Traditional Chinese formula, lubricating gut pill, stimulates cAMP-dependent Cl− secretion across rat distal colonic mucosa

Aim of the studyLubricating gut pill (LGP), a traditional Chinese formula, had been conformed to improve the loperamide-induced rat constipation by stimulation of Cl− secretion, but its mechanism has not been fully explored. Thus, the purpose of this study was to identify the action sites of LGP-stimulated Cl− secretion across rat distal colonic mucosa.Materials and methodsRat distal colonic mucosa was mounted in Ussing chambers and short circuit current (ISC), apical Cl− current and basolateral K+ current were recorded. Intracellular cyclic adenosine monophosphate (cAMP) content and protein kinase A (PKA) activity were determined with ELISA kit and the non-radioactive PepTag test, respectively.ResultsLGP at 800 μg/ml elicited a sustained increase in Cl− secretory response, which was inhibited by CFTRinh172, a cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor. Permeabilizing apical membrane with nystatin revealed that LGP-stimulated basolateral K+ current was significantly inhibited by KCNQ1 K+ channel inhibitor chromanol 293B. LGP-stimulated ISC was markedly reduced by pretreatment with cis-N-[2-phenylcyclopentyl]-azacyclotridec-1-en-2amine (MDL-12,330A) and N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), but not with inhibitors of Ca2+-dependent signaling pathway. Treatment of tissue with LGP resulted in an increase in intracellular cAMP level and the activation in protein kinase A. The E-prostanoid4 (EP)4 receptor antagonist L-161,982 completely eliminated LGP-induced response.ConclusionsThe results showed that LGP enhances Cl− and fluid secretion via prostanoid receptor signaling and also cAMP and protein kinase A pathway, subsequently triggering the activation of apical Cl− channels mostly CFTR and basolateral cAMP-dependent K+ channel.