The G-protein on cholesterol-rich membrane microdomains mediates mucosal sensing of short- chain fatty acid and secretory response in rat colon

Aim: Short-chain fatty acids (SCFA) stimulate colonic contraction and secretion, which are mediated by an enteric reflex via a mucosal sensing and cholinergic mechanisms. The involvement of G-protein signal transduction was examined in the secretory response to luminal propionate sensing in rat distal colon. Methods: Mucosa-submucosa and mucosa preparations were used to measure short-circuit current (I _sc) and acetylcholine (ACh) release respectively. Cholesterol-rich membrane microdomains, lipid rafts/caveolae, were fractionated using a sucrose gradient ultra-centrifugation after detergent-free extraction of the isolated colonic crypt. Results: Luminal addition of methyl-β-cyclodextrin (10mm) and mastoparan (30μm), lipid rafts/caveolae disruptors, significantly inhibited luminal propionate-induced (0.5mm) increases in I _sc, but did not affect increases in I _sc induced by serosal ACh (0.05mm) or electrical field stimulation (EFS). Luminal addition of YM-254890 (10μm), a Gα _q/11-selective inhibitor, markedly inhibited propionate-induced increase in I _sc, but did not affect I _sc responses to ACh and EFS. Both methyl-β-cyclodextrin and YM-254890 significantly inhibited luminal propionate-induced non-neuronal release of ACh from colonocytes. Real-time PCR demonstrated that in mRNA expression of SCFA receptors, GPR 43 was far higher than that of GPR41 in the colon. Western blotting analysis revealed that the cholesterol-rich membrane microdomains that fractionated from colonic crypt cells were associated with caveolin-1, flotillin-1 and Gα _q/11, but not GPR43. Uncoupling of Gα _q/11 from flotillin-1 in lipid rafts occurred under desensitization of the I _sc response to propionate. Conclusions: These data demonstrate that the secretory response to luminal propionate in rat colon is mediated by G-protein on cholesterol-rich membrane microdomains, provably via Gα _q/11.