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 (H(2)S) have an interaction on the pacemaker activities of interstitial cells of Cajal (ICC) from the mouse small intestine. The actions of NO and H(2)S on pacemaker activities were investigated by using the whole-cell patch-clamp technique and intracellular Ca(2+) 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 H(2)S, 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 Ca(2+) ([Ca(2+)](i)) oscillations in cultured ICC. In conclusion, this study describes the enhanced inhibitory effects of NO plus H(2)S on ICC in the mouse small intestine. NO+H(2)S inhibited the pacemaker activity of ICC by modulating intracellular Ca(2+). These results may be evidence of a physiological interaction of NO and H(2)S in ICC for modulating gastrointestinal motility.

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.

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 M(3) receptor antagonist, but not by methotramine, a muscarinic M(2) receptor antagonist. Intracellular GDP-beta-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 M(3) receptors by a G-protein dependent intracellular Ca2+ release mechanism.

Involvement of thromboxane a(2) 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.

(-)-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.

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+ currents 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) (GDPbetaS) 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.

Action of imipramine on activated ATP-sensitive K(+) channels in interstitial cells of Cajal from murine small intestine.

Tricyclic antidepressants have been widely used for the treatment of depression and as a therapeutic agent for the altered gastrointestinal (GI) motility of irritable bowel syndrome (IBS). The aim of this study was to clarify whether antidepressants directly modulate pacemaker currents in cultured interstitial cells of Cajal (ICC). We used the whole-cell patch-clamp techniques at 30 degrees C in cultured ICC from the mouse small intestine. Treatment of pinacidil, an ATP-sensitive K(+) channel opener, in the ICC using the current clamping mode, produced hyperpolarization of the membrane potential and decreased the amplitude of the pacemaker potentials. With the voltage clamp mode, we observed a decrease in the frequency and amplitude of pacemaker currents and increases in the resting outward currents. These effects of pinacidil on pacemaker potentials and currents were completely suppressed by glibenclamide, an ATP-sensitive K(+) channel blocker. Also, with the current clamp mode, imipramine blocked the affect of pinacidil on the pacemaker potentials. Observations of the voltage clamp mode with imipramine, desipramine and amitryptyline suppressed the action of pinacidil in the ICC. Next, we examined whether protein kinase C (PKC) and the G protein are involved in the action of imipramine on pinacidil induced pacemaker current inhibition. We used chelerythrine, a potent PKC inhibitor and GDPbetaS, a nonhydrolyzable guanosine 5-diphosphate (GDP) analogue that permanently inactivates GTP-binding proteins. We found that pretreatment with chelerythrine and intracellular application of GDPbetaS had no influence on the blocking action of imipramine on inhibited pacemaker currents by pinacidil. We conclude that imipramine inhibited the activated ATP-sensitive K(+) channels in ICC. This action does not appear to be mediated through the G protein and protein kinase C. Furthermore, this study may suggest another possible mechanism for tricyclic antidepressants related modulation of GI motility.

Effects of pine needle extract on pacemaker currents in interstitial cells of Cajal from the murine small intestine.

Extracts of pine needles (Pinus densiflora Sieb. et Zucc.) have diverse physiological and pharmacological actions. In this study we show that pine needle extract alters pacemaker currents in interstitial cells of Cajal (ICC) by modulating ATP-sensitive K+ channels and that this effect is mediated by prostaglandins. In whole cell patches at 30 degrees , ICC generated spontaneous pacemaker potentials in the current clamp mode (I = 0), and inward currents (pacemaker currents) in the voltage clamp mode at a holding potential of -70 mV. Pine needle extract hyperpolarized the membrane potential, and in voltage clamp mode decreased both the frequency and amplitude of the pacemaker currents, and increased the resting currents in the outward direction. It also inhibited the pacemaker currents in a dose-dependent manner. Because the effects of pine needle extract on pacemaker currents were the same as those of pinacidil (an ATP-sensitive K+ channel opener) we tested the effect of glibenclamide (an ATP-sensitive K+ channels blocker) on ICC exposed to pine needle extract. The effects of pine needle extract on pacemaker currents were blocked by glibenclamide. To see whether production of prostaglandins (PGs) is involved in the inhibitory effect of pine needle extract on pacemaker currents, we tested the effects of naproxen, a non-selective cyclooxygenase (COX-1 and COX-2) inhibitor, and AH6809, a prostaglandin EP1 and EP2 receptor antagonist. Naproxen and AH6809 blocked the inhibitory effects of pine needle extract on ICC. These results indicate that pine needle extract inhibits the pacemaker currents of ICC by activating ATP-sensitive K+ channels via the production of PGs.

[Effects of tamoxifen on the voltage-dependent ionic currents in mouse colonic smooth muscle cells].

BACKGROUND/AIMS: Tamoxifen is a widely used anticancer drug for breast cancer with frequent gastrointestinal side effects. Changes in gastrointestinal motility is associated with altered activities of membrane ion channels. Ion channels have important role in regulating membrane potential and cell excitability. This study was performed to investigate the effects of tamoxifen on the membrane ionic currents in colonic smooth muscle cells. METHODS: Murine colonic smooth muscle cells were isolated from the proximal colon using collagenase, and the membrane currents were recorded using a whole-cell patch clamp technique. RESULTS: Two types of voltage-dependent K(+) currents were recorded (A-type and delayed rectifier K(+) currents). Tamoxifen inhibited both types of voltage-dependent K(+) currents in a dose-dependent manner. However, tamoxifen did not change the half-inactivation potential and the recovery time of voltage-dependent K(+) currents. Chelerythrine, a protein kinase C inhibitor or phorbol 12, 13-dibutyrate, a protein kinase C activator did not affect the voltage-dependent K(+) currents. Guanosine 5'-O-(2-thio-diphosphate) did not affect the tamoxifen-induced inhibition of voltage-dependent K(+) currents. Tamoxifen inhibited voltage-dependent Ca(2+) currents completely in whole-test ranges. CONCLUSIONS: These results suggest that tamoxifen can alter various membrane ionic currents in smooth muscle cells and cause some adverse effects on the gastrointestinal motility.