Cellular mechanisms underlying the inhibitory effect of flufenamic acid on chloride secretion in human intestinal epithelial cells

Intestinal Cl− secretion is involved in the pathogenesis of secretory diarrheas including cholera. We recently demonstrated that flufenamic acid (FFA) suppressed Vibrio cholerae El Tor variant-induced intestinal fluid secretion via mechanisms involving AMPK activation and NF-κB-suppression. The present study aimed to investigate the effect of FFA on transepithelial Cl− secretion in human intestinal epithelial (T84) cells. FFA inhibited cAMP-dependent Cl− secretion in T84 cell monolayers with IC50 of ∼8 μM. Other fenamate drugs including tolfenamic acid, meclofenamic acid and mefenamic acid exhibited the same effect albeit with lower potency. FFA also inhibited activities of CFTR, a cAMP-activated apical Cl− channel, and KCNQ1/KCNE3, a cAMP-activated basolateral K+ channel. Mechanisms of CFTR inhibition by FFA did not involve activation of its negative regulators. Interestingly, FFA inhibited Ca2+-dependent Cl− secretion with IC50 of ∼10 μM. FFA inhibited activities of Ca2+-activated Cl− channels and KCa3.1, a Ca2+-activated basolateral K+ channels, but had no effect on activities of Na+–K+–Cl− cotransporters and Na+–K+ ATPases. These results indicate that FFA inhibits both cAMP and Ca2+-dependent Cl− secretion by suppressing activities of both apical Cl− channels and basolateral K+ channels. FFA and other fenamate drugs may be useful in the treatment of secretory diarrheas

Natural statin derivatives as potential therapy to reduce intestinal fluid loss in cholera.

As a leading cause of death in children under 5 years old, secretory diarrheas including cholera are characterized by excessive intestinal fluid secretion driven by enterotoxin-induced cAMP-dependent intestinal chloride transport. This study aimed to identify fungal bioactive metabolites possessing anti-secretory effects against cAMP-dependent chloride secretion in intestinal epithelial cells. Using electrophysiological analyses in human intestinal epithelial (T84) cells, five fungus-derived statin derivatives including α,β-dehydrolovastatin (DHLV), α,β-dehydrodihydromonacolin K, lovastatin, mevastatin and simvastatin were found to inhibit the cAMP-dependent chloride secretion with IC50 values of 1.8, 8.9, 11.9, 11.4 and 5 μM, respectively. Being the most potent statin derivatives, DHLV was evaluated for its pharmacological properties including cellular toxicity, mechanism of action, target specificity and in vivo efficacy. DHLV at concentrations up to 20 μM did not affect cell viability and barrier integrity of T84 cells. Electrophysiological analyses indicated that DHLV inhibited cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-dependent apical chloride channel, via mechanisms not involving alteration of intracellular cAMP levels or its negative regulators including AMP-activated protein kinases and protein phosphatases. DHLV had no effect on Na+-K+ ATPase activities but inhibited Ca2+-dependent chloride secretion without affecting intracellular Ca2+ levels. Importantly, intraperitoneal (2 mg/kg) and intraluminal (20 μM) injections of DHLV reduced cholera toxin-induced intestinal fluid secretion in mice by 59% and 65%, respectively without affecting baseline intestinal fluid transport. This study identifies natural statin derivatives as novel natural product-derived CFTR inhibitors, which may be beneficial in the treatment of enterotoxin-induced secretory diarrheas including cholera

Galactomannan Pentasaccharide Produced from Copra Meal Enhances Tight Junction Integration of Epithelial Tissue through Activation of AMPK

Mannan oligosaccharide (MOS) is well-known as an effective fed supplement for livestock to increase their nutrients absorption and health status. Pentasaccharide of mannan (MOS5) was reported as a molecule that possesses the ability to increase tight junction of epithelial tissue, but the structure and mechanism of action remains undetermined. In this study, the mechanism of action and structure of MOS5 were investigated. T84 cells were cultured and treated with MOS5 compared with vehicle and compound C, a 5′-adenosine monophosphate-activated protein kinase (AMPK) inhibitor. The results demonstrated that the ability of MOS5 to increase tight junction integration was inhibited in the presence of dorsomorphine (compound C). Phosphorylation level of AMPK was elevated in MOS5 treated group as determined by Western blot analysis. Determination of MOS5 structure was performed using enzymatic mapping together with 1H, 13C NMR, and 2D-NMR analysis. The results demonstrated that the structure of MOS5 is a β-(1,4)-mannotetraose with α-(1,6)-galactose attached at the second mannose unit from non-reducing end

Antidiarrheal application of diclofenac.

<p>(A) Effect of diclofenac on cholera toxin (CT)-induced Cl⁻ secretion in T84 cells. Short-circuit current measurements were performed in T84 cells. After stimulation of Cl⁻ secretion by CT (1 µg/ml), diclofenac was added into both apical and basolateral solutions (n = 5). (B) Effect of diclofenac on cAMP-induced Cl⁻ secretion in mouse intestinal sheets. Mouse intestinal sheets were mounted in Ussing chambers and Cl⁻ secretion was stimulated by forskolin (20 µM). Diclofenac was added into both apical and basolateral solutions (n = 4). (C) Effect of diclofenac on CT-induced intestinal fluid secretion in mice. Ileal loops were instilled with PBS or PBS containing CT (1 µg/loop) with or without concomitant intraperitoneal administration of diclofenac (30 mg/kg). Four h later, ileal loops were removed for loop weight/length ratio measurements; (left) representative photographs of ileal loops, (right) summary of data. Data are expressed as means of loop weight/length ratio ± S.E.M. *, p<0.05 compared with CT-treated control (n = 8). (D) Effect of diclofenac on <i>V. cholerae</i>-induced intestinal fluid secretion in mice. Ileal loops were inoculated with PBS or PBS containing <i>V. cholerae</i> (10⁷ CFU/loop) with or without concomitant intraperitoneal administration of diclofenac (30 mg/kg). Twelve hours post-inoculation, ileal loops were removed for measurements of loop weight/length ratio; (left) representative photographs of ileal loops, (right) summary of data. Data are expressed as means of loop weight/length ratio ± S.E.M. **, p<0.01 compared with <i>V. cholerae</i>-inoculated control (n = 6) (E) Effect of diclofenac on intestinal fluid absorption. Ileal loops were instilled with PBS with or without intraperitoneal administration of diclofenac (30 mg/kg). Twenty or forty min later, ileal loops were removed for loop weight/length ratio measurements; (left) representative photographs of ileal loops, (right) summary of data. Ileal loops at 1 min after PBS instillation and ileal loops without PBS instillation (-PBS) were shown for comparisons. Data are expressed as means of loop weight/length ratio ± S.E.M. (n = 6–8). NS, non-statistical difference compared with control at the same time point.</p

Effects of diclofenac on CaCC, IRC and Ca²⁺-activated basolateral K⁺ channel.

<p>(A) Inhibition of CaCC-mediated Cl⁻ transport by diclofenac. (left) Representative tracing of CaCC-mediated apical Cl⁻ current with basolateral membrane permeabilization. CFTR_inh-172 (5 µM) and ATP (100 µM) were added into apical solutions before addition of diclofenac into both apical and basolateral solutions (n = 5). (right) Diclofenac had no effect on ATP-induced CaMKII phosphorylation. T84 cells were incubated for 20 min with vehicle (control), ATP (100 µM), or ATP (100 µM) plus diclofenac (20 µM). CaMKII phosphorylation was investigated using immunoblot analysis of phosphorylated CaMKII. Results of band intensity analysis are expressed as relative band intensity. NS, non-statistical difference; *, p<0.05 compared with control (n = 3). (B) Inhibition of IRC-mediated Cl⁻ transport by diclofenac. In this experiment, apical Cl⁻ current analysis was performed. CFTR_inh-172 (5 µM) was added into apical solution before IRC activation by forskolin (20 µM). Diclofanac was added into both apical and basolateral solutions (n = 5). (C) Inhibition of Ca²⁺-activated basolateral K⁺ channel (K_Ca3.1) by diclofenac. In this experiment, basolateral K⁺ current measurements were performed in the presence of BaCl₂ (5 mM) in the apical solution. DMSO (control) or diclofenac was added into both apical and basolateral solutions before activation of K_Ca3.1 by ATP (100 µM) (n = 4–6).</p

Evaluation of cytotoxic potential of diclofenac in T84 cells.

<p>(A) Effect of diclofenac on T84 cell viability. T84 cells grown in 96-well plates were incubated for 24 h with diclofenac at the indicated concentrations. MTT assays were used to determine T84 cell viability. Data are expressed as means of % control ± S.E.M. (n = 5). (B) Effect of diclofenac on barrier function. T84 cells grown on permeable support were exposed for 24 h to diclofenac at the indicated concentrations before measuring flux of FITC-dextran (molecular weight ∼4.4 kDa). Data are expressed as means of % control ± S.E.M. EGTA (3 mM) was used as a positive control. NS, non-statistical difference; *, p<0.05 compared with control (n = 4).</p

Mechanism of CFTR inhibition by diclofenac in T84 cells.

<p>(A) Schematic diagram illustrating the regulatory mechanisms of CFTR Cl⁻ channel activity in T84 cells. Inhibitors/activators used in this study are shown. PP, protein phosphatase; AMPK, AMP-activated protein kinase; PDE, phosphodiesterase; PKA, protein kinase A; AC, adenylate cyclase; MRP4, multidrug resistance-associated protein 4 (B) Involvements of PDE, MRP4, AMPK and protein phosphatase in the inhibition of CFTR-mediated Cl⁻ transport. Dose-inhibition studies of diclofenac were performed after the indicated treatments. Representative current tracings are shown. (C) Summary of dose-inhibition studies. Data are fitted to Hill equation and expressed as means of % agonist-stimulated apical Cl⁻ current ± S.E.M. (n = 6–8).</p

Effect of diclofenac on intracellular cAMP content in T84 cells.

<p>T84 cells were incubated for an hour with DMSO (vehicle), diclofenac (200 µM), forskolin (20 µM) or forskolin (20 µM) plus diclofenac (200 µM), followed by cell lysis and cAMP measurement using cAMP immunoassay kit. Data are expressed as means ± S.E.M. NS, non-statistical difference (n = 3).</p

Effect of diclofenac on NKCC1 activity in T84 cells.

<p>(A) Diagrammatic protocol for Tl⁺ influx-based assay of NKCC1 activity. After loading with Tl⁺-sensitive dye, T84 cells were incubated for 15 min in the Cl⁻-free buffer containing Tl₂SO₄ and clotrimazole (to block K⁺ channels) with or without diclofenac (200 µM). Then, NaCl solution (final concentration of NaCl = 135 mM) was added to stimulate NKCC1-mediated Tl⁺ influx causing an increase in fluorescence from Tl⁺-sensitive dye. NKCC1 activity was analyzed from the slope of linear increases in fluorescent intensity within 15 s after NaCl addition. (B) Representatives of relative fluorescent signals from 5 separate experiments, without (control) and with diclofenac (200 µM). Bumetanide (100 µM), a known inhibitor of NKCC1, was used as a positive control.</p

Effect of statin derivatives isolated from soil fungus <i>Aspergillus sclerotiorum</i> and statin drugs on cAMP-dependent Cl^- secretion in T84 cell monolayers.

(A) Effect of α,β-dehydrolovastatin (DHLV), lovastatin, α,β-dehydrodihydromonacolin K, mevastatin, simvastatin, and pravastatin on cAMP-dependent Cl- secretion determined by ISC analysis. (B) Effect of CFTRinh-172 (CFTR inhibitor) on cAMP-dependent Cl- secretion determined by ISC analysis. All of compounds were added accumulatively in both apical and basolateral solutions at the indicated concentrations. Representative ISC tracings are shown. (C) Summary of concentration- inhibition studies. Data are fitted to Hill’s equation and expressed as means of % forskolin-stimulated ISC ± S.E.M. (n = 3–7).</p