Involvement of intramuscular interstitial cells of Cajal in neuroeffector transmission in the gastrointestinal tract

Specialized cells known as interstitial cells of Cajal (ICC) are distributed in specific locations within the tunica muscularis of the gastrointestinal (GI) tract. ICC serve as electrical pacemakers, provide pathways for the active propagation of slow waves, are mediators of enteric motor neurotransmission and play a role in afferent neural signalling. Morphological studies have provided evidence that motor neurotransmission in the GI tract does not occur through poorly defined structures between nerves and smooth muscle, but rather via specialized synapses that exist between enteric nerve terminals and intramuscular ICC or ICC-IM. ICC-IM are coupled to smooth muscle cells via gap junctions and post-junctional responses elicited in ICC-IM are conducted to neighbouring smooth muscle cells. Electrophysiological studies from the stomachs and sphincters of wild-type and mutant animals that lack ICC-IM have provided functional evidence for the importance of ICC in cholinergic excitatory and nitrergic inhibitory motor neurotransmission. Intraperitoneal injection of animals with Kit neutralizing antibody or organ culture of gastrointestinal tissues in the presence of neutralizing antibody, which blocks the development and maintenance of ICC, has provided further evidence for the role of ICC in enteric motor transmission. ICC-IM also generate an ongoing discharge of unitary potentials in the gastric fundus and antrum that contributes to the overall excitability of the stomach.     

长爪沙鼠胃肠道Cajal间质细胞的形态学

目的 探讨长爪沙鼠胃肠道Cajal间质细胞（ICCs）的形态和分布规律。 方法 采用10只成年长爪沙鼠,体重50~70g,取胃、小肠、结肠制作冷冻切片,结合全层铺片的c-Kit免疫荧光染色。结果 ICCs呈网络状分布于整个胃肠道,不同部位ICCs的分布及形态有所不同。在胃底部,仅见肌内ICCs(ICC-IM）,而在胃体和胃窦部除ICC-IM外,可见肌间ICCs(ICC-MY)分布在肌间神经丛周围;其细胞密度胃底ICC-IM最多,由胃底至胃窦逐渐减少,而ICC-MY由胃体至胃窦逐渐增多。在小肠可见ICC-IM, ICC-MY和深肌层ICCs(ICC-DMP)3个亚群,结肠管壁内也分布有ICC-IM、ICC-MY和黏膜下ICCs(ICC-SM)3个亚群。结论 沙鼠可用于有关ICCs正常形态、结构及功能的研究。     

Ultrastructural characterization of the interstitial cells of Cajal

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.     

The interstitial cells of Cajal and a gastroenteric pacemaker system

In spite of a claim by Kobayashi (1990) that they do not correspond to the cells originally depicted by CAJAL, a particular category of fibroblast-like cells have been identified in the gut by electron microscopy (Faussone-Pellegrini, 1977; Thuneberg, 1980) and by immunohistochemistry for Kit protein (Maeda et al., 1992) under the term of the "interstitial cells of Cajal (ICC)". Generating electrical slow waves, the ICC are intercalated between the intramural neurons and the effector smooth muscular cells, to form a gastroenteric pacemaker system. ICC at the level of the myenteric plexus (IC-MY) are multipolar cells forming a reticular network. The network of IC-MY which is believed to be the origin of electrical slow waves is morphologically independent from but associated with the myenteric plexus. On the other hand, intramuscular ICC (IC-IM) usually have spindle-shaped contours arranged in parallel with the bulk smooth muscle cells. Associated with nerve bundles and blood vessels, the IC-IM possess receptors for neurotransmitters and such circulating hormones as cholecystokinin, suggesting their roles in neuromuscular and hormone-muscular transmissions. In addition, gap junctions connect the IC-MY and IC-IM, thereby realizing the electrically synchronized integrity of ICC as a pacemaker system in the gut. The smooth muscle cells are also coupled with ICC via gap junctions, and the functional unit thus formed enables rhythmically synchronized contractions and relaxations. It has recently been found that a lack of Kit-expressing cells may induce hyper-contractility of the tunica muscularis in vitro, whereas a decrease in Kit expression within the muscle wall causes dysmotility-like symptoms in vivo. The pacemaker system in the gut thus seems to play a critical role in the maintenance of both moderate and normal motility of the digestive tract. A loss of Kit positive cells has been detected in several diseases with an impaired motor activity, including diabetic gastroenteropathy. Pathogenesis of these diseases is thought to be accounted for by impaired slow waves and neuromuscular transmissions; a pacemaker disorder may possibly induce a dysmotility-like symptom called 'gastroenteric arrhythmia'. A knowledge of the structure and function of the ICC and the pacemaker system provides a basis for clarifying the normal mechanism and the pathophysiology of motility in the digestive tract.     

Kit mutants and gastrointestinal physiology

There has been considerable speculation about the function of interstitial cells of Cajal (ICC) since their discovery more than 100 years ago. It has been difficult to study these cells under native conditions, but great insights about the function of ICC have come from studies of genetic models with loss-of function mutations in the Kit signalling pathway. First it was discovered that signalling via Kit (a receptor tyrosine kinase) was vital for the development and maintenance of the ICC phenotype in gastrointestinal (GI) muscles. In compound heterozygotes ( W/W^V and Sl/Sl^d animals), where there are partial loss-of-function mutations in Kit receptors or Kit ligand (stem cell factor), ICC failed to develop in various regions of the GI tract, but no major changes in the smooth muscle layers or enteric nervous system occurred in the absence of these cells. Animals with these mutations provided an unprecedented opportunity to understand the role of ICC in GI motor function, and it is now clear from these studies that ICC serve as: (i) pacemaker cells, generating the spontaneous electrical rhythms of the gut known as slow waves; (ii) a propagation pathway for slow waves so that large areas of the musculature can be entrained to a dominant pacemaker frequency; (iii) mediators of excitatory cholinergic and inhibitory nitrergic neural inputs from the enteric nervous system, and (iv) stretch receptors that modulate membrane potential and electrical slow wave frequency. This review describes the use of genetic models to understand the important physiological role of ICC in the GI tract.     

Ultrastructural studies of gastrointestinal stromal tumors

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors in the gastrointestinal tract (GIT). Although interstitial cells of Cajal has been suggested as origin of this tumor, the cytological and ultrastructural features of GISTs are heterogeneous and unclear. A total 10 cases of normal gastrointestinal tissue (control), 13 GISTs of the stomach (8), small intestine (3), mesocolon (1) and liver (1), and 2 gastrointestinal autonomic nervous tumor (GANT) of small intestine were ultrastructurally studied. Normal interstitial cells of Cajal (ICC) were abundantly present around the myenteric plexuses or individually scattered through the wall of GIT. ICC was characterized by slender cytoplasmic processes, well-developed endoplasmic reticulum (ER), mitochondria, Golgi apparatus, caveolae and intermediate filaments. The GISTs and GANTs had overlapping ultrastructures. The most common and important ultrastructural features of GISTs were rich villous cytoplasmic processes, dispersed intermediate filaments and abundant SER, and those of GANTs were neurosecretory granules and skenoid fibers. Compared with ICC, the GISTs and GANTs had remarkably reduced caveolae and gap junctions. Our study suggested that ultrastructural analysis gives much information to investigate lineage differentiation of neoplastic cells and make a differential diagnosis of these tumors from other mesenchymal tumors and between GISTs and GANTs.     

Interstitial cells of Cajal: primary targets of enteric motor innervation

For many years morphologists have noted the close relationship between interstitial cells of Cajal (ICC) and nerve fibers within the tunica muscularis of gastrointestinal (GI) organs. These observations led to speculations about a role for ICC in mediating neural inputs to the GI tract. Immunohistochemical and functional studies demonstrated the presence of receptors for the neurotransmitters utilized by enteric motor neurons, and changes in second messengers in ICC after field stimulation of intrinsic enteric neurons showed that ICC were functionally innervated in GI muscles. Recent double labeling experiments have shown that both excitatory and inhibitory enteric motor neurons are closely associated with ICC in the deep muscular plexus (IC-DMP) of the small intestine and intramuscular ICC (IC-IM) of the proximal and distal GI tract. Enteric motor neurons form synaptic-like structures with IC-IM and IC-DMP. Far fewer close contacts are found between enteric motor neurons and smooth muscle cells. Experiments on W/W(V) mutants that lack IC-IM in the stomach, lower esophageal sphincter, and pylorus have shown that these ICC are critical components of the neuromuscular junction. Cholinergic excitatory and nitrergic inhibitory neurotransmission are severely decreased in tissues lacking IC-IM, yet there is no loss of cholinergic or nitrergic neurons in W/W(V) mutants. These data suggest that either the post-junctional mechanisms responsible for receiving and transducing neurotransmitter signals are specifically expressed by ICC, or that the large extracellular spaces typically between nerve terminals and smooth muscle cells may not allow effective concentrations of neurotransmitters to reach receptors expressed by smooth muscle cells. These findings indicate an important role for certain classes of ICC in enteric neurotransmission and predict that loss of ICC in human motor disturbances may significantly compromise neural regulation of GI motility.     

Immunohistological characterization of intercellular junction proteins in rhesus macaque intestine

Epithelial junctions play an important role in regulating paracellular permeability and intercellular adhesion. It has been reported that changes in the density of epithelial junctions and/or distribution pattern can contribute to various gastrointestinal (GI) disorders. In this study, we investigated the distribution of the tight junction (Claudins. 1, 3, 4, 5, 7, 10, Zonula Occludens (ZO-1), Occludin), adherens junction (E-cadherin), desmosome (Desmoglein 2, Desmocollin 2) and gap junction (Connexin 43) proteins in the jejunum, ileum and colonic epithelium of healthy rhesus macaques (RM) using immunofluorescence labeling. While proteins in these respective junctions were expressed throughout the jejunum, ileum and colon of RM, we observed differential labeling in epithelial cells from these sites. Claudins 1, 3, 4, 7, E-cadherin and Desmoglein 2 were distributed in the respective intercellular junctions with additional labeling in the lateral membrane of epithelial cells in both small and large intestine. However, claudin 5, claudin 10, ZO-1 and occludin showed uniform distribution in the intercellular junctions of crypt and surface epithelial cells of the intestine. Desmocollin 2 localized predominantly in the upper two thirds along the lateral membrane while desmoglein 2 was distributed along the entire lateral membrane of intestinal epithelial cells. In contrast, connexin 43 exhibited punctate lateral labeling in crypt epithelial cells of the small and large intestine. Our results show diverse localization of epithelial intercellular junction proteins along the intestinal tract of RM. These findings may correlate with differences in paracellular permeability and adhesion along the intestinal tract and could correlate with pathologic disease in these regions of the intestine.     

Postnatal development of interstitial cells of Cajal in mouse colon in response to Kit signal blockade with Imatinib (Glivec)

This study investigated the response of interstitial cells of Cajal (ICC) in postnatal mouse colon to treatment with Imatinib (Glivec®, a potent inhibitor of Kit receptor). ICC were revealed by immunofluorescent staining on frozen cross-sections and whole-mount preparations by anti-Kit and DOG1 antibodies. Kit and p-Kit protein were also evaluated by Western blot. After administration of Imatinib for 4 days beginning at 8 days post-partum (P8), the mean density of Kit⁺ ICC, which were localized around the myenteric nerve plexus (ICC-MY), within smooth muscle layers (ICC-IM) and in the connective tissue beneath the serosa (ICC-SS), was dramatically decreased to about 50% when compared with controls, but those Kit⁺ cells located at the submucosal border of circular smooth muscle layer (ICC-SM) seemed to be unchanged in both cell number and morphology. A small number of DOG1⁺/Kit⁻ cells appeared during Imatinib administration. However, these Kit⁺ ICC were not changed in mice even after 12 days of Imatinib treatment from P24. When Imatinib was discontinued, the number of ICC recovered to normal within 4 days. Our results indicate that the postnatal development of ICC in the mouse colon is Kit dependent, but ICC-SM are unlikely, and the Kit dependence of ICC development is also age-dependent.     

Distribution and ultrastructure of interstitial cells of Cajal in the gastric antrum of wild-type and Ws/Ws rats

Interstitial cells of Cajal (ICC) in the stomach of wild-type and Ws/Ws mutant rats that are deficient in c-kit were studied by immunohistochemistry and electron microscopy to elucidate their regional specialization in the gastric antrum. Immunohistochemistry for Kit protein demonstrated that in wild-type rats ICC were located at the submucosal border of the circular muscle layer (ICC-SM) in a limited extension of the antrum from the pyloric sphincter towards the corpus, as well as within both the circular (ICC-CM) and longitudinal (ICC-LM) muscle layers and in the myenteric plexus region (ICC-AP). In c-kit mutant Ws/Ws rats while ICC-CM and ICC-LM were not observed, but unexpectedly, a few ICC-SM and ICC-AP were found. By electron microscopy, ICC-SM and ICC-AP were characterized by abundant mitochondria, many caveolae, a distinct basal lamina and formed gap junctions with other ICC or with smooth muscle cells and make close contacts with nerves. Thus, ICC-SM and ICC-AP of the rat antrum were classified as Type 3 ICC, the type most similar to smooth muscle cells. The functional significance of ICC-SM and their survival in the c-kit mutant animals is discussed in reference to the role of the c-kit/stem cell factor system for their cellular maturation.     

The interstitial cells of Cajal of the rat stomach. A light and electron microscope study

Interstitial cells of Cajal (ICC), presumed to have a smooth muscle-like nature and to play a pacemaker role, are usually identified for their peculiar ultrastructural features and specific location throughout the gut muscle wall. A Zinc-Iodide-Osmium (ZIO) impregnation for ICC identification under the light microscope has been proposed. However, controversies as to certain ICC identification under both light and electron microscopes are still present, due to their ultrastructural features somewhat similar to the fibroblast ones and the low specificity of the ZIO-staining. The rat stomach has been studied. Some specimens have been routinely processed for electron microscopy, some others have been ZIO-impregnated and further routinely processed for both light and electron microscopy, in order to assure that all the cells presumed to be ICC for their ZIO-staining affinity are the same cells identified as ICC with routine electron microscope procedures, and not fibroblasts. The routine electron microscope examination made it possible to identify within the rat gastric muscle coat two cell populations, one with the same location and morphology as those reported in literature for the gastric ICC, and a second one with a similar location, but showing undoubted fibroblastic features. ZIO-staining, under both light and electron microscopes, revealed ZIO-stained cells distributed within the muscle coat in a manner identical to that of the ultrastructurally identified ICC. Under electron microscope examination, this cell type only was fully impregnated by the zinciodide deposits, whereas all other cell types, including fibroblast-like cells, were devoid of them. These data confirm that ICC can be electively ZIO-stained and that these cells and fibroblasts are two distinct cell types, as ultrastructural and physiological reports had previously suggested.     

Developmental Changes in Postnatal Murine Intestinal Interstitial Cell of Cajal Network Structure and Function

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.     

Muscarinic M(2) acetylcholine receptor distribution in the guinea-pig gastrointestinal tract

In the enteric nervous system, acetylcholine is the most common neurotransmitter to induce gastrointestinal smooth muscle contractions. Cholinergic signaling is mediated by muscarinic acetylcholine receptors on the surface of smooth muscle cells. Five different muscarinic receptor subtypes (M(1)-M(5)) have been identified and characterized, all of which belong to the superfamily of the G-protein-coupled receptor. The muscarinic M(2) acetylcholine receptor is the major muscarinic receptor subtype expressed by smooth muscle tissues in the gastrointestinal tract, where it is coexpressed with a smaller population of M(3) receptor. In this study, we examined the immunohistochemical distribution of the M(2) receptor using a specific antibody in the guinea-pig gastrointestinal tract. M(2) receptor-like immunoreactivity was mainly observed as associated with smooth muscle cells in the gastrointestinal tract. M(2) receptor-like immunoreactivity in smooth muscle cells was distributed throughout the cell membrane associated with caveolae. In the proximal colon, M(2) receptor-like immunoreactivity in the smooth muscle cells was weak. In the small intestine, interstitial cells of Cajal that possessed neurokinin 1 receptor-like immunoreactivity had intense M(2) receptor-like immunoreactivity. In the proximal colon, intramuscular and myenteric interstitial cells of Cajal exhibited M(2) receptor-like immunoreactivity. These findings indicate that, in the gastrointestinal musculature, M(2) receptors are distributed both in the smooth muscle cells and interstitial cells of Cajal, suggesting that the M(2) receptor elicits smooth muscle cell contraction and the interstitial cells of Cajal are the sites of innervation by enteric cholinergic neurons.     

GABAA receptor subtype specific enhancement of inhibition in human motor cortex

Interstitial cells of Cajal (ICC) were described more than 100 years ago by Ramon y Cajal. For many years these cells were identified only by non-specific histological stains and later, more reliably, by electron microscopy. Ultrastructural features and the anatomical locations of ICC suggested important physiological roles for these cells. A breakthrough occurred in our ability to study ICC when it was recognized that antibodies for Kit could be used to identify ICC, even in living tissues. Signalling via Kit, a receptor tyrosine kinase, is also necessary for ICC development and maintenance of phenotype. Thus, blocking Kit, by a variety of techniques, caused loss of ICC in experimental animals and demonstrated the critical physiological functions of these cells in gastrointestinal motility. Loss of ICC in human gastrointestinal diseases may contribute to the motor pathologies observed. Unrestrained Kit signalling leads to the transformation of ICC and the development of gastrointestinal stromal tumours. Now ICC-like cells have been identified in a variety of smooth muscle tissues, and the race is on to discover whether these cells have equivalent or even novel functions in organs outside the gastrointestinal tract. This perspectives article gives a short overview of the history of ICC research and directions for future investigation.     

消化道蠕动的起搏器与Cajal间质细胞

消化道蠕动是消化道平滑肌群的节律性收缩活动。越来越多的研究证据表明,这种活动是由一类叫做(pacem aker)起搏器的特殊细胞引起的。这些细胞可能就是分布在消化道平滑肌层间的(interstitialcells of Cajal)ICC。它们因分布的部位不同而形态各异。但它们都含有丰富的线粒体,呈c-K it免疫染色阳性,并伸出很多突起形成网络。他们相互之间及与周围的平滑肌细胞之间以缝隙结合相连接。这篇综述主要介绍此类细胞的形态结构及分布特点。     

Interstitial cells of Cajal are innervated by nitrergic nerves and express nitric oxide-sensitive guanylate cyclase in the guinea-pig gastrointestinal tract

Nitric oxide (NO) is a major signaling molecule in the gastrointestinal tract, and released NO inhibits muscular contraction. The actions of NO are mediated by stimulation of soluble guanylate cyclase (sGC, NO-sensitive GC) and a subsequent increase in cGMP concentration. To elucidate NO targets in the gastrointestinal musculature, we investigated the immunohistochemical localization of the beta1 and alpha1 subunits of sGC and the distribution of neuronal NO synthase (nNOS) -containing nerves in the guinea-pig gastrointestinal tract. Distinct immunoreactivity for sGCbeta1 and sGCalpha1 was observed in the interstitial cells of Cajal (ICC), fibroblast-like cells (FLC) and enteric neurons in the musculature. Double immunohistochemistry using anti-c-Kit antibody and anti-sGCbeta1 antibody revealed sGCbeta1 immunoreactivity in almost all intramuscular ICC throughout the entire gastrointestinal tract. Immunoelectron microscopy revealed that sGCbeta1-immunopositive cells possessed some of the criteria for intramuscular ICC: presence of caveolae; frequently associated with nerve bundles; and close contact with smooth muscle cells. sGCbeta1-immunopositive ICC were closely apposed to nNOS-containing nerve fibers in the muscle layers. Immunohistochemical and immunoelectron microscopical observations revealed that FLC in the musculature also showed sGCbeta1 immunoreactivity. FLC were often associated with nNOS-immunopositive nerve fibers. In the myenteric layer, almost all myenteric ganglia contained nNOS-immunopositive nerve cells and were surrounded by myenteric ICC and FLC. Myenteric ICC in the large intestine and FLC in the entire gastrointestinal tract showed sGCbeta1 immunoreactivity in the myenteric layer. Smooth muscle cells in the stomach and colon showed weak sGCbeta1 immunoreactivity, and those in the muscularis mucosae and vasculature also showed evident immunoreactivity. These data suggest that ICC are primary targets for NO released from nNOS-containing enteric neurons, and that some NO signals are received by FLC and smooth muscle cells in the gastrointestinal tract.     

Plasticity of Interstitial Cells of Cajal: A Study in the Small Intestine of Adult Guinea Pigs

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, α‐smooth muscle actin, (α‐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 α‐SMA and a few ICC‐like cells were only stained with α‐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 α‐SMA⁺ cells, perhaps a phenotype of smooth muscle cells, when there is a loss‐of‐function of Kit. Anat Rec, 292:985–993, 2009. © 2009 Wiley‐Liss, Inc.     

Differential expression of connexin 43 in gastrointestinal stromal tumours of gastric and small intestinal origin

Gastrointestinal stromal tumours (GISTs) are considered to originate from interstitial cells of Cajal (ICCs). ICCs are classified into several subtypes according to their location or roles. Several reports indicate that GISTs of the small intestine appear to have different clinical and pathological characteristics from gastric GISTs. We previously found using a cDNA expression chip that connexin 43, a component of gap junctions, is expressed specifically in small intestinal GISTs but not in gastric GISTs. To confirm the specificity of connexin 43 expression, we analysed 10 small intestinal GISTs and 15 gastric GISTs by northern blotting, western blotting and immunohistochemistry in this study. Northern blotting was performed in five small intestinal GISTs and five gastric GISTs, and revealed connexin 43 mRNA expression in all of the five small intestinal GISTs, but in none of the gastric GISTs. By western blotting, bands corresponding to connexin 43 were easily detected in all of the five small intestinal GISTs studied but were absent in all five gastric GISTs analysed. Immunohistochemistry showed that all of the 10 small intestinal GISTs were positive for connexin 43 but only one of 15 gastric GISTs, which exhibited a mutation in exon 9 of the KIT gene, was connexin 43-positive. We also examined the localization of connexin 43 in the normal stomach and small intestine. Immunoreactivity for connexin 43 was present in both normal gastric and small intestinal circular muscle layers, but it was unclear which cell type was positive. These results suggest that GISTs are divided into at least two groups, namely the gastric subtype and the small intestinal subtype, through phenotype but not location. Furthermore, these data indicate that the gastric and the small intestinal subtypes of GIST may originate from different subtypes of ICC.