Zuojin Pill enhances gastrointestinal motility by modulating the pacemaker potentials in interstitial cells of Cajal through multiple signaling pathways

Zuojin Pill (ZJP) is a traditional herbal preparation used to treat various gastrointestinal (GI) disorders. The purpose of this study was to elucidate the underlying cellular and molecular mechanisms by evaluating the effects of ZJP on the pacemaker activity of isolated interstitial cells of Cajal (ICCs) and on <i>in vivo</i> GI motility in mice. We isolated ICCs from mouse small intestine and measured pacemaker potentials by whole-cell patch clamping as well as intracellular calcium signaling by microfluorometry. Intestinal transit rate (ITR) was measured by Evans Blue migration. Administration of ZJP depolarized ICCs and reduced the amplitude and frequency of pacemaker potentials. Pretreatment with the 5-HT4 receptor antagonist RS39604, administration of the muscarinic M3 receptor antagonist 4-DAMP, or intracellular perfusion of the G-protein inhibitor GDP-β-S blocked ZJP-induced ICCs depolarization. In addition, external calcium-free medium and administration of the Ca²⁺-ATPase inhibitor thapsigargin, which depletes intracellular calcium stores, also blocked ZJP-induced ICCs depolarization. Moreover, ZJP-induced ICCs depolarization was inhibited in the presence of the phospholipase C (PLC) inhibitor U-73122, IP₃-dependent Ca²⁺ release inhibitor xestospongin C, or various mitogen-activated protein kinase (MAPK) inhibitors. Alternatively, ZJP-induced ICCs depolarization in the presence of protein kinase C (PKC) inhibitors. Furthermore, ZJP reversed the reduction of ITR caused by loperamide (Imodium) and normalized the ITR abnormality of two etiologically distinct GI motility disorder (GMD) mouse models. Finally, ZJP increased serum concentrations of the pro-peristalsis factors motilin and substance P. Our results suggest that ZJP depolarizes ICCs via 5-HT4 or muscarinic M3 receptor activation and G-protein dependent calcium-, PLC-, inositol triphosphate-, and MAPK signaling pathways (but not PKC-dependent pathways), leading to enhanced GI motility.