Kazuhide Horiguchi

Loss of interstitial cells of Cajal and development of electrical dysfunction in murine small bowel obstruction

1 Partial obstruction of the murine ileum led to changes in the gross morphology and ultrastructure of the tunica muscularis. Populations of interstitial cells of Cajal (ICC) decreased oral, but not aboral, to the site of obstruction. Since ICC generate and propagate electrical slow waves in gastrointestinal muscles, we investigated whether the loss of ICC leads to loss of function in partial bowel obstruction. 2 Changes in ICC networks and electrical activity were monitored in the obstructed murine intestine using immunohistochemistry, electron microscopy and intracellular electrophysiological techniques. 3 Two weeks following the onset of a partial obstruction, the bowel increased in diameter and hypertrophy of the tunica muscularis was observed oral to the obstruction site. ICC networks were disrupted oral to the obstruction, and this disruption was accompanied by the loss of electrical slow waves and responses to enteric nerve stimulation. These defects were not observed aboral to the obstruction. 4 Ultrastructural analysis revealed no evidence of cell death in regions where the lesion in ICC networks was developing. Cells with a morphology intermediate between smooth muscle cells and fibroblasts were found in locations that are typically populated by ICC. These cells may have been the redifferentiated remnants of ICC networks. 5 Removal of the obstruction led to the redevelopment of ICC networks and recovery of slow wave activity within 30 days. Neural responses were partially restored in 30 days. 6 These data describe the plasticity of ICC networks in response to partial obstruction. After obstruction the ICC phenotype was lost, but these cells regenerated when the obstruction was removed. This model may be an important tool for evaluating the cellular/molecular factors responsible for the regulation and maintenance of the ICC phenotype.

Loss of Enteric Motor Neurotransmission in the Gastric Fundus of Sl/Sld Mice

Studies of W/WV mice, which lack intramuscular interstitial cells of Cajal (IC‐IM), have suggested that IC‐IM act as mediators of enteric motor neurotransmission in the gastrointestinal tract. We have studied Sl/Sld mice, which lack the ability to make membrane‐bound stem cell factor, to determine the consequences of inappropriate stem cell factor expression on IC‐IM populations and on enteric motor neurotransmission. IC‐IM were found within the circular and longitudinal muscles of the gastric fundus of wild‐type mice. IC‐IM were intimately associated with motor nerve terminals and nerve varicosities formed synaptic structures with these cells. IC‐IM were also connected with neighbouring smooth muscle cells via gap junctions. Immunohistochemistry and electron microscopy showed that IC‐IM were absent from fundus muscles of Sl/Sld mice, but the density of excitatory and inhibitory nerves was not significantly different than in wild‐type muscles. Loss of IC‐IM was associated with decreased membrane noise (unitary potentials) and significant reductions in post‐junctional excitatory and inhibitory enteric nerve responses. Reductions in neural responses were not due to defects in smooth muscle cells as responses to exogenous ACh and K+‐induced depolarization were normal in Sl/Sld mice. Responses to neurally released ACh were revealed in Sl/Sld mice by inhibiting ACh breakdown with the acetylcholinesterase inhibitor neostigmine. Inhibitory nerve stimulation elicited inhibitory junction potentials (IJPs) and relaxations in wild‐type mice. IJPs were reduced in amplitude and relaxation responses were absent in Sl/Sld mice. These observations suggest that membrane‐bound stem cell factor is essential for development of IC‐IM and that the close, synaptic‐like relationship between nerve terminals and IC‐IM may be the primary site of innervation by enteric motor neurons in gastric muscles.

Distribution of pacemaker function through the tunica muscularis of the canine gastric antrum

1 Interstitial cells of Cajal (ICC) have been shown to generate pacemaker activity in gastrointestinal (GI) muscles. Experiments were performed to characterize the ICC within the canine gastric antrum and to determine the site(s) of pacemaker activity and whether active propagation pathways exist within the thick‐walled tunica muscularis of large mammals. 2 Immunohistochemistry and electron microscopy revealed four populations of ICC within the antral muscularis on the basis of anatomical location. Typical ICC were found in the myenteric region of the small intestine (IC‐MY). Intramuscular ICC (IC‐IM) were intermingled between muscle fibres of circular and longitudinal muscle layers. ICC were also found within septa (IC‐SEP) between muscle bundles and along the submucosal surface of the circular muscle layer (IC‐SM). ICC were identified in each location by ultrastructural features. 3 Intracellular electrical recordings demonstrated nifedipine‐insensitive slow waves throughout the circular muscle layer. Separation of interior and submucosal circular muscle strips from the dominant (myenteric) pacemaker region dramatically slowed frequency but did not block spontaneous slow waves, suggesting that pacemaker cells populate all regions of the circular muscle. 4 Slow waves could be evoked in interior and submucosal circular muscles at rates above normal antral frequency by electrical pacing or by acetylcholine (0.3 μm). Active slow wave propagation occurred in all regions of the circular muscle, and propagation velocities were similar in each region. 5 In summary, antral muscles of the canine stomach have pacemaker capability throughout the circular muscle. Normally, a dominant pacemaker near the myenteric plexus drives slow waves that actively propagate throughout the circular layer. Pacemaker activity and the active propagation pathway may occur in networks of ICC that are distributed in the region of the myenteric plexus and throughout the circular muscle layer.

c-Kit-negative fibroblast-like cells express platelet-derived growth factor receptor α in the murine gastrointestinal musculature

Platelet-derived growth factor receptors (PDGFRs) belong to the same kinase group as c-Kit receptor tyrosine kinase that is specifically expressed in the interstitial cells of Cajal (ICC) in the gastrointestinal tract. In this study, we examined PDGFRα immunoreactivity in the murine gastrointestinal tract. PDGFRα-immunopositive (PDGFRα-ip) cells were observed in the musculature in all parts of the gastrointestinal tract. Although PDGFRα-ip cells were distinct from ICC and neurons, these cells were closely associated with intramuscular ICC and enteric nerve fibers. In the myenteric layer, PDGFRα-ip cells formed a cellular network with their ramified processes and encompassed myenteric ganglia. Numerous PDGFRα-ip cells were observed in the subserosal plane and showed a multipolar shape. The distribution pattern of the PDGFRα-ip cells in the ICC-deficient Wv/Wv mutant mice was the same as that in normal mice. PDGFRα-ip cells that showed intense immunoreactivity of SK3 potassium channel were considered to correspond to fibroblast-like cells or non-Cajal interstitial cells. Our observations suggest that PDGFRα-ip cells are basic cellular elements throughout the gastrointestinal musculature and are involved in the gastrointestinal functions.

Enteric motor neurons form synaptic-like junctions with interstitial cells of Cajal in the canine gastric antrum

Morphological studies have shown synaptic-like structures between enteric nerve terminals and interstitial cells of Cajal (ICC) in mouse and guinea pig gastrointestinal tracts. Functional studies of mice lacking certain classes of ICC have also suggested that ICC mediate enteric motor neurotransmission. We have performed morphological experiments to determine the relationship between enteric nerves and ICC in the canine gastric antrum with the hypothesis that conservation of morphological features may indicate similar functional roles for ICC in mice and thicker-walled gastrointestinal organs of larger mammals. Four classes of ICC were identified based on anatomical location within the tunica muscularis. ICC in the myenteric plexus region (IC-MY) formed a network of cells that were interconnected to each other and to smooth muscle cells by gap junctions. Intramuscular interstitial cells (IC-IM) were found in muscle bundles of the circular and longitudinal layers. ICC were located along septa (IC-SEP) that separated the circular muscle into bundles and were also located along the submucosal surface of the circular muscle layer (IC-SM). Immunohistochemistry revealed close physical associations between excitatory and inhibitory nerve fibers and ICC. These contacts were synaptic-like with pre- and postjunctional electron-dense regions. Synaptic-like contacts between enteric neurons and smooth muscle cells were never observed. Innervated ICC formed gap junctions with neighboring smooth muscle cells. These data show that ICC in the canine stomach are innervated by enteric neurons and express similar structural features to innervated ICC in the murine GI tract. This morphology implies similar functional roles for ICC in this species.

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.

Possible involvement of muscularis resident macrophages in impairment of interstitial cells of Cajal and myenteric nerve systems in rat models of TNBS-induced colitis

Resident macrophages are distributed in the network of interstitial cells of Cajal (ICC) and the myenteric nerve within the myenteric plexus. We evaluated changes in chemoattractant protein mRNA expression in macrophages and neutrophils, the ICC, nerve and macrophages in the myenteric plexus of model rats with TNBS-induced colitis. Chemoattractant proteins, MCP-1, GRO, MIP-2 and CINC-2α were upregulated in the colonic muscle layer after inflammation. Leukocyte infiltration and MPO activity were increased in the muscle layer. Electron microscopy indicated an irregular contour of the myenteric ganglia into which numerous macrophages had penetrated. Macrophages were also distributed near the ICC in the inflamed myenteric plexus. Immunohistochemistry showed that the ICC network and myenteric nerve system had disappeared from the inflamed region, whereas the number of resident macrophages was increased. TTX-insensitive, possibly ICC-mediated, rhythmic contractions of circular smooth muscle strips and enteric neuron-mediated TTX-sensitive peristalsis in the whole proximal colon tissue were significantly inhibited in the inflamed colon, indicating that the ICC-myenteric nerve system was dysfunctional in the inflamed muscle layer. Their accumulation around the myenteric nerve plexus and the ICC network suggests that macrophages play an important role in inducing intestinal dysmotility in gut inflammation.

Reconciling neuronally and nonneuronally derived acetylcholine in the regulation of immune function

Immune cells, including lymphocytes, express muscarinic and nicotinic acetylcholine (ACh) receptors (mAChRs and nAChRs, respectively), and agonist stimulation of these AChRs causes functional and biochemical changes in the cells. The origin of the ACh that acts on immune cell AChRs has remained unclear until recently, however. In 1995, we identified choline acetyltransferase mRNA and protein in human T cells, and found that immunological T cell activation potentiated lymphocytic cholinergic transmission by increasing ACh synthesis and AChR expression. We also found that M1/M5 mAChR signaling upregulates IgG1 and proinflammatory cytokine production, whereas α7 nAChR signaling has the opposite effect. These findings suggest that ACh synthesized by T cells acts as an autocrine and/or paracrine factor via AChRs on immune cells to modulate immune function. In addition, a recently discovered endogenous allosteric α7 nAChR ligand, SLURP‐1, also appears to be involved in modulating normal T cell function.

Ultrastructural characterization of interstitial cells of Cajal in the rat small intestine using control and Ws/Ws mutant rats

Abstract Interstitial cells in the myenteric plexus and the deep muscular plexus of the small intestine of the c-kit mutant rats (Ws/Ws) and their normal siblings (+/+) were studied. c-Kit immunoreactivity was detected in two regions corresponding to the myenteric plexus and the deep muscular plexus in the jejunum of +/+ rats, while no immunoreactivity was detected in Ws/Ws rats. Using electron microscopy, two types of gap junction-forming interstitial cells were found in association with the myenteric plexus in +/+ rats: one type characterized by a typical fibroblastic ultrastructure, and the other characterized by numerous mitochondria and less electron-dense cytoplasm. Since the latter were greatly reduced in Ws/Ws rats, it was suggested that these cells correspond to c-kit-expressing cells, i.e. interstitial cells of Cajal in the myenteric plexus region. In contrast, two types of interstitial cells in the region of the deep muscular plexus were observed with no difference between +/+ and Ws/Ws rats. Probable interstitial cells of Cajal in this region were characterized by a basal lamina and numerous caveolae as well as large gap junctions that interconnect with each other and with the smooth muscle cells. We concluded that interstitial cells of Cajal in the rat intestine are heterogeneous in ultrastructure, c-kit dependency in the cell maturation, and functional role.

Interstitial cells of cajal are involved in neurotransmission in the gastrointestinal tract.

Interstitial cells of Cajal (ICC) are important cells which coordinate gastrointestinal motility. ICC express Kit receptor tyrosine kinase, and Kit immunohistochemistry reveals ICC morphology and distribution in the gastrointestinal musculature. ICC show a highly branched morphology and form unique networks. Myenteric ICC (ICC-MY) are located at the layer of the myenteric plexus and serve as electrical pacemakers. Intramuscular ICC (ICC-IM) and ICC in the deep muscular plexus (ICC-DMP) are distributed within the muscular layers, and are densely innervated by excitatory and inhibitory enteric motor neurons and in close contact with nerve terminals. Recent studies combined with morphological and functional techniques directly revealed that ICC-IM and ICC-DMP are mediators of enteric motor neurotransmission. These types of ICC express several receptors for neurotransmitters such as acetylcholine and substance P and show responses to excitatory nerve stimulations. ICC also express receptive mechanisms for nitric oxide, which is an inhibitory neurotransmitter in the gastrointestinal tract. They can respond to nitrergic nerve stimulation by cyclic GMP production. Kit mutant mice lack ICC-IM and show attenuated postsynaptic responses after intrinsic nerve stimulation. These findings indicate the importance for ICC in neurotransmission in the gastrointestinal tract.