The effect of PRR ligands on the membrane potential of intestinal epithelial cells

BackgroundDisproportionate signaling through intestinal epithelial pattern recognition receptors (PRR) plays a role in IBD (inflammatory bowel disease) pathophysiology. Diarrhea is a clinical trademark of IBD and altered activity of K+ channels (KC) may contribute to the low sodium absorption state. Here we sought to study the impact of PRR activation on the membrane potential of human intestinal epithelial cells and the role of KC in it.MethodsAll assays were performed in cultured HT-29 cells. KC activity was assessed by spectrofluorometry, measuring changes in cell membrane potential (MP) with the anionic fluorophore DiBAC4(3). PRRs were activated by specific ligands (MDP, LTA, MPLA, flagellin, loxoribine and ODN2216). KC modulators employed were BaCl2, pinacidil, noxiustoxin and AMP-PNP.ResultsActivation of NOD2, TLR5, TLR7 and TLR9 hyperpolarized the membrane (at 103 ng/ml, the normalized AUC of the fluorescence intensity variation from the control were respectively (mean ± SEM): −725.3 ± 111.5; −1517.4 ± 95.0; −857.8 ± 61.1 and −995.6 ± 53.6), while TLR2 and TLR4 stimulation induced membrane depolarization (1110.4 ± 73.1 and 3890.3 ± 342.7 at 103 ng/ml, respectively). MPD effect on MP was abolished by BaCl2, partially reversed by AMP-PNP (a KATP channel inhibitor) and insensitive to noxiustoxin (a voltage-gated KC inhibitor).ConclusionIt was shown for the first time that PRR activation affects MP in human intestinal epithelial cells. KC appear to be important mediators in this phenomenon; in particular, KATP channels may partake in NOD2-derived effects.