The short-circuit current (I(SC)) technique was used to examine the effects of cAMP-evoking agents, forskolin/IBMX, and a Chinese medicinal formula, Huoxiang-zhengqi liquid (HZL) on HCO(3)(-) secretion by intact porcine distal airway epithelium. The freshly isolated airway epithelial tissue displayed a transepithelial basal current of (94.9±8.2) μA/cm(2), 16.6% and 62.7% of which was inhibited by amiloride (epithelial Na(+) channel blocker, 100 μmol/L) and NPPB (cystic fibrosis transmembrane conductance regulator Cl(-) channel blocker, 100 μmol/L). Substitution of Cl(-) with impermeable gluconate(-) in the K-H bath solution resulted in a basal current of (54.0±6.7) μA/cm(2), which could be abolished by further removal of HCO(3)(-) in the solution, indicating HCO(3)(-) secretion under unstimulated conditions. Application of forskolin/IBMX (10 μmol/L/100 μmol/L) stimulated an increase of (13.8±1.9) μA/cm(2) in I(SC) which could be blocked by Cl(-) channel inhibitor DPC. With Cl(-) and Cl(-)/HCO(3)(-) substitution, forskolin/IBMX evoked an increase of (7.3±0.5) μA/cm(2) in HCO(3)(-)-dependent, DPC-inhibitable I(SC) (I(HCO(3))). Noticeably, basolateral application of HZL (10 μL/mL) in normal K-H solution evoked an I(SC) of (15.9±2.4) μA/cm(2). The EC(50) of this I(SC) was (6.1±1.4) μL/mL. When substituting Cl(-), HZL stimulated an increase of (7.4±1.9) μA/cm(2) in I(HCO(3)), suggesting HZL-induced HCO(3)(-) secretion. After pretreating the epithelial tissues with forskolin/IBMX in Cl(-)-free K-H solution, HZL induced a further increase of (8.4±0.9) μA/cm(2) in I(HCO(3)), and pretreating tissues with HZL did not significantly affect the subsequent forskolin/IBMX-induced I(HCO(3)) response, indicating that HZL- and forskolin/IBMX-induced I(HCO(3)) responses appeared to be independent and be most likely mediated via different cellular mechanisms. Our results suggest that HCO(3)(-) can be secreted by porcine distal airway epithelium under unstimulated and stimulated conditions, and the stimulatory effect of HZL on HCO(3)(-) secretion in the distal airway epithelium shows HZL to be a hopeful new agonist for distal airway HCO(3)(-) secretion that could be of therapeutic significance.
Amiloride ; pharmacology ; Animals ; Bicarbonates ; metabolism ; Biological Transport ; Colforsin ; pharmacology ; Cystic Fibrosis Transmembrane Conductance Regulator ; antagonists & inhibitors ; Drugs, Chinese Herbal ; pharmacology ; Epithelium ; drug effects ; metabolism ; Respiratory System ; drug effects ; metabolism ; Swine

OBJECTIVETo study the impact of the chloride channel dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) on the cytoskeleton of Sertoli cells in the mouse.

METHODSTM4 Sertoli cells were cultured and treated with CFTR(inh)-172 at the concentrations of 1, 5, 10 and 20 μmol/L for 48 hours. Then the cytotoxicity of CFT(inh)-172 was assessed by CCK-8 assay, the expressions of F-actin and Ac-tub in the TM4 Sertoli cells detected by immunofluorescence assay, and those of N-cadherin, vimentin and vinculin determined by qPCR.

RESULTSCFTR(inh)-172 produced cytotoxicity to the TM4 Sertoli cells at the concentration of 20 μmol/L. The expressions of F-actin and Ac-tub were decreased gradually in the TM4 Sertoli cells with the prolonging of treatment time and increasing concentration of CFTR(inh)-172 (P < 0.05). The results of qPCR showed that different concentrations of CFTR(inh)-172 worked no significant influence on the mRNA expressions of N-cadherin, vimentin and vinculin in the Sertoli cells.

CONCLUSIONThe CFTR chloride channel plays an important role in maintaining the normal cytoskeleton of Sertoli cells. The reduced function and expression of the CFTR chloride channel may affect the function of Sertoli cells and consequently spermatogenesis of the testis.

Actins ; metabolism ; Animals ; Benzoates ; pharmacology ; Chloride Channels ; physiology ; Cystic Fibrosis Transmembrane Conductance Regulator ; antagonists & inhibitors ; Cytoskeleton ; drug effects ; Male ; Mice ; Sertoli Cells ; drug effects ; metabolism ; Spermatogenesis ; Thiazolidines ; pharmacology ; Time Factors

Hyperoxic ventilation induces detrimental effects on the respiratory system, and ambient oxygen may be harmful unless compensated by physiological anti-oxidants, such as vitamin C. Here we investigate the changes in electrolyte transport of airway epithelium in mice exposed to normobaric hyperoxia and in gulonolacton oxidase knock-out (gulo[-/-]) mice without vitamin C (Vit-C) supplementation. Short-circuit current (Isc) of tracheal epithelium was measured using Ussing chamber technique. After confirming amiloride-sensitive Na+ absorption (DeltaIsc,amil), cAMP-dependent Cl- secretion (DeltaIsc,forsk) was induced by forskolin. To evaluate Ca2+-dependent Cl- secretion, ATP was applied to the luminal side (DeltaIsc,ATP). In mice exposed to 98% PO2 for 36 hr, DeltaIsc,forsk decreased, DeltaIsc,amil and DeltaIsc,ATP was not affected. In gulo(-/-) mice, both DeltaIsc,forsk and DeltaIsc,ATP decreased from three weeks after Vit-C deprivation, while both were unchanged with Vit-C supplementation. At the fourth week, tissue resistance and all electrolyte transport activities were decreased. An immunofluorescence study showed that the expression of cystic fibrosis conductance regulator (CFTR) was decreased in gulo(-/-) mice, whereas the expression of KCNQ1 K+ channel was preserved. Taken together, the CFTR-mediated Cl- secretion of airway epithelium is susceptible to oxidative stress, which suggests that supplementation of the antioxidant might be beneficial for the maintenance of airway surface liquid.
Animals ; Ascorbic Acid Deficiency/*metabolism ; Biological Transport/drug effects ; Chlorides/*metabolism ; Cystic Fibrosis Transmembrane Conductance Regulator/antagonists & inhibitors/drug ; Forskolin/pharmacology ; Hyperbaric Oxygenation ; Hyperoxia/*physiopathology ; Ion Transport/drug effects ; Mice ; Mice, Inbred C57BL ; Mice, Inbred ICR ; Mice, Knockout/metabolism ; Mice, Transgenic ; Microscopy, Fluorescence ; Oxidative Stress ; Oxygen/adverse effects/pharmacology ; Potassium Channels/metabolism ; Respiratory Mucosa/drug effects/*metabolism/secretion ; Sodium ; Sugar Acids/metabolism

Aim of the present study is to investigate activation effect of nobiletin on cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel activity. CFTR-mediated iodide influx assay and patch-clamp tests were done on FRT cells stably co-transfected with human CFTR and EYFP/H148Q. Nobiletin potently activated CFTR chloride channel activity in a dose- and time-dependent manner. The CFTR blocker CFTR(inh)-172 could completely reverse the effect. Preliminary mechanism study indicated that nobiletin activated CFTR chloride channel through a direct binding way. In addition, ex vivo tests done on mice trachea showed that nobiletin time-dependently stimulated submucosal gland fluid secretion. Nobiletin may be a therapeutic lead compound in treating CFTR-related diseases including disseminated bronchiectasis.
Animals ; Benzoates ; pharmacology ; Cystic Fibrosis Transmembrane Conductance Regulator ; antagonists & inhibitors ; drug effects ; metabolism ; Dose-Response Relationship, Drug ; Epithelial Cells ; metabolism ; Exocrine Glands ; secretion ; Flavones ; administration & dosage ; pharmacology ; Humans ; Mice ; Patch-Clamp Techniques ; Rats ; Rats, Inbred F344 ; Thiazolidines ; pharmacology ; Thyroid Gland ; cytology ; Time Factors ; Trachea ; secretion