Cellular mechanism for herbal medicine Junchoto to facilitate intestinal Cl\u2212/water secretion that involves cAMP-dependent activation of CFTR

Tomohiro Numata,Yasunobu Okada,Kaori Sato-Numata,Ryuji Inoue

doi:10.1007/s11418-018-1207-9

Abstract

Constipation is a common symptom frequently compromising the quality of daily life. Several mechanistically different drugs have been used to mitigate constipation, including Japanese herbal (Kampo) medicines. However, the mechanisms of their actions are often not well understood. Here we aimed to investigate the molecular mechanisms underlying the effects of Junchoto (JCT), a Kampo medicine empirically prescribed for chronic constipation. Cl− channel activity was measured by the patch-clamp method in human cystic fibrosis transmembrane conductance regulator (CFTR)-expressing HEK293T cells and human intestinal Caco-2 cells. cAMP was measured by a luciferase-based assay. Cell volume change was measured by a particle-sizing and particle-counting analyzer and video-microscopic measurement. In both CFTR-expressing HEK293T and Caco-2 cells, JCT dose-dependently induced whole-cell currents showing typical biophysical and pharmacological features of CFTR. Robust expression of CFTR was confirmed by RT-PCR and Western blotting in Caco-2 cells. Luciferase-based measurement revealed that JCT increases intracellular cAMP levels. Administration of the adenylate cyclase inhibitor SQ22536 or CFTR inhibitor-172, or treatment with small interfering RNAs (siRNA) targeting CFTR, abolished JCT-induced whole-cell currents, suggesting that elevated intracellular cAMP by JCT causes activation of CFTR in Caco-2 cells. Finally, blockade of CFTR activity by CFTR inhibitor-172 or siRNA-knockdown of CFTR or application of SQ22536 markedly reduced the degree of cell volume decrease induced by JCT. JCT can induce a Cl− efflux through the CFTR channel to promote water secretion, and this effect is likely mediated by increased cAMP production.

Highlights

Constipation affects multiple aspects of a person’s health, including health-related quality of life
Since this trans-epithelial current was effectively reduced by the CFTR inhibitor-172 [9], it was concluded that the current arose from CFTR activation
The JCT-induced current responded to step voltage pulses with almost instantaneous activation and deactivation time courses (Fig. 1b, upper traces), and showed a linear current–voltage (I–V) relationship (Fig. 1c, open circles). These electrophysiological properties are characteristic of heterologously expressed CFTRmediated Cl− channels, suggesting that JCT is a robust activator of the CFTR channel

Summary

Introduction

Constipation affects multiple aspects of a person’s health, including health-related quality of life It is one of the most frequently reported functional gastrointestinal disorders. In the intestinal epithelia of both mice and human, endogenous expression of CFTR is restricted to the apical membrane while that of ClC-2 is localized largely in the basolateral membrane, and, only the former can be activated by lubiprostone [6]. It still remains controversial what type of ion channels/ transporters are involved in lubiprostone’s laxative actions. It is reported that guanylate cyclase-C (GC-C) receptor activators, linaclotide and plecanatide, exert similar gastrokinetic actions, through enhanced intracellular cGMP synthesis and subsequent phosphorylation of CFTR protein by cGMPdependent protein kinase II (PKG II), which facilitates luminal chloride secretion and paracellular movement of sodium and water [3, 7]

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