RESUMO
BACKGROUND: Previous observations demonstrate that Cftr-null cells and tissues exhibit alterations in cholesterol processing including perinuclear cholesterol accumulation, increased de novo synthesis, and an increase in plasma membrane cholesterol accessibility compared to wild type controls. The hypothesis of this study is that membrane cholesterol accessibility correlates with CFTR genotype and is in part influenced by de novo cholesterol synthesis. METHODS: Electrochemical detection of cholesterol at the plasma membrane is achieved with capillary microelectrodes with a modified platinum coil that accepts covalent attachment of cholesterol oxidase. Modified electrodes absent cholesterol oxidase serves as a baseline control. Cholesterol synthesis is determined by deuterium incorporation into lipids over time. Incorporation into cholesterol specifically is determined by mass spectrometry analysis. All mice used in the study are on a C57Bl/6 background and are between 6 and 8 weeks of age. RESULTS: Membrane cholesterol measurements are elevated in both R117H and DeltaF508 mouse nasal epithelium compared to age-matched sibling wt controls demonstrating a genotype correlation to membrane cholesterol detection. Expression of wt CFTR in CF epithelial cells reverts membrane cholesterol to WT levels further demonstrating the impact of CFTR on these processes. In wt epithelial cell, the addition of the CFTR inhibitors, Gly H101 or CFTRinh-172, for 24 h surprisingly results in an initial drop in membrane cholesterol measurement followed by a rebound at 72 h suggesting a feedback mechanism may be driving the increase in membrane cholesterol. De novo cholesterol synthesis contributes to membrane cholesterol accessibility. CONCLUSIONS: The data in this study suggest that CFTR influences cholesterol trafficking to the plasma membrane, which when depleted, leads to an increase in de novo cholesterol synthesis to restore membrane content.
Assuntos
Membrana Celular/metabolismo , Colesterol/biossíntese , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/metabolismo , Células Epiteliais/metabolismo , Animais , Benzoatos/farmacologia , Sítios de Ligação , Linhagem Celular , Colesterol Oxidase/metabolismo , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/antagonistas & inibidores , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Modelos Animais de Doenças , Técnicas Eletroquímicas/instrumentação , Células Epiteliais/efeitos dos fármacos , Genótipo , Humanos , Hidroximetilglutaril-CoA Sintase/genética , Hidroximetilglutaril-CoA Sintase/metabolismo , Cinética , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos CFTR , Microeletrodos , Mutação , Mucosa Nasal/metabolismo , Fenótipo , Regiões Promotoras Genéticas , Mucosa Respiratória/metabolismo , Resveratrol , Estilbenos/farmacologia , Tiazolidinas/farmacologia , TransfecçãoRESUMO
Cyst expansion in polycystic kidney disease (PKD) involves progressive fluid accumulation, which is believed to require chloride transport by the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Herein is reported that small-molecule CFTR inhibitors of the thiazolidinone and glycine hydrazide classes slow cyst expansion in in vitro and in vivo models of PKD. More than 30 CFTR inhibitor analogs were screened in an MDCK cell model, and near-complete suppression of cyst growth was found by tetrazolo-CFTR(inh)-172, a tetrazolo-derived thiazolidinone, and Ph-GlyH-101, a phenyl-derived glycine hydrazide, without an effect on cell proliferation. These compounds also inhibited cyst number and growth by >80% in an embryonic kidney cyst model involving 4-d organ culture of embryonic day 13.5 mouse kidneys in 8-Br-cAMP-containing medium. Subcutaneous delivery of tetrazolo-CFTR(inh)-172 and Ph-GlyH-101 to neonatal, kidney-specific PKD1 knockout mice produced stable, therapeutic inhibitor concentrations of >3 microM in urine and kidney tissue. Treatment of mice for up to 7 d remarkably slowed kidney enlargement and cyst expansion and preserved renal function. These results implicate CFTR in renal cyst growth and suggest that CFTR inhibitors may hold therapeutic potential to reduce cyst growth in PKD.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/antagonistas & inibidores , Glicina/análogos & derivados , Rim Policístico Autossômico Dominante/tratamento farmacológico , Tiazolidinas/uso terapêutico , Animais , Caderinas/genética , Linhagem Celular , Cães , Glicina/farmacologia , Glicina/uso terapêutico , Integrases/genética , Integrases/metabolismo , Rim/patologia , Camundongos , Camundongos Endogâmicos C57BL , Rim Policístico Autossômico Dominante/patologia , Canais de Cátion TRPP , Tiazolidinas/farmacologiaRESUMO
Secretory diarrhea is the leading cause of infant death in developing countries and a major cause of morbidity in adults. The cystic fibrosis transmembrane conductance regulator (CFTR) protein is required for fluid secretion in the intestine and airways and, when defective, causes the lethal genetic disease cystic fibrosis. We screened 50,000 chemically diverse compounds for inhibition of cAMP/flavone-stimulated Cl(-) transport in epithelial cells expressing CFTR. Six CFTR inhibitors of the 2-thioxo-4-thiazolidinone chemical class were identified. The most potent compound discovered by screening of structural analogs, CFTR(inh)-172, reversibly inhibited CFTR short-circuit current in less than 2 minutes in a voltage-independent manner with K(I) approximately 300 nM. CFTR(inh)-172 was nontoxic at high concentrations in cell culture and mouse models. At concentrations fully inhibiting CFTR, CFTR(inh)-172 did not prevent elevation of cellular cAMP or inhibit non-CFTR Cl(-) channels, multidrug resistance protein-1 (MDR-1), ATP-sensitive K(+) channels, or a series of other transporters. A single intraperitoneal injection of CFTR(inh)-172 (250 micro g/kg) in mice reduced by more than 90% cholera toxin-induced fluid secretion in the small intestine over 6 hours. Thiazolidinone CFTR inhibitors may be useful in developing large-animal models of cystic fibrosis and in reducing intestinal fluid loss in cholera and other secretory diarrheas.