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Anti-c-Kit protein immunoreactive cells corresponding to the interstitial cells of Cajal in the guinea pig small intestine
Abstract
Interstitial cells of Cajal (ICC) of the guinea-pig small intestine were studied with whole-mount preparations by using the zinc iodide-osmic acid method (ZIO) and immunohistochemistry for vimentin and c-kit receptor tyrosine kinase, and by electron microscopy. The myenteric ICC visualized with ZIO staining are immunopositive to both anti-c-kit antibody (ACK-2) and anti-vimentin antibody (V9), and constitute an independent cellular network from the myenteric plexus. Those cells are characterized by many mitochondria, abundant intermediate filaments, and surface cell membranes not covered with a basal lamina. They are connected with each other by gap junctions at tips of the cytoplasmic processes. It is concluded that the myenteric ICC of the guinea-pig intestine are fibroblast-like cells and that they correspond to the c-kit expressing cells regarded as the intestinal pacemaker.
... Kit signaling is considered to be essential for the development and functional maintenance of ICCs (Maeda et al., 1992;Huizinga et al., 1995). Moreover, this receptor has been used as a special marker to identify ICCs in the GI tract for more than a decade (Komuro and Zhou, 1996). ...
... To obtain whole-mount preparations, the stomach, small intestine and colon (n ¼ 3 for each segment) were placed in acetone for 30 min and the smooth muscle layer containing ICCs was prepared with the aid of a dissection microscope. The immunolabeling procedures used have been previously described (Komuro and Zhou, 1996). Briefly, ICCs were identified using a rat monoclonal antibody raised against Kit (ACK2, diluted 1:100; eBioscience) and a mouse monoclonal antibody raised against vimentin (V9, diluted 1:50; Dako). ...
This study investigates whether CD44 immunopositive cells truly correspond to the interstitial cells of Cajal (ICCs) in the muscular layers of the gastrointestinal tract in guinea pigs and Balb/c mice using immunohistochemistry with antibodies directed against CD44, Kit, vimentin and neurofilament 200 (NF200). All the sub-groups of ICCs were immunopositive for the anti-Kit antibody in the muscular layers of stomach, small intestine and colon in both cross sections and whole-mount preparations. Kit/CD44/vimentin triple immunolabeling showed that all the ICCs in different segments and muscular layers of the digestive tract were CD44, Kit and vimentin immunopositive. Kit/CD44/NF200 triple immunolabeling revealed that neither enteric nerves nor other major cells were CD44 immunopositive in the muscular layers, apart from the ICCs. CD44 and Kit were co-localized in the same group of cells, apart from a very small number (0.6%) of CD44 immunopositive cells that were not Kit immunopositive. Our results indicate that these CD44 immunopositive cells truly correspond to ICCs, thus immunolocalisation of CD44 can be used as a special marker, in addition to Kit, to identify ICCs in the digestive tract in adult guinea pigs and mice.
... The small intestine was inflated with acetone for 30 min (room temperature), then opened along the mesenteric border, then the mucosa was removed, and the longitudinal smooth muscle layer containing the ICC-MY (also referred to as the ICC associated with Auerbach's plexus or ICC-MP by some authors) and the circular smooth muscle layer associated with ICC around the deep muscular plexus were prepared with the aid of a dissection microscope. The immunostaining procedures have been described previously [26]. Briefly, ICC were identified by using a rat monoclonal antibody raised against Kit (ACK2, 5 μg ml −1 ; eBioscience) and immunoreactivity was detected by using a Cy3-conjugated secondary antibody (antirat IgG, 1:100; Zymed). ...
... We have confirmed these previous results showing cell death by using TUNEL methodology and about 10-15% of the apoptotic Kit+ cells are at an early stage after I/R. Vimentin, structural protein, is colocalized with Kit in ICC [26]. The Kit/vimentin/TUNEL labeling cells further confirm a true loss of ICC which suggests that the apoptosis of ICC is also involved in the cell number reduction as well as the reduction of Kit expression. ...
This study aimed at evaluating whether apoptosis of interstitial cells of Cajal (ICC), smooth muscle cells (SMC), and enteric neurons was involved in a guinea pig model of intestinal ischemia and reperfusion injury. The small intestinal segments were resected at either 6 (I60/R6h) and 12 h (I60/R12h) or 7 (I60/R7d) to 14 (I60/R14d) days after 60 min intestinal ischemia in the adult guinea pigs and studied by immunohistochemistry with anti-Kit, 5-bromo-2'-deoxyuridine (BrdU), alpha-smooth muscle actin, vimentin, and beta-tublin III antibodies. Also, apoptosis was tested by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method. In the I60/R12h injury, there was a approximately 50% decrease of Kit+ cells in cell numbers at the level of myenteric plexus and a number of Kit-/vimentin-positive cells were labeled by TUNEL. Also, a few SMC and enteric neurons were TUNEL positive. The Kit+ ICC recovered to normal and a number of Kit-/BrdU-double-positive cells were observed in the I60/R14d group. Our results indicated that the intestinal I/R injury could lead to apoptosis of ICC, SMC, and enteric neurons which may contribute to the gastrointestinal motility disorders, and proliferation was involved in the recovery of ICC.
... [63,64], and guinea-pigs [65,66]. These studies and further investigations have increased our understanding of the complex architecture of ICC networks in relation to the ENS and the intestinal smooth muscle layers [67]. Recently, Ano1 has been identified as a highly specific marker for all subtypes of ICCs within the gastrointestinal tract of mice and humans, and its expression has been associated with the generation of electric slow waves [12] (Figs. 1, 2 and 3). ...
Interstitial cells of Cajal (ICCs) are pacemaker cells of gastrointestinal motility that generate and transmit electrical slow waves to smooth muscle cells in the gut wall, thus inducing phasic contractions and coordinated peristalsis. Traditionally, tyrosine-protein kinase Kit (c-kit), also known as CD117 or mast/stem cell growth factor receptor, has been used as the primary marker of ICCs in pathology specimens. More recently, the Ca ²⁺ -activated chloride channel, anoctamin-1, has been introduced as a more specific marker of ICCs. Over the years, various gastrointestinal motility disorders have been described in infants and young children in which symptoms of functional bowel obstruction arise from ICC-related neuromuscular dysfunction of the colon and rectum. The current article provides a comprehensive overview of the embryonic origin, distribution, and functions of ICCs, while also illustrating the absence or deficiency of ICCs in pediatric patients with Hirschsprung disease intestinal neuronal dysplasia, isolated hypoganglionosis, internal anal sphincter achalasia, and congenital smooth muscle cell disorders such as megacystis microcolon intestinal hypoperistalsis syndrome.
... The existence of interstitial cells that were exclusively positive for c-Kit or CD34 labelling may indicate functional differences between such cells. C-kit had been established as a marker for interstitial cells, the ICCs, since before the characterization of telocytes [38]. The primary function assigned to these cells was a pacemaker function in the contraction of smooth muscle [19,20]. ...
Telocytes are CD34-positive interstitial cells, known to exert several functions, one of which is a role in tissue organisation, previously demonstrated by telocytes in the myocardium. The existence of telocytes in the prostate has recently been reported, however, there is a lack of information regarding the function of these cells in prostate tissue, and information regarding the possible role of these cells in prostatic development. This study used immunofluorescence techniques in prostate tissue and prostatic telocytes in culture to determine the relationship between telocytes and prostate morphogenesis. Furthermore, immunofluorescent labelling of telocytes was performed on prostate tissue at different stages of early postnatal development. Initially, CD34-positive cells are found at the periphery of the developing alveoli, later in the same region, c-kit-positive cells and cells positive for both factors are verified and CD34-positive cells were predominantly observed in the interalveolar stroma and the region surrounding the periductal smooth muscle. Fluorescence assays also demonstrated that telocytes secrete TGF-β1 and are ER-Beta (ERβ) positive. The results suggest that telocytes play a changing role during development, initially supporting the differentiation of periductal and perialveolar smooth muscle, and later, producing dense networks that separate alveoli groups and form a barrier between the interalveolar region and periurethral smooth muscle. We conclude that telocytes play a relevant role in prostate tissue organisation during postnatal development.
... Many studies of ICC utilized KIT antibodies (aka anti-CD117) to identify these cells in tissues and in cultures [8][9][10][11][12]. While immuno-localization has been shown to be useful, detection of ICC within fresh dispersions of GI muscles was limited. ...
Transcriptome-scale data can reveal essential clues into understanding the underlying molecular mechanisms behind specific cellular functions and biological processes. Transcriptomics is a continually growing field of research utilized in biomarker discovery. The transcriptomic profile of interstitial cells of Cajal (ICC), which serve as slow-wave electrical pacemakers for gastrointestinal (GI) smooth muscle, has yet to be uncovered. Using copGFP-labeled ICC mice and flow cytometry, we isolated ICC populations from the murine small intestine and colon and obtained their transcriptomes. In analyzing the transcriptome, we identified a unique set of ICC-restricted markers including transcription factors, epigenetic enzymes/regulators, growth factors, receptors, protein kinases/phosphatases, and ion channels/transporters. This analysis provides new and unique insights into the cellular and biological functions of ICC in GI physiology. Additionally, we constructed an interactive ICC genome browser (http://med.unr.edu/physio/transcriptome) based on the UCSC genome database. To our knowledge, this is the first online resource that provides a comprehensive library of all known genetic transcripts expressed in primary ICC. Our genome browser offers a new perspective into the alternative expression of genes in ICC and provides a valuable reference for future functional studies.
... Many studies of ICC utilized KIT antibodies (aka anti-CD117) to identify these cells in tissues and in cultures [8][9][10][11][12]. While immuno-localization has been shown to be useful, detection of ICC within fresh dispersions of GI muscles was limited. ...
... The fact is that the guinea pig intestine exhibits robust slow wave activity but this activity is not omnipresent, it develops in response to a stimulus such as distention . There is no doubt in my mind that the rhythmic peristaltic activity of the guinea pig small intestine, already shown by Trendelenburg to be able to occur without neural infl uence is governed by slow wave activity generated by interstitial cells of Cajal (Komuro and Zhou 1996 ) with the ENS being the major excitatory force for smooth muscle depolarization. The guinea pig intestine can switch form peristalsis to segmentation with segmentation still occurring at the slow wave frequency (Gwynne and Bornstein 2007 ). ...
Myogenic control mechanisms play a role in all motor activities of the gut. Myogenic control systems are defined here as control systems that are intrinsic to the smooth muscle cells and/or interstitial cells of Cajal (ICC) and that can operate without an essential contribution of the intrinsic (ENS) and extrinsic nervous systems. In vivo however, the ENS and the myogenic control systems always work in cooperation. Although myogenic control plays a role in every gut organ, this review focuses on the peristaltic and segmentation activity of the small intestine. It provides some historical perspectives and some discussion on the development of our understanding of the cooperative nature of the myogenic and neurogenic control mechanisms. It highlights how some influential papers inadvertently provided hindrance to full understanding, it discusses how the guinea pig model has hampered acceptance of myogenic control systems and it provides some background into the genesis of our understanding of control mechanisms involving ICC.
... Since that time ICC were detected in many other tissues, always in the vicinity of smooth muscle cells. ICC were identified in alimentary tract of many species, i.e. mice [3], rats [4], guinea pigs [5]. In humans ICC were found in the wall of alimentary tract [6], pancreas [7], in the muscle of atria and ventricles [8,9,10], vagina [11], mammary gland [12,13], oviduct [14], ductus deferens [15], urinary tract [16,17,18] [26,28]. ...
This paper reviews the distribution of interstitial cells of Cajal (ICC) in the human gastrointestinal (GI) tract, based on ultrastructural and immunohistochemical evidence. The distribution and morphology of ICC at each level of the normal GI tracts is addressed from the perspective of their functional significance. Alterations of ICC reported in as well as in gastrointestinal stromal tumors are reviewed, with emphasis on the place of ICC in the pathophysiology of disease.
... Interstitial cells were detected in many other tissues, always in the vicinity of smooth muscle cells. ICC were identified in gastrointestinal tract of many species, i.e. mice [3], rats [4], guinea pigs [5]. In humans ICC were found in the wall of alimentary tract [6], pancreas [7], in the muscle of atria and ventricles [8][9][10], vagina [11], mammary gland [12,13] To conclude, the role of ICC is associated with: generation and modulation of slow waves within gastrointestinal tract, facilitation of propagation of slow waves, their coordination and mediation in transmission of excitatory or inhibitory stimu-lation (neurotransmission) between autonomic nervous system and muscular cells. ...
Based on the review of current literature and on their own studies authors postulate that decreased number of interstitial cells in the wall of gallbladder may be pathognomonic for gallstone disease (cholelithiasis).
... ICC network imaging data was collected using methods as described previously 17,23 . All experiments were performed in accordance with the Health Guide for the Care and Use of Laboratory Animals of the Third Military Medical University (Chongqing, China). ...
The mammalian gastrointestinal (GI) tract undergoes rapid development during early postnatal life in order to transition from a milk to solid diet. Interstitial cells of Cajal (ICC) are the pacemaker cells that coordinate smooth muscle contractility within the GI tract, and hence we hypothesized that ICC networks undergo significant developmental changes during this early postnatal period. Numerical metrics for quantifying ICC network structural properties were applied on confocal ICC network imaging data obtained from the murine small intestine at various postnatal ages spanning birth to weaning. These imaging data were also coupled to a biophysically-based computational model to simulate pacemaker activity in the networks, to quantify how changes in structure may alter function. The results showed a pruning-like mechanism which occurs during postnatal development, and the temporal course of this phenomenon was defined. There was an initial ICC process overgrowth to optimize network efficiency and increase functional output volume. This was followed by a selective retaining and strengthening of processes, while others were discarded to further elevate functional output volume. Subsequently, new ICC processes were formed and the network was adjusted to its adult morphology. These postnatal ICC network developmental events may be critical in facilitating mature digestive function.
... The immunostaining procedures for detecting ICC networks and proliferation have been previously described [22]. Ki67, a nuclear protein expressed during all the active phases of cell and vanished in the resting cell, has been applied to detect the proliferative ICC [23]. ...
Depletion of interstitial cells of Cajal (ICC) is certified in the stomach of diabetic patients. Though electroacupuncture (EA) at ST36 is an effective therapy to regulate gastric motility, the mechanisms of EA at ST36 on gastric emptying and networks of ICC remain to be elucidated. The aims of this study were to investigate the effects of EA on gastric emptying and on the alterations of ICC networks. Rats were randomized into the control, diabetic rats (DM), diabetic rats with sham EA (DM+SEA), diabetic rats with low frequency EA (DM+LEA) and diabetic rats with high frequency EA groups (DM+HEA). The expression of c-kit in each layer of gastric wall was assessed by western blotting. The proliferation of ICC was identified by immunolabeling of c-kit and Ki67 as the apoptosis of ICC was examined by TUNEL staining. The results were as follows: (1) Gastric emptying was severely delayed in the DM group, but accelerated in the LEA and HEA group, especially in the LEA group. (2) The expression of c-kit in each layer was reduced apparently in the DM group, but also up-regulated in the LEA and HEA group. (3) Plentiful proliferated ICC (c-kit+/Ki67+) forming bushy networks with c-kit+ cells were observed in the LEA and HEA group, while the apoptotic cells (c-kit+/TUNEL+) were hardly captured in the LEA and HEA group. Collectively, low and high frequency EA at ST36 rescue the damaged networks of ICC by inhibiting the apoptosis and enhancing the proliferation in the stomach of diabetic rats, resulting in an improved gastric emptying.
... The identification of the expression of the gene product c-kit in ICCs was a major breakthrough in ICCs research. C-kit is a protooncogene that encodes the receptor tyrosine kinase kit, which is expressed in ICCs and mast cells within the gastrointestinal tract [35][36][37][38][39]. Consequently many studies have been performed showing the expression of c-kitpositive ICCs in the gastrointestinal tract of several species, including humans [40][41][42][43], mice [44], rats [45,46], and guinea pigs [47,48]. These studies and further investigations have increased our understanding of the complex architecture of ICCs networks in relation to the ENS and the intestinal smooth muscles [49]. ...
Hirschsprung disease (HD) is the most prevalent congenital gastrointestinal motility disorder. The pathogenesis of HD is defined as a functional intestinal obstruction resulting from a defect in the intrinsic innervation of the distal bowel. In addition to the enteric nervous system, the interstitial cells of Cajal (ICC) play an important role in the generation of coordinated gastrointestinal peristalsis. The major function of the ICCs is the generation of slow waves that allow these cells to act as specialised pacemaker cells within various tissues. ICCs have additional functions in the gastrointestinal tract as regulators of mechanical activity and neurotransmission. Due to the central role of ICCs in gastrointestinal peristalsis, it has been suggested that defects or impairments of the ICCs may contribute to motility dysfunction in several gastrointestinal motility disorders. This review describes the distribution and functions of ICCs in the normal gut and in Hirschsprung disease.
... Vimentin is the major cytoskeletal component of mesenchymal cells and is often used as a marker of mesenchymally derived cells. Because of this it is also often used as an ICC marker and has been reported in guinea pig bladder ICC and ICC in other organs [12,40,41,42]. Immunostaining showed co-localization of NTPDase2-positive cells with vimentin (Fig. 6A–C). ...
Background:
Interstitial cells of Cajal (ICC) have been identified in urinary bladder of several species, but their presence in mice remains uncertain. Meanwhile, dozens of reports indicate that dysregulation of connexin 43 plays an important role in bladder overactivity, but its localization has not been clearly defined, with reports of expression in either the smooth muscle or in myofibroblasts. We recently identified a population of ectonucleoside triphosphate diphosphohydrolase 2 (NTPDase2) positive cells that resemble ICC and are distinct from smooth muscle, fibroblasts, myofibroblasts and neurons. Thus we sought to define more clearly the molecular signature of ICC and in doing so resolve some of these uncertainties.
Principle findings:
Immunofluorescent localization revealed that NTPDase2-positive cells lie closely adjacent to smooth muscle but are separate from them. NTPDase2 positive cells exhibited co-localization with the widely accepted ICC marker - c-kit. They were further shown to co-localize with other ICC markers CD34 and Ano1, but not with mast cell marker tryptase. Significantly, they show convincing co-localization with connexin 43, which was not present in smooth muscle. The identity of these cells as ICC was further confirmed by the presence of three mesenchymal markers - vimentin, desmin, and PDGFβ receptor, which indicates their mesenchymal origin. Finally, we observed for the first time, the presence of merlin/neurofibromin 2 in ICC. Normally considered a neuronal protein, the presence of merlin suggests ICC in bladder may have a role in neurotransmission.
Conclusions:
NTPDase2 positive cells in mice bladder are ICC, which can be defined by the presence of c-Kit, CD34, Ano1, NTPDase2, connexin 43, vimentin, desmin, PDGFβ receptor and merlin/NF2. These data establish a definitive molecular expression profile, which can be used to assist in explorations of their functional roles, and the presence of NTPDase2 suggests that purinergic signaling plays a role in regulation of ICC function.
... Then the mucosa was removed and the longitudinal smooth muscle layer containing the ICC-MY and the circular smooth muscle layer containing the ICC-DMP were prepared with the aid of a dissection microscope respectively. The immunostaining procedures have been described previously (Komuro and Zhou, 1996). Briefly, the specimens were incubated with rat monoclonal anti-Kit antibody (ACK2, 1:100; eBiosicence) or mouse monoclonal anti-a-SMA antibody (1:100; Santa Cruz), and immunoreactivity was detected by using a Cy3-conjugated secondary antibody (anti-rat IgG, 1:100; Zymed) or a FITC-conjugated secondary antibody (anti-mouse IgG, 1:100; DAKO). ...
Although it is well known that the reduction of interstitial cells of Cajal (ICCs) is associated with several gastrointestinal motility disorders in clinic, it is unknown whether the mature ICCs still have an active plasticity in adult mammals. This study focused on the issues of the reduction of ICCs during Imatinib administration and the recovery of ICCs following drug withdrawal in the small intestine of adult guinea pigs. ICCs were revealed by immunofluorescence on whole mount preparations with anti-Kit, alpha-smooth muscle actin, (alpha-SMA), and 5-bromo-2'-deoxyuridine (BrdU) antibodies. Moreover, the occurrence of apoptosis was also assayed. Imatinib treatment led to a gradual reduction of ICCs in number around the myenteric plexus and deep muscular plexus, which was dependent on the time but no apoptosis of ICCs was detected with the TUNEL method. During Imatinib treatment, some ICC-like cells were double labeled for Kit and alpha-SMA and a few ICC-like cells were only stained with alpha-SMA. When Imatinib was discontinued, the number of ICCs recovered to normal within 32 days. During this time, some proliferating ICCs were demonstrated by double labeling with Kit and BrdU antibodies. Our results indicated that Kit signaling was essential for the maintenance of survival and proliferation of the mature ICCs in the small intestine of adult guinea pigs. Moreover, ICCs might transdifferentiate to a type of alpha-SMA(+) cells, perhaps a phenotype of smooth muscle cells, when there is a loss-of-function of Kit.
... After 30 min Wxation, and the longitudinal smooth muscle layer containing the ICC-MY was prepared with the aid of a dissection microscope. The immunostaining procedures have been previously described (Komuro and Zhou 1996). BrieXy, after the specimens were incubated with primary antibody (Table 1) for 8 h at 4°C, the immunoreactivity was detected by using a Cy3-conjugated secondary antibody (anti-rat IgG, 1:100; Zymed), or FITC-conjugated secondary antibody (anti-mouse IgG, 1:100; DAKO), or Cy5-conjugated secondary antibody (anti-rabbit IgG, 1:100; Zymed). ...
This paper aimed at investigating the alterations in interstitial cells of Cajal (ICCs) in the murine small intestine from 0-day to 56-day post-partum (P0-P56) by immunohistochemistry. The Kit+ ICCs, which were situated around myenteric nerve plexus (ICC-MY) formed a loose cellular network at P0 which changed into an intact one before P32. The density of ICC-MY increased from P0 to P12, and then decreased until P32. In contrast, the estimated total amount increased more than 15-fold at P32 than that at P0. Some Kit+/BrdU+ cells were observed at 24 h after one BrdU injection to the different-aged mice, and the number decreased from P2 to P24 and vanished at P32. Actually a few Kit+/BrdU+ cells can be observed at 1 h after one BrdU injection at P10, and the amount doubled at 24 h along with paired Kit+/BrdU+ cells. A number of BrdU+ ICCs were also labeled with CD34, CD44 and insulin-like growth factor I receptor. About 65% ICCs were BrdU+ at P32 after daily BrdU injection from P0. Our results indicate that an age-dependent proliferation is involved in the postnatal development of ICC-MY which increase greatly in cell numbers and proliferative ICCs may originate from ICCs progenitor cells.
... Correlations with electron microscopy have made it likely that at least most Kit-positive cells within the gut musculature are ICCs (Komuro and Zhou, 1996). Kitpositive cells are markedly diminished in number in the afflicted section of the human colon in Hirschsprung's disease (Vanderwinden et al., 1996c) and in the circular muscle layer of the pylorus in infantile pyloric stenosis (Vanderwinden et al., 1996a). ...
Intestinal motor patterns are not well developed in premature infants. Similarly, in neonatal mice, irregular motor patterns were observed. Pacemaker cells, identified in the small intestine as interstitial cells of Cajal (ICCs) associated with Auerbach's plexus (ICC-APs), contribute to the generation of peristaltic movements. The objective of the present study was to assess the hypothesis that abnormal gut motor activity in (preterm) newborns can be associated with underdeveloped ICCs. Specifically, the aim was to identify at which point the electrical pacemaker activity is fully developed and whether or not the development of pacemaker activity has a structural correlation with the developmental stage of ICCs. Pacemaker activity was identified as that component of the slow wave that is insensitive to L-type calcium (Ca2+) channel blockers and displays a characteristic reduction in frequency in the presence of cyclopiazonic acid (CPA), a specific inhibitor of the endoplasmic reticulum Ca2+ pump. In newborn, unfed neonates, action potentials occurred that were irregular in frequency and amplitude and sensitive to verapamil. CPA (5 microM) abolished all action potentials. Quiescent spots were observed in approximately 50% of impalements. Six hours after birth, slow-wave activity appeared at a regular frequency and amplitude, and a well-defined plateau phase was observed. Verapamil did not affect the frequency, 5 microM CPA decreased it. The effect of CPA on the pacemaker frequency 2 days after birth was identical to that observed in adult mice. In 2-hr-old neonates, ICCs could be identified through selective uptake of methylene blue, but ultrastructural features were not fully developed. At 48 hr, a complete ICC network covering Auerbach's plexus was formed, confirmed by electron microscopy. In summary, the pacemaker component of the slow waves can be identified in neonates as early as 6 hr after birth. The pacemaker component was fully developed 2 days after birth. These electrophysiological observations correlated with the development of full network characteristics of ICC-APs and the development of fully differentiated ICC-APs from "blast-like" cells.
... 11,12 Our understanding of the physiology of ICC has been greatly accelerated by the discovery that ICC express c-kit, the proto-ocogene that encodes the receptor tyrosine kinase, Kit. 8±10,13,14 Labelling of Kit receptors or c-kit mRNA has provided an ef®cient means of identifying ICC at the light level in a variety of preparations, including humans, 15±17 guinea-pig, 18,19 mouse, 8±10 rat 20±22 and birds. 23 Simple and reliable immunohistochemical methods have quickly enhanced our understanding of the structure and distribution of the networks formed by ICC, developed a more complete understanding of the relationships between ICC and smooth muscle cells and ICC and neurones, and allowed ef®cient screening of ICC in a variety of pathophysiological conditions. ...
Interstitial cells of Cajal (ICC) are the pacemakers in gastrointestinal (GI) muscles, and these cells also mediate or transduce inputs from the enteric nervous system. Different classes of ICC are involved in pacemaking and neurotransmission. ICC express specific ionic conductances that make them unique in their ability to generate and propagate slow waves in GI muscles or transduce neural inputs. Much of what we know about the function of ICC comes from developmental studies that were made possible by the discoveries that ICC express c-kit and proper development of ICC depends upon signalling via the Kit receptor pathway. Manipulating Kit signalling with reagents to block the receptor or downstream signalling pathways or by using mutant mice in which Kit or its ligand, stem cell factor, are defective has allowed novel studies into the specific functions of the different classes of ICC in several regions of the GI tract. Kit is also a surface antigen that can be used to conveniently label ICC in GI muscles. Immunohistochemical studies using Kit antibodies have expanded our knowledge about the ICC phenotype, the structure of ICC networks, the interactions of ICC with other cells in the gut wall, and the loss of ICC in some clinical disorders. Preparations made devoid of ICC have also allowed analysis of the consequences of losing specific classes of ICC on GI motility. This review describes recent advances in our knowledge about the development and plasticity of ICC and how developmental studies have contributed to our understanding of the functions of ICC. We have reviewed the clinical literature and discussed how loss or defects in ICC affect GI motor function.
... Cells in the deep muscular plexus (Fig. 1 I) displayed SUR1 immunoreactivity. These cells were identified as interstitial cells of Cajal, by the presence of c-Kit immunoreactivity (Fig. 1 I, inset) (Komuro and Zhou, 1996). As demonstrated previously in the mouse pancreas (Suzuki et al., 1997), Kir6.2 immunoreactivity was found in guinea pig islets (Fig. 2 A). ...
We tested the hypothesis that a subset of enteric neurons is glucoresponsive and expresses ATP-sensitive K(+) (K(ATP)) channels. The immunoreactivities of the inwardly rectifying K(+) channel 6.2 (Kir6.2) and the sulfonylurea receptor (SUR), now renamed SUR1, subunits of pancreatic beta-cell K(ATP) channels, were detected on cholinergic neurons in the guinea pig ileum, many of which were identified as sensory by their costorage of substance P and/or calbindin. Glucoresponsive neurons were distinguished in the myenteric plexus because of the hyperpolarization and decrease in membrane input resistance that were observed in response to removal of extracellular glucose. The effects of no-glucose were reversed on the reintroduction of glucose or by the K(ATP) channel inhibitor tolbutamide. No reversal of the hyperpolarization was observed when D- mannoheptulose, a hexokinase inhibitor, was present on the reintroduction of glucose. Application of the K(ATP) channel opener diazoxide or the ob gene product leptin mimicked the effect of glucose removal in a reversible manner; moreover, hyperpolarizations evoked by either agent were inhibited by tolbutamide. Glucoresponsive neurons displayed leptin receptor immunoreactivity, which was widespread in both enteric plexuses. Superfusion of diazoxide inhibited fast synaptic activity in myenteric neurons, via activation of presynaptic K(ATP) channels. Diazoxide also produced a decrease in colonic motility. These experiments demonstrate for the first time the presence of glucoresponsive neurons in the gut. We propose that the glucose-induced excitation of these neurons be mediated by inhibition of K(ATP) channels. The results support the idea that enteric K(ATP) channels play a role in glucose-evoked reflexes.
... The blots were blocked with skimmed milk and immunoreacted with the mouse antibody KM 1112 for type 1, KM 1083 for type 2 and KM 1082 for type 3 (Sugiyama et al. 1994), and then with horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Amersham). Immunostaining for c-kit was performed by the methods reported previously (Komuro & Zhou, 1996;Seki et al. 1998). Briefly, segments of antrum muscles were moderately inflated with injections of OCT compounds and immediately frozen with liquid nitrogen in the embedding medium. ...
Membrane potential recordings, made from the circular smooth muscle layer of the gastric antrum taken from mutant mice which lacked the inositol trisphosphate (InsP3) type 1 receptor, were compared with those obtained from the stomach of control (wild-type) mice. Immunostaining of gastric muscles indicated that the distribution and form of c-kit positive cells were similar in wild-type and mutant mice. Smooth muscles from wild-type mice generated slow waves that in turn initiated spike potentials, while those from mutant mice were either quiescent or generated irregular bursts of spike potentials. In the presence of nifedipine, slow waves with reduced amplitude were generated in wild-type mice, while all electrical activity was abolished in mutant mice. Acetylcholine depolarized and sodium nitroprusside hyperpolarized the membrane in muscles from both types of mice, being more effective in wild-type mice. Noradrenaline produced similar hyperpolarizations in both types of mice. Transmural nerve stimulation evoked inhibitory junction potentials (IJPs) in both wild-type and mutant mice. In wild-type mice, the IJPs were reduced in amplitude by nitroarginine and converted to a cholinergic excitatory junction potential (EJP) by apamin. In mutant mice, the IJPs were unaffected by nitroarginine or atropine but were abolished by apamin. It is concluded that in antral smooth muscle, the expression of InsP3 type 1 receptors may be causally related to the generation of slow waves but not to the generation of action potentials. A lack of InsP3 receptors attenuates cholinergic excitatory and nitrergic inhibitory responses but does not alter the response to noradrenaline.
... Two populations of morphologically distinct immunoreactive c-kit cells have been identified in the digestive tract of mammals and humans, namely the mastocytes (Vliagoftis et al. 1997) and the interstitial cells (Sanders 1996). C-kit immunoreactive interstitial cells (c-kit ICs) have been found to exist in various species, such as mice (Maeda et al. 1992; Huizinga et al. 1995; Torihashi et al. 1997; Ward et al. 1997), rats (Isozaki et al. 1995; Ishikawa et al. 1997; Horiguchi and Komuro 1998), and guinea pigs (Komuro and Zhou 1996; Burns et al. 1997; Seki et al. 1998), as well as humans (Horie et al. 1993; Matsuda et al. 1993; Rumessen 1994; Vanderwinden et al. 1996a,b; Hagger et al. 1997 Hagger et al. , 1998 Horisawa et al. 1998; Romert and Mikkelsen 1998; Torihashi et al. 1999; Wester et al. 1999 ). All in all, these studies have shown that the distribution and morphology of the c-kit ICs vary from one species to another, as well as from one region of the digestive tract to another, within a given species. ...
C-kit immunocytochemistry was performed on ultrathin sections of human distal colon. Our attention was focused on relationships between c-kit immunoreactive interstitial cells (c-kit ICs) and muscular cells and nervous elements located in the external muscular layers of the colonic wall. C-kit ICs established membrane apposition with both nerve fibers and smooth muscle cells of, respectively, the longitudinal and circular muscle layers, the myenteric area, and the extremus submucosus plexus. C-kit ICs also surrounded the external submucosus plexus and established membrane appositions with nerve elements located inside the myenteric ganglia. These membrane appositions were observed either at the level of the c-kit IC bodies or at that of their cytoplasmic processes. In some cases, membrane appositions were observed concomitantly between the c-kit ICs, nerve fibers, and smooth muscle cells. In all the regions studied, the c-kit ICs were also found to be located in the close vicinity of blood vessels and to have established close contacts with non-immunoreactive fibroblast-like cells. The results of the present study shed essential light on the relationships of c-kit ICs with the neighboring muscle cells and nerve elements, and confirm that the intercalated c-kit ICs well fit with the so-called "interstitial cells of Cajal".
Over the past three to four decades, the molecular pathogenesis of gastrointestinal stromal tumors (GISTs) has been elucidated in great detail. In this review, we discuss the biological genesis of GISTs, identification of the various primary activating driver mutations (focusing on KIT and PDGFRA), oncogene addiction and targeted therapies with imatinib and other tyrosine kinase inhibitors, and the subsequent characterization of the various mechanisms of drug resistance. We illustrate how GIST has become a quintessential paradigm for personalized medicine.
Expected final online publication date for the Annual Review of Pathology: Mechanisms of Disease, Volume 17 is January 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
Over the past three to four decades, the molecular pathogenesis of gastroin-testinal stromal tumors (GISTs) has been elucidated in great detail. In this review, we discuss the biological genesis of GISTs, identification of the various primary activating driver mutations (focusing on KIT and PDGFRA), oncogene addiction and targeted therapies with imatinib and other tyrosine kinase inhibitors, and the subsequent characterization of the various mechanisms of drug resistance. We illustrate how GIST has become a quintessen-tial paradigm for personalized medicine.
Background:
Gastroparesis is a complex clinical entity; many aspects of which remain unknown. Although most patients have idiopathic, diabetic, or postsurgical gastroparesis, many are thought to have measurable neuromuscular abnormalities. Immunotherapy has recently been utilized to treat suspected autoimmune gastrointestinal dysmotility.
Methods:
Fourteen patients with symptoms of gastroparesis (Gp) who were refractory to drug/device were selected from 443 Gp patients from 2013 to 2015 who were treated at the University of Louisville motility center. All patients underwent a structural and psychiatric evaluation along with detailed psychological and behavioral examination to rule out eating disorders. We performed detailed neuromuscular evaluation and all 14 patients received at least 12 weeks of intravenous immunoglobulin (400 mg/kg infusion weekly). Response was defined subjectively (symptomatic improvement) using standardized IDIOM score system.
Key results:
All 14 patients had serological evidence and/or tissue evidence of immunological abnormality. Post-IVIG therapy, there was a significant improvement in symptoms scores for nausea, vomiting, early satiety, and abdominal pain.
Conclusions and inferences:
Although limited by the absence of placebo group, the data illustrate the role of autoimmunity and neuromuscular evaluation in patients with gastroparesis and support the utility of a diagnostic trial of immunotherapy in an effort to improve therapeutic outcomes for such patients.
The telocytes have recently been described in the prostate gland. In mature gland, they exist in close association with the acini and their telopodes form networks whose functions remain unclear. In this chapter, our group gives a brief introduction to telocytes and explores the history that led to such a concept and then discusses hypotheses and presents new evidences about the roles exerted by telocytes in the prostate. First is given emphasis on the role that these cells possibly play in paracrine signaling employed in the differentiation of smooth muscle periacinar are then discussed other roles potentially performed by telocytes in the prostate, such as the organizational, where these cells would act in order to delimit stromal microenvironments, thereby assisting the differentiation of the prostatic anatomical components. In addition, the pacemaker function of smooth muscle cells contraction, as evidenced by the presence of caveolae and gap-type junction and, finally, the role of telocytes in prostate remodeling and the possible action as adult progenitor cells. Generally speaking, the chapter reaffirms the existence of telocytes as distinct cells of other stromal cells and the importance of this new cell type for normal metabolism and prostate development.
Objective: To explore the possible effect of cajal, a kind of special cell in bladder, on the detrusor instability by studying the electrophysiology of cajal, so as to clarity the pathogenesy of detrusor instability and provide theoretical and experimental evidence for the new treatment. Data sources: A computer-based online search of Plumbed was undertaken to identify English articles about cajal interstitial cells and urodynamics dated from 1996 to 2006 with the keywords of "electrical characterization, bladder, interstitial cells of cajal. Study selection: Only articles about 1 the morphology and function of cajal in gastrointestinal tract; 2 morphology and function of cajal in urinary system, and 3 electrophysiology of cajal were selected. Repetitive researches were excluded. Data extraction: Data were firstly selected, and the full-texts of articles met the requirement were looked over. Finally, 30 articles were included. Data synthesis: Cajal interstitial cells, which play the pacing and conducting roles in the spontaneous peristaltic contraction of smooth muscle of digestive tract, have specific and changeable morphological features. And positive cajal markers with similar morphous are found in many parts of urinary system. The transport of urine from upper urinary tract to bladder is based on the peristalsis of pelvis and ureter. Detrusor muscle has spontaneous myogenic activity. cajal as the pacer and conductor of slow-wave activity of urinary system plays a regulating role in neuromuscular signal transmission of nerve and muscle, and correlates with the occurrence of certain dynamic diseases in urinary system. Conclusion: Detrusor instability is the most common disease of urinary dysfunction, and its pathogenesy is still uncertain. Cajal-like cells in urinary system may be the pacer of peristalsis of smooth muscle, and play a role in regulating neuromuscular signal transmission.
Slow waves (slow wavesICC) were recorded from myenteric interstitial cells of Cajal (ICC-MY) in situ in the rabbit small intestine, and their properties were compared with those of mouse small intestine. Rabbit slow wavesICC consisted of an upstroke depolarization followed by a distinct plateau component. Ni(2+) and nominally Ca(2+)-free solutions reduced the rate-of-rise and amplitude of the upstroke depolarization. Replacement of Ca(2+) with Sr(2+) enhanced the upstroke component, but decreased the plateau component of rabbit slow wavesICC. In contrast, replacing Ca(2+) with Sr(2+) decreased both components of mouse slow wavesICC. The plateau component of rabbit slow wavesICC was inhibited in low[Cl(－)]o solutions and by 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS), an inhibitor of Cl(－) channels, cyclopiazonic acid (CPA), an inhibitor of internal Ca(2+) pumps, or bumetanide, an inhibitor of Na(+)-K(+)-2Cl(－) cotransporter (NKCC1). Bumetanide also inhibited the plateau component of mouse slow wavesICC. NKCC1-like immunoreactivity was observed mainly in ICC-MY in the rabbit small intestine. Membrane depolarization with a high-K(+) solution reduced the upstroke component of rabbit slow wavesICC. In cells depolarized with elevated external K(+), DIDS, CPA and bumetanide blocked slow wavesICC. These results suggest that the upstroke component of rabbit slow wavesICC is partially mediated by voltage-dependent Ca(2+) influx, whereas the plateau component is dependent upon Ca(2+)-activated Cl(－) efflux. NKCC1 is likely to be responsible for Cl(－) accumulation in ICC-MY. The results also suggest that the mechanism of the upstroke component differs in rabbit and mouse slow wavesICC in the small intestine.
Copyright © 2014, American Journal of Physiology- Gastrointestinal and Liver Physiology.
The purpose of this study was to investigate the mechanism by which magnolol treatment prevents lipopolysaccharide (LPS)-induced septic dysmotility in mice. Sepsis was induced by intravenous tail vein injection of LPS (4 mg/kg body weight). Animals were divided into three groups: the magnolol-treated septic group, the placebo-treated septic group, and the control group. Intestinal transit and circular smooth muscle contraction were measured 12 h after LPS injection, and immunocytochemisty was performed to study the morphology of interstitial cells of Cajal (ICCs). Stem cell factor (SCF) expression and c-kit phosphorylation were determined by Western blot analysis, and the mRNA levels of inducible NO synthase (iNOS) were determined by RT-PCR. Nitric oxide (NO) content, superoxide dismutase (SOD) activity, and malondialdehyde (MDA) concentration were detected using commercial kits. Intestinal transit and muscular contractility were significantly lower in the LPS-treated group than in the control group. Immunocytochemical experiments showed that the total number of ICCs, and the total and average lengths of the ICC processes were significantly decreased in the LPS-treated group compared with those in the control group. In LPS-treated animals, magnolol pretreatment significantly accelerated intestinal transit, increased circular muscle contraction, and prevented ICC morphology changes. Phosphorylation of c-kit and expression of SCF were significantly downregulated in LPS-treated animals compared with control animals. Magnolol pretreatment prevented sepsis-induced decreases in c-kit phosphorylation and SCF expression in LPS-treated animals. Magnolol pretreatment prevented the sepsis-induced increase in NO concentration, iNOS expression, and MDA concentration, and decrease in SOD activity in LPS-treated animals. Our results suggest that magnolol treatment prevents sepsis-induced intestinal dysmotility by regulating SCF/c-kit and NO signaling to maintain functional ICCs.
Peristalsis is a propulsive motor pattern orchestrated by neuronal excitation and inhibition in cooperation with intrinsic muscular control mechanisms, including those residing in interstitial cells of Cajal (ICC). Interstitial cells of Cajal form a network of cells in which electrical slow waves originate and then propagate into the musculature initiating rhythmic contractile activity upon excitaton by enteric nerves. Interstitial cells of Cajal have now been isolated and their intrinsic properties reveal the presence of rhythmic inward currents not found in smooth muscle cells. In tissues where classical slow waves are not present, enteric cholinergic excitation will evoke slow wave-like activity that forces action potentials to occur in a rhythmic manner. Intrinsic and induced slow wave activity directs many of the peristaltic motor patterns in the gut. Microsc. Res. Tech. 47:239–247, 1999. © 1999 Wiley-Liss, Inc.
Cryosections and whole-mount preparations of the guinea pig small intestine and colon were single or double immunolabeled
using the anti-c-Kit and protein gene product 9.5 antibodies. Immunolabeled specimens were observed under a confocal laser
scanning microscope. The main findings of the present study are: (1) the distribution and profiles of three-dimensional structures
of c-Kit-positive cellular networks in the small intestine and colon, and (2) the anatomical relations of c-Kit-positive cells
to the enteric nerves in the layers. In the small intestine, c-Kit-positive cellular networks were observed at levels of the
deep muscular plexus and myenteric plexus. The c-Kit-positive cellular networks ran along or overlay the nerve fibers at the
deep muscular plexus, while they showed the reticular structures intermingled with the nerve elements at the myenteric plexus.
In the colon, c-Kit-positive cellular networks were observed at levels of the submuscular plexus and myenteric plexus, and
were further identified within the circular and longitudinal muscle layers as well as in the subserosal layer. In the circular
muscle layer, c-Kit-positive cells surrounded the associated nerve fibers and extended several long processes toward the adjacent
c-Kit-positive cells. The c-Kit-positive cellular networks within the longitudinal muscle layer as well as in the subserosal
layer were not associated with the nerve fibers. In the layers of the intestinal wall with c-Kit-positive cells, the cellular
networks of the interstitial cells were identified in ultrastructure. The characteristic profiles of c-Kit-positive cellular
networks provide a morphological basis upon which to investigate the mechanisms regulating intestinal movement.
The present work describes the distribution of S-100 protein in the intestinal tract of a Chinese soft-shelled turtle specimen (Pelodiscus sinensis). S-100 protein positive cells were located in the intestinal tract, from the proximal small to distal large intestine. S-100 protein positive dendritic cells had irregular shape and were positive in both cytoplasm and nucleus. Most of them were located both lamina propria and submucosa in the small intestine, while few were found in the large intestine. S-100 protein, C-kit positive ICCs and Silver staining glial cells were predominantly observed in three locations: (1) in the interspace between the submucosa and circular muscle layer; (2) in the circular muscle layer; and (3) between the circular and longitudinal muscle layers of the intestine. Fewer were found in the large intestinal lamina propria and submucosa. Three types of positive cells (S-100 protein positive cells, C-kit positive ICCs and Silver staining glial cells) with 1-2 long or 2-3 short processes were distributed as lace-like or surrounding blood vessels in the different locations mentioned above. In the lamina propria, all the positive cells with irregular processes were connected with each other and formed a network. In the submucosa, all the positive cells were found surrounding the blood vessels.
Pathologic assessment of gastric tissue in patients with gastroparesis is limited. Aims To evaluate gastric histopathology in patients with gastroparesis.
Full-thickness antral biopsies were obtained in 28 patients with gastroparesis. Control specimens were obtained from patients undergoing gastric resection. H&E and immunohistochemical stained slides were reviewed for the presence of inflammation, ganglion cells, and interstitial cells of Cajal (ICCs).
A mild lymphocytic infiltrate in the myenteric plexus was present in 6 out of 14 patients with diabetic gastroparesis (DG), one of 14 idiopathic gastroparesis (IG) and 0 of eight controls. Significant reductions in total nerve cell bodies were seen in gastroparesis patients (2.2 +/- 0.3 per hpf; p = 0.0002) compared to controls (3.2 +/- 0.12). This was seen in both DG (2.4 +/- 0.32) and IG (2.0 +/- 0.2). Sixteen patients (ten IG and six DG) had a reduction of ganglion cells (<2.3 cells/hpf). C-kit staining showed a reduction of ICCs in six patients (five IG and one DG). Four patients (three IG and one DG) had abnormal ICC morphology on C-kit staining with more rounded morphology and less dendritic processes.
This study shows several pathologic abnormalities in the gastric tissue in some patients with refractory gastroparesis. An inflammatory infiltrate was present in nearly half of the patients with diabetic gastroparesis. There was a reduction in nerve cell bodies in both idiopathic and diabetic gastroparesis. A reduced number of ICCs were found in the myenteric plexus. Thus, histologic abnormalities in gastroparesis are heterogeneous and include myenteric inflammation, decreased innervation, and reduction of ICCs.
Interstitial cells of Cajal (ICCs) have recently been identified as the pacemaker cells for contractile activity of the gastrointestinal tract. These cells generate the electrical 'slow-wave' activity that determines the characteristic frequency of phasic contractions of the stomach, intestine and colon. Slow waves also determine the direction and velocity of propagation of peristaltic activity, in concert with the enteric nervous system. Characterization of receptors and ion channels in the ICC membrane is under way, and manipulation of slow-wave activity markedly alters movement of contents through the gut organs. Here Jan Huizinga, Lars Thuneberg, Jean-Marie Vanderwinden and Jüri Rumessen, suggest that, as ICCs are unique to the gut, they might be ideal targets for pharmacological intervention in gastrointestinal motility disorders, which are very common and costly.
The distribution of the c-kit receptor expressing cells and gap junction protein, connexin (Cx) 43 in the guinea-pig stomach (antrum), small intestine (jejunum) and colon (ascending) was studied by immunohistochemistry. The anti-c-kit protein immunopositive cells were regularly observed in the myenteric region throughout all three organs. The immunopositive cells were also sparsely distributed in the circular muscle layer of both the stomach and the colon, but not in the small intestine. They were densely located in the regions of the deep muscular plexus (DMP) of the small intestine and submuscular plexus (SMP) of the colon. In contrast, strong immunoreactivity to anti-Cx 43 antibody was observed in almost the entire thickness of the circular muscle layer of the stomach and the small intestine, but not in the colon. Dense immunoreaction deposits were observed in the region of the DMP and SMP. However, only very weak immunoreactivity to anti-Cx 43 antibody was detected in the myenteric region of all three organs. These results suggest that the c-kit receptor expressing cells or interstitial cells of Cajal (ICC) in the myenteric region of the three organs, and in the SMP of the colon, are poorly coupled with the bulk of circular muscle tissue by gap junctions, while ICC in the DMP and in the circular muscle layer of the stomach couple well with the surrounding muscle tissue.
Networks of interstitial cells of Cajal embedded in the musculature of the gastrointestinal tract are involved in the generation of electrical pacemaker activity for gastrointestinal motility. This pacemaker activity manifests itself as rhythmic slow waves in membrane potential, and controls the frequency and propagation characteristics of gut contractile activity. Mice that lack a functional Kit receptor fail to develop the network of interstitial cells of Cajal associated with Auerbach's plexus in the mouse small intestine and do not generate slow wave activity. These cells could provide an essential component of slow wave activity (for example, a biochemical trigger that would be transferred to smooth muscle cells), or provide an actual pacemaker current that could initiate slow waves. Here we provide direct evidence that a single cell, identified as an interstitial cell of Cajal by light microscopy, electron microscopy and expression of Kit mRNA, generates spontaneous contractions and a rhythmic inward current that is insensitive to L-type calcium channel blockers. Identification of the pacemaker of gut motility will aid in the elucidation of the pathophysiology of intestinal motor disorders, and provide a target cell for pharmacological treatment.
Partial obstruction of the ileum causes a notable hypertrophy of smooth muscle cells and enteric neurones in the proximally located intestine.
To study the expression of neuromessengers in the hypertrophic ileum of rat as little is known about neuromessenger plasticity under these conditions. To investigate the presence of interstitial cells of Cajal (ICC) in hypertrophic ileum.
Ileal hypertrophy was induced by circumferential application of a strip of plastic film for 18-24 days. Immunocytochemistry, in situ hybridisation, nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase histochemistry, and ethidium bromide staining were used to investigate the number of enteric neurones expressing neuropeptides and nitric oxide synthase, and the frequency of ICC.
In the hypertrophic ileum several neuronal populations showed changes in their expression of neuromessengers. Myenteric neurones expressing vasoactive intestinal peptide (VIP), pituitary adenylate cyclase activating peptide, and galanin were notably increased in number. In submucous ganglia the number of VIP immunoreactive neurones decreased while those expressing VIP mRNA increased. NADPH diaphorase positive submucous neurones increased dramatically while the number of neuronal type nitric oxide synthase expressing ones was unchanged. The number of ICC decreased notably in hypertrophic ileum.
Enteric neurones change their levels of expression of neuromessengers in hypertrophic ileum. ICC are also affected. The changes are presumably part of an adaptive response to the increased work load.
The aims of this work were to determine whether cells that are similar to the interstitial cells of Cajal (ICC) and have immunoreactivity for the neurokinin 1 (NK1) receptor are indeed ICC; to determine whether the agonist, substance P, binds to and activates the receptor on presumptive ICC; and to investigate the relationship between substance P-immunoreactive nerve fibres and ICC. ICC at the level of the myenteric plexus and in the deep muscular plexus in the duodenum and ileum of the guinea-pig were investigated. Immunoreactivities for the ICC marker, Kit, and the NK1 receptor were colocalised in ICC of the myenteric and deep muscular plexuses. In tissue fixed immediately after its removal from the animal, NK1 receptor-immunoreactive ICC were found at the level of the myenteric plexus in the duodenum, but not in the ileum, and in the deep muscular plexus in the duodenum and ileum. The majority of receptor immunoreactivity was on the cell surface. ICC were exposed to substance P (10(-7) M), initially at 4 degrees C for 1 h to allow the agonist to bind, followed by incubation at 37 degrees C to allow receptor internalisation to proceed. Exposure to substance P caused the NK1 receptor immunoreactivity to aggregate in clumps in the cytoplasm of ICC of the myenteric and deep muscular plexuses, including the ICC of the myenteric plexus of the ileum, where NK1 receptor immunoreactivity was not seen if tissue was not exposed to substance P. Substance P, to which the fluorescent label, cyanine 3.18 (Cy-3), was coupled, bound to the ICC. The Cy-3-substance P was internalised with the receptor following warming to 37 degrees C. Many, but not all, ICC were closely apposed by nerve fibres with immunoreactivity for substance P. It is concluded that the NK1 receptor immunoreactivity on ICC represents receptor that is functional in the sense that it binds the natural agonist substance P and undergoes agonist-induced internalisation. ICC are likely to receive excitatory innervation from the close approaches of tachykinin-containing nerve fibres.
Recent studies have suggested that enteric inhibitory neurotransmission is mediated via interstitial cells of Cajal in some gastrointestinal tissues. This study describes the physical relationships between enteric neurons and interstitial cells of Cajal in the deep muscular plexus (IC-DMP) of the guinea-pig small intestine. c-Kit and vimentin were colocalized in the cell bodies and fine cellular processes of interstitial cells of the deep muscular plexus. Anti-vimentin antibodies were subsequently used to examine the relationships of interstitial cells with inhibitory motor neurons (as identified by nitric oxide synthase-like immunoreactivity) and excitatory motor neurons (using substance P-like immunoreactivity). Neurons with nitric oxide synthase- and substance P-like immunoreactivities were closely associated with the cell bodies of interstitial cells and ramified along their processes for distances greater than 300 micrometer. With transmission electron microscopy, we noted close relationships between interstitial cells and the nitric oxide synthase- and substance P-like immunoreactive axonal varicosities. Varicosities of nitric oxide synthase and substance P neurons were found as close as 20 and 25 nm from interstitial cells, respectively. Specialized junctions with increased electron density of pre- and postsynaptic membranes were observed at close contact points between nitric oxide synthase- and substance P-like immunoreactive neurons and interstitial cells. Close structural relationships (approximately 25 nm) were also occasionally observed between either nitric oxide synthase- and substance P-like immunoreactive varicosities and smooth muscle cells of the outer circular muscle layer. The data suggest that interstitial cells in the deep muscle plexus are heavily innervated by excitatory and inhibitory enteric motor neurons. Thus, these interstitial cells may provide an important, but probably not exclusive, pathway for nerve-muscle communication in the small intestine.
The interstitial cells of Cajal (ICC) are c-kit immunoreactive cells of the gastrointestinal tract which are suggested to have a role in the control of intestinal motility. Cells with c-kit immunoreactivity have not been previously described in the gastrointestinal tract of the horse. Immunoreactivity for c-kit was revealed using immunohistochemical labelling with an anti-c-kit polyclonal antibody. Sections of normal gastrointestinal tissue were examined from 13 anatomically defined sites from stomach to small colon taken from horses free from gastrointestinal disease. Three types of c-kit immunoreactive cells were identified: spindle-shaped cells in the region of the myenteric plexus, stellate or bipolar cells in the circular muscle layer, and round cells in the submucosa. The round cells were shown to be mast cells with the use of toluidine blue staining, whereas the other c-kit immunoreactive cells did not exhibit metachromasia and were classified as ICC. This study will serve as a basis for future pathological studies in the horse.
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.
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.
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.
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.
The tissue-specific expression of connexin subtypes in gap junctions between the interstitial cells and smooth muscle cells in the submuscular plexus of the colon has a functional importance in relation to intestinal pacemaker activity. Immunocytochemical observations of two types of connexin molecules, connexin43 and connexin45, were made with a confocal laser scanning microscope on cryosections of freshly frozen dog, guinea pig, mouse and rat proximal colon. Connexin43 immunoreactivity appeared as a series of dots along the submuscular plexus of guinea pig and dog. In contrast, connexin43 immunoreactivity was not found in mouse and rat colon. Connexin43 immunoreactivity was not observed in the colon muscular layer in the four animal species examined. In double-stained materials with a marker for either vimentin or smooth muscle actin, connexin43 immunoreactivity was colocalized with vimentin immunoreactivity, whereas it was not with either smooth muscle actin immunoreactivity or phalloidin reactivity. This indicated that the connexin43-expressing cells possess a vimentin-positive fibroblast-like nature rather than a smooth muscle-like one. In addition, in guinea pig colon, connexin43 immunoreactivity colocalized with c-Kit immunoreactivity. In conclusion, network-forming cells are connected by connexin43 gap junctions in the submuscular plexus of guinea pig and dog colon, most likely indicating that interstitial cells act as an intestinal pacemaker and conductive system.
Recent studies on the interstitial cells of Cajal (ICC) have determined ultrastructural criteria for the identification of these previously enigmatic cells. This review deals with the electron microscopic findings obtained by the author's research group in different tissue regions of the gut in mice, rats and guinea-pigs, comparing these with reports from other groups in different species and in humans. ICC are characterized by the following morphological criteria: numerous mitochondria, abundant intermediate filaments and large gap junctions which connect the cells with each other and with smooth muscle cells. Due to their location in the gut and the specific species, the ICC are markedly heterogeneous in appearance, ranging from cells closely resembling smooth muscle cells to those similar to fibroblasts (Table 1). Nevertheless, the above-mentioned morphological features are shared by all types of ICC and serve in identifying them. Recent discoveries on a significant role of c- kit in the maturation of the ICC and their specific immunoreactivity to anti-c-Kit antibody have confirmed the view that the ICC comprise an independent and specific entity of cells. This view is reinforced by the findings of the author's group that the ICC characteristically possess vimentin filaments and are stained with the zinc iodide-osmium tetroxide method which provides a staining affinity similar to methylene blue, the dye used in the original work by Cajal, (1911). Developmental studies indicate that the ICC are derived from a non-neuronal, mesenchymal origin. This paper further reviews advances in the physiological studies on the ICC, in support of the hypothesis by THUNEBERG (1982) that they function as a pacemaker in the digestive tract and a mediator transmitting impulses from the nerve terminals to the smooth muscle cells.
This paper reviews the distribution of interstitial cells of Cajal (ICC) in the human gastrointestinal (GI) tract, based on ultrastructural and immunohistochemical evidence. The distribution and morphology of ICC at each level of the normal GI tracts is addressed from the perspective of their functional significance. Alterations of ICC reported in achalasia of cardia, infantile hypertrophic pyloric stenosis, chronic intestinal pseudoobstruction, Hirschsprung's disease, inflammatory bowel diseases, slow transit constipation, and some other disorders of GI motility as well as in gastrointestinal stromal tumors are reviewed, with emphasis on the place of ICC in the pathophysiology of disease.
This manuscript reviews gap junctions' roles in control of intestinal motility. Gap junctions (GJs) of small intestine (SmIn) are found between circular muscle (CM) cells, between interstitial cells of Cajal (ICC) of deep muscular plexus (DMP) and between them and adjacent outer circular muscle (OCM). GJs between longitudinal muscle (LM) cells or between cells of inner circular muscle (ICM) have not been reported. Occasional GJs have been reported between ICC of the myenteric plexus (MyP) and rarely between these ICC and adjacent LM or CM cells, or between ICC within CM and smooth muscle cells. In the colon (Co) of several species a special network of ICC lines the inner border of CM, the submuscular plexus (SP). GJs are found between ICCs and between them and CM cells. The ICC of MyP of Co are associated with LM and CM; occasional GJs exist between ICC and each muscle layer. Small GJs are missed by electron microscopy or light microscopic Immunocytochemistry. Therefore, GJ coupling may exist without demonstrated GJs. The consequences for the pacemaking functions of ICC networks of varied densities of GJ between ICC and between ICC of MyP or DMP or of SP and CM are considered. Connexins (Cxs) that compose intestinal GJs may affect coupling, but are incompletely known. Understanding of the role of GJs in coordinating intestinal motility requires knowing: (1) what passes through gap junctions to couple ICC to smooth muscle cells; (2) what Cx with what conductances and what modulatory controls connect ICC and smooth muscle cells; (3) whether smooth muscles can generate slow waves independent of ICC networks; and (4) what happens to motility, slow waves, and IJPs when GJs are selectively uncoupled.
The shape, distribution, and ultrastructural features of interstitial cells of Cajal (ICC) of different tissue layers and organs of the rat and guinea-pig digestive tract were described and compared with the corresponding cells in other species including mice, dogs, and humans, as reported in the literature. By light microscopy, the best marker for ICC appeared to be immunoreactivity for c-Kit. Ultrastructurally, ICC were characterized by the presence of many mitochondria, bundles of intermediate filaments, and gap junctions, which linked ICC with each other. However, ICC were morphologically heterogeneous and had particular features, depending on their tissue and organ location and species. ICC in the deep muscular plexus of the small intestine and in the submuscular plexus of the colon were the most like smooth muscle cells, and had a distinct basal lamina and numerous caveolae. In contrast, ICC of Auerbach's plexus at all levels of the gastrointestinal tract were the least like smooth muscle cells. They most closely resembled unremarkable fibroblasts. ICC within the circular muscle layer were intermediate in form. In addition to the tissue specificity, some organ and species specificity could be distinguished. The structural differences between ICC may be determined by their microenvironment, including the effects of mechanical force, type of nerve supply, and spacial relationship with smooth muscle cells.
The interstitial cell of Cajal, abbreviated ICC, is a specific cell type with a characteristic distribution in the smooth muscle wall throughout the alimentary tract in humans and laboratory mammals. The number of publications relating to ICC is rapidly increasing and demonstrate a rich variation in the structure and organization of these cells. This variation is species-, region-, and location-dependent. We have chosen to define a "reference ICC," basically the ICC in the murine small intestine, as a platform for discussion of variability. The growing field of ICC markers for light and electron microscopy is reviewed. Although there is a rapidly increasing number of approaches applicable to bright field and fluorescence microscopy, the location of markers by electron microscopy still suffers from inadequate preservation of ultrastructural detail. Finally, we summarize evidence related to ICC ultrastructure under conditions differing from those of the normal, adult individual (during differentiation, in pathological conditions, transplants, mutants, and in cell culture).
The roles of the interstitial cells of Cajal in the stomach and intestine are becoming increasingly clear. Interstitial cells of Cajal in the colon are less well known, however. We studied the development and distribution of the interstitial cells of Cajal in the mouse colon, using the tyrosine kinase receptor Kit as a marker. Sections and whole mounts were studied by confocal microscopy after double immunofluorescence with specific antibodies. The ultrastructure of Kit-expressing cells was examined by electron microcopy in Kit
W-lacz
/+ transgenic mice, which carry the lacz gene inserted in place of the first exon of the Kit gene. In the subserosa, the interstitial cells of Cajal formed a two-dimensional plexus. In the myenteric area, the interstitial cells of Cajal formed a dense plexus that gradually merged with the interstitial cells of Cajal in the outer half of the circular muscle. The inner half of the circular layer was devoid of interstitial cells of Cajal whereas in the submuscular region the interstitial cells of Cajal formed a two-dimensional plexus. Tertiary nerves with various chemical codings closely followed interstitial cell of Cajal processes. By electron microscopy, Kit-expressing cells in the outer parts of the musculature had scattered caveolae, inconspicuous basal lamina and numerous mitochondria, whereas in the submuscular region they had more pronounced myoid features. Kit-expressing cells in the mouse colon are identifiable as interstitial cells of Cajal by their ultrastructure. The interstitial cells of Cajal in the mouse colon mature postnatally. They are organized into a characteristic plexus, close to the nerves with various chemical codings.
In an in vitro model for distention-induced peristalsis in the guinea pig small intestine, the electrical activity, intraluminal pressure, and outflow of contents were studied simultaneously to search for evidence of myogenic control activity. Intraluminal distention induced periods of nifedipine-sensitive slow wave activity with superimposed action potentials, alternating with periods of quiescence. Slow waves and associated high intraluminal pressure transients propagated aborally, causing outflow of content. In the proximal small intestine, a frequency gradient of distention-induced slow waves was observed, with a frequency of 19 cycles/min in the first 1 cm and 11 cycles/min 10 cm distally. Intracellular recording revealed that the guinea pig small intestinal musculature, in response to carbachol, generated slow waves with superimposed action potentials, both sensitive to nifedipine. These slow waves also exhibited a frequency gradient. In addition, distention and cholinergic stimulation induced high-frequency membrane potential oscillations (~55 cycles/min) that were not associated with distention-induced peristalsis. Continuous distention produced excitation of the musculature, in part neurally mediated, that resulted in periodic occurrence of bursts of distally propagating nifedipine-sensitive slow waves with superimposed action potentials associated with propagating intraluminal pressure waves that caused pulsatile outflow of content at the slow wave frequency.
A discovery that the protooncogene encoding the receptor tyrosine kinase, c-kit, is allelic with the Dominant white spotting (W) locus establishes that c-kit plays a functional role in the development of three cell lineages, melanocyte, germ cell, and hematopoietic cell which are defective in W mutant mice. Recent analyses of c-kit expression in various tissues of mouse, however, have demonstrated that c-kit is expressed in more diverse tissues which are phenotypically normal in W mutant mice. Thus, whether or not c-kit expressed outside the three known cell lineages plays a functional role is one of the important questions needing answering in order to fully elucidate the role of c-kit in the development of the mouse. Here, we report that some of the cells in smooth muscle layers of developing intestine express c-kit. Blockade of its function for a few days postnatally by an antagonistic anti-c-kit monoclonal antibody (mAb) results in a severe anomaly of gut movement, which in BALB/c mice produces a lethal paralytic ileus. Physiological analysis indicates that the mechanisms required for the autonomic pacing of contraction in an isolated gut segment are defective in the anti-c-kit mAb-treated mice, W/Wv mice and even W/+ mice. These findings suggest that c-kit plays a crucial role in the development of a component of the pacemaker system that is required for the generation of autonomic gut motility.
The pacemaker activity in the mammalian gut is responsible for generating anally propagating phasic contractions. The cellular basis for this intrinsic activity is unknown. The smooth muscle cells of the external muscle layers and the innervated cellular network of interstitial cells of Cajal, which is closely associated with the external muscle layers of the mammalian gut, have both been proposed to stimulate pacemaker activity. The interstitial cells of Cajal were identified in the last century but their developmental origin and function have remained unclear. Here we show that the interstitial cells of Cajal express the Kit receptor tyrosine kinase. Furthermore, mice with mutations in the dominant white spotting (W) locus, which have cellular defects in haematopoiesis, melanogenesis and gametogenesis as a result of mutations in the Kit gene, also lack the network of interstitial cells of Cajal associated with Auerbach's nerve plexus and intestinal pacemaker activity.
Incubation with 50 microM methylene blue (MB) and subsequent intense illumination resulted in abolition of the slow-wave activity in the submuscular interstitial cells of Cajal-circular muscle (ICC-CM) preparations of canine colon. This was often accompanied by a decrease in resting membrane potential. Repolarization of cells back to -70 mV did not restore the slow-wave activity, indicating that MB plus light directly interrupted the generation mechanism of slow waves. After MB incubation, a 2-min illumination consistently changed the mitochondrial conformation in ICCs from very condensed to orthodox, without inducing any obvious changes in smooth muscle cells. After 4- to 10-min illumination, ICCs became progressively more damaged with swollen and ruptured mitochondria, loss of cytoplasmic contrast and detail, loss of caveolae, and rupture of the plasma membrane. No damage was seen in smooth muscle cells or nerves. Gap junctional ultrastructure was preserved. Intense illumination without preincubation with MB left the slow waves and the ultrastructure of ICC-CM preparations unaffected. In CM preparations, without the submuscular ICC-smooth-muscle network, MB plus light induced no changes in electrical activity. We conclude that the correlation between selective damage to the submuscular ICCs (relative to smooth muscle) and selective loss of the slow-wave activity (relative to other electrical activity of the CM) strongly indicates that the ICCs play an essential role in the generation of slow waves.
Previous morphological and electrophysiological studies have supported the hypothesis that interstitial cells of Cajal have important regulatory (pacemaker) functions in the gut. In the current study, interstitial cells of Cajal associated with Auerbach's plexus in human small intestine were studied. Freshly resected intestine was examined by light and electron microscopy. The interstitial cells of Cajal resembled modified smooth muscle cells. They had caveolae and dense bodies, an incomplete basal lamina, a very well-developed smooth endoplasmic reticulum, and abundant intermediate (10 nm) filaments. Myosin filaments were not seen. Fibroblast-like cells were distinguished by their lack of caveolae and dense bodies, the relative scarcity of smooth cisternae and intermediate filaments, and the abundant granular endoplasmic reticulum. Interstitial cells of Cajal were arranged in networks of bundles containing processes of two to seven cells with fibroblastlike cells interspersed in the bundles. The bundles were innervated by nerve elements of Auerbach's plexus and extended into both layers of smooth muscle, between muscle cells, and into septa. The bundles were closely associated with elastin fibers. The organization shown in this study strongly supports the concept of interstitial cells of Cajal as important regulatory cells also in the human small intestine. The characteristic cytology and organization of interstitial cells of Cajal may provide a basis for future morphological, electrophysiological, and pathological studies of these cells in human small intestine.
The three-dimensional organization of the mucosa and submucosa of the rat intestine was analysed by scanning electron microscopy (SEM) aided by micro-dissection methods. New functional aspects raised by the SEM observations were examined by transmission electron microscopy (TEM). Morphogenesis of the intestinal villi was also illustrated three dimensionally. Removal of the epithelium by osmic acid maceration revealed the presence of many round fenestrations averaging 3 microns in diameter over the villous basal lamina. TEM confirmed that they were not artifacts but represented passages or tracks of cells of the immune system such as lymphocytes, eosinophils and macrophages. Penetration of the epithelial processes into the lamina propria was also observed. Close contacts between these free cells and the epithelial cells suggest an intercellular communication between these different cell types. The basal lamina is thus appears as a structure that allows dynamic interaction between the epithelial layer and the lamina propria, though it is generally regarded as a rigid structure acting as a barrier. Beneath the basal lamina, a cellular reticulum of fibroblast-like cells overlies the capillary network in the villi. These cells are characterized by bundles of actin filaments and contact with each other by gap junctions. This cellular reticulum probably influences the absorption of nutrients from the villi by its contractile ability in addition to its supportive role. A similar cellular network occurs beneath the epithelium of the intestinal glands. These cells may also mechanically support the glandular organization, maintaining the delicate microvascular bed. The submucosa is considered the skeleton of the intestine. SEM reveals the main framework of the submucosa is as being composed of two sets of collagen fibers running diagonally around the intestinal wall, one set in a clockwise direction, the other, counterclockwise. These fibers--in different arrays--interweave to form a lattice sheet, which presumably provides the tissue with a high resistance to mechanical forces, particularly in respect to radial forces. The diagonal orientation of the collagen fibers is essential for the flexibility of the submucosa in allowing deformation of the intestinal wall during peristalsis. Despite the nonelastic nature of the collagen fibers, the submucosa can adapt to the various shapes of the intestinal lumen by simply changing the angles formed by these fibers. Structures of the villous microcirculation, the muscularis mucosae and of the submucous plexus are also discussed.
Scanning electron microscopic observations of connective tissue cells show a new aspect of the nature of fibroblasts, and the subsequent broad survey of references makes clear that fibroblasts of many tissues have various features which are regarded as atypical of fibroblasts, and at the same time that various connective tissue cells in different organs have features typical of fibroblasts. Both morphological and functional features of fibroblasts are more or less common to those of fibroblast-like cells, and differences among these cells are quantitative rather than qualitative. Therefore, it is almost impossible to set clear-cut criteria for distinguishing genuine fibroblasts from a large population of fibroblast-like cells. The majority of cells sharing features of fibroblasts, if not all, seem to belong to the same population of cells. They are probably adapted to special functional needs in their own micro-environment that are peculiar to local or pathological or experimental conditions. It is proposed to categorize these cells into subtypes depending on their main functions: 1, fibrogenesis; 2, tissue skeleton or barrier; 3, intercellular communication system; 4, gentle contractile machinery; 5, endocrine activity; and 6, vitamin A-storing. Re-evaluation of fibroblasts and fibroblast-like cells is required to facilitate their better understanding.
The plane between longitudinal and circular muscle of human colon, as revealed on examination with light and electron microscopes, has no clear-cut border. Some groups of smooth muscle cells, obliquely oriented and with features similar to both circular and longitudinal ones--the connecting muscle bundles--run from one muscle layer to another. Other groups of smooth muscle cells, possessing their own specific ultrastructural features--the myenteric muscle sheaths--, make up envelopes of variable thickness around some myenteric ganglia and nerve strands, partially or completely embedding them in one or other muscle layer. Non-neuronal, non-muscular cells (interstitial cells of Cajal, covering cells, fibroblast-like and macrophage-like cells) complicate the texture of the myenteric muscle sheaths, creating an intricate, interconnected cellular network inside them, widespread among nerve bundles and smooth muscle cells; however, only interstitial cells have cell-to-cell junctions also with the smooth muscle cells and nerve endings. These data document the existence in this colonic area of two different types of muscle cell arrangements, one of which, the myenteric muscle sheath, only contains putative pacemaker cells.
Mice carrying mutations at the W locus located on chromosome 5 are characterized by severe macrocytic anaemia, lack of hair pigmentation and sterility. Mutations at this locus appear to affect the proliferation and/or migration of cells during early embryogenesis and result in an intrinsic defect in the haematopoietic stem cell hierarchy. An understanding of the molecular basis of the complex and pleiotropic phenotype in W mutant mice would thus provide insights into the important developmental processes of gametogenesis, melanogenesis and haematopoiesis. Here we show that the mouse mutant W has a deletion of the c-kit proto-oncogene. Interspecific backcross analysis demonstrates that the W locus is very tightly linked to c-kit and that the two loci cannot be segregated at this level of analysis. c-kit is the cellular homologue of the oncogene v-kit of the HZ4 feline sarcoma virus and encodes a transmembrane protein tyrosine kinase receptor that is structurally similar to the receptors for colony-stimulating factor-1 (CSF-1) and platelet derived growth factor. The co-localization of c-kit with W provides a molecular entry into this important region of the mouse genome. In addition, these observations provide the first example of a germ-line mutation in a mammalian proto-oncogene and implicate the c-kit gene as a candidate for the W locus.
Mutations at the W locus in the mouse have pleiotropic effects on embryonic development and hematopoiesis. The characteristic phenotype of mutants at this locus, which includes white coat color, sterility, and anemia, can be attributed to the failure of stem cell populations to migrate and/or proliferate effectively during development. Mapping experiments suggest that the c-kit proto-oncogene, which encodes a putative tyrosine kinase receptor, is a candidate for the W locus. We show here that the c-kit gene is disrupted in two spontaneous mutant W alleles, W44 and Wx. Genomic DNA that encodes amino acids 240 to 342 of the c-kit polypeptide is disrupted in W44; the region encoding amino acids 342 to 791 is disrupted in Wx. W44 homozygotes exhibit a marked reduction in levels of c-kit mRNA. These results strongly support the identification of c-kit as the gene product of the W locus.
Slow oscillations of the membrane potential difference of circular smooth muscle cells of the colon, slow waves, arise at the interface between the submucosa and muscularis externa of the colon. These studies test the hypothesis that the generation of slow waves in the circular muscle layer of the colon of the cat is dependent upon an intact interface between the submucosa and muscularis externa. Electrical recordings were made from 3 types of tissue preparations from the proximal colon: (a) submucosa and muscularis externa intact, (b) submucosa isolated from the circular muscle, and (c) muscularis externa isolated from the submucosa. The cellular make-up of each tissue was determined by the osmic acid-zinc iodide method (Champy-Maillet), and the Masson's trichrome stain. Continuous slow waves were recorded from intact tissues. Removal of the submucosa abolished slow waves from the underlying muscularis externa. Either irregular electrical transients or waxing and waning slow wave-like activity were recorded from the isolated submucosa. Submucosal tissues removed from the muscularis externa contained no circular smooth muscle, but did contain elements of the plexus submucosus extremus.
Spontaneous electrical activity was recorded with intracellular microelectrodes from cells within the circular muscle of isolated, 2 cm long, intact segments of guinea-pig ileum that were unstretched, and in segments that had been slit open along the entire length of either their mesenteric or antimesenteric border and pinned flat under a minimum of tension. Intact segments usually exhibited fast spontaneous irregular oscillations in membrane potential (mean 1.6 Hz) which were unaffected by hyoscine (0.5 microM), the substance P antagonist D-Arg1, D-Pro2, D-Trp7.9, Leu11-substance P (10 microM), hexamethonium (100 microM), propranolol (1 microM) or phentolamine (1 microM) but were blocked by tetrodotoxin (0.4 microM) or apamin (0.4 microM). This irregular spontaneous activity is deduced to be due to ongoing firing of inhibitory motor neurons. After blockade with apamin or tetrodotoxin, a slow wave-like activity with a mean frequency of 16.4 cycles/min and maximum amplitude 2-14 mV was observed in 47% of intact segments. The amplitude of slow waves waxed and waned with a mean frequency of 0.9 cycles/min. Spontaneous cholinergic (hyoscine-sensitive) excitatory junction potentials were observed in some preparations. In contrast, in the majority of opened segments the resting membrane potential was quite stable, although slow waves that were similar to those in intact segments were observed in 14% of preparations. These studies indicate that spontaneous inhibitory junction potentials and slow waves can be recorded in intact segments of guinea-pig ileum. Their relative absence in opened segments suggests their normal expression is facilitated by the circumferential integrity of the intestine.
The effect of heptanol on electrical coupling between submucosal circular muscle cells of the dog colon and consequences for slow-wave activity were investigated. Electrotonic potentials showed exponential decay giving a length constant of 2.6 +/- 0.5 mm and a time constant of 157 +/- 48 ms. Heptanol reversibly abolished electrotonic current spread, and subsequently no slow-wave activity was recorded. The length constant decreased to less than 0.2 mm. The input resistance increased from 3 to 36 M omega, suggesting a change from tissue syncytium to electrically isolated cells. D600 (5 X 10(-6) M) also abolished slow wave activity but had opposite effects on electrotonic current spread. The data are consistent with the hypothesis that heptanol reversibly inhibits intercellular coupling, resulting in loss of spread of extracellularly applied current, uncoupling of cells, and loss of pacemaker activity. Regulation of intercellular communication may be important in the control of intestinal motility.
Intracellular recordings were made from cells located in the longitudinal, inner and outer circular muscle layers of the dog, cat, rabbit, opossum and human small intestine. In whole-thickness preparations in all five species, longitudinal muscle cells generated slow waves and spikes. However, in isolated longitudinal muscle preparations, all cells tested were electrically silent. In whole-thickness and in isolated preparations, cells in the inner circular muscle layer generated spontaneous spikes superimposed on slow potentials. However, the occurrence of spikes and slow potentials was more regular in whole-thickness preparations. In whole-thickness preparations, cells in the outer circular muscle layer generated slow waves which were coupled with phasic contractions. However, in isolated outer circular muscle preparations, all cells tested were electrically silent and spontaneous phasic contractions were absent. In whole-thickness preparations, non-neural cells located on the serosal side of the outer circular muscle layer generated slow waves. The data suggest that spontaneous slow waves of the small intestine of the dog, cat, rabbit, opossum and human are generated in non-neural cells located between the longitudinal and outer circular muscle layer and by non-neural cells located between the outer and inner circular muscle layers.
Electrical slow waves were recorded by intracellular electrodes and by quasi-intracellular pressure and suction electrodes from muscle fibers at different levels in edgewise preparations of cat jejunum. Simultaneous recordings from longitudinal and circular muscle layers showed similar resting potentials from either muscle layer near the boundary zone, and lower resting potentials in cells of circular muscle near the submucosa. Slow waves were maximal in amplitude at the boundary between the two layers and spread electrotonically away from the boundary in both layers. Bipolar recordings were of opposite polarity on the two sides of the boundary. Amplitudes of slow waves from inner circular fibers were significantly lower than from outer circular fibers. Small strips of each muscle layer were prepared with or without the attached interstitial cells of Cajal plexus as identified by methylene blue staining. Either muscle layer showed slow waves from regions where interstitial cells of Cajal (ICC) were observed after the recording. No slow waves were recorded from either layer from regions where ICC were not observed. Strips containing ICC but not strips lacking ICC could be driven electrically. Since blocking of neurons does not abolish slow waves and since regions of muscle lacking ICC do not have slow waves, it is concluded that the interstitial cells (ICC-I) are most likely the boundary elements essential for slow waves in either layer of intestinal muscle.
The zinc iodide/osmic acid (ZIO) method was used in a modification that selectively stained nerves and associated interstitial cells of Cajal (ICC) of muscularis externa. Due to its selectivity the method allowed a detailed stereoscopical analysis of whole mounts with respect to the topography and morphology of these elements. The method thus assisted and expanded our ultrastructural studies. The ZIO staining allowed a distinction of four morphologically different interstitial cell types (ICC-I-IV) confined to four compartments. The stained components were: (1) A rich plexus of highly ramified interstitial cells (ICC-II) in the subserous layer. (2) Auerbach's plexus with an associated extensive plexus of interstitial cells (ICC-I) in close contact with tertiary fasciculi. (3) Nerve fasciculi of the outer division of the circular muscle layer. These formed a nerve plexus in a well-defined plane in the outermost cell layers (plexus muscularis superficialis), with few fasciculi located internal to this plexus. A few bipolar interstitial cells (ICC-IV) were associated with nerve fasciculi of this region. (4) A nerve plexus located in the region between the two subdivisions of the circular muscle, plexus muscularis profundus (PMP). PMP was revealed throughout the small intestine as a continuous network of elongated, circularly oriented meshes. The pattern of connections between PMP and the other enteric plexuses was studied stereoscopically. Ganglion cells intrinsic to PMP occurred widely scattered. Interstitial cells associated with PMP (ICC-III) were arranged in a plexiform manner; their morphology and relations to nerves were investigated in great detail. A selective innervation of ICC-III via axons of PMP was strongly supported.
Interstitial cells of Cajal (ICCs) are believed to initiate the basic contractile activity of the gastrointestinal tract. Because ICCs in the intestine of mice express c-kit receptor tyrosine kinase and because rats are more commonly used than mice for pathophysiological investigations of the gastrointestinal tract, the number of the c-kit messenger RNA-expressing cells was compared with gastrointestinal movement in rats.
The c-kit messenger RNA-expressing cells were detected by in situ hybridization. The autonomous contraction of excised segments of the ileum was recorded. The function of the pyloric sphincter was evaluated by measuring the content of bile acids in the stomach.
The c-kit messenger RNA-expressing cells were not detectable in the stomach of Ws/Ws mutant rats with a small deletion at the tyrosine kinase domain of c-kit, and the number of c-kit messenger RNA-expressing cells decreased to 7% that of normal control rats in the ileum of Ws/Ws rats. The contractile activity of the ileum was apparently impaired, and the content of bile acids in the stomach was significantly increased in Ws/Ws rats.
The abnormalities in the ileal movement and pyloric sphincter function in Ws/Ws rats were attributable to the deficiency of c-kit messenger RNA-expressing cells.
This study has demonstrated that cells stained using the zinc iodide-osmic acid (ZIO) method have the same fine structural features as those of fibroblasts. They have a well developed Golgi apparatus, granular endoplasmic reticulum and many mitochondria. They have no basal lamina. They are distributed in association with the deep muscular plexus, within the outer circular muscle layer and in the space between the circular and longitudinal muscle layers. Since this staining method is believed to co-stain nerves and interstitial cells of Cajal, we concluded that these ZIO-positive, fibroblast-like cells represent at least some, if not all, of the interstitial cells which appeared in the original description by Cajal.
1. Interstitial cells of Cajal (ICs) have been proposed as pacemakers in the gastrointestinal tract. We studied the characteristics and distribution of ICs and electrical activity of small intestinal muscles from mice with mutations at the dominant-white spotting/c-kit (W) locus because the tyrosine kinase function of c-kit may be important in the development of the IC network. 2. W/WV mutants (days 3-30 postpartum) had few ICs in the myenteric plexus region compared with wild type (+/+) siblings. The few ICs present were associated with neural elements and lay between myenteric ganglia and the longitudinal muscle layer. 3. Electrical recordings from intestinal muscle strips showed that electrical slow waves were always present in muscles of +/+ siblings, but were absent in W/WV mice. 4. Muscles from W/WV mice responded to stimulation of intrinsic nerves. Neural responses, attributed to the release of acetylcholine, nitric oxide and other unidentified transmitters, were recorded. 5. These findings are consistent with the hypothesis that ICs are a critical element in the generation of electrical rhythmicity in intestinal muscles. The data also show that neural regulation of gastrointestinal muscles can develop independently of the IC network. 6. W locus mutants provide a powerful new model for studies of the physiological role of ICs and the significance of electrical rhythmicity to normal gastrointestinal motility.
1. Voltage-dependent ionic currents of isolated interstitial cells were characterized using the whole-cell voltage clamp technique, and compared with currents recorded from circular muscle cells. Both cell types were isolated from the submucosal pacemaking region in the canine distal colon. 2. Upon depolarization, interstitial cells and smooth muscle cells generated transient inward, followed by slowly inactivating outward, currents. 3. After blocking inward current and much of the Ca(2+)-dependent outward current, interstitial cells displayed voltage-dependent outward current that rapidly activated, reached a peak, and then inactivated. This current was resistant to 4-aminopyridine(4-AP; 1 mM). Smooth muscle cells expressed a similar current but it was reduced by about 40% at a test potential of +20 mV by 4-AP (1 mM). 4. The inactivation characteristics of the voltage-dependent outward currents of interstitial cells and smooth muscle cells were compared. The outward current of interstitial cells inactivated at more negative potentials; half-inactivation occurred at -53 mV, whereas half-inactivation occurred at -20 mV in smooth muscle cells. 5. Inward currents were not strikingly different in the two cell types when dialysing pipettes were used. When the perforated patch technique (using Amphotericin-B) was used, a negatively activating inward current was observed in interstitial cells that had a resolution threshold of -70 to -60 mV. This current peaked at -10 mV. Inward currents in smooth muscle cells were resolved at test potentials positive to -50 mV and peaked at 0 to +10 mV. 6. When interstitial cells were held at -40 mV, inward current could not be resolved with test depolarization negative to -30 mV. From this holding potential, peak amplitude was reduced by 85% with test depolarizations to -10 mV. Holding smooth muscle cells at -40 mV also reduced inward current, but the peak current in these cells was reduced by only 39% at 0 mV. 7. Ni2+ partially inhibited peak inward current in interstitial cells and abolished a 'hump' in the I-V curve that occurred at negative potentials. In dialysed cells where this 'hump' was not apparent, addition of nifedipine unmasked a 'hump'. The presence of both nifedipine and Ni2+ abolished inward current. 8. A portion of the inward current in smooth muscle cells was sustained and persisted for the duration of test pulses. Very little sustained inward current was observed in interstitial cells. 9. The time course of inactivation of inward current in interstitial cells was fitted with two exponentials.(ABSTRACT TRUNCATED AT 400 WORDS)
Observation of whole mount stretch preparations using the zinc-iodide-osmic acid method reveals a wide variety of interstitial cells in different tissue layers of the guinea-pig small intestine. And a subsequent electron-microscopic examination and survey of references makes clear that the interstitial cells of Cajal (ICC) depicted in original drawings of Cajal are heterogeneous and correspond to different types of interstitial cells. The myenteric ICC are characterized by long dichotomous branching processes which constitute cellular networks independent from the nerve plexus and form many gap junctions at their tips. Their ultrastructure is similar to that of fibroblasts and they have no basal lamina. The myenteric ICC show strong immunoreactivity for vimentin and the c-kit receptor, and probably correspond to the intestinal pacemaker cells. Within the circular muscle layer, ICC are represented by the cells that are closely associated with fine nerve bundles. The ICC have various shapes, ranging from bipolar to stellate, depending on the running pattern of the nerve fibers that they are associated with. They show fibroblast-like ultrastructure and have no basal lamina. They form gap junctions with smooth muscle cells and are immunoreactive for vimentin. On the other hand, ICC associated with the deep muscular plexus described in the guinea-pig by Cajal could not be clearly identified. However, it is suggested that the ICC in this location may correspond to glycogen-rich cells possessing a basal lamina. Although they show a fairly well-developed rough endoplasmic reticulum, Golgi apparatus and immunoreactivity for vimentin, ICC of the deep muscular plexus are probably specialized smooth muscle cells in nature.
Identification of the intersti-tial cells of Cajal (Abstracts). XIV Federative Int
- T Komuro
- D S Zhou
- K Tokui
Komuro, T., Zhou, D.S. and Tokui, K., Identification of the intersti-tial cells of Cajal (Abstracts). XIV Federative Int. Congr. of Anat. (1994) p. 169.
Interstitial cells of Cajal Handbook of Physiology
- L Thuneberg
Thuneberg, L., Interstitial cells of Cajal. In J.D. Wood (Ed.), Handbook of Physiology. The Gastrointestinal System, Vol 1. Amer-ican Physiology Society, Bethesda Maryland, 1989, pp. 349-386.
c-kit dependent development of interstitial cells and electrical activity in the murine gastrointestinal tract
- Torihashi