Ionic dependency, sensitivity to transport inhibitors and signal transduction of propionate-induced anion secretion in rat caecum

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Although the caecum is the most important fermentation chamber in non-ruminant animals, the epithelial responses evoked by microbially produced short-chain fatty acids such as propionate are not well understood. Short-circuit currents (I sc ) across rat caecum were measured in Ussing chamber experiments, where anionic conditions, transport mechanisms and underlying signaling pathways regarding propionate-induced I sc (I Prop ) were analyzed. These findings were complemented with immunofluorescence and RT-PCR. I Prop was dependent on the simultaneous presence of Cl - and HCO 3 - . This contrasts with the I sc evoked by the cAMP- or Ca 2+ -dependent secretagogues, forskolin and carbachol, where replenishment of Cl - was sufficient to induce a strong increase in I sc . Blocking the cystic fibrosis transmembrane regulator (CFTR) with CFTR inh -172 inhibited I Prop in the oral caecum, whereas in the aboral caecum the additional presence of 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS), an inhibitor of anion exchangers, was necessary to reduce I Prop significantly. Since propionate can activate free-fatty acid receptors type 2 (coupled to G q and G i proteins) and 3 (coupled to G i proteins), downstream signaling was further elucidated. Inhibition of G qα , phospholipase C, inositol-3,4,5-trisphosphate receptors, or phosphatidyl inositol kinases reduced I Prop significantly. Kinases of the ‘with no lysine’ family (WNK), known to mediate the switch between Cl - to HCO 3 - secretion in other cells, are also involved in propionate-induced anion secretion, since I Prop was reduced by panWNK and WNK1 inhibition. Propionate stimulates anion secretion, probably in order to buffer the caecal luminal content, by a mechanism exhibiting a unique anion dependency different from that of other secretagogues. Overview about the underlying signaling cascade and involvement of transporters and channels in the propionate-dependent anion secretion, for which the presence of HCO 3 - and Cl - on both sides of the epithelium is necessary. Assumed signaling and findings from previous studies are shown in gray; main results of the current study are shown in red; enzymes are shown in bold letters (normal arrows = activation of signaling molecules; dotted arrows = enzymatic conversion, dashed arrows = assumed pathways).Abbreviations: CFTR (cystic fibrosis transmembrane conductance regulator), FFAR (free-fatty acid receptor), IP3 (inositol-1,4,5-trisphosphate), IP3R (IP3 receptor), M1 / M3 (muscarinic acetylcholine receptor subtype 1 and 3), NKCC (Na+-K+-2Cl- cotransporter), PI (phosphatidylinositol), PIP2 (phosphatidylinositol-4,5-bisphosphate), PIP3 (phosphatidylinositol-3,4,5-trisphosphate), PI3K (phosphatidylinositol 3-kinase), PI4K (phosphatidylinositol 4-kinase), PI5K (phosphatidylinositol 5-kinase), PKA (protein kinase A), PKC (protein kinase C), PLC (phospholipase C), WNK (with no lysine kinase).