RESUMO
Epithelia of humans and mice with cystic fibrosis are unable to secrete chloride in response to a chloride gradient or to cAMP-elevating agents. Bioelectrical properties measured using the nasal transepithelial potential difference (TEPD) assay are believed to reflect these cystic fibrosis transmembrane conductance regulator (CFTR)-dependent chloride transport defects. Although the response to forskolin is CFTR mediated, the mechanisms responsible for the response to a chloride gradient are unknown. TEPD measurements performed on inbred mice were used to compare the responses to low chloride and forskolin in vivo. Both responses show little correlation between or within inbred strains of mice, suggesting they are mediated through partially distinct mechanisms. In addition, these responses were assayed in the presence of several chloride channel inhibitors, including DIDS, diphenylamine-2-carboxylate, glibenclamide, and 5-nitro-2-(3-phenylpropylamino)-benzoic acid, and a protein kinase A inhibitor, the Rp diastereomer of adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS). The responses to low chloride and forskolin demonstrate significantly different pharmacological profiles to both DIDS and Rp-cAMPS, indicating that channels in addition to CFTR contribute to the low chloride response.
Assuntos
Cloretos/metabolismo , AMP Cíclico/análogos & derivados , Fibrose Cística/metabolismo , Potenciais da Membrana , Mucosa Nasal/fisiologia , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/farmacologia , Amilorida/farmacologia , Animais , Canais de Cloreto/antagonistas & inibidores , Colforsina/farmacologia , AMP Cíclico/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Transporte de Íons , Camundongos , Camundongos Endogâmicos , Mucosa Nasal/efeitos dos fármacos , Tionucleotídeos/farmacologiaRESUMO
Recent evidence has shown that the inducible form of nitric oxide (NO) synthase (NOS2) has reduced expression in airway epithelia of patients with cystic fibrosis (CF) despite the presence of chronic inflammation. The goal of this paper is to determine whether NOS2 expression is regulated by the presence of functional CF transmembrane conductance regulator (CFTR). Using a human trachea epithelial cell line in which CFTR activity is blocked by the overexpression of the CFTR regulatory domain, we found that loss of CFTR activity reduces NOS2 messenger RNA expression as determined by reverse transcriptase/polymerase chain reaction and reduces overall NO production compared with mock-transfected controls. An in vivo model using mice lacking CFTR expression (cftr -/-), wild-type mice (cftr +/+), and cftr -/- mice that have had human CFTR introduced to the intestinal epithelium using the fatty acid binding protein (FABP) promoter (FABP-hcftr) was also examined. Electrical characterization confirmed that FABP-hcftr mice had corrected electrophysiologic properties compared with cftr -/- mice in the ileum, but FABP-hcftr nasal transepithelial potential difference measurements were identical to cftr -/- values showing specific intestinal correction. NOS2-specific immunostaining revealed that NOS2 expression is evident in sections of ileum and nasal epithelium of cftr +/+ mice but is absent in both tissues in cftr -/- mice. FABP-hcftr mice, however, show strong NOS2 staining in epithelial cells of the ileum but reduced staining in the nasal epithelium, suggesting a CFTR-related influence in the regulation of NOS2 expression in epithelial cells.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Células Epiteliais/enzimologia , Proteínas de Neoplasias , Proteínas do Tecido Nervoso , Óxido Nítrico Sintase/biossíntese , Proteínas Supressoras de Tumor , Animais , Proteínas de Transporte/genética , Linhagem Celular , Regulador de Condutância Transmembrana em Fibrose Cística/deficiência , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Proteína 7 de Ligação a Ácidos Graxos , Proteínas de Ligação a Ácido Graxo , Humanos , Íleo/enzimologia , Imuno-Histoquímica , Potenciais da Membrana/genética , Camundongos , Camundongos Knockout , Proteína P2 de Mielina/genética , Mucosa Nasal/fisiologia , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , Regiões Promotoras Genéticas , Estrutura Terciária de Proteína/genética , Coloração e RotulagemRESUMO
Transepithelial ion transport is regulated by a variety of cellular factors. In light of recent evidence that nitric oxide (NO) production is decreased in cystic fibrosis airways, we examined the role of NO in regulating sodium and chloride transport in murine nasal epithelium. Acute intervention with the inducible NO synthase (iNOS)-selective inhibitor S-methylisothiourea resulted in an increase of amiloride-sensitive sodium absorption observed as a hyperpolarization of nasal transepithelial potential difference. Inhibition of iNOS expression with dexamethasone also hyperpolarized transepithelial potential difference, but only a portion of this increase proved to be amiloride sensitive. Chloride secretion was significantly inhibited in C57BL/6J mice by the addition of both S-methylisothiourea and dexamethasone. Mice lacking iNOS expression [NOS2(-/-)] also had a decreased chloride-secretory response compared with control mice. These data suggest that constitutive NO production likely plays some role in the downregulation of sodium absorption and leads to an increase in transepithelial chloride secretion.