(-)-epigallocatechin gallate inhibits the pacemaker activity of interstitial cells of cajal of mouse small intestine

Abstract

The effects of (-)-epigallocatechin gallate (EGCG) on pacemaker activities of cultured interstitial cells of Cajal (ICC) from murine small intestine were investigated using whole-cell patch-clamp technique at 30 and Ca(2+) image analysis. ICC generated spontaneous pacemaker currents at a holding potential of -70 mV. The treatment of ICC with EGCG resulted in a dose-dependent decrease in the frequency and amplitude of pacemaker currents. SQ-22536, an adenylate cyclase inhibitor, and ODQ, a guanylate cyclase inhibitor, did not inhibit the effects of EGCG. EGCG-induced effects on pacemaker currents were not inhibited by glibenclamide, an ATP-sensitive K(+) channel blocker and TEA, a Ca(2+)-activated K(+) channel blocker. Also, we found that EGCG inhibited the spontaneous [Ca(2+)](i) oscillations in cultured ICC. In conclusion, EGCG inhibited the pacemaker activity of ICC and reduced [Ca(2+)](i) oscillations by cAMP-, cGMP-, ATP-sensitive K+ channel-independent manner.

Keywords: (-)-epigallocatechin gallate (EGCG); Interstitial cells of Cajal (ICC); Intestinal motility; Pacemaker currents.

Fig. 1

Effects of catechins on pacemaker currents recorded in cultured ICC from mouse small intestine (A) Pacemaker currents of ICC recorded at a holding potential of -70 mV, when exposed to polyphenon 60 (200µM). (B) Pacemaker currents of ICC recorded at a holding potential of -70 mV when exposed to epicatechin gallate (200µM). (C) Pacemaker currents of ICC recorded at a holding potential of -70 mV which were exposed to epigallocatechin gallate (200µM). Vertical solid line scales amplitude of pacemaker current and horizontal solid line scales duration of recording (s) pacemaker currents. ECG: (-)-epicatechin gallate, EGCG: (-)-epigallocatechin gallate.

Fig. 2

Dose-dependent effects of (-)-epigallocatechin gallate on pacemaker currents in cultured ICC of mouse small intestine. (A), (B), and (C) show pacemaker currents of ICC exposed to (-)-epigallocatechin gallate (50, 100, or 200 µM respectively) at a holding potential of -70 mV. Vertical solid line scales amplitude of pacemaker current and horizontal solid line scales duration of recording (s) pacemaker currents. (-)-epigallocatechin gallate at 100 and 200 µM concentrations inhibited spontaneous pacemaker currents of ICC. (D), (E), and (F) summarize the inhibitory effects of (-)-epigallocatechin gallate on pacemaker currents in ICC. Bars represents means±SE (n=5/group). ^*Asterisks mean significantly different from the controls (p<0.05). EGCG: (-)-epigallocatechin gallate, CON: control.

Fig. 3

Effects of SQ-22536, an inhibitor of adenylate cyclase, and ODQ, an inhibitor of guanylate cyclase, on (-)-epigallocatechin gallate-induced response in cultured ICC from mouse small intestine. (A) Pretreatment with SQ-22536 (10µM) did not affect the inhibitory effects of (-)-epigallocatechin gallate (200µM) on spontaneous inward currents. (B) Pretreatment with ODQ (10µM) did not affect the inhibitory effects of (-)-epigallocatechin gallate (200µM) on spontaneous inward currents. Vertical solid line scales amplitude of pacemaker current and horizontal solid line scales duration of recording (s) pacemaker currents. EGCG: (-)-epigallocatechin gallate.

Fig. 4

Effects of tetraethylammonium chloride, a Ca²⁺-activated K⁺ channel blocker, and glibenclamide, an ATP-sensitive K⁺ channel blocker, on (-)-epigallocatechin gallate-induced response in cultured ICC from mouse small intestine. (A) Pretreatment with tetraethylammonium chloride (2 mM) did not affect the inhibitory effects of (-)-epigallocatechin gallate (200µM) on spontaneous inward currents. (B) Pretreatment with glibenclamide (10µM) did not affect the inhibitory effects of (-)-epigallocatechin gallate (200µM) on spontaneous inward currents. Vertical solid line scales amplitude of pacemaker current and horizontal solid line scales duration of recording (s) pacemaker currents. EGCG: (-)-epigallocatechin gallate, TEA: tetraethylammonium chloride.

Fig. 5

Effects of (-)-epigallocatechin gallate on intrcellular Ca²⁺ oscillation in cultured ICC from mouse small intestine. (A) Sequential fluorescence intensity images of fluo-3-loaded cultured ICC in control condition. (B) Sequential fluorescence intensity images of fluo-3-loaded cultured ICC in the presence of (-)-epigallocatechin gallate (200µM). The images of basal (1 and 2) and peak (1 and 2) in (A) and (B) acquired from indicators in (B). (C) Fluorescence intensity change plotted in (A) and (B) red marker.