Small Intestinal Interstitial Cells Of Cajal 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.

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.

Trypsin Depolarizes Pacemaker Potentials in Murine Small Intestinal Interstitial Cells of Cajal

Interstitial cells of Cajal (ICCs) generate pacemaker potentials in the gastrointestinal (GI) tract. In this study, the effects of trypsin on pacemaker potentials in murine small intestinal ICCs were examined. We used whole-cell patch-clamp analysis. The results of whole-cell patch-clamp analysis revealed that trypsin dose-dependently depolarized pacemaker potentials and decreased their amplitude. Treatments with the antagonists of neurokinin1 (NK1) and NK2 receptors (SR-140333 and SR-48968, respectively) slightly inhibited the trypsin-induced responses. However, treatment with the combination of SR-140333 and SR-48968 completely inhibited trypsin-induced responses. Trypsin slightly depolarized pacemaker potentials and increased their amplitude after the intracellular application of GDP-β-S. Additionally, incubation in external Ca2+-free solution inhibited trypsin-induced responses. In the presence of U-73122, staurosporine, Go6976, or xestospongin C, trypsin did not depolarize the pacemaker’s potentials. However, trypsin depolarized the pacemaker potentials in the presence of rottlerin. Finally, HC067047, a TRPV4 inhibitor, did not affect the trypsin-induced responses. These results suggest that trypsin depolarized pacemaker potentials through NK1 and NK2 receptors in the murine small intestinal ICCs, with this effect being dependent on the G protein, phospholipase C, protein kinase C, inositol triphosphate pathways, and extracellular Ca2+ but being independent of the TRPV4 pathway. Hence, trypsin-mediated GI motility regulation must be considered for prokinetic drug developments.

HAND1 and BARX1 Act as Transcriptional and Anatomic Determinants of Malignancy in Gastrointestinal Stromal Tumor

Gastrointestinal stromal tumor (GIST) arises from interstitial cells of Cajal (ICC) or their precursors, which are present throughout the gastrointestinal tract. Although gastric GIST is commonly indolent and small intestine GIST more aggressive, a molecular understanding of disease behavior would inform therapy decisions in GIST. Although a core transcription factor (TF) network is conserved across GIST, accessory TFs HAND1 and BARX1 are expressed in a disease state-specific pattern. Here, we characterize two divergent transcriptional programs maintained by HAND1 and BARX1, and evaluate their association with clinical outcomes. We evaluated RNA sequencing and TF chromatin immunoprecipitation with sequencing in GIST samples and cultured cells for transcriptional programs associated with HAND1 and BARX1. Multiplexed tissue-based cyclic immunofluorescence and IHC evaluated tissue- and cell-level expression of TFs and their association with clinical factors. We show that HAND1 is expressed in aggressive GIST, modulating KIT and core TF expression and supporting proliferative cellular programs. In contrast, BARX1 is expressed in indolent and micro-GISTs. HAND1 and BARX1 expression were superior predictors of relapse-free survival, as compared with standard risk stratification, and they predict progression-free survival on imatinib. Reflecting the developmental origins of accessory TF programs, HAND1 was expressed solely in small intestine ICCs, whereas BARX1 expression was restricted to gastric ICCs. Our results define anatomic and transcriptional determinants of GIST and molecular origins of clinical phenotypes. Assessment of HAND1 and BARX1 expression in GIST may provide prognostic information and improve clinical decisions on the administration of adjuvant therapy.

Transient Receptor Potential Canonical 4 and 5 Channel Antagonist ML204 Depolarized Pacemaker Potentials of Interstitial Cells of Cajal.

Background/AimsTo investigate an effect of ML204 (an inhibitor of transient receptor potential canonical 4 and 5 [TRPC4/5] channels) on interstitial cells of Cajal (ICCs) and therefore determine whether TRPC4/5 channels act on ICC-generated pacemaker activity.MethodsWe enforced whole cell patch clamp analysis, measurements of the intracellular Ca2+ concentration, and reverse transcription polymerase chain reaction to determine the effect of ML204 (10 μM) or englerin A (a selective activator of TRPC4/5 channeles, 10 μM) and the existence of TRPC4/5 in mouse small intestinal ICC.ResultsTreatment of ICCs with ML204 or englerin A caused the membrane potentials to depolarize. This depolarization effect of membrane potentials by ML204 in ICCs was observed to be concentration-dependent. After treating Ca2+- and Na+-free solutions or flufenamic acid (a non-selective cation channel blocker), the pacemaker potentials in the ICCs were abolished. A specific anoctamin 1 channel blocker did not have any effect on the pacemaker activity in ML204-untreated control cells; however, they blocked ML204-induced pacemaker activity in ICCs. Specific primers designed against TRPC4 and TRPC5 detected the presence of TRPC4/5 in small intestinal ICCs, and the application of ML204 increased raise the frequency of Ca2+ oscillations in ICCs, as assessed using Fluo-4 AM.ConclusionThe results implied that ML204 could not inhibit the pacemaker activity but depolarized the membrane potential of ICCs by regulating intracellular Ca2+ oscillations and anoctamin 1 channels.

Differential sensitivity of gastric and small intestinal muscles to inducible knockdown of anoctamin 1 and the effects on gastrointestinal motility.

Electrical pacemaking in gastrointestinal muscles is generated by specialized interstitial cells of Cajal that produce the patterns of contractions required for peristalsis and segmentation in the gut. The calcium-activated chloride conductance anoctamin-1 (Ano1) has been shown to be responsible for the generation of pacemaker activity in GI muscles, but this conclusion is established from studies of juvenile animals in which effects of reduced Ano1 on gastric emptying and motor patterns could not be evaluated. Knocking down Ano1 expression using Cre/LoxP technology caused dramatic changes in in gastric motor activity, with disrupted slow waves, abnormal phasic contractions and delayed gastric emptying; modest changes were noted in the small intestine. Comparison of the effects of Ano1 antagonists on muscles from juvenile and adult small intestinal muscles suggests that conductances in addition to Ano1 may develop with age and contribute to pacemaker activity. Interstitial cells of Cajal (ICC) generate slow waves and transduce neurotransmitter signals in the gastrointestinal (GI) tract, facilitating normal motility patterns. ICC express a Ca2+ -activated Cl- conductance (CaCC), and constitutive knockout of the channel protein anoctamin-1 leads to loss of slow waves in gastric and intestinal muscles. These knockout experiments were performed on juvenile mice. However, additional experiments demonstrated significant differences in the sensitivity of gastric and intestinal muscles to antagonists of anoctamin-1 channels. Furthermore, the significance of anoctamin-1 and the electrical and mechanical behaviours facilitated by this conductance have not been evaluated on the motor behaviours of adult animals. Cre/loxP technology was used to generate cell-specific knockdowns of anoctamin-1 in ICC (KitCreERT2/+ ;Ano1tm2jrr/+ ) in GI muscles. The recombination efficiency of KitCreERT was evaluated with an eGFP reporter, molecular techniques and immunohistochemistry. Electrical and contractile experiments were used to examine the consequences of anoctamin-1 knockdown on pacemaker activity, mechanical responses, gastric motility patterns, gastric emptying and GI transit. Reduced anoctamin-1 caused loss of gastric, but not intestinal slow waves. Irregular spike complexes developed in gastric muscles, leading to uncoordinated antral contractions, delayed gastric emptying and increased total GI transit time. Slow waves in intestinal muscles of juvenile mice were more sensitive to anoctamin-1 antagonists than slow waves in adult muscles. The low susceptibility to anoctamin-1 knockdown and weak efficacy of anoctamin-1 antagonists in inhibiting slow waves in adult small intestinal muscles suggest that a conductance in addition to anoctamin-1 may develop in small intestinal ICC with ageing and contribute to pacemaker activity.

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.

The impact of inflammatory cells in malignant ascites on small intestinal ICCs' morphology and function.

Malignant ascites is one of the common complication at the late stage of abdominal cancers, which may deteriorate the environment of abdominal cavity and lead to potential damage of functional cells. Interstitial cells of Cajal (ICCs) are mesoderm-derived mesenchymal cells that function normal gastrointestinal motility. The pathological changes of ICCs or the reduced number may lead to the motility disorders of gastrointestinal tract. In this study, through analysis of malignant ascites which were obtained from cancer patients, we found that inflammatory cells, including tumour-infiltrating lymphocytes, accounted for 17.26 ± 1.31% and tumour-associated macrophages, occupied 19.06 ± 2.27% of total cells in the ascites, suggesting these inflammatory cells, in addition to tumour cells, may exert important influence on the tumour environment of abdominal cavity. We further demonstrated that the number of mice ICCs were significant decreased, as well as morphological and functional damage when ICCs were in the simulated tumour microenvironment in vitro. Additionally, we illustrated intestinal myoelectrical activity reduced and irregular with morphological changes of ICCs using the mice model of malignant ascites. In conclusion, our data suggested that inflammatory cells in malignant ascites may damage ICCs of the small intestine and lead to intestinal motility disorders.

Regulatory Roles of Endogenous Mitogen-Activated Protein Kinases and Tyrosine Kinases in the Pacemaker Activity of Colonic Interstitial Cells of Cajal

Background and Purpose: Mitogen-activated protein (MAP) and tyrosine kinases play an important role in regulating smooth muscle contraction of the gastrointestinal (GI) tract. Interstitial cells of Cajal (ICCs) are pacemaker cells that regulate GI smooth muscle activity. Thus, the role of MAP and tyrosine kinases on the pacemaker potentials of colonic ICCs was investigated. Methods: Cultured ICCs were prepared from mice colons, and their pacemaker potentials were recorded using whole-cell patch clamping. Results: In current-clamping mode, colonic ICCs displayed spontaneous pacemaker potentials. SB203580 (a p38 MAP kinase inhibitor), SP600125 (a c-jun NH₂-terminal kinase (JNK) inhibitor), genistein and herbimycin A (tyrosine kinase inhibitors) blocked the generation of pacemaker potentials. However, PD98059 (a p42/44 MAP kinase inhibitor) had no effects on pacemaker potentials. LY-294002 (phosphoinositide 3-kinase inhibitor) also reduced the pacemaker potential frequency but calphostin C and chelerythrine (protein kinase C inhibitors) had no effects. However, PD98059, SB203589, SP600125, genistein, herbimycin A, LY-294002, and calphostin C had no effect on normal pacemaker activity in small intestinal ICCs. Conclusions: Endogenous p38 MAP kinases, JNKs, tyrosine kinases, and PI3-kinases participate in the generation of pacemaker potentials in colonic ICCs but not in ICCs of the small intestine.

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.

Computational modeling of anoctamin 1 calcium-activated chloride channels as pacemaker channels in interstitial cells of Cajal

Interstitial cells of Cajal (ICC) act as pacemaker cells in the gastrointestinal tract by generating electrical slow waves to regulate rhythmic smooth muscle contractions. Intrinsic Ca(2+) oscillations in ICC appear to produce the slow waves by activating pacemaker currents, currently thought to be carried by the Ca(2+)-activated Cl(-) channel anoctamin 1 (Ano1). In this article we present a novel model of small intestinal ICC pacemaker activity that incorporates store-operated Ca(2+) entry and a new model of Ano1 current. A series of simulations were carried out with the ICC model to investigate current controversies about the reversal potential of the Ano1 Cl(-) current in ICC and to predict the characteristics of the other ion channels that are necessary to generate slow waves. The model results show that Ano1 is a plausible pacemaker channel when coupled to a store-operated Ca(2+) channel but suggest that small cyclical depolarizations may still occur in ICC in Ano1 knockout mice. The results predict that voltage-dependent Ca(2+) current is likely to be negligible during the slow wave plateau phase. The model shows that the Cl(-) equilibrium potential is an important modulator of slow wave morphology, highlighting the need for a better understanding of Cl(-) dynamics in ICC.

Poncirus trifoliate fruit modulates pacemaker activity in interstitial cells of Cajal from the murine small intestine

Ethnopharmacological relevancePoncirus fructus (PF) has been widely used as a traditional medicine in Eastern Asia, especially to ameliorate the symptoms of gastrointestinal (GI) disorders related to abnormal GI motility. Aim of the studyPoncirus fructus (PF), also known as Poncirus trifoliata (L.) Raf. (Rutaceae), is widely used as a traditional medicine in Eastern Asia mainly to ameliorate the symptoms of gastrointestinal (GI) disorders related to abnormal GI motility. In a previous study, a methanol extract of PF was found to have particularly potent gastroprokinetic effects. Interstitial cells of Cajal (ICCs) are pacemaker cells in the gastrointestinal tract, but the action mechanisms of PF extract in mouse small intestinal ICCs have not been investigated. Therefore, in the present study, we investigated the effects of a methanol extract of PF (MPF) in mouse small intestinal ICCs. In addition, we sought to identify the receptors involved. Materials and MethodsEnzymatic digestions were used to dissociate ICCs from small intestines. The whole-cell patch-clamp configuration was used to record potentials (current clamp) from cultured ICCs. In addition, we analyzed intracellular Ca2+ concentrations ([Ca2+]i). ResultsMPF decreased the amplitudes of pacemaker potentials in ICCs, and depolarized resting membrane potentials in a concentration dependent manner. Y25130 (a 5-HT3 receptor antagonist) and RS39604 (a 5-HT4 receptor antagonist) blocked MPF-induced membrane depolarizations, whereas SB269970 (a 5-HT7 receptor antagonist) did not. Pretreatment with Na+ or Ca2+-free solution or thapsigargin (a Ca2+-ATPase inhibitor in endoplasmic reticulum) abolished the generation of pacemaker potentials and suppressed MPF-induced activity. [Ca2+]i analysis showed that MPF increased [Ca2+]i. Furthermore, treatments with PD 98059, SB203580, or JNK II inhibitor blocked MPF-induced membrane depolarizations in ICCs. ConclusionThese results suggest that MPF modulates pacemaker potentials through 5-HT3 and 5-HT4 receptor-mediated pathways via external Na+ and Ca2+ influx, and via Ca2+ release from internal stores in a mitogen-activated protein kinase dependent manner. The study shows MPF is a good candidate for the development of a gastroprokinetic agent. In view of the effects of MPF on ICCs, further research is required, particularly to identify the active compound(s) involved and to determine their action mechanisms.

Effects of Histamine on Cultured Interstitial Cells of Cajal in Murine Small Intestine

Interstitial cells of Cajal (ICCs) are the pacemaker cells in the gastrointestinal tract, and histamine is known to regulate neuronal activity, control vascular tone, alter endothelial permeability, and modulate gastric acid secretion. However, the action mechanisms of histamine in mouse small intestinal ICCs have not been previously investigated, and thus, in the present study, we investigated the effects of histamine on mouse small intestinal ICCs, and sought to identify the receptors involved. Enzymatic digestions were used to dissociate ICCs from small intestines, and the whole-cell patch-clamp configuration was used to record potentials (in current clamp mode) from cultured ICCs. Histamine was found to depolarize resting membrane potentials concentration dependently, and whereas 2-PEA (a selective H1 receptor agonist) induced membrane depolarizations, Dimaprit (a selective H2-agonist), R-alpha-methylhistamine (R-alpha-MeHa; a selective H3-agonist), and 4-methylhistamine (4-MH; a selective H4-agonist) did not. Pretreatment with Ca2+-free solution or thapsigargin (a Ca2+-ATPase inhibitor in endoplasmic reticulum) abolished the generation of pacemaker potentials and suppressed histamine-induced membrane depolarization. Furthermore, treatments with U-73122 (a phospholipase C inhibitor) or 5-fluoro-2-indolyl des-chlorohalopemide (FIPI; a phospholipase D inhibitor) blocked histamine-induced membrane depolarizations in ICCs. On the other hand, KT5720 (a protein kinase A inhibitor) did not block histamine-induced membrane depolarization. These results suggest that histamine modulates pacemaker potentials through H1 receptor-mediated pathways via external Ca2+ influx and Ca2+ release from internal stores in a PLC and PLD dependent manner.

Neurotensin modulates pacemaker activity in interstitial cells of Cajal from the mouse small intestine.

Neurotensin, a tridecapeptide localized in the gut to discrete enteroendocrine cells of the small bowel mucosa, is a hormone that plays an important role in gastrointestinal secretion, growth, and motility. Neurotensin has inhibitory and excitatory effects on peristaltic activity and produces contractile and relaxant responses in intestinal smooth muscle. Our objective in this study is to investigate the effects of neurotensin in small intestinal interstitial cells of Cajal (ICC) and elucidate the mechanism. To determine the electrophysiological effects of neurotensin on ICC, whole-cell patch clamp recordings were performed in cultured ICC from the small intestine. Exposure to neurotensin depolarized the membrane of pacemaker cells and produced tonic inward pacemaker currents. Only neurotensin receptor1 was identified when RT-PCR and immunocytochemistry were performed with mRNA isolated from small intestinal ICC and c-Kit positive cells. Neurotensin-induced tonic inward pacemaker currents were blocked by external Na⁺-free solution and in the presence of flufenamic acid, an inhibitor of non-selective cation channels. Furthermore, neurotensin-induced action is blocked either by treatment with U73122, a phospholipase C inhibitor, or thapsigargin, a Ca²⁺-ATPase inhibitor in ICC. We found that neurotensin increased spontaneous intracellular Ca²⁺ oscillations as seen with fluo4/AM recording. These results suggest that neurotensin modulates pacemaker currents via the activation of non-selective cation channels by intracellular Ca²⁺-release through neurotensin receptor1.

Pharmacological Differences of Endothelin Receptors-mediated Modulation in Cultured Interstitial Cells of Cajal from the Murine Small and Large Intestine

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. Under current clamping, ICCs had a mean resting membrane potential of -58 ± 3 mV and externally applied ET produced membrane depolarization in a dosedependent manner. These effects were reduced by intracellular GDP beta S. A comparison of the concentration-dependent membrane depolarizations on pacemaker potentials to ET-1, ET-2 and ET-3 showed a rank order of potency ET-1≥ET-2≥ET-3 in cultured murine small intestinal ICCs. The pretreatment with Ca²⁺-free solution and thapsigargin, a Ca²⁺-ATPase inhibitor in endoplasmic reticulum, abolished the generation of pacemaker potentials and suppressed the ET-1 induced membrane depolarizations. Chelerythrine and calphostin C, protein kinase C inhibitors or naproxen, an inhibitor of cyclooxygenase, did not block the ET-1 induced effects on pacemaker potentials. Pretreatment with BQ-123 (ET_Areceptor antagonist) or BQ-788 (ET_Breceptor antagonist) blocked the ET-1 induced effects on pacemaker potentials in cultured murine small intestinal ICCs. However, pretreatment with BQ-788 selectively did not block the ET-1 induced effects on pacemaker potentials in cultured murine large intestinal ICCs. Also, only externally applied selective ET_Breceptor agonist, IRL 1620 did not show any influence on pacemaker potentials in cultured murine large intestine ICCs. RT-PCR results indicated the presence of the ET_Aand ET_Breceptor in ICCs. These results suggested that ET-1 modulates pacemaker potentials through ET_Aand ET_Breceptor activation in murine small intestinal ICCs and ET_Areceptor activation in murine large intestinal ICCs by external Ca²⁺ influx and internal Ca²⁺ release via protein kinase C or cyclooxygenase-independent mechanism. Therefore, the ICCs are targets for ET and their interaction can affect intestinal motility.

Purification of interstitial cells of Cajal by fluorescence-activated cell sorting.

Interstitial cells of Cajal (ICC) in the gastrointestinal tract generate and propagate slow waves and mediate neuromuscular neurotransmission. Although damages to ICC have been described in several gastrointestinal motor disorders, analysis of their gene expression in health and disease has been problematic because of the difficulties in isolating these cells. Our goal was to develop techniques for large-scale purification of ICC. Murine ICC were identified in live gastrointestinal muscles with fluorescent Kit antibodies. Because this technique also labels resident macrophages nonspecifically, we attempted to separate ICC from these cells by fluorescence-activated cell sorting with or without immunomagnetic presorting. Efficacy and specificity of ICC purification were tested by quantitative RT-PCR of cell-specific markers. Fluorescence-based separation of small intestinal ICC from unlabeled cells and macrophages tagged with F4/80 antibodies yielded 30,000-40,000 cells and approximately 60-fold enrichment of c-kit mRNA. However, the macrophage marker CD68 was also enriched approximately 6-fold. Magnetic presorting of ICC did not significantly improve selectivity. After labeling contaminating cells with additional paramagnetic (anti-CD11b, -CD11c) and fluorescent antibodies (anti-CD11b) and depleting them by magnetic presorting, we harvested approximately 2,000-4,000 cells from single gastric corpus-antrum muscles and detected an approximately 30-fold increase in c-kit mRNA, no enrichment of mast cells, and an approximately 4-fold reduction of CD68 expression. Adding labeled anti-CD45 antibody to our cocktail further increased c-kit enrichment and eliminated mast cells and macrophages. Smooth muscle cells and myenteric neurons were also depleted. We conclude that immunofluorescence-based sorting can yield ICC in sufficiently high numbers and purity to permit detailed molecular analyses.