title: The G-protein on cholesterol-rich membrane microdomains mediates mucosal sensing of short- chain fatty acid and secretory response in rat colon.
abstract: OBJECTIVE
  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 (10 mm) and mastoparan (30 μm), lipid rafts/caveolae disruptors, significantly inhibited luminal propionate-induced (0.5 mm) increases in I(sc) , but did not affect increases in I(sc) induced by serosal ACh (0.05 mm) or electrical field stimulation (EFS). Luminal addition of YM-254890 (10 μm), 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) .
authors.affiliation: Meiji Dairies Research Chair, Creative Research Institution, Hokkaido University, Sapporo, Japan. yajimata@cris.hokudai.ac.jp
authors.forename: T
authors.initials: T
authors.lastname: Yajima
authors.forename: R
authors.initials: R
authors.lastname: Inoue
authors.forename: M
authors.initials: M
authors.lastname: Yajima
authors.forename: T
authors.initials: T
authors.lastname: Tsuruta
authors.forename: S
authors.initials: S
authors.lastname: Karaki
authors.forename: T
authors.initials: T
authors.lastname: Hira
authors.forename: A
authors.initials: A
authors.lastname: Kuwahara
mesh_terms: D000109:Acetylcholine / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D002499:Centrifugation, Density Gradient; D002784:Cholesterol / Q000378:metabolism*; D003106:Colon / Q000187:drug effects / Q000294:innervation / Q000378:metabolism*; D004558:Electric Stimulation; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D036341:Intercellular Signaling Peptides and Proteins; D007413:Intestinal Mucosa / Q000187:drug effects / Q000294:innervation / Q000378:metabolism*; D007419:Intestinal Secretions / Q000378:metabolism*; D008297:Male; D021962:Membrane Microdomains / Q000187:drug effects / Q000378:metabolism*; D008564:Membrane Potentials; D008565:Membrane Proteins / Q000378:metabolism; D010455:Peptides / Q000494:pharmacology; D010456:Peptides, Cyclic / Q000494:pharmacology; D011422:Propionates / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D060888:Real-Time Polymerase Chain Reaction; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D014862:Wasp Venoms / Q000494:pharmacology; D047392:beta-Cyclodextrins / Q000494:pharmacology
chemical_list: C518630:Ffar2 protein, rat; D036341:Intercellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D010455:Peptides; D010456:Peptides, Cyclic; D011422:Propionates; D043562:Receptors, G-Protein-Coupled; D014862:Wasp Venoms; C475455:YM-254890; D047392:beta-Cyclodextrins; C106354:flotillins; C108732:methyl-beta-cyclodextrin; C037500:mastoparan; D002784:Cholesterol; C514135:sodium propionate; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D000109:Acetylcholine