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

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.

Visualization and bibliometric analysis of cAMP signaling system research trends and hotspots in cancer

“…Cyclic adenosine monophosphate (cAMP) is an important cellular second messenger that firstly discovered in 1956 [1] and widely distributed among prokaryotic and eukaryotic organisms. cAMP mediates a multitude of cellular processes and biological functions [2], including gene regulation, cell proliferation and differentiation, apoptosis, energy metabolism, memory formation, neurotransmission, intestinal secretion, retinal phototransduction and immune functions [3][4][5][6][7][8][9]. cAMP is formed from ATP by adenylyl cyclases (ACs) and hydrolyzed by phosphodiesterases (PDEs) [10,11].…”

Cell Volume-Activated and Volume-Correlated Anion Channels in Mammalian Cells: Their Biophysical, Molecular, and Pharmacological Properties

“…A flavonoid, genistein, which is a known potent PTK inhibitor, can PTK-independently activate CFTR from the extracellular (not intracellular) side (French et al, 1997;Wang et al, 1998;Zhou et al, 1998;Niisato et al, 1999). CFTR was also activated by some herbs such as menthol (Morise et al, 2010), which is a cyclic terpene alcohol produced by the peppermint herb; mashiningan (Harada et al, 2017), which is a Japanese herbal (Kampo) medicine used to treat constipation; as well as Junchoto, another Kampo medicine empirically prescribed for chronic constipation (Numata et al, 2018).…”

Herbal components of Japanese Kampo medicines exert laxative actions in colonic epithelium cells via activation of BK and CFTR channels

“…Previous studies performed by short-circuit current measurements with Ussing chambers suggested that MSG and JCT produce membrane currents sensitive to a CFTR blocker, CFTR inhibitor-172 (CFTR-inh) 12,13 , across the polarized human bronchial epithelial cell layer 9,14 . In addition, we previously showed that JCT facilitates intestinal Cl − secretion by activation of CFTR 10 . We therefore next investigated the effects of these four herbal components on CFTR activity in HEK/CFTR cells by conventional whole-cell recordings.…”

“…Solute secretion from cells drives the transfer of osmotically obliged water, resulting in a cell volume reduction, which is called a secretory volume decrease (SVD) 10,11 . We therefore investigated herbal components in two types of Japanese Kampo medicines, JCT and MSG, which are widely used as laxatives, by measuring the SVD in human colonic epithelial Caco-2 cells.…”

“…Traditional Japanese Kampo medicines Junchoto (JCT) and Mashiningan (MSG) have been recently shown to induce cAMP production in the intestinal epithelium and activate CFTR Cl − channels, thereby inducing intestinal fluid secretion 9,10 . Kampo medicines are mixtures of numerous herbal plant extracts.…”