bronchial epithelial cells (BE). Cl- currents activated by hypotonic shock were poorly blocked by extracellular nedocromil or cromoglycate. The block was voltage-dependent since it was observed only at positive membrane potentials. At the concentration of 5 mM, the current inhibition by both chromones at +80 mV was about 40% for 9HTEo- and only 20% for BE. Intracellular application of chromones elicited a voltage-independent inhibition in 9HTEo- cells. Under this condition, volume-sensitive Cl- currents were reduced at all membrane potentials (60 and 45% inhibition by 2 mM nedocromil and cromoglycate respectively). In contrast intracellular chromones were ineffective in BE cells. The relative refractoriness to chromones, in contrast with the high sensitivity shown by other Cl- channels, suggests that the epithelial volume-sensitive Cl- channel is not involved in asthma.
Assuntos
Antiasmáticos/farmacologia , Canais de Cloreto/antagonistas & inibidores , Cloretos/fisiologia , Cromolina Sódica/farmacologia , Nedocromil/farmacologia , Traqueia/efeitos dos fármacos , Células 3T3/efeitos dos fármacos , Células 3T3/fisiologia , Animais , Células Cultivadas , Canais de Cloreto/fisiologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/fisiologia , Humanos , Cinética , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Camundongos , Sensibilidade e Especificidade , Traqueia/fisiologiaRESUMO
We performed Ussing chamber experiments on cultured human bronchial epithelial cells to look for the presence of electrogenic dibasic amino acid transport. Apical but not basolateral L-arginine (10-1, 000 microM) increased the short-circuit current. Maximal effect and EC50 were approximately 3.5 microA/cm2 and 80 microM, respectively, in cells from normal subjects and cystic fibrosis patients. The involvement of nitric oxide was ruled out because a nitric oxide synthase inhibitor (NG-nitro-L-arginine methyl ester) did not decrease the arginine-dependent current. Apical L-lysine, L-alanine, and L-proline, but not aspartic acid, were also effective in increasing the short-circuit current, with EC50 values ranging from 26 to 971 microM. Experiments performed with radiolabeled arginine demonstrated the presence of an Na+-dependent concentrative transporter on the apical membrane of bronchial cells. This transporter could be important in vivo to maintain a low amino acid concentration in the fluid covering the airway surface.
Assuntos
Aminoácidos/farmacologia , Brônquios/fisiologia , Proteínas de Transporte/metabolismo , Membrana Celular/fisiologia , Células Epiteliais/fisiologia , Alanina/farmacologia , Amilorida/farmacologia , Sistemas de Transporte de Aminoácidos , Arginina/farmacologia , Membrana Celular/efeitos dos fármacos , Células Cultivadas , Células Epiteliais/efeitos dos fármacos , Humanos , Cinética , Lisina/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Doadores de Óxido Nítrico/farmacologia , Penicilamina/análogos & derivados , Penicilamina/farmacologia , Prolina/farmacologia , S-Nitroso-N-AcetilpenicilaminaRESUMO
The question of whether a single molecule can account for every observed swelling-activated Cl- current deserves to be addressed and biophysical description seems to be an adequate criterion to classify these channels. We studied the biophysical properties of swelling-activated Cl- currents in 9HTEo-cells using whole-cell and outside-out patch clamp recordings. Hypotonic shock activated outwardly rectifying currents that inactivated at potentials higher than 20 mV. The decay phase of the current was well fitted by two exponential functions and both time constants were voltage-dependent. Two voltage-dependent time constants were also necessary to describe reactivation. The midpoint of current inactivation was 54 mV. The voltage dependence of kinetics did not significantly change by modifying the extracellular NaCl concentration while the inactivation midpoint slightly shifted. In conclusion, our results indicate that the voltage-dependent properties of the swelling-activated Cl- currents in 9HTEo- cells are largely independent from the extracellular ionic strength and the extracellular Cl- concentration. Excised patches from cells exposed to hypotonic shock showed single channel currents that inactivated at positive membrane potentials and displayed chord conductance of approximately 60 pS at 100 mV and of approximately 20 pS at -80 mV. The permeability sequence for the single channel was I- > Br- > Cl- > gluconate and currents were blocked by Reactive blue 2. These properties indicate that intermediate conductance outwardly rectifying channels are responsible for the macroscopic swelling-activated current.
Assuntos
Canais de Cloreto/fisiologia , Células Epiteliais/fisiologia , Linhagem Celular Transformada , Membrana Celular/fisiologia , Humanos , Potenciais da Membrana/fisiologia , Concentração Osmolar , Técnicas de Patch-Clamp , Vírus 40 dos Símios/genética , Cloreto de Sódio/farmacologia , TraqueiaRESUMO
Transforming growth factor beta (TGFbeta) determines a nearly complete inhibition of cystogenesis by MDCK cells grown in collagen I-enriched matrices in vitro. In order to elucidate the mechanism implicated in this phenomenon, we performed a series of experiments aimed at discovering a relevant role of extracellular matrix. TGFbeta (2 ng/ml) played a marked stimulatory effect on the expression of extracellular matrix by MDCK with a selective effect on collagen V (three to fourfold increase of protein and mRNA) and in parallel inhibited cystogenesis by 95%. Cotreatment with TGFbeta and anti-collagen V antibodies restored a normal cystogenesis. In analogy, when MDCK cells were grown in three-dimensional matrices containing collagen I and minor (10%) amounts of collagen V, cystogenesis was once again inhibited by 95%. To characterize the molecular mechanism activated by TGFbeta and collagen V, we looked at the electrophysiological characteristics of MDCK monolayers and found a drastic fall of transepithelial electrical resistance (TER) in both conditions. In parallel with the decrease in TER, TGFbeta and collagen V also induced the leakage of two high molecular weight tracers, i.e., [3H]-inulin and 150 kD FITC-Dextran, suggesting a perturbation of the paracellular permeability. Finally, TGFbeta at the relevant concentration did not stimulate apoptosis in our cellular model, as judged by propidium iodide staining and by in situ end labeling of DNA fragments. These observations suggest that TGFbeta inhibits cystogenesis by MDCK cells in vitro by altering the collagenic composition of the three-dimensional milieu where MDCK cells grow and form cysts. The molecular mechanism responsible for inhibition of cystogenesis is the increase of paracellular flux which overcomes the active transport of solutes and water inside cysts.
Assuntos
Colágeno/fisiologia , Células Epiteliais/fisiologia , Matriz Extracelular/fisiologia , Doenças Renais Císticas/etiologia , Fator de Crescimento Transformador beta/farmacologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Transporte Biológico/efeitos dos fármacos , Linhagem Celular , Membrana Celular/ultraestrutura , Cães , Sinergismo Farmacológico , Matriz Extracelular/efeitos dos fármacos , Inulina/metabolismo , Rim/citologia , Doenças Renais Císticas/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Fator de Crescimento Transformador beta/fisiologia , TrítioRESUMO
Electrophysiological studies of human bronchial epithelial cells in vitro are limited by the scarcity of biological material available for primary culture. To overcome this problem, we set up a protocol in which the cell number is first enlarged in LHC9/RPMI 1640 serum-free medium for up to six passages, each passage giving a four- to eightfold amplification. The cells are then plated at high density on permeable supports. Cell differentiation, monitored by measuring transepithelial potential difference (PD) and electrical resistance (R), is induced with a medium containing serum and a cocktail of different supplements and hormones. Maximal values of PD and R, obtained after 4-7 d of culture on permeable supports, are around -50 mV and 3000-4000 omega/cm2, respectively. Ussing chamber experiments show that basal short-circuit current (Isc) is partially inhibited by the epithelial Na+ channel blocker amiloride. Stimulation with a cAMP-elevating agent induces a Isc increase that is inhibited by the cystic fibrosis transmembrane conductance regulator (CFTR) blocker glibenclamide. Our culture protocol provides a large number of differentiated bronchial epithelial cell monolayers starting from a low amount of material. This characteristic is useful for in vitro studies of ion transport in airway epithelium.
Assuntos
Técnicas de Cultura de Células/métodos , Células Epiteliais/citologia , Brônquios/citologia , Diferenciação Celular , Meios de Cultura , Meios de Cultura Livres de Soro , Células Epiteliais/fisiologia , HumanosRESUMO
Expression cloning revealed a chloride channel (ICln) that we found to be fundamental for the regulatory volume decrease in a variety of cells. The chromosomal localization of the human ICln-gene showed two loci, one at chromosome 11 in position q13.5-q14.1, termed CLNS1A, and a second one at chromosome 6 at position p12.1-q13, termed CLNS1B. In this study, we offer a detailed characterization of the CLNS1A gene and provide the exact position (6p12) and sequence data of CLNS1B, an intronless gene 91.3% homologous to the coding region of CLNS1A.