Effects of Prunus mume Siebold & Zucc. in the pacemaking activity of interstitial cells of Cajal in murine small intestine

Abstract

Interstitial cells of Cajal (ICCs) function as pacemaker cells in the gastrointestinal (GI) tract and therefore, serve an important role in regulating GI motility. The effects of a species of plum (Prunus mume Siebold & Zucc.) on cultured ICC cluster-induced pacemaker potentials in the mouse small intestine were investigated, and the effects of a methanolic extract of Prunus mume (m-PM) on ICC pacemaker activities were examined using the whole-cell patch-clamp technique. ICC pacemaker membrane potentials were depolarized by m-PM in a concentration dependent manner in current clamp mode. 4-Diphenylacetoxy-N-methyl-piperidine methiodide, which is a muscarinic 3 (M₃) receptor antagonist, was able to block m-PM-induced pacemaker potential increases, whereas methoctramine, which is a muscarinic 2 (M₂) receptor antagonist, was not. When 1 mM guanosine diphosphate β-5 was present in the pipette solution, m-PM induced slight pacemaker depolarization. Following pretreatment in bath solution of Ca²⁺-free solution or a Ca²⁺-ATPase inhibitor in endoplasmic reticulum, the pacemaker currents were inhibited. Furthermore, pretreatment with PD98059, SB203580 or SP600125, which is a c-jun NH2-terminal kinase inhibitor, blocked m-PM-induced ICC potential depolarization. Furthermore, m-PM inhibited transient receptor potential melastatin (TRPM) 7 channels, but did not affect Ca²⁺-activated Cl^- channels. These results suggest that m-PM is able to modulate pacemaker potentials through the muscarinic M₃ receptor, via G-protein and external and internal Ca²⁺, in a mitogen-activated protein kinase and TRPM7-dependent manner. Therefore, m-PM may provide a basis for the development of a novel gastroprokinetic agent.

Keywords: Prunus mume Siebold & Zucc.; gastrointestinal tract; interstitial cells of Cajal; plum.

Figure 1.

Effects of m-PM on the pacemaker potentials of cultured ICCs from murine small intestine. (A-E) Pacemaker potentials of ICCs exposed to m-PM (50–500 µg/ml) in current clamping mode (I=0). Responses to m-PM are summarized in (F and G). Bars represent mean values ± standard deviation. *P<0.05 vs. control, **P<0.01 vs. control. m-PM, methanoic extract of the fruits of Prunus mume Siebold & Zucc.; ICCs, interstitial cells of Cajal; CTRL, control.

Figure 2.

Effects of muscarinic receptor subtype antagonists on m-PM-induced pacemaker potential depolarizations in cultured ICCs. (A) Pacemaker potentials of ICCs were depolarized following exposure to m-PM (300 µg/ml) in the presence of methoctramine (a muscarinic M₂ receptor antagonist; 10 µM). (B) Pacemaker potentials of ICCs were not affected by exposure to m-PM in the presence of 4-DAMP (a muscarinic M₃ receptor antagonist; 10 µM). Responses to m-MP in the presence of different receptor antagonists are summarized in (C). Bars represent mean values ± standard error of the mean. **P<0.01 vs. control. m-PM, methanoic extract of the fruits of Prunus mume Siebold & Zucc.; ICCs, interstitial cells of Cajal; 4-DAMP, 4-diphenylacetoxy-N-methyl-piperidine methiodide; CTRL, control; Methoct., methoctramine.

Figure 3.

Effects of GDP-β-S on m-PM-induced pacemaker potential depolarization in cultured ICCs. (A) Pacemaker potentials of ICCs exposed to m-PM (300 µg/ml) in the presence of GDP-β-S (1 mM) in the pipette were depolarized. (B) Summary of responses to m-PM in the presence of GDP-β-S. Bars represent mean values ± standard error of the mean. **P<0.01 vs. control. GDP-β-S, Guanosine 5′-O-(2-thiodiphosphate); m-PM, methanoic extract of the fruits of Prunus mume Siebold & Zucc.; ICCs, interstitial cells of Cajal; CTRL, control.

Figure 4.

Effects of an external Ca²⁺-free solution and of thapsigargin (a Ca²⁺-ATPase inhibitor in endoplasmic reticulum) on m-PM-induced pacemaker potential depolarizations in cultured ICCs. (A) External Ca²⁺-free solution abolished the generation of pacemaker potentials and blocked m-PM-induced pacemaker depolarizations. (B) Thapsigargin (5 µM) abolished pacemaker depolarizations and blocked m-PM-induced pacemaker depolarizations. (C) Responses to m-PM in external Ca²⁺-free solution and in the presence of thapsigargin are summarized. Bars represent mean values ± standard error of the mean. **P<0.01 vs. control. m-PM, methanoic extract of the fruits of Prunus mume Siebold & Zucc.; ICCs, interstitial cells of Cajal; CTRL, control.

Figure 5.

Effects of various MAPK inhibitors on m-PM-induced pacemaker potential responses in cultured ICCs. (A) Pacemaker potentials of cultured ICCs exposed to m-PM (300 µg/ml) in the presence of 10 µM PD98059 (a p42/44 MAPK inhibitor). (B) Pacemaker potentials of cultured ICCs exposed to m-PM in the presence of 10 µM SB203580 (a p38 MAPK inhibitor). (C) Pacemaker potentials of ICCs exposed to m-PM in the presence of 10 µM JNK II inhibitor. Responses to m-PM in the presence of different MAPK inhibitors are summarized in (D). Bars represent mean values ± standard error of the mean. **P<0.01 vs. control. MAPK, mitogen-activated protein kinase; m-PM, methanoic extract of the fruits of Prunus mume Siebold & Zucc.; JNK II, c-jun NH2-terminal kinase; CTRL, Control.

Figure 6.

Effects of m-PM on overexpressed TRPM7 and ANO1 in HEK293 cells. (A) TRPM7 currents were recorded in mock transfected HEK293 cells. (B) Ca²⁺-activated Cl-currents were recorded in mock transfected HEK293 cells. (C) Representative I–V relationship of the effect of m-PM on TRPM7 currents in HEK293 cells. (D) Representative I–V relationship of the effect of m-PM on Ca²⁺-activated Cl⁻ currents in HEK293 cells. A voltage ramp from 100 to −100 mV was applied from a holding potential of −60 mV. (E and F) Summary of responses to m-PM during TRPM7 or ANO1 currents. Bars represent mean values ± SE. **P<0.01 vs. TRPM7 in the absence of m-PM. m-PM, methanoic extract of the fruits of Prunus mume Siebold & Zucc.; TRPM7, transient receptor potential melastatin type 7; ANO1, anoctamin-1, a Ca²⁺-activated Cl⁻ channel; HEK 293, human embryonic kidney 293 cells; I–V, current-voltage.