The mechanism and spread of pacemaker activity through myenteric interstitial cells of Cajal in human small intestine

BACKGROUND & AIMS: It has been generally assumed that interstitial cells of Cajal (ICC) in the human gastrointestinal tract have similar functions to those in rodents, but no direct experimental evidence exists to date for this assumption. This is an important question because pathologists have noted decreased numbers of ICC in patients with a variety of motility disorders, and some have speculated that loss of ICC could be responsible for motor dysfunction. Our aims were to determine whether myenteric ICC (ICC-MY) in human jejunum are pacemaker cells and whether these cells actively propagate pacemaker activity. METHODS: The mucosa and submucosa were removed, and strips of longitudinal muscle were peeled away to reveal the ICC-MY network. ICC networks were loaded with the Ca(2+) indicator fluo-4, and pacemaker activity was recorded via high-speed video imaging at 36.5 degrees C +/- 0.5 degrees C. RESULTS: Rhythmic, biphasic Ca(2+) transients (6.03 +/- 0.33 cycles/min) occurred in Kit-positive ICC-MY. These consisted of a rapidly propagating upstroke phase that initiated a sustained plateau phase, which was associated with Ca(2+) spikes in neighboring smooth muscle. Pacemaker activity was dependent on inositol 1,4,5-triphosphate receptor-operated stores and mitochondrial function. The upstroke phase of Ca(2+) transients in ICC-MY appeared to result from Ca(2+) influx through dihydropyridine-resistant Ca(2+) channels, whereas the plateau phase was attributed to Ca(2+) release from inositol 1,4,5-triphosphate receptor-operated Ca(2+) stores. CONCLUSIONS: Each ICC-MY in human jejunum generates spontaneous pacemaker activity that actively propagates through the ICC network. Loss of these cells could severely disrupt the normal function of the human small intestine.     

Decreased interstitial cells of Cajal in the sigmoid colon of patients with slow transit constipation

Background and aims Slow transit constipation (STC) is a colonic motor disorder that is characterized by measurably delayed movement of materials through the colon. Although abnormalities in the neuronal networks of the colon have been demonstrated in patients with STC, the etiology of STC remains unclear. Interstitial cells of Cajal (ICC) have been shown to be the pacemaker cells of the intestine and have been implied in the pathogenesis of a number of gastrointestinal motility dysfunctions, including idiopathic STC. This study aimed to determine the normal distribution of ICC within the colon of the Chinese and also to determine if ICC are decreased in Chinese STC patients.Patients and methods Twelve patients with STC and eight age-matched normal controls were studied. Specimens of sigmoid colon were obtained immediately after resection. ICC were identified with a monoclonal antibody to c-kit by an indirect immunofluorescence method. Immunostained tissues were examined with a laser scanning confocal microscope and the area occupied by ICC was calculated with an image analysis system.Results ICC were located in the external muscle layers including myenteric plexus (MP) and submucosal border (SMB). Two types of Kit-positive ICC were observed: bipolar cells characterized by one or two long processes and multipolar cells characterized by long stellate processes extending in various directions. A higher percentage of ICC was present in the MP regions and circular muscle (CM) layers compared with the SMB and longitudinal muscle (LM) layers. Tissues from STC patients showed a considerable decrease in the number of ICC located in the four regions (ICC-LM, ICC-MP, ICC-CM, ICC-SMB), especially the ICC-SMB, in which ICC almost completely disappeared.Conclusions Similar distribution of ICC was observed in the normal sigmoid colon of the Chinese. Decreased area of c-kit+ ICC may play an important role in the pathophysiology of STC. It remains to be determined whether the loss of ICC is primary or secondary to another lesion.     

Deficiency of KIT-positive cells in the colon of patients with diabetes mellitus

BACKGROUND: Diabetes mellitus is a well-known cause of gastrointestinal dysmotility. The pathogenesis of diabetic gastroenteropathy is mainly considered to be a neuropathy, but the cause of dysmotility remains unknown. Interstitial cells of Cajal (ICC), which express c-kit receptor tyrosine kinase (KIT), are considered to be pacemaker cells for the gastrointestinal movement. Therefore, we investigated a possible involvement of ICC in the pathogenesis of diabetic gastroenteropathy in humans. METHODS: The KIT-positive cells in the proper muscle layer of the colon were detected by immunohistochemistry in patients with diabetes mellitus and normal control subjects. Mast cells, which are also known to express KIT, were detected by staining with Alcian blue. The numbers of KIT-positive cells and Alcian blue-positive cells in the proper muscle layer were counted under the microscope and the number of KIT-positive cells apart from Alcian blue-positive cells was calculated. RESULTS: In the normal control subjects, KIT-positive cells were located at the myenteric plexus region and in the circular muscle layer of the colon. Their distribution pattern was similar to that of ICC. The average number of KIT-positive cells, apart from mast cells (which reflects the number of ICC), in patients with diabetes mellitus was approximately 40% of that found in normal subjects. CONCLUSIONS: Deficiency of ICC might be related to the pathogenesis of diabetic gastroenteropathy in humans.     

胰岛素对2型糖尿病患者结肠Cajal间质细胞及乙酰胆碱变化的影响

目的 比较接受不同治疗的2型糖尿病患者与非糖尿病对照的结肠cajal间质细胞(ICC)及乙酰胆碱(Ach)的形态、分布及数量,了解糖尿病患者结肠ICC及Ach的变化以及胰岛素治疗对其的影响.方法 取结肠癌手术标本切缘的正常结肠组织共81例,其中非糖尿病对照29例,2型糖尿病52例(胰岛素治疗24例,口服降糖药治疗28例).免疫组化检测c-kit阳性细胞及Ach表达;甲苯胺蓝染色肥大细胞. c-kit阳性细胞与肥大细胞数之差为ICC数目,图像处理软件分析Ach表达及密度.结果 结肠的黏膜下层、环行肌及纵行肌层内(IM)、肌间(MY)神经丛均有c-kit阳性细胞分布.糖尿病患者的ICC细胞突起明显减少、分布松散,较多空泡形成,这些变化在口服降糖药组更明显.ICC数目比较:对照组>胰岛素组>口服降糖药组(ICC-MY:60.12比23.95比16.49, P=0.000; ICC.IM:41.79比33.18比25.88, P=0.000).结肠黏膜下神经丛和肌间神经丛均有Ach表达,糖尿病患者Ach阳性细胞分布松散,较多空泡形成,口服降糖药组更明显. Ach阳性表达分析,面密度:对照组>胰岛素组>口服降糖药组(147.50比103.82比86.38, P=0.000).对非糖尿病对照组的分析表明,ICC数目及Ach表达与年龄、性别无相关性.结论 糖尿病患者结肠ICC及Ach较对照组分布松散,较多空泡形成,ICC数量及Ach表达明显减少.胰岛素对上述变化具有保护作用.结肠ICC数目及Ach表达与年龄及性别无关.     

Novel human and mouse genes encoding a shank-interacting protein and its upregulation in gastric fundus of W/WV mouse

BACKGROUND AND AIMS: A division of labor exists between different classes of interstitial cells of Cajal (ICC) in the gastrointestinal tract. In the stomach and small intestine, ICC at the level of the myenteric plexus (IC-MY) act as slow wave pacemaker cells, whereas intramuscular ICC (IC-IM) in the stomach act as intermediaries in enteric motor neurotransmission. The muscle layers of the gastric fundus do not have IC-MY, therefore electric slow waves are not generated. Intramuscular ICC are absent in the gastric fundus of W/WV mutant mice, and excitatory and inhibitory motor nerve responses are reduced in these tissues. The absence of IC-IM in W/WV mutants in the fundus provides a unique opportunity to study the molecular changes that are associated with the loss of these cells. METHODS: The tissue gene expression of wild-type and W/WV mice from gastric fundus was assayed using a murine microarray chip analysis displaying a total of 8734 elements. RESULTS: Twenty-one queries were differentially expressed in wild-type and W/WV mice. One candidate gene, encoding a novel protein homologous to rat Shank-interacting protein (Sharpin) was significantly upregulated in fed and starved W/WV mice. The full-length clone of the murine gene and its human counterpart were isolated and designated as Shank-interacting protein-like 1 (SIPL1). Human SIPL1 complementary DNA encodes a protein of 345 amino acids. This gene was localized to chromosome 8. SIPL1 was abundantly expressed in human stomach and small intestine, and scarcely expressed in cecum and rectum. CONCLUSIONS: Gene analysis showed that SIPL1 differentially express in the gastric fundus of normal and W/WV mice. The upregulation of SIPL1 in the fundus of W/WV mice, and expression in the upper gastrointestinal tract suggest that the SIPL1 gene could be associated with ICC function in mice and humans.     

Studies on expression and function of the TMEM16A calcium-activated chloride channel

Calcium-activated chloride channels (CaCC) with similar hallmark features are present in many cell types and mediate important physiological functions including epithelial secretion, sensory signal transduction, and smooth muscle contraction. Having identified TMEM16A of the transmembrane proteins with unknown function (TMEM) 16 family as a CaCC subunit, we have developed antibodies specific for mouse TMEM16A, as evidenced by the absence of immunoreactivity in TMEM16A knockout mice. Here, we show that TMEM16A is located in the apical membranes of epithelial cells in exocrine glands and trachea. In addition, TMEM16A is expressed in airway smooth muscle cells and the smooth muscle cells of reproductive tracts, the oviduct and ductus epididymis. In the gastrointestinal (GI) tract, TMEM16A is absent from smooth muscle cells, but present in the interstitial cells of Cajal (ICC), the pacemaker cells that control smooth muscle contraction. The physiological importance of TMEM16A is underscored by the diminished rhythmic contraction of gastric smooth muscle from TMEM16A knockout mice. The TMEM16A expression pattern established in this study thus provides a roadmap for the analyses of physiological functions of calcium-activated chloride channels that contain TMEM16A subunits.     

The Importance of Interstitial Cells of Cajal in the Gastrointestinal Tract

Gastrointestinal (GI) motility function and its regulation is a complex process involving collaboration and communication of multiple cell types such as enteric neurons, interstitial cells of Cajal (ICC), and smooth muscle cells. Recent advances in GI research made a better understanding of ICC function and their role in the GI tract, and studies based on different types of techniques have shown that ICC, as an integral part of the GI neuromuscular apparatus, transduce inputs from enteric motor neurons, generate intrinsic electrical rhythmicity in phasic smooth muscles, and have a mechanical sensation ability. Absence or improper function of these cells has been linked to some GI tract disorders. This paper provides a general overview of ICC; their discovery, subtypes, function, locations in the GI tract, and some disorders associated with their loss or disease, and highlights some controversial issues with regard to the importance of ICC in the GI tract.     

An enteric occult reflex underlies accommodation and slow transit in the distal large bowel

BACKGROUND & AIMS: Transit of fecal material through the human colon takes > or =30 hours, whereas transit through the small intestine takes 24 hours. The mechanisms underlying colonic storage and slow transit have yet to be elucidated. Our aim was to determine whether an intrinsic neural mechanism underlies these phenomena. METHODS: Recordings were made from circular muscle (CM) cells and myenteric neurons in the isolated guinea pig distal colon using intracellular recordings and Ca(2+) imaging techniques. Video imaging was used to determine the effects of colonic filling and pellet transit. RESULTS: Circumferential stretch generated ongoing oral excitatory and anal inhibitory junction potentials in the CM. The application of longitudinal stretch inhibited all junction potentials. N-omega-nitro-L-arginine (100 micromol/L) completely reversed the inhibitory effects of longitudinal stretch suggesting that nitric oxide (NO) inhibited interneurons controlling peristaltic circuits. Ca(2+) imaging in preparations that were stretched in both axes revealed ongoing firing in nNOS +ve descending neurons, even when synaptic transmission was blocked. Inhibitory postsynaptic potentials were evoked in mechanosensitive interneurons that were blocked by N-omega-nitro-L-arginine (100 micromol/L). Pellet transit was inhibited by longitudinal stretch. Filling the colon with fluid led to colonic elongation and an inhibition of motility. CONCLUSIONS: Our data support the novel hypothesis that slow transit and accommodation are generated by release of NO from descending (nNOS +ve) interneurons triggered by colonic elongation. We refer to this powerful inhibitory reflex as the intrinsic occult reflex (hidden from observation) because it withdraws motor activity from the muscle.     

Cajal间质细胞与糖尿病胃轻瘫的关系

Cajal间质细胞（ICC）是胃肠道中与肠神经细胞和平滑肌密切相关的间质细胞,网络状分布于整个胃肠组织,对胃肠慢波的产生及胃肠动力的维持起关键作用。研究表明,糖尿病胃轻瘫（DGP）患者及其动物模型均存在ICC数目明显减少、结构破坏、且与神经末梢及平滑肌之间的连接减少。因此,ICC的异常可能是DGP的重要原因之一。本文就ICC与DGP的研究现状进行探讨。     

Interstitial cells of Cajal are present in human extrahepatic bile ducts

Background and Aims: Interstitial cells of Cajal (ICC) are distributed with smooth muscle throughout the gastrointestinal tract and are involved in regulating motility. ICC were recently discovered in the wall of the human gallbladder. This study sought to determine whether ICC are present in human bile ducts. Methods: Biliary tract samples were obtained from several sources: surgical specimens (n = 16, 11 women, mean age 61 years); archival post‐mortem specimen (n = 1, 86 years, man); and cadavers (n = 2, 68 and 80 years, men). Paraffin‐embedded sections (3 µm) from the gallbladder (fundus, body and neck) and both extrahepatic and intrahepatic bile ducts were investigated. A double immunofluorescence protocol using polyclonal and monoclonal c‐kit antibodies and mast cell tryptase was used to distinguish c‐kit‐positive cells with typical ICC morphology from c‐kit‐positive mast cells. Small bowel samples were used as positive controls. ICC in the gallbladder were confirmed by ultrastructural study. Results: c‐kit‐positive cells with characteristic ICC morphology were identified in the subepithelial and muscular layers of the gallbladder and extrahepatic bile ducts. They were most prominent within the muscle layer of the extrahepatic bile ducts where they were organized into loosely arranged laminae running parallel to circular smooth muscle fibers. ICC were not found in intrahepatic bile ducts. Conclusion: This study demonstrates for the first time that ICC are present in human extrahepatic bile ducts where they are more densely aggregated than in the gallbladder. This cellular network is likely to be involved in biliary tract motility and its related disorders.     

急性坏死性胰腺炎大鼠肠道Cajal间质细胞以及c-kit mRNA表达的改变

目的 检测急性坏死性胰腺炎(ANP)大鼠小肠组织中Cajal间质细胞(ICC)的改变,探讨其参与胃肠道动力障碍的机制.方法 24只雄性SD大鼠按数字表法随机分为ANP组和对照组,各12只.采用胆总管逆行注射5%牛磺胆酸钠建立ANP大鼠模型.术后23 h自胃管注入美蓝1.0 ml,1 h后处死大鼠,测量幽门至蓝染距离,计算活体肠道推进率;胰腺和小肠组织常规病理检查;应用免疫组化和实时PCR法检测ICC标志物酪氨酸激酶(c-kit)蛋白及mRNA的表达;应用透射电镜观察ICC超微结构改变.结果 ANP组胰腺腺泡细胞小叶结构破坏,片状出血,灶性坏死,大量炎症细胞浸润;小肠黏膜水肿、糜烂,炎症细胞浸润.ANP组肠道推进率为(41.55±3.85)%,较对照组的(68.66±2.66)%显著下降(P＜0.05);c-kit阳性细胞数较对照组减少(56.11±5.09对88.47±4.49,P＜0.05);c-kit mRNA表达亦较对照组下降(0.64±0.33对1.29±0.91,P＜0.05).ANP组ICC的线粒体空泡变,周围突起减少,与平滑肌细胞之间的连接结构不清.结论 ICC减少和超微结构改变是ANP时胃肠道动力障碍的重要机制之一,而ICC的这些改变可能是由于c-kit基因表达下降所致.     

胃肠道神经-Cajal间质细胞-平滑肌网络研究进展

Cajal间质细胞是胃肠起搏细胞,并具有传导神经递质的作用.近年来的研究表明胃肠神经-ICC-平滑肌网络间存在密切的联系.此文就近期胃肠神经-ICC-平滑肌网络的研究进展作一综述.     

小鼠空肠cajal间质细胞的分离与培养

目的研究CD1小鼠空肠cajal间质细胞(ICC)的分离和培养方法,为进一步研究其生理学特征提供基础。方法无菌条件下取小鼠空肠组织,分离出平滑肌肌条,采取组织块培养法,在24孔培养板中进行原代培养,光镜下观察其形态,用荧光标记的特异性C_Kit抗体进行鉴定。结果倒置显微镜下观察ICC有多个突起,并有次级分支,相互之间形成网络。免疫荧光法可见ICC呈阳性。结论组织块培养法可以简便、有效地培养ICC。所培养的ICC细胞生物学特性有待进一步研究。     

In Vivo Gastric and Intestinal Slow Waves in W/W<Superscript>v</Superscript> Mice

The aim was to investigate whether there are regular gastric and intestinal slow waves in conscious W/W^v mice. Eleven W/W^v mice and 11 wild-type mice were implanted with two pairs of electrodes in the stomach and small intestine. Gastrointestinal slow waves were recorded both under anesthesia and in the conscious state. Atropine and verapamil were given separately to an additional 10 W/W^v mice. Results were as follows. (1) The conscious W/W^v mice showed lower rhythmic slow waves in the small intestine (77.1 vs 93.5%; P < 0.001). However, the frequency (10.7 vs 18.8 cpm; P < 0.0001) and the antregrade propagation of intestinal slow waves in W/W^v mice were significantly lower than in the controls. In the stomach, regular slow waves were recorded in both groups, with no difference between the two groups. (2) Anesthesia significantly impaired both gastric and intestinal slow waves in both groups. (3) Atropine and verapamil had no effects on the rhythmicity of intestinal slow waves. We conclude that ICC-MY may not be the sole pacemaker cells for slow waves in the small intestine.There may be some abnormality of smooth muscle cells in W/W^v mice that causes a reduction in the frequency, rhythmicity, and antegrade propagation of slow waves.Xiaohua Hou is a Visiting Scientist from the Division of Gastroenterology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.     

Interstitial cells of Cajal, the Maestro in health and disease

Interstitial cells of Cajal (ICC) are important players in the symphony of gut motility. They have a very signif icant physiological role orchestrating the normal peristaltic activity of the digestive system. They are the pacemaker cells in gastrointestinal (GI) muscles. Absence, reduction in number or altered integrity of the ICC network may have a dramatic effect on GI system motility. More understanding of ICC physiology will foster advances in physiology of gut motility which will help in a future break...     

Blockade of kit signaling induces transdifferentiation of interstitial cells of cajal to a smooth muscle phenotype.

BACKGROUND & AIMS: Interstitial cells of Cajal (ICC) serve as pacemaker cells and mediators of neurotransmission from the enteric nervous system to gastrointestinal muscles. ICC develop from mesenchymal cells that express c-Kit, and signaling via Kit receptors is necessary for normal development of ICC. We studied the fate of functionally developed ICC after blockade of Kit receptors to determine whether ICC undergo cell death or whether the phenotype of the cells is modified. The fate of undeveloped ICC was also investigated. METHODS: Neutralizing, anti-Kit monoclonal antibody (ACK2) was administered to mice for 8 days after birth. ICC in the small intestine were examined by immunohistochemistry and electron microscopy. Occurrence of apoptosis was also assayed. RESULTS: When Kit receptors were blocked, ICC nearly disappeared from the small intestine. Apoptosis was not detected in regions where ICC are normally distributed. Remaining Kit-immunopositive cells in the pacemaker region of the small intestine developed ultrastructural features similar to smooth muscle cells, including prominent filament bundles and expression of the muscle-specific intermediate filament protein, desmin, and smooth muscle myosin. ICC of the deep muscular plexus normally develop after birth in the mouse. Precursors of these cells remained in an undifferentiated state when Kit was blocked. CONCLUSIONS: These data, along with previous studies showing that ICC in the pacemaker region of the small intestine and longitudinal muscle cells develop from the same Kit-immunopositive precursor cells, suggest inherent plasticity between the ICC and smooth muscle cells that is regulated by Kit-dependent cell signaling.     

Interstitial cells of cajal and inflammation-induced motor dysfunction in the mouse small intestine

BACKGROUND & AIMS: Interstitial cells of Cajal (ICC) play an important role in the control of gastrointestinal motility. We aimed to determine a potential role for ICC in the pathophysiology of inflammation-induced motor disorders. METHODS: Effects of Trichinella spiralis infection on electrical pacemaker activity, the structure of ICC associated with Auerbach's plexus, and in vivo motor patterns were studied in the mouse small intestine. RESULTS: Between days 1 and 15 after infection, structural damage occurred in the network of ICC, particularly in the processes connecting ICC to each other and to smooth muscle cells. This was associated with desynchronization of electrical pacemaker activity. Abnormal slow wave activity occurred, including doubling of frequency and electrical quiescence, leading to the development of ectopic pacemaker activity in vivo. In vivo motor patterns in the small intestine changed from consistent peristaltic contractile activity in control animals to periods of quiescence alternating with both orally and aborally propagating contractile activity in the presence of inflammation. Sixty days after infection, all parameters studied had returned to normal values. CONCLUSIONS: Inflammation-induced alterations in the network of ICC of the small intestine associated with Auerbach's plexus lead to disorganization of motor patterns. Because of the strong temporal correlation between damage to the ICC network, electrical uncoupling, the appearance of ectopic pacemaker activity, and the occurrence of retrograde peristalsis, it is concluded that ICC can play a major role in inflammation-induced motor disturbances.     

Plasticity of electrical pacemaking by interstitial cells of Cajal and gastric dysrhythmias in W/W mutant mice

BACKGROUND & AIMS: Interstitial cells of Cajal (ICC) generate and propagate slow waves in the stomach. Gastric peristalsis depends on a proximal-to-distal gradient in slow wave frequency. We tested whether the gastric frequency gradient was an intrinsic property of ICC and whether dysrhythmias result from disruptions of ICC networks. METHODS: We studied wild-type (WT) and W/W(V) mice, which have only myenteric (pacemaker) ICC in the stomach. ICC distributions were analyzed by Kit immunofluorescence. Pacemaking in tissues was studied by intracellular electrophysiologic recording and in cultured ICC by monitoring mitochondrial [Ca(2+)] oscillations with rhod-2 fluorescence or membrane potential with DiBAC(4)(3) fluorescence. RESULTS: Slow wave frequencies were constant throughout WT gastric muscle sheets containing corpus and antrum. Separating the antrum from the corpus caused a significant drop in antral slow wave frequency. ICC from WT antrums also displayed significantly slower pacemaker frequencies than corpus ICC, but the corpus pacemaker frequency dominated in cocultures of corpus and antrum ICC. Myenteric ICC networks were reduced in W/W(V) mice, particularly in the corpus. In W/W(V) mice, separating the antrum from the corpus failed to reduce antral slow wave frequency. Antral pacemaker frequency in ICC from W/W(V) stomachs was the same as in corpus ICC. CONCLUSIONS: The proximal-to-distal slow wave frequency gradient and entrainment of distal electrical activity by proximal pacemakers are fundamental properties of gastric ICC. Chronic depletion of ICC networks disrupts the proximal-to-distal frequency gradient, and emergence of ectopic pacemakers in the antrum may be caused by "reprogramming" of the ICC pacemaker apparatus.