• Biomedical Science Letters
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• v.18 no.3
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• pp.218-226
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• 2012

Dopamine is an enteric neurotransmitter that regulates gastrointestinal motility. This study was done to investigate whether dopamine modulates spontaneous pacemaker activity in cultured interstitial cells of Cajal (ICCs) from mouse using whole cell patch clamp technique, RT-PCR and live $Ca^{2+}$ imaging analysis. ICCs generate pacemaker inward currents at a holding potential of -70 mV and generate pacemaker potentials in current-clamp mode. Dopamine did not change the frequency and amplitude of pacemaker activity in small intestinal ICCs. On the contrary dopamine reduced the frequency and amplitude of pacemaker activity in large intestinal ICCs. RT-PCR analysis revealed that Dopamine2 and 4-receptors are expressed in c-Kit positive ICCs. Dopamine2 and 4 receptor agonists inhibited pacemaker activity in large intestinal ICCs mimicked those of dopamine. Domperidone, dopamine2 receptor antagonist, increased the frequency of pacemaker activity of large intestinal ICCs. In $Ca^{2+}$-imaging, dopamine inhibited spontaneous intracellular $Ca^{2+}$ oscillations of ICCs. These results suggest that dopamine can regulate gastrointestinal motility through modulating pacemaker activity of large intestinal ICCs and dopamine effects on ICCs are mediated by dopamine2 receptor and intracellular $Ca^{2+}$ modulation.

• The Korean Journal of Physiology and Pharmacology
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• v.18 no.4
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• pp.341-346
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• 2014

Lubiprostone is a chloride ($Cl^-$) channel activator derived from prostaglandin $E_1$ and used for managing constipation. In addition, lubiprostone affects the activity of gastrointestinal smooth muscles. Interstitial cells of Cajal (ICCs) are pacemaker cells that generate slow-wave activity in smooth muscles. We studied the effects of lubiprostone on the pacemaker potentials of colonic ICCs. We used the whole-cell patch-clamp technique to determine the pacemaker activity in cultured colonic ICCs obtained from mice. Lubiprostone hyperpolarized the membrane and inhibited the generation of pacemaker potentials. Prostanoid $EP_1$, $EP_2$, $EP_3$, and $EP_4$ antagonists (SC-19220, PF-04418948, 6-methoxypyridine-2-boronc acid N-phenyldiethanolamine ester, and GW627368, respectively) did not block the response to lubiprostone. L-NG-nitroarginine methyl ester (L-NAME, an inhibitor of nitric oxide synthase) and 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ, an inhibitor of guanylate cyclase) did not block the response to lubiprostone. In addition, tetraethylammonium (TEA, a voltage-dependent potassium [$K^+$] channel blocker) and apamin (a calcium [$Ca^{2+}$]-dependent $K^+$ channel blocker) did not block the response to lubiprostone. However, glibenclamide (an ATP-sensitive $K^+$ channel blocker) blocked the response to lubiprostone. Similar to lubiprostone, pinacidil (an opener of ATP-sensitive $K^+$ channel) hyperpolarized the membrane and inhibited the generation of pacemaker potentials, and these effects were inhibited by glibenclamide. These results suggest that lubiprostone can modulate the pacemaker potentials of colonic ICCs via activation of ATP-sensitive $K^+$ channel through a prostanoid EP receptor-independent mechanism.

• The Korean Journal of Physiology and Pharmacology
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• v.14 no.2
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• pp.83-89
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• 2010

In this study, we studied whether hydrogen sulfide ($H_2S$) has an effect on the pacemaker activity of interstitial cells of Cajal (ICC), in the small intestine of mice. The actions of $H_2S$ on pacemaker activity were investigated using whole-cell patch-clamp technique, intracellular $Ca^{2+}$ analysis at $30^{\circ}C$ and RT-PCR in cultured mouse intestinal ICC. Exogenously applied sodium hydrogen sulfide (NaHS), a donor of hydrogen sulfide, caused a slight tonic inward current on pacemaker activity in ICC at low concentrations (50 and $100{\mu}m$), but at high concentration ($500{\mu}m$ and 1 mM) it seemed to cause light tonic inward currents and then inhibited pacemaker amplitude and pacemaker frequency, and also an increase in the resting currents in the outward direction. Glibenclamide or other potassium channel blockers (TEA, $BaCl_2$, apamin or 4-aminopydirine) did not have an effect on NaHS-induced action in ICC. The exogenous application of carbonilcyanide p-triflouromethoxyphenylhydrazone (FCCP) and thapsigargin also inhibited the pacemaker activity of ICC as NaHS. Also, we found NaHS inhibited the spontaneous intracellular $Ca^{2+}$ ($[Ca^{2+}]_i$) oscillations in cultured ICC. In doing an RT-PCR experiment, we found that ICC enriched population lacked mRNA for both CSE and CBS, but was prominently detected in unsorted muscle. In conclusion, $H_2S$ inhibited the pacemaker activity of ICC by modulating intracellular $Ca^{2+}$. These results can serve as evidence of the physiological action of $H_2S$ as acting on the ICC in gastrointestinal (GI) motility.

• The Korean Journal of Physiology
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• v.16 no.2
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• pp.119-128
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• 1982

The frog truncus arterious were studied with conventional glass microelectrode technique in order to elucidate the underlying mechanism of spontaneous pacemaker activity. The analyses were focussed on the ionic nature of pacemaker current by changing the concentrations of extracellular $K^+$ and, $Na^+$, or by using blockers of K- and Ca-current and chronotropic transmitters. 1) The action potential of the spontaneously active truncus arteriosus has some characteristic feature of maximal distolic potential ranged from -65 to -75 mV, resting potential from -45 to -50 mV and overshoot voltage about +30 mV, respectively. Duration of the action potential taken from rapid upstroke to maximal diastolic potential was about 600 msec. Usual discharge rate was $25{\sim}30/min$ at room temperature $(18{\sim}20^{\circ}C)$. 2) The sensitivity of the resting membrane potential to change extracellular potassium concentrations $(0{\sim}12\;mM)$ was relatively low. Transient hyperpolarization was appeared in the 12 mM K Ringer after 10 min exposure to 0 mM K and it could be related to Na-pump reactivation by high potassium. 3) Reduction of extracellular sodium concetrations diminished the amplitude and frequency of the action potential. In Ringer solution containing 30% Na (substituted by equimolar Tris), spontaneous activity stopped but reappeared as very slow and small action potential. There was no spotaneous activity in zero Na Ringer solution. 4) Caesium(10 mM), K-current blocker decreased the frequency of the action potential and also pacemaker depolarization. Manganese (2 mM) known to be Ca-current antagonist, blocked spontaneous activity completely. 5) Adrenaline and acetylcholine had no chronotropic effect. But adrenaline increased the duration of plateau phase and the magnitude of the action potential in the follower cell. It is concluded that K-, Na-and Ca-current components are involved in the genesis of spontaneous activity of the frog truncus arteriosus like cardiac pacemaker tissues. But the insensitivity of truncus arteriosus to adrenaline and acetylcholine indicates that there are some different control mechanisms of spontaneous rhythm in two tissues.

• Molecules and Cells
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• v.21 no.3
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• pp.337-342
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• 2006

Interstitial cells of Cajal (ICCs) are pacemaker cells that activate the periodic spontaneous depolarization (pacemaker potentials) responsible for the production of slow waves in gastrointestinal smooth muscle. The effects of vasoactive intestinal polypeptide (VIP) on the pacemaker potentials in cultured ICCs from murine small intestine were investigated by whole-cell patch-clamp techniques. Addition of VIP (50 nM-$1{\mu}M$) decreased the amplitude of pacemaker potentials and depolarized resting membrane potentials. To examine the type of receptors involved in ICC, we examined the effects of the $VIP_1$ agonist and found that it had no effect on pacemaker potentials. Pretreatment with $VIP_1$ antagonist ($1{\mu}M$) for 10 min also did not block the VIP (50 nM)-induced effects. On the other hand exposure to 1H-(1,2,4)oxadiazolo(4,3-A)quinoxalin-1-one (ODQ, $100{\mu}M$), an inhibitor of guanylate cyclase, prevented VIP inhibition of pacemaker potentials. Similarly KT-5823 ($1{\mu}M$) or RP-8-CPT-cGMPS ($10{\mu}M$), inhibitors of protein kinase G (PKG) blocked the effect of VIP (50 nM) on pacemaker potentials as did N-nitro-L-arginine (L-NA, $100{\mu}M$), a non-selective nitric oxide synthase (NOS) inhibitor. These results imply that the inhibition of pacemaker activity by VIP depends on the NO-cGMP-PKG pathway.

• The Korean Journal of Physiology and Pharmacology
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• v.19 no.5
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• pp.435-440
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• 2015

This study aimed to investigate the effect of pituitary adenylate cyclase-activating peptide (PACAP) on the pacemaker activity of interstitial cells of Cajal (ICC) in mouse colon and to identify the underlying mechanisms of PACAP action. Spontaneous pacemaker activity of colonic ICC and the effects of PACAP were studied using electrophysiological recordings. Exogenously applied PACAP induced hyperpolarization of the cell membrane and inhibited pacemaker frequency in a dose-dependent manner (from 0.1 nM to 100 nM). To investigate cyclic AMP (cAMP) involvement in the effects of PACAP on ICC, SQ-22536 (an inhibitor of adenylate cyclase) and cell-permeable 8-bromo-cAMP were used. SQ-22536 decreased the frequency of pacemaker potentials, and cell-permeable 8-bromo-cAMP increased the frequency of pacemaker potentials. The effects of SQ-22536 on pacemaker potential frequency and membrane hyperpolarization were rescued by co-treatment with glibenclamide (an ATP-sensitive $K^+$ channel blocker). However, neither $N^G$-nitro-L-arginine methyl ester (L-NAME, a competitive inhibitor of NO synthase) nor 1H-[1,2,4]oxadiazolo[4,3-${\alpha}$]quinoxalin-1-one (ODQ, an inhibitor of guanylate cyclase) had any effect on PACAP-induced activity. In conclusion, this study describes the effects of PACAP on ICC in the mouse colon. PACAP inhibited the pacemaker activity of ICC by acting through ATP-sensitive $K^+$ channels. These results provide evidence of a physiological role for PACAP in regulating gastrointestinal (GI) motility through the modulation of ICC activity.

• Journal of Ginseng Research
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• v.39 no.4
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• pp.314-321
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• 2015

Background: Ginseng belongs to the genus Panax. Its main active ingredients are the ginsenosides. Interstitial cells of Cajal (ICCs) are the pacemaker cells of the gastrointestinal (GI) tract. To understand the effects of ginsenoside Re (GRe) on GI motility, the authors investigated its effects on the pacemaker activity of ICCs of the murine small intestine. Methods: Interstitial cells of Cajal were dissociated from mouse small intestines by enzymatic digestion. The whole-cell patch clamp configuration was used to record pacemaker potentials in cultured ICCs. Changes in cyclic guanosine monophosphate (cGMP) content induced by GRe were investigated. Results: Ginsenoside Re ($20-40{\mu}M$) decreased the amplitude and frequency of ICC pacemaker activity in a concentration-dependent manner. This action was blocked by guanosine 50-[${\beta}-thio$]diphosphate [a guanosine-5'-triphosphate (GTP)-binding protein inhibitor] and by glibenclamide [an adenosine triphosphate (ATP)-sensitive $K^{+}$ channel blocker]. To study the GRe-induced signaling pathway in ICCs, the effects of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (a guanylate cyclase inhibitor) and RP-8-CPT-cGMPS (a protein kinase G inhibitor) were examined. Both inhibitors blocked the inhibitory effect of GRe on ICC pacemaker activity. L-NG-nitroarginine methyl ester ($100{\mu}M$), which is a nonselective nitric oxide synthase (NOS) inhibitor, blocked the effects of GRe on ICC pacemaker activity and GRe-stimulated cGMP production in ICCs. Conclusion: In cultured murine ICCs, GRe inhibits the pacemaker activity of ICCs via the ATP-sensitive potassium ($K^{+}$) channel and the cGMP/NO-dependent pathway. Ginsenoside Re may be a basis for developing novel spasmolytic agents to prevent or alleviate GI motility dysfunction.

• The Korean Journal of Physiology and Pharmacology
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• v.15 no.3
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• pp.129-135
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• 2011

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-$HT_7$ 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.

• The Korean Journal of Physiology and Pharmacology
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• v.12 no.3
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• pp.111-115
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• 2008

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 $30^{\circ}C$ 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.

• The Korean Journal of Physiology and Pharmacology
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• v.10 no.1
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• pp.7-11
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• 2006

Interstitial cells of Cajal (ICCs) are pacemakers in gastrointestinal tracts, regulating rhythmicity by activating nonselective cation channels (NSCCs). In the present study, we investigated the general characteristics and pH-mediated regulation of pacemaker activity in cultured interstitial cells of Cajal. Under voltage clamp mode and at the holding potential of -60 mV, the I-V relationships and difference current showed that there was no reversal potential and voltage-independent inward current. Also, when the holding potentials were changed from +20 mV to -80 mV with intervals of 20 mV, there was little difference in inward current. In pacemaker activity, the resting membrane potential (RMP) was depolarized (In pH 5.5, $23{\pm}1.5$ mV depolarized) and the amplitude was decreased by a decrease of the extracellular pH. However, in case of increase of extracellular pH, the RMP was slightly hyperpolarized and the amplitude was decreased a little. The melastatin type transient receptor potential (TRPM) channel 7 has been suggested to be required for intestinal pacemaking activity. TRPM7 produced large outward currents and small inward currents by voltage ramps, ranging from +100 to -100 mV from a holding potential of -60 mV. The inward current of TRPM7 was dramatically increased by a decrease in the extracellular pH. At pH 4.0, the average inward current amplitude measured at -100 mV was increased by about 7 fold, compared with the current amplitude at pH 7.4. Changes in the outward current (measured at +100 mV) were much smaller than those of the inward current. These results indicate that the resting membrane potential of pacemaking activity might be depolarized by external acidic pH through TRPM7 that is required for intestinal pacemaking activity.