Pacemaker Activity In Interstitial Cells Research Articles

5-Hydroxytryptamine Enhances the Pacemaker Activity of Interstitial Cells of Cajal in Mouse Colon.

We examined the localization of the 5-hydroxytryptamine (5-HT) receptor and its effects on mouse colonic interstitial cells of Cajal (ICCs) using electrophysiological techniques. Treatment with 5-HT increased the pacemaker activity in colonic ICCs with depolarization of membrane potentials in a dose-dependent manner. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channel blockers blocked pacemaker activity and 5-HT-induced effects. Moreover, an adenylate cyclase inhibitor inhibited 5-HT-induced effects, and cell-permeable 8-bromo-cAMP increased the pacemaker activity. Various agonists of the 5-HT receptor subtype were working in colonic ICCs, including the 5-HT4 receptor. In small intestinal ICCs, 5-HT depolarized the membrane potentials transiently. Adenylate cyclase inhibitors or HCN blockers did not show any influence on 5-HT-induced effects. Anoctamin-1 (ANO1) or T-type Ca2+ channel blockers inhibited the pacemaker activity of colonic ICCs and blocked 5-HT-induced effects. A tyrosine protein kinase inhibitor inhibited pacemaker activity in colonic ICCs under controlled conditions but did not show any influence on 5-HT-induced effects. Among mitogen-activated protein kinase (MAPK) inhibitors, a p38 MAPK inhibitor inhibited 5-HT-induced effects on colonic ICCs. Thus, 5-HT's effect on pacemaker activity in small intestinal and colonic ICCs has excitatory but variable patterns. ANO1, T-type Ca2+, and HCN channels are involved in 5-HT-induced effects, and MAPKs are involved in 5-HT effects in colonic ICCs.

Network Pharmacological Analysis and Experimental Validation of the Effect of Smilacis Glabrae Rhixoma on Gastrointestinal Motility Disorder.

Gastrointestinal motility disorder (GMD) is a disease that causes digestive problems due to inhibition of the movement of the gastrointestinal tract and is one of the diseases that reduce the quality of life of modern people. Smilacis Glabrae Rhixoma (SGR) is a traditional herbal medicine for many diseases and is sometimes prescribed to improve digestion. As a network pharmacological approach, we searched the TCMSP database for SGR, reviewed its constituents and target genes, and analyzed its relevance to gastrointestinal motility disorder. The effects of the SGR extract on the pacemaker activity in interstitial cells of Cajal (ICC) and gastric emptying were investigated. In addition, using the GMD mouse model through acetic acid (AA), we investigated the locomotor effect of SGR on the intestinal transit rate (ITR). As a result of network pharmacology analysis, 56 compounds out of 74 candidate compounds of SGR have targets, the number of targets is 390 targets, and there are 904 combinations. Seventeen compounds of SGR were related to GMD, and as a result of comparing the related genes with the GMD-related genes, 17 genes (active only) corresponded to both. When looking at the relationship network between GMD and SGR, it was confirmed that quercetin, resveratrol, SCN5A, TNF, and FOS were most closely related to GMD. In addition, the SGR extract regulated the pacemaker activity in ICC and recovered the delayed gastric emptying. As a result of feeding the SGR extract to AA-induced GMD mice, it was confirmed that the ITR decreased by AA was restored by the SGR extract. Through network pharmacology, it was confirmed that quercetin, resveratrol, SCN5A, TNF, and FOS were related to GMD in SGR, and these were closely related to intestinal motility. Based on these results, it is suggested that SGR in GMD restores digestion through the recovery of intestinal motility.

Effect of adenosine-1 receptor activation on pacemaker activity of interstitial cells of Cajal from mouse colon.

Adenosine plays an important role on gastrointestinal (GI) motility through adenosine receptors. Interstitial cells of Cajal (ICC) are pacemaker cells that regulate GI smooth muscle activity. The functional role and its signal mechanism of adenosine on the pacemaker activity were investigated using whole-cell patch clamp, RT-PCR, and intracellular Ca2+-imaging with ICC from mouse colon. Adenosine depolarized the membrane potentials and increased the pacemaker potential frequency, which was blocked by a selective A1-receptor antagonist, but not A2a-, A2b, or A3-receptor antagonist. A selective A1 receptor agonist represented similar effects as those of adenosine and mRNA transcript of A1-receptor was expressed in ICC. The adenosine-induced effects were blocked by phospholipase C (PLC) and a Ca2+-ATPase inhibitor. Adenosine increased spontaneous intracellular Ca2+ oscillations, as seen fluo4/AM. Both hyperpolarization-activated cyclic nucleotide (HCN) channel inhibitors and adenylate cyclase inhibitors blocked the adenosine-induced effects. And adenosine increased the basal cellular adenylate cyclase activity in colonic ICC. However, adenosine and adenylate cyclase inhibitors did not show any influence on pacemaker activity in small intestinal ICC for a comparison with that of the small intestine. These results suggest adenosine modulates the pacemaker potentials by acting HCN channels- and intracellular Ca2+- dependent mechanisms through A1-receptor. Therefore, adenosine may be a therapeutic target in colonic motility disorders.

Na+/Ca2 + Exchange and Pacemaker Activity of Interstitial Cells of Cajal.

Interstitial cells of Cajal (ICC) are pacemaker cells that generate electrical slow waves in gastrointestinal (GI) smooth muscles. Slow waves organize basic motor patterns, such as peristalsis and segmentation in the GI tract. Slow waves depend upon activation of Ca2+-activated Cl– channels (CaCC) encoded by Ano1. Slow waves consist of an upstroke depolarization and a sustained plateau potential that is the main factor leading to excitation-contraction coupling. The plateau phase can last for seconds in some regions of the GI tract. How elevated Ca2+ is maintained throughout the duration of slow waves, which is necessary for sustained activation of CaCC, is unknown. Modeling has suggested a role for Na+/Ca2+ exchanger (NCX) in regulating CaCC currents in ICC, so we tested this idea on murine intestinal ICC. ICC of small and large intestine express NCX isoforms. NCX3 is closely associated with ANO1 in ICC, as shown by immunoprecipitation and proximity ligation assays (PLA). KB-R7943, an inhibitor of NCX, increased CaCC current in ICC, suggesting that NCX, acting in Ca2+ exit mode, helps to regulate basal [Ca2+]i in these cells. Shifting NCX into Ca2+ entry mode by replacing extracellular Na+ with Li+ increased spontaneous transient inward currents (STICs), due to activation of CaCC. Stepping ICC from −80 to −40 mV activated slow wave currents that were reduced in amplitude and duration by NCX inhibitors, KB-R7943 and SN-6, and enhanced by increasing the NCX driving force. SN-6 reduced the duration of clustered Ca2+ transients that underlie the activation of CaCC and the plateau phase of slow waves. Our results suggest that NCX participates in slow waves as modeling has predicted. Dynamic changes in membrane potential and ionic gradients during slow waves appear to flip the directionality of NCX, facilitating removal of Ca2+ during the inter-slow wave interval and providing Ca2+ for sustained activation of ANO1 during the slow wave plateau phase.

Disruption of the pacemaker activity of interstitial cells of Cajal via nitric oxide contributes to postoperative ileus.

Interstitial cells of Cajal (ICC) serve as intestinal pacemakers. Postoperative ileus (POI) is a gastrointestinal motility disorder that occurs following abdominal surgery, which is caused by inflammation-induced dysfunction of smooth muscles and enteric neurons. However, the participation of ICC in POI is not well understood. In this study, we investigated the functional changes of ICC in a mouse model of POI. Intestinal manipulation (IM) was performed to induce POI. At 24h or 48h after IM, the field potential of the intestinal tunica muscularis was investigated. Tissues were also examined by immunohistochemistry and electron microscopic analysis. Gastrointestinal transit was significantly decreased with intestinal tunica muscularis inflammation at 24h after IM, which was ameliorated at 48h after IM. The generation and propagation of pacemaker potentials were disrupted at 24h after IM and recovered to the control level at 48h after IM. ICC networks, detected by c-Kit immunoreactivity, were remarkably disrupted at 24h after IM. Electron microscopic analysis revealed abnormal vacuoles in the ICC cytoplasm. Interestingly, the ICC networks recovered at 48h after IM. Administration of aminoguanidine, an inducible nitric oxide synthase inhibitor, suppressed the disruption of ICC networks. Ileal smooth muscle tissue cultured in the presence of nitric oxide donor, showed disrupted ICC networks. The generation and propagation of pacemaker potentials by ICC are disrupted via nitric oxide after IM, and this disruption may contribute to POI. When inflammation is ameliorated, ICC can recover their pacemaker function.

Effects of ATP on Pacemaker Activity of Interstitial Cells of Cajal from the Mouse Small Intestine.

Purinergic receptors play an important role in regulating gastrointestinal (GI) motility. Interstitial cells of Cajal (ICCs) are pacemaker cells that regulate GI smooth muscle activity. We studied the functional roles of external adenosine 5′-triphosphate (ATP) on pacemaker activity in cultured ICCs from mouse small intestines by using the whole-cell patch clamp technique and intracellular Ca2+ ([Ca2+]i) imaging. External ATP dose-dependently depolarized the resting membrane and produced tonic inward pacemaker currents, and these effects were antagonized by suramin, a purinergic P2 receptor antagonist. ATP-induced effects on pacemaker currents were suppressed by an external Na+-free solution and inhibited by the nonselective cation channel blockers, flufenamic acid and niflumic acid. The removal of external Ca2+ or treatment with thapsigargin (inhibitor of Ca2+ uptake into endoplasmic reticulum) inhibited the ATP-induced effects on pacemaker currents. Spontaneous [Ca2+]i oscillations were enhanced by external ATP. These results suggest that external ATP modulates pacemaker activity by activating nonselective cation channels via external Ca2+ influx and [Ca2+]i release from the endoplasmic reticulum. Thus, it seems that activating the purinergic P2 receptor may modulate GI motility by acting on ICCs in the small intestine.

Effects of Ca2+-Activated Cl- Channel ANO1inhibitors on Pacemaker Activity in Interstitial Cells of Cajal

Background/Aims: Anoctamin1 (Ca²⁺-activated Cl^- channel, ANO1) is a specific marker of the interstitial cells of Cajal (ICC) in the gastrointestinal tract, and are candidate proteins that can function as pacemaker channels. Recently, novel selective ANO1 inhibitors were discovered and used to study Ca²⁺-activated Cl^- channels. Therefore, to investigate whether ANO1 channels function as pacemaker channels, selective ANO1 inhibitors were tested with respect to the pacemaker potentials in ICC. Methods: Whole-cell patch-clamp recording, RT-PCR, and intracellular Ca²⁺ ([Ca²⁺]_i) imaging were performed in cultured ICC obtained from mice. Results: Though CaCCinh-A01 (5 µM), T16Ainh-A01 (5 µM), and MONNA (5 µM) (selective ANO1 inhibitors) blocked the generation of pacemaker potentials in colonic ICC, they did not do so in small intestinal ICC. Though nifulmic acid (10 µM) and DIDS (10 µM) (classical Ca²⁺-activated Cl^- channel inhibitors) also had no effect in small intestinal ICC, they suppressed the generation of pacemaker potentials in colonic ICC. In addition, knockdown of ANO1 reduced the pacemaker potential frequency in colonic ICC alone. Though ANO1 inhibitors suppressed [Ca²⁺]_i oscillations in colonic ICC, they did not do so in small intestinal ICC. T-type Ca²⁺ channels were expressed in the both the small intestinal and colonic ICC, but mibefradil (5 µM) and NiCl₂ (30 µM) (T-type Ca²⁺ channel inhibitors) inhibited the generation of pacemaker potentials in colonic ICC alone. Conclusion: These results indicate that though ANO1 and T-type Ca²⁺ channels participate in generating pacemaker potentials in colonic ICC, they do not do so in small intestinal ICC. Therefore, the mechanisms underlying pacemaking in ICC might be different in the small intestine and the colon.

ATP-sensitive K+ channels maintain resting membrane potential in interstitial cells of Cajal from the mouse colon

To investigate the role of ATP-sensitive K+(KATP) channels on pacemaker activity in interstitial cells of Cajal (ICC), whole-cell patch clamping, RT-PCR, and intracellular Ca2+([Ca2+]i) imaging were performed in cultured colonic ICC. Pinacidil (a K+ channel opener) hyperpolarized the membrane and inhibited the generation of pacemaker potential, and this effect was reversed by glibenclamide (a KATP channel blocker). RT-PCR showed that Kir 6.1 and SUR2B were expressed in Ano-1 positive colonic ICC. Glibenclamide depolarized the membrane and increased pacemaker potential frequency. However, 5-hydroxydecanoic acid (a mitochondrial KATP channel blocker) had no effects on pacemaker potentials. Phorbol 12-myristate 13-acetate (PMA; a protein kinase C activator) blocked the pinacidil-induced effects, and PMA alone depolarized the membrane and increased pacemaker potential frequency. Cell-permeable 8-bromo-cyclic AMP also increased pacemaker potential frequency. Recordings of spontaneous intracellular Ca2+([Ca2+]i) oscillations showed that glibenclamide increased the frequency of [Ca2+]i oscillations. In small intestinal ICC, glibenclamide alone did not alter the generation of pacemaker potentials, and Kir 6.2 and SUR2B were expressed in Ano-1 positive ICC. Therefore, KATP channels in colonic ICC are activated in resting state and play an important role in maintaining resting membrane potential.

Orai1 mediates tumor-promoting store-operated Ca2+ entry in human gastrointestinal stromal tumors via c-KIT and the extracellular signal-regulated kinase pathway.

Gastrointestinal stromal tumors originate from interstitial cells of Cajal, the pacemaker cells of the gut. Ca2+ regulates the pacemaker activity of interstitial cells of Cajal. Store-operated Ca2+ entry mediates the majority of Ca2+ entry in most cancer cells and may be a factor in regulating intracellular Ca2+ in interstitial cells of Cajal and gastrointestinal stromal tumors. Therefore, a blockade of this mechanism may affect the progression of gastrointestinal stromal tumors. Orai1 is the pore subunit of store-operated Ca2+ channels. Here, we reported that Orai1 was overexpressed in gastrointestinal stromal tumor tissues and was positively correlated with a high-risk grade in gastrointestinal stromal tumor patients. Furthermore, upon Orai1 silencing, the functional store-operated Ca2+ entry in gastrointestinal stromal tumor cells was decreased, indicating that the function of store-operated Ca2+ entry was mediated by Orai1. Inhibition of Orai1-mediated store-operated Ca2+ entry by Orai1 silencing or store-operated Ca2+ entry blockers (SKF-96365 and 2-aminoethyl diphenylborate) induced obvious cell proliferation suppression, cell-cycle distribution, and apoptosis stimulation in GIST-T1 cells. Conversely, Orai1 overexpression increased store-operated Ca2+ entry and cell proliferation in GIST882 cells. In addition, we found that activation of c-KIT and the extracellular signal-regulated kinase pathway participated in the oncogenic functions of Orai1-mediated store-operated Ca2+ entry in gastrointestinal stromal tumor cells. These results revealed that Orai1-mediated store-operated Ca2+ entry is critical for gastrointestinal stromal tumor cell proliferation via c-KIT and ERK signaling pathway activation. Orai1-mediated store-operated Ca2+ entry plays an oncogenic role and may be a novel prognostic factor and therapeutic target for patients with gastrointestinal stromal tumors.

Na+-K+-Cl- cotransporter (NKCC) maintains the chloride gradient to sustain pacemaker activity in interstitial cells of Cajal.

Interstitial cells of Cajal (ICC) generate electrical slow waves by coordinated openings of ANO1 channels, a Ca2+-activated Cl- (CaCC) conductance. Efflux of Cl- during slow waves must be significant, as there is high current density during slow-wave currents and slow waves are of sufficient magnitude to depolarize the syncytium of smooth muscle cells and PDGFRα+ cells to which they are electrically coupled. We investigated how the driving force for Cl- current is maintained in ICC. We found robust expression of Slc12a2 (which encodes an Na+-K+-Cl- cotransporter, NKCC1) and immunohistochemical confirmation that NKCC1 is expressed in ICC. With the use of the gramicidin permeabilized-patch technique, which is reported to not disturb [Cl-]i, the reversal potential for spontaneous transient inward currents (ESTICs) was -10.5 mV. This value corresponds to the peak of slow waves when they are recorded directly from ICC in situ. Inhibition of NKCC1 with bumetanide shifted ESTICs to more negative potentials within a few minutes and reduced pacemaker activity. Bumetanide had no direct effects on ANO1 or CaV3.2 channels expressed in HEK293 cells or L-type Ca2+ currents. Reducing extracellular Cl- to 10 mM shifted ESTICs to positive potentials as predicted by the Nernst equation. The relatively rapid shift in ESTICs when NKCC1 was blocked suggests that significant changes in the transmembrane Cl- gradient occur during the slow-wave cycle, possibly within microdomains formed between endoplasmic reticulum and the plasma membrane in ICC. Recovery of Cl- via NKCC1 might have additional consequences on shaping the waveforms of slow waves via Na+ entry into microdomains.

Regulation of the Pacemaker Activity of Colonic Interstitial Cells of Cajal by Protease-Activated Receptors: Involvement of Hyperpolarization-Activated Cyclic Nucleotide Channels

Background and Purpose: The exact mechanism of protease-activated receptors (PARs) on pacemaker activity of interstitial cells of Cajal (ICCs) has not been reported. We investigated the effects on pacemaker activity by the activation of PARs and its signal mechanisms in colonic ICCs. Methods: The whole-cell patch-clamp technique, RT-PCR and Ca²⁺ imaging were used in cultured ICCs from mouse colon. Results: PAR-1 and PAR-2 were expressed in Ano-1 positive ICCs. TFLLR-NH₂ (a PAR-1 agonist) and trypsin (a PAR-2 agonist) depolarized the membrane and increased the pacemaker potential frequency. U-73122 (a phospholipase C (PLC) inhibitor) and thapsigargin (a Ca²⁺ ATPase inhibitor) suppressed the TFLLR-NH₂- and trypsin-induced effects on pacemaker potential. TFLLR-NH₂ and trypsin also increased intracellular Ca²⁺ ([Ca²⁺]_i) intensity with increasing of Ca²⁺ oscillations. Genistein (a tyrosine kinase inhibitor), SP600125 (a JNK inhibitor), CsCl, ZD7288, clonidine (hyperpolarization-activated cyclic nucleotide (HCN) channel blockers), SQ-22536 and dideoxyadenosine (adenylate cyclase inhibitors) suppressed the increased pacemaker potential frequency without effects on depolarization of the membrane induced by TFLLR-NH₂ and trypsin. Conclusion: These results suggest that activation of PAR-1 and PAR-2 modulates the pacemaker activity of colonic ICCs through the PLC-dependent [Ca²⁺]_i release pathway. The increased pacemaker potential frequency by PAR-1 and PAR-2 was also dependent on tyrosine kinase, JNK, and HCN activation.

Stimulus-induced pacemaker activity in interstitial cells of Cajal associated with the deep muscular plexus of the small intestine.

The ICC-DMP have been proposed to generate stimulus-dependent pacemaker activity, rhythmic transient depolarizations, that take part in orchestrating segmentation and clustered propulsive motor patterns in the small intestine. However, little is known about the fundamental properties of ICC-DMP. This study was undertaken to increase our understanding of intrinsic properties of the ICC-DMP through calcium imaging and intracellular electrical recordings. Without stimulation, most ICC-DMP were quiescent. In some preparations ICC-DMP generated rhythmic low-frequency calcium oscillations (<10 cpm) with or without high frequency activity superimposed (>35 cpm). Immunohistochemistry proved the existence of NK1R on the ICC-DMP and close contacts between ICC-DMP and substance P-positive nerves. Substance P (25 nM) induced low-frequency calcium oscillations that were synchronized across the ICC-DMP network. Substance P also induced low frequency rhythmic transient depolarizations (<10cpm) in circular muscle cells close to the ICC-DMP. An intracellular recording from a positively identified ICC-DMP showed rhythmic transient depolarizations with superimposed high frequency activity. To investigate if quiescent ICC-DMP were chronically inhibited by nitrergic activity, nNOS was inhibited, but without effect. Substance P changes non-synchronized high frequency flickering or quiescence in ICC-DMP into strong rhythmic calcium transients that are synchronized within the network; they are associated with rhythmic transient depolarizations within the same frequency range. We hypothesize that Substance P, released from nerves, can evoke rhythmicity in ICC-DMP, thereby providing it with potential pacemaker activity.

Functional effects of β3-adrenoceptor on pacemaker activity in interstitial cells of Cajal from the mouse colon

We investigated the presence of β3-adrenoceptor and its functional effects on pacemaker potentials in colonic interstitial cells of Cajal (ICCs) from mice. The whole-cell patch clamp technique was used to record pacemaker potentials in cultured ICCs and reverse transcription polymerase chain reaction (RT-PCR) was performed to detect the mRNA transcript levels β-adrenoceptors. The β3-adrenoceptor agonist, BRL37344, reduced the frequency of pacemaker potentials in a concentration-dependent manner. The inhibitory effects of BRL37344 were blocked by the pretreatment of propranolol, a nonspecific β-adrenoceptor antagonist, but not by the selective β1-adrenoceptor antagonist atenolol and the selective β2-adrenoceptor antagonist butoxamine. β3-adrenoceptor antagonists SR59230A and L748337 blocked the inhibitory effects of BRL37344. RT-PCR revealed mRNA transcripts of β1- and β3-adrenoceptor, but not β2-adrenoceptor, in c-kit- and Ano-1-positive colonic ICCs. The K+ channel blockers tetraethylammonium, apamin, and glibenclamide did not block the effects of BRL37344. Nω-Nitro-l-arginine methyl ester hydrochloride (L-NAME), an NO synthase inhibitor, and chelerythrine, a protein kinase C inhibitor, also did not block the effects of BRL37344. Noradrenaline mimicked the effects of BRL37344 in colonic ICCs. However, the inhibitory effects of noradrenaline on pacemaker potentials were blocked only by pretreatment with atenolol but not by butoxamine, SR59230A, or L748337. In small intestinal ICCs, BRL37344 had no effect on pacemaker potentials and mRNA transcripts of β1-and β2-adrenoceptor, but not β3-adrenoceptor were detected. These results suggest that β3-adrenoceptors are present in colonic ICCs and may play a role in regulating gastrointestinal motility by the inhibition of pacemaker potentials.

Intracellular Ca2+ release from endoplasmic reticulum regulates slow wave currents and pacemaker activity of interstitial cells of Cajal

Interstitial cells of Cajal (ICC) provide pacemaker activity in gastrointestinal muscles that underlies segmental and peristaltic contractions. ICC generate electrical slow waves that are due to large-amplitude inward currents resulting from anoctamin 1 (ANO1) channels, which are Ca(2+)-activated Cl(-) channels. We investigated the hypothesis that the Ca(2+) responsible for the stochastic activation of ANO1 channels during spontaneous transient inward currents (STICs) and synchronized activation of ANO1 channels during slow wave currents comes from intracellular Ca(2+) stores. ICC, obtained from the small intestine of Kit(+/copGFP) mice, were studied under voltage and current clamp to determine the effects of blocking Ca(2+) uptake into stores and release of Ca(2+) via inositol 1,4,5-trisphosphate (IP3)-dependent and ryanodine-sensitive channels. Cyclocpiazonic acid, thapsigargin, 2-APB, and xestospongin C inhibited STICs and slow wave currents. Ryanodine and tetracaine also inhibited STICs and slow wave currents. Store-active compounds had no direct effects on ANO1 channels expressed in human embryonic kidney-293 cells. Under current clamp, store-active drugs caused significant depolarization of ICC and reduced spontaneous transient depolarizations (STDs). After block of ryanodine receptors with ryanodine and tetracaine, repolarization did not restore STDs. ANO1 expressed in ICC has limited access to cytoplasmic Ca(2+) concentration, suggesting that pacemaker activity depends on Ca(2+) dynamics in restricted microdomains. Our data from studies of isolated ICC differ somewhat from studies on intact muscles and suggest that release of Ca(2+) from both IP3 and ryanodine receptors is important in generating pacemaker activity in ICC.

Pituitary Adenylate Cyclase-activating Polypeptide Inhibits Pacemaker Activity of Colonic Interstitial Cells of Cajal.

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 NG-nitro-L-arginine methyl ester (L-NAME, a competitive inhibitor of NO synthase) nor 1H-[1,2,4]oxadiazolo[4,3-α]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.

Quercetin Inhibits Pacemaker Potentials via Nitric Oxide/cGMP-Dependent Activation and TRPM7/ANO1 Channels in Cultured Interstitial Cells of Cajal from Mouse Small Intestine

Background: Quercetin regulates gastrointestinal (GI) motor activity but the molecular mechanism involved has not been determined. The authors investigated the effects of quercetin, a flavonoid present in various foods, on the pacemaker activities of interstitial cells of Cajal (ICCs) in murine small intestine in vitro and on GI motility in vivo. Materials and Methods: Enzymatic digestion was used to dissociate ICCs from mouse small intestines. The whole-cell patch-clamp configuration was used to record pacemaker potentials in cultured ICCs in the absence or presence of quercetin and to record membrane currents of transient receptor potential melastatin (TRPM) 7 or transmembrane protein 16A (Tmem16A, anoctamin1 (ANO1)) overexpressed in human embryonic kidney (HEK) 293 cells. The in vivo effects of quercetin on GI motility were investigated by measuring the intestinal transit rates (ITRs) of Evans blue in normal mice. Results: Quercetin (100-200 μM) decreased the amplitudes and frequencies of pacemaker activity in a concentration-dependent manner in current clamp mode, but this action was blocked by naloxone (a pan-opioid receptor antagonist) and by GDPβS (a GTP-binding protein inhibitor). However, potassium channels were not involved in these inhibitory effects of quercetin. To study the quercetin signaling pathway, we examined the effects of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of guanylate cyclase, and of RP-8-CPT-cGMPS, an inhibitor of protein kinase G (PKG). These inhibitors blocked the inhibitory effects of quercetin on pacemaker activities. Also, L-NAME (100 μM), a non-selective NO synthase (NOS) inhibitor, blocked the effects of quercetin on pacemaker activity and quercetin stimulated cGMP production. Furthermore, quercetin inhibited both Ca²⁺-activated Cl^- channels (TMEM16A, ANO1) and TRPM7 channels. In vivo, quercetin (10-100 mg/kg, p.o.) decreased ITRs in normal mice in a dose-dependent manner. Conclusions: Quercetin inhibited ICC pacemaker activities by inhibiting TRPM7 and ANO1 via opioid receptor signaling pathways in cultured murine ICCs. The study shows quercetin attenuates GI tract motility, and suggests quercetin be considered the basis for the development of novel spasmolytic agents for the prevention or alleviation of GI motility dysfunctions.

Effects of San-Huang-Xie-Xin-tang, a traditional Chinese prescription for clearing away heat and toxin, on the pacemaker activities of interstitial cells of Cajal from the murine small intestine

Ethnopharmacological relevanceSan-Huang-Xie-Xin-Tang (SHXXT) is a traditional Chinese medicinal formula composed of Coptidis rhizoma (Coptis chinesis Franch), Scutellariae radix (Scutellaria baicalensis Georgi), and Rhei rhizoma (Rheum officinale Baill) and is widely used in Eastern Asia, especially to ameliorate the symptoms of gastrointestinal (GI) disorders related to gastritis, gastric bleeding, peptic ulcers, and abnormal GI motility Aim of the studyInterstitial cells of Cajal (ICCs) are pacemaker cells in the GI tract that generate rhythmic oscillations in membrane potentials known as slow waves. Because GI disorders, especially abnormal GI motility, are major lifelong problems, the authors investigated the effects of SHXXT on mouse small intestine ICCs, and sought to identify the receptors and the action mechanisms involved. Materials and methodsEnzymatic digestions were used to dissociate ICCs from small intestines, and the whole-cell patch-clamp configuration was used to record potentials generated by cultured ICCs. ResultsSHXXT produced membrane depolarization in current-clamp mode, and Y25130 (a 5-HT3 receptor antagonist) and RS39604 (a 5-HT4 receptor antagonist) blocked SHXXT-induced membrane depolarizations, whereas SB269970 (a 5-HT7 receptor antagonist) did not. However, during external Ca2+ free conditions or in the presence of thapsigargin, SHXXT did not exhibit membrane depolarization. Furthermore, the application of flufenamic acid (a nonselective cation channel (NSCC) blocker) or DIDS (a chloride channel blocker) abolished pacemaker potential generation and blocked SHXXT-induced membrane depolarizations. In addition, SHXXT-induced membrane depolarizations, which are dependent on G-protein, in ICCs were blocked by PD 98059 (a p42/44 mitogen-activated protein kinase (MAPK) inhibitor), SB203580 (a p38 MAPK inhibitor), and by a c-jun NH2-terminal kinase (JNK) II inhibitor. Regarding the components of SHXXT, Coptidis rhizome and Rhei rhizoma modulated ICC pacemaking activity, whereas Scutellariae radix did not. ConclusionSHXXT modulates pacemaker potentials via 5-HT3 and 5-HT4 receptor-mediated pathways, external Ca2+ influx, and Ca2+ release from internal stores. Furthermore, NSCCs and Cl- channels play important roles in the regulation of pacemaking activity in a MAPK dependent manner in ICCs. The regulation of pacemaking activity by SHXXT may be due to the activity of Coptidis rhizome and Rhei rhizome. The study shows SHXXT can modulate the pacemaking activity of ICCs in the GI tract, and thus, suggests SHXXT has potential pharmacological relevance for the treatment of GI motility disorders.

Basal cGMP regulates the resting pacemaker potential frequency of cultured mouse colonic interstitial cells of Cajal

Cyclic guanosine 3',5'-monophosphate (cGMP) inhibited the generation of pacemaker activity in interstitial cells of Cajal (ICCs) from the small intestine. However, cGMP role on pacemaker activity in colonic ICCs has not been reported yet. Thus, we investigated the role of cGMP in pacemaker activity regulation by colonic ICCs. We performed a whole-cell patch-clamp and Ca(2+) imaging in cultured ICCs from mouse colon. 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, an inhibitor of guanylate cyclase) increased the pacemaker potential frequency, whereas zaprinast (an inhibitor of phosphodiesterase) and cell-permeable 8-bromo-cGMP decreased the pacemaker potential frequency. KT-5823 (an inhibitor of protein kinase G [PKG]) did not affect the pacemaker potential. L-N(G)-nitroarginine methyl ester (L-NAME, an inhibitor of nitric oxide [NO] synthase) increased the pacemaker potential frequency, whereas (±)-S-nitroso-N-acetylpenicillamine (SNAP, a NO donor) decreased the pacemaker potential frequency. Glibenclamide (an ATP-sensitive K(+) channel blocker) did not block the effects of cell-permeable 8-bromo-cGMP and SNAP. Recordings of spontaneous intracellular Ca(2+) ([Ca(2+)]i) oscillations revealed that ODQ and L-NAME increased [Ca(2+)]i oscillations. In contrast, zaprinast, 8-bromo cGMP, and SNAP decreased the [Ca(2+)]i oscillations. Basal cGMP levels regulate the resting pacemaker potential frequency by the alteration on Ca(2+) release via a PKG-independent pathway. Additionally, the endogenous release of NO seems to be responsible maintaining basal cGMP levels in colonic ICCs.

Characteristics of Diprophylline-Induced Bidirectional Modulation on Rat Jejunal Contractility

In this study, we propose that diprophylline exerts bidirectional modulation (BM) on the isolated rat jejunal segment depending on its contractile state. The results supported the hypothesis. Diprophylline (20 µM) exerted stimulatory effects on the contractility of jejunal segment in six low contractile states while inhibitory effects in six high contractile states, showing the characteristics of BM. Diprophylline-induced stimulatory effect was significantly blocked by atropine, indicating the correlation with cholinergic activation. Diprophylline-induced inhibitory effect was partially blocked by phentolamine, propranolol, and L-N-Nitro-Arginine respectively, indicating their correlation with sympathetic activation and nitric oxide-mediated relaxing mechanisms. Diprophylline-induced BM was abolished by tetrodotoxin or in a Ca2+ free condition or pretreated with tyrosine kinase inhibitor imatinib, suggesting that diprophylline-induced BM is Ca2+ dependent, and that it requires the presence of enteric nervous system as well as pacemaker activity of interstitial cells of Cajal. Diprophylline significantly increased the reduced MLCK expression and myosin extent in constipation-prominent rats and significantly decreased the increased MLCK expression and myosin extent in diarrhea-prominent rats, suggesting that the change of MLCK expression may also be involved in diprophylline-induced BM on rat jejunal contractility. In summary, diprophylline-exerted BM depends on the contractile states of the jejunal segments, requires the presence of Ca2+, enteric nervous system, pacemaker activity of interstitial cells of Cajal, and MLCK-correlated myosin phosphorylation. The results suggest the potential implication of diprophylline in relieving alternative hypo/hyper intestinal motility.

Effects of lubiprostone on pacemaker activity of interstitial cells of cajal from the mouse colon.

Lubiprostone is a chloride (Cl-) channel activator derived from prostaglandin E1 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 EP1, EP2, EP3, and EP4 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 [Ca2+]-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.