Pyung Jin Yoon

Carbachol regulates pacemaker activities in cultured interstitial cells of Cajal from the mouse small intestine

We studied the effect of carbachol on pacemaker currents in cultured interstitial cells of Cajal (ICC) from the mouse small intestine by muscarinic stimulation using a whole cell patch clamp technique and Ca2+-imaging. ICC generated periodic pacemaker potentials in the current-clamp mode and generated spontaneous inward pacemaker currents at a holding potential of–70 mV. Exposure to carbachol depolarized the membrane and produced tonic inward pacemaker currents with a decrease in the frequency and amplitude of the pacemaker currents. The effects of carbachol were blocked by 1-dimethyl-4-diphenylacetoxypiperidinium, a muscarinic M3 receptor antagonist, but not by methotramine, a muscarinic M2 receptor antagonist. Intracellular GDP-β-S suppressed the carbachol-induced effects. Carbachol-induced effects were blocked by external Na+-free solution and by flufenamic acid, a non-selective cation channel blocker, and in the presence of thapsigargin, a Ca2+-ATPase inhibitor in the endoplasmic reticulum. However, carbachol still produced tonic inward pacemaker currents with the removal of external Ca2+. In recording of intracellular Ca2+ concentrations using fluo 3-AM dye, carbachol increased intracellular Ca2+ concentrations with increasing of Ca2+ oscillations. These results suggest that carbachol modulates the pacemaker activity of ICC through the activation of non-selective cation channels via muscarinic M3 receptors by a G-protein dependent intracellular Ca2+ release mechanism.

5-Hydroxytryptamine Generates Tonic Inward Currents on Pacemaker Activity of Interstitial Cells of Cajal from Mouse Small Intestine

In this study we determined whether or not 5-hydroxytryptamine (5-HT) has an effect on the pacemaker activities of interstitial cells of Cajal (ICC) from the mouse small intestine. The actions of 5-HT on pacemaker activities were investigated using a whole-cell patch-clamp technique, intracellular Ca(2+) ([Ca(2+)](i)) analysis, and RT-PCR in ICC. Exogenously-treated 5-HT showed tonic inward currents on pacemaker currents in ICC under the voltage-clamp mode in a dose-dependent manner. Based on RT-PCR results, we found the existence of 5-HT(2B, 3, 4, and 7) receptors in ICC. However, SDZ 205557 (a 5-HT(4) receptor antagonist), SB 269970 (a 5-HT7 receptor antagonist), 3-tropanylindole - 3 - carboxylate methiodide (3-TCM; a 5-HT(3) antagonist) blocked the 5-HT-induced action on pacemaker activity, but not SB 204741 (a 5-HT(2B) receptor antagonist). Based on [Ca(2+)](i) analysis, we found that 5-HT increased the intensity of [Ca(2+)](i). The treatment of PD 98059 or JNK II inhibitor blocked the 5-HT-induced action on pacemaker activity of ICC, but not SB 203580. In summary, these results suggest that 5-HT can modulate pacemaker activity through 5-HT(3, 4, and 7) receptors via [Ca(2+)](i) mobilization and regulation of mitogen-activated protein kinases.

Interplay of hydrogen sulfide and nitric oxide on the pacemaker activity of interstitial cells of cajal from mouse small intestine.

We studied whether nitric oxide (NO) and hydrogen sulfide (H2S) have an interaction on the pacemaker activities of interstitial cells of Cajal (ICC) from the mouse small intestine. The actions of NO and H2S on pacemaker activities were investigated by using the whole-cell patch-clamp technique and intracellular Ca2+ analysis at 30℃ in cultured mouse ICC. Exogenously applied (±)-S-nitroso-N-acetylpenicillamine (SNAP), an NO donor, or sodium hydrogen sulfide (NaHS), a donor of H2S, showed no influence on pacemaker activity (potentials and currents) in ICC at low concentrations (10 µM SNAP and 100 µM NaHS), but SNAP or NaHS completely inhibited pacemaker amplitude and pacemaker frequency with increases in the resting currents in the outward direction at high concentrations (SNAP 100 µM and NaHS 1 mM). Co-treatment with 10 µM SNAP plus 100 µM NaHS also inhibited pacemaker amplitude and pacemaker frequency with increases in the resting currents in the outward direction. ODQ, a guanylate cyclase inhibitor, or glibenclamide, an ATP-sensitive K+ channel inhibitor, blocked the SNAP+NaHS-induced inhibition of pacemaker currents in ICC. Also, we found that SNAP+NaHS inhibited the spontaneous intracellular Ca2+ ([Ca2+]i) oscillations in cultured ICC. In conclusion, this study describes the enhanced inhibitory effects of NO plus H2S on ICC in the mouse small intestine. NO+H2S inhibited the pacemaker activity of ICC by modulating intracellular Ca2+. These results may be evidence of a physiological interaction of NO and H2S in ICC for modulating gastrointestinal motility.

Imipramine inhibits A-type delayed rectifier and ATP-sensitive K+ currents independent of G-protein and protein kinase C in murine proximal colonic myocytes

The effects of imipramine on A-type delayed rectifier K+ curents and ATP-sensitive K+ (KATP) currents were studied in isolated murine proximal colonic myocytes using the whole-cell patch-clamp technique. Depolarizing test pulses between −80 mV and +30 mV with 10 mV increments from the holding potential of −80 mV activated voltage-dependent outward K+ currents that peaked within 50 ms followed by slow decreasing sustained currents. Early peak currents were inhibited by the application of 4-aminopyridine, whereas sustained currents were inhibited by the application of TEA. The peak amplitude of A-type delayed rectifier K+ currents was reduced by external application of imipramine. The half-inactivation potential and the half-recovery time of A-type delayed rectifier K+ currents were not changed by imipramine. With 0.1 mM ATP and 140 mM K+ in the pipette and 90 mM K+ in the bath solution and a holding potential of −80 mV, pinacidil activated inward currents; this effect was blocked by glibenclamide. Imipramine also inhibited KATP currents. The inhibitory effects of imipramine in A-type delayed rectifier K+ currents and KATP currents were not changed by guanosine 5-O-(2-thiodiphosphate) (GDPβS) and chelerythrine, a protein kinase C inhibitor. These results suggest that imipramine inhibits A-type delayed rectifier K+ currents and KATP currents in a manner independent of G-protein and protein kinase C.

epigallocatechin gallate inhibits the pacemaker activity of interstitial cells of cajal of mouse small intestine.

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.

Involvement of Thromboxane A2 in the Modulation of Pacemaker Activity of Interstitial Cells of Cajal of Mouse Intestine

Although many studies show that thromboxane A(2) (TXA(2)) has the action of gastrointestinal (GI) motility using GI muscle cells and tissue, there are no reports on the effects of TXA(2) on interstitial cells of Cajal (ICC) that function as pacemaker cells in GI tract. So, we studied the modulation of pacemaker activities by TXA(2) in ICC with whole cell patch-clamp technique. Externally applied TXA(2) (5microM) produced membrane depolarization in current-clamp mode and increased tonic inward pacemaker currents in voltage-clamp mode. The tonic inward currents by TXA(2) were inhibited by intracellular application of GDP-beta-S. The pretreatment of ICC with Ca(2+) free solution and thapsigargin, a Ca(2+)-ATPase inhibitor in endoplasmic reticulum, abolished the generation of pacemaker currents and suppressed the TXA(2)-induced tonic inward currents. However, chelerythrine or calphostin C, protein kinase C inhibitors, did not block the TXA(2)-induced effects on pacemaker currents. These results suggest that TXA(2) can regulate intestinal motility through the modulation of ICC pacemaker activities. This modulation of pacemaker activities by TXA(2) may occur by the activation of G protein and PKC independent pathway via extra and intracellular Ca(2+) modulation.