The present study examined the effects and mechanisms of actions of penta-m-digalloyl-glucose (PDG), a hydrolysable tannin extracted from Chinese gallnut, on cystic fibrosis transmembrane conductance regulator protein (CFTR).
Fisher rat thyroid cells stably expressing human CFTR (FRT cells) and human intestinal T84 cells were used as cell models to investigate the effects of PDG on chloride secretion using short-circuit current analysis. The mechanisms by which PDG affected chloride secretion were also examined. Finally, in vivo antidiarrheal efficacy and effects of PDG on intestinal fluid absorption were evaluated in mouse closed-loop models.
In FRT cells, apical chloride current induced by forskolin, CPT-cAMP and apigenin were reversibly inhibited by PDG (IC50 approximately 10microM) without effects on intracellular cAMP content and cell viability. Similarly, in T84 cells, PDG effectively inhibited chloride secretion induced by forskolin and cholera toxin. However, it had no effect on calcium-induced chloride secretion. In mice, a single intraluminal injection of PDG (0.6 mg/kg) reduced cholera toxin-induced intestinal fluid secretion by 75% with no effect on intestinal fluid absorption.
PDG represents a new class of CFTR inhibitors. Further development of this class of compounds may provide a new therapeutic intervention for diarrhea.
"Immunofluorescence analyses were carried out following the protocol of previous study [9, 40]. Tissue sections (5 μm) of colon and HT-29 cells were treated with 0.2% Triton in PBS prior to blocking with 5% normal goat serum (NGS) in TPBS-BSA (PBS containing 0.05% Tween-20 and 0.1% bovine serum albumin). "
[Show abstract][Hide abstract] ABSTRACT: Tannins, a group of major active components of Chinese rhubarb and widely distributed in nature, have a significant antidiarrhoeal activity. Aquaporins (AQPs) 2 and 3 play important roles in regulating water transfer during diarrhoea. The present study aims to determine the effect of the total tannins extract of rhubarb on aquaporins (AQPs) 2 and 3 in diarrhoea mice and HT-29 cells both induced by magnesium sulphate (MgSO4). Our results showed that rhubarb tannins extract (RTE) significantly decreased the faecal water content in colon and evaluation index of defecation of diarrhoea mice. Interestingly, RTE could markedly reduce the mRNA and protein expression levels of AQPs 2 and 3 in apical and lateral mucosal epithelial cells in the colons of diarrhoea mice and HT-29 cells both induced by MgSO4 in a dose-dependent manner. Furthermore, RTE suppressed the production of cyclic monophosphate- (cAMP-) dependent protein kinase A catalytic subunits α (PKA C-α) and phosphorylated cAMP response element-binding protein (p-CREB, Ser133) in MgSO4-induced HT-29 cells. Our data showed for the first time that RTE inhibit AQPs 2 and 3 expression in vivo and in vitro via downregulating PKA/p-CREB signal pathway, which accounts for the antidiarrhoeal effect of RTE.
BioMed Research International 08/2014; 2014:619465. DOI:10.1155/2014/619465 · 3.17 Impact Factor
"FRT cell viability after exposure to DQA was determined using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. FRT cells were seeded in 96-well plate at a density of 2 × 104 cells/well, grown for 24 h in a humidified incubator under 5% CO2/95% O2 at 37°C, followed by incubation for a further 24 h in serum-free culture medium containing vehicle or various concentrations of DQA. "
[Show abstract][Hide abstract] ABSTRACT: This study aimed to evaluate the antidiarrheal efficacy and pharmacological properties of ethyl 2-(4-oxo-3-o-tolyl-3,4-dihydroquinazolin-2-ylthio)acetate (DQA) as an inhibitor of cystic fibrosis transmembrane conductance regulator protein (CFTR) both in vitro and in vivo.
The effects of DQA on CFTR function and cell viability were investigated in Fisher rat thyroid (FRT) cells expressing human CFTR and human intestinal epithelial T84 cells by short-circuit current measurements and MTT assays, respectively. In vivo antidiarrheal efficacy of DQA was evaluated in a closed loop model of cholera in mice.
In permeabilized FRT cells, apical chloride current induced by CFTR agonists (10 μM forskolin, 100 μM CPT-cAMP, and 20 μM apigenin) was inhibited by DQA with IC(50) ~ 20 μM and complete inhibition at 200 μM. The inhibitory effect was reversible and not associated with cytotoxicity to FRT cells (5-500 μM DQA for 24 h). Likewise, DQA effectively inhibited both forskolin and cholera toxin-induced transepithelial chloride secretion in T84 cells. In mice, intraluminal injection of 100 μM DQA reduced cholera toxin (1 μg/closed loop)-induced intestinal fluid secretion by 85% without affecting intestinal fluid absorption.
DQA represents a new class of small molecule CFTR inhibitor with potential application in treatment of cholera.
Indian Journal of Pharmacology 09/2012; 44(5):619-23. DOI:10.4103/0253-7613.100392 · 0.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Many aspects of design automation software have similar requirements for representing, manipulating, and storing design information. The recognition of these common requirements in CAD tools, allows the Flexible Architecture Compilation Environment's (FACE) Core Environment to provide a suite of high level tools for the CAD developer. The Core Environment software has been developed using object-oriented software technology, and may be readily adapted to specific applications. The focus of the core environment is to improve the productivity of CAD tool developers through better tool integration and a state-of-the-art software development environment. This Core Environment software has been used in the development of an integrated tool set covering algorithm specification, structural synthesis, and physical assembly of digital hardware systems. The focus of this paper is on the Core Environment's organization and its use in application tool development.
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