Influences of influenza virus on cystic fibrosis transmembrane conductance regulator ( CFTR) / 中华微生物学和免疫学杂志

Objective To evaluate the differentiation of mouse tracheal epithelial cells ( MTEC) at an air-liquid interface and to investigate the influences of influenza virus on the cystic fibrosis transmem-brane conductance regulator ( CFTR) in primary cultured MTEC for further elucidating the possible mecha-nism of imbalanced fluid and salt transportation in respiratory system caused by influenza virus infection . Methods The morphology of primarily cultured MTEC was observed under inverted microscope .Trans epi-thelial electrical resistance ( TEER) was measured by a resistance meter to evaluate the integrity of cultured MTEC.An Ussing chamber apparatus was used to record the short-circuit current of primary cultured MTEC . Results The primarily cultured MTEC clustered together and had a tight pavement-like appearance under light microscope .The TEER was greater than 1000 Ωafter 6 days of culture .Influenza virus could reduce the short-circuit current of CFTR to (52.77±10.30)％in intact cell membrane and to (41.50±1.09)％in monolayer MTEC after increasing the permeability of basement membrane .It had been proved that CFTR was essential to maintaining the balance of fluid and salt transportation in respiratory system.Conclusion Mouse MTEC are efficiently cultured at a air-liquid interface and the primarily cultured cells are highly simi-lar to those in a normal physiologic state .Influenza virus may block the secretion of anions through inhibiting the function of CFTR , which may induce the development of chronic obstructive pulmonary disease and the incidence of asthma .

Study on the relationship between CFTR physiological secretion pathway and intracellular calcium signaling in rabbit cornea epithelium / 中华实验眼科杂志

Background Researches showed that cystic fibrosis transmembrane conductance regulator protein(CFTR) is a channel secreting anion and water,and it plays an important role in tear secreting.Traditional conception thought that CFTR pathway is cAMP-PKA-dependent without the participation of intracellular calcium.However,studies disclosed that elevating intracellular cAMP could not open the CFTR channel.So whether calcium signal is associated with CFTR-related corneal epithelial secretion is controversial.Objective This study was to investigate the association between CFTR secretion and intracellular calcium signaling in rabbit corneal epithelium.Methods Sixteen New Zealand white rabbits were randomized into odd number group and even number group by computer randomized number method.The corneas were obtained under the general anesthesia and placed in Ussing Chamber for the record of short cricuit current (Isc).The right eyes of rabbits in the odd number group were stimulated with ATP and served as ATP stimulating group.The left eyes were pretreated with CFTRinh-172 prior to ATP stimulation and served as CFTRinh-172 pretreated group.In the even number group,the left eyes of rabbits were pretreated with BMPTA/AM before ATP stimulation and served as BMPTA/AM pretreated group,and the right eyes of the rabbits were used to isolate and culture corneal epithelial cells by explant adherent method,the level of intracellular calcium were evaluated using Leica SP5 laser scan confocal microscope.Results The ATP-induced ΔIsc of corneal epithelium was (5.73 ± 1.36),(1.30 ± 0.95) and (2.47 ± 0.55) μA/cm2 in the ATP stimulating group,CFTRinh.172 pretreated group and BMPTA/AM pretreated group,respectively,and the AIsc was significantly reduced in the CFTRinh.172 pretreated group and BMPTA/AM pretreated group compared with ATP stimulating group (t=11.201,5.508,both at P ＜ 0.001).The fluorescence intensity of intracellular calcium release after ATP stimulation was 3.25 folds more than that before ATP stimulation.Conclusions ATP promotes rapid short circuit current of corneal epithelium.CFTRinh-172 depresses the ATP-induced corneal epithelium AIsc,and BMPTA/AM suppresses intracellular calcium release.It is suggested that intracellular calcium signaling secretion probably participates in the functional CFTR activity in rabbit corneal epithelium.

Evaluation of endoscopically obtained duodenal biopsy samples from cats and dogs in an adapter-modified Ussing chamber

This study was conducted to evaluate an adapter-modified Ussing chamber for assessment of transport physiology in endoscopically obtained duodenal biopsies from healthy cats and dogs, as well as dogs with chronic enteropathies. 17 duodenal biopsies from five cats and 51 duodenal biopsies from 13 dogs were obtained. Samples were transferred into an adapter-modified Ussing chamber and sequentially exposed to various absorbagogues and secretagogues. Overall, 78.6% of duodenal samples obtained from cats responded to at least one compound. In duodenal biopsies obtained from dogs, the rate of overall response ranged from 87.5% (healthy individuals; n = 8), to 63.6% (animals exhibiting clinical signs of gastrointestinal disease and histopathological unremarkable duodenum; n = 15), and 32.1% (animals exhibiting clinical signs of gastrointestinal diseases and moderate to severe histopathological lesions; n = 28). Detailed information regarding the magnitude and duration of the response are provided. The adapter-modified Ussing chamber enables investigation of the absorptive and secretory capacity of endoscopically obtained duodenal biopsies from cats and dogs and has the potential to become a valuable research tool. The response of samples was correlated with histopathological findings.

Helicobacter pylori Vacuolating Toxin Exhibits Polar Activity of Cl Secretion and Secretory Response to Carbachol in T84 Cells

To investigate whether VacA (vacuolating toxin) produced by Helicobacter pylori Korean stain 99 induces intestinal secretion, purified VacA was added to T84 cell monolayers mounted in Ussing chambers, and electrical parameters were monitored. Mucosal addition of low pH-pretreated VacA increased short circuit current (Isc). The effect was time- and dose-dependent and saturable. The time-to-peak Isc was concentration-dependent. Chloride channel inhibitors, niflumic acid or 5- nitro-2- (3-phenylpropylamino) -benzoate (NPPB), inhibited VacA-stimulated Isc. Carbachol (CCh) -induced increase of Isc was prolonged by the addition of VacA to the mucosal side only. The effect was unaltered by the addition of niflumic acid. VacA did not show cytopathic effects. These studies indicate that VacA is a nonlethal toxin that acts in a polar manner on T84 monolayers to potentiate Cl secretion and the response to CCh secretion without decrease in monolayer resistance. VacA may contribute to diarrhea diseases in human intestinal epithelial cells.

Asymmetrical distribution of P2Y nucleotide receptors in rabbit inner medullary collecting duct cells

We cultured the rabbit inner medullary collecting duct (IMCD) cells as monolayers on collagen-coated membrane filters, and investigated distribution of the P2Y receptors by analyzing nucleotide-induced short circuit current (Isc) responses. Exposure to different nucleotides of either the apical or basolateral surface of cell monolayers stimulated Isc. Dose-response relationship and cross-desensitization studies suggested that at least 3 distinct P2Y receptors are expressed asymmetrically on the apical and basolateral membranes. A P2Y2-like receptor, which responds to UTP and ATP, is expressed on both the apical and basolateral membranes. In addition, a uracil nucleotide receptor, which responds to UDP and UTP, but not ATP, is expressed predominantly on the apical membrane. In contrast, a P2Y1-like receptor, which responds to ADP and 2-methylthio-ATP, is expressed predominantly on the basolateral membrane. These nucleotides stimulated intracellular cAMP production with an asymmetrical profile, which was comparable to that in the stimulation of Isc. Our results suggest that the adenine and uracil nucleotides can interact with different P2Y nucleotide receptors that are expressed asymmetrically on the apical and basolateral membranes of the rabbit IMCD cells, and that both cAMP- and Ca2+-dependent signaling mechanisms underlie the stimulation of Isc.

Effects of Hydrogen Peroxide on Rabbit Corneal Bioelectric Properties

The rabbit cornea was studied in vitro in modified Ussing chambers to determine the effects of ion transport inhibitors and hydrogen peroxide(H2O2) on ion transport through the cornea by measuring the bioelectric properties. Apical(tear side, T side) addition of furosemide, bumetanide and SITS were ineffective on resting Isc(short circuit current). Apical addition of 1.0mM amiloride(Na+/H+ antiport inhibitor) and NPAA(Cl- channel blocker) markedly reduced the resting Isc, but basolateral(stromal side, S side) addition of amiloride was ineffective. The site of action of these agents was the apical membrane. H2O2, an oxygen free radical, markedly increased the lsc when was added to the T side, but S side addition of the H2O2 was ineffective. To determine the degree of cellular catalase participation in protection against H2O2 induced injury the cornea was pretreated with ATAZ for 30 min prior to H2O2 action. The increase of lsc by H2O2 was markedly potentiated after pretreatment with ATAZ on T side compared to that of S side addition. This result indicates that the corneal endothelial H2O2 may be largely degraded by catalase. When H2O2 was added to the T side, Isc was raised by increased ion transport. All ion transport inhibitors that were used inhibited the H2O2 effect on Isc. Moreover, amiloride and NPAA markedly inhibited induced lsc by H2O2. These results suggest that H2O2 stimulates the corneal epithelial ion transport and that its site of action is apical membrane Na+/H+ antiport system and CI- channel system.