Satoshi Iino

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.

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.

GABA-containing sympathetic preganglionic neurons in rat thoracic spinal cord send their axons to the superior cervical ganglion.

γ‐Aminobutyric acid (GABA)‐containing fibers have been observed in the rat superior cervical ganglion (SCG) and, to a lesser extent, in the stellate ganglion (STG). The aim of present study is to clarify the source of these fibers. No cell body showed mRNAs for glutamic acid decarboxylases (GADs) or immunoreactivity for GAD of 67 kDa (GAD67) in the cervical sympathetic chain. Thus, GABA‐containing fibers in the ganglia are suggested to be of extraganglionic origin. GAD67‐immunoreactive fibers were found not in the dorsal roots or ganglia, but in the ventral roots, so GABA‐containing fibers in the sympathetic ganglia were considered to originate from the spinal cord. Furthermore, almost all GAD67‐immunoreactive fibers in the sympathetic ganglia showed immunoreactivity for vesicular acetylcholine transporter, suggesting that GABA was utilized by some cholinergic preganglionic neurons. This was confirmed by the following results. 1) After injection of Sindbis/palGFP virus into the intermediolateral nucleus, some anterogradely labeled fibers in the SCG were immunopositive for GAD67. 2) After injection of fluorogold into the SCG, some retrogradely labeled neurons in the thoracic spinal cord were positive for GAD67 mRNA. 3) When the ventral roots of the eighth cervical to the fourth thoracic segments were cut, almost all GAD67‐ and GABA‐immunoreactive fibers disappeared from the ipsilateral SCG and STG, suggesting that the vast majority of GABA‐containing fibers in those ganglia were of spinal origin. Thus, the present findings strongly indicate that some sympathetic preganglionic neurons are not only cholinergic but also GABAegic. J. Comp. Neurol. 502:113–125, 2007.

Muscarinic M2 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.

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.

Interstitial cells of Cajal contain signalling molecules for transduction of nitrergic stimulation in guinea pig caecum

Abstract  Nitric oxide (NO) is an inhibitory signalling molecule in the gastrointestinal (GI) tract that is released from neurons and from leucocytes during inflammation. NO stimulates soluble guanylate cyclase (sGC), elevates cyclic guanosine 3′,5′‐monophospate (cGMP), and subsequently activates cGMP‐dependent protein kinase (PKG). Targets for NO in the guinea pig caecum were investigated by characterizing the cellular distribution of sGC, cGMP and PKG. Immunoreactivity for both isoforms of sGC, sGCα1 and sGCβ1, was observed in the interstitial cells of Cajal (ICC) and enteric neurons in the tunica muscularis. Double labelling with anti‐Kit and anti‐sGC antibodies showed sGCα1 and sGCβ1‐like immunoreactivity (LI) in almost all intramuscular (IM) and myenteric ICC. Neuronal processes with neuronal NO synthase were closely apposed to ICC expressing sGC‐LI. Cells with sGC‐LI possessed ultrastructural features of ICC‐IM: caveolae, close association with nerve bundles and contacts with smooth muscle cells (SMC). Sodium nitroprusside, added with the phosphodiesterase inhibitors (3‐isobutyl‐1‐methylxanthine and zaprinast), enhanced cGMP‐LI in almost all ICC and in some enteric neurons. Nerve stimulation also increased cGMP‐LI in ICC and enteric neurons. In contrast, no resolvable increase in cGMP‐LI was observed in any cells when the sGC inhibitor 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one was present. ICC and SMC also expressed PKG type I‐LI. These data show that ICC express the downstream signalling molecules necessary to transduce nitrergic signals and activate inhibitory pathways and thus are primary targets for NO released from neurons and other cells in the GI tract.

Serotonin Augments Gut Pacemaker Activity via 5-HT3 Receptors

Serotonin (5-hydroxytryptamine: 5-HT) affects numerous functions in the gut, such as secretion, muscle contraction, and enteric nervous activity, and therefore to clarify details of 5-HTs actions leads to good therapeutic strategies for gut functional disorders. The role of interstitial cells of Cajal (ICC), as pacemaker cells, has been recognised relatively recently. We thus investigated 5-HT actions on ICC pacemaker activity. Muscle preparations with myenteric plexus were isolated from the murine ileum. Spatio-temporal measurements of intracellular Ca2+ and electric activities in ICC were performed by employing fluorescent Ca2+ imaging and microelectrode array (MEA) systems, respectively. Dihydropyridine (DHP) Ca2+ antagonists and tetrodotoxin (TTX) were applied to suppress smooth muscle and nerve activities, respectively. 5-HT significantly enhanced spontaneous Ca2+ oscillations that are considered to underlie electric pacemaker activity in ICC. LY-278584, a 5-HT3 receptor antagonist suppressed spontaneous Ca2+ activity in ICC, while 2-methylserotonin (2-Me-5-HT), a 5-HT3 receptor agonist, restored it. GR113808, a selective antagonist for 5-HT4, and O-methyl-5-HT (O-Me-5-HT), a non-selective 5-HT receptor agonist lacking affinity for 5-HT3 receptors, had little effect on ICC Ca2+ activity. In MEA measurements of ICC electric activity, 5-HT and 2-Me-5-HT caused excitatory effects. RT-PCR and immunostaining confirmed expression of 5-HT3 receptors in ICC. The results indicate that 5-HT augments ICC pacemaker activity via 5-HT3 receptors. ICC appear to be a promising target for treatment of functional motility disorders of the gut, for example, irritable bowel syndrome.

Therapeutic action of 5-HT3 receptor antagonists targeting peritoneal macrophages in post-operative ileus

Post‐operative ileus (POI) is induced by intestinal inflammation. Here, we aimed to clarify the effects of 5‐HT3 receptor antagonists against POI.

PDGF and TGF‐β promote tenascin‐C expression in subepithelial myofibroblasts and contribute to intestinal mucosal protection in mice

Tenascin‐C (TnC) is a multi‐domain extracellular matrix glycoprotein that is expressed at a high level during embryogenesis but is almost absent during normal postnatal life. This multi‐domain complex molecule is reported to associate with both pro‐inflammatory and anti‐inflammatory signalling cascades. In this study, we examined how TnC modulated intestinal inflammation.

Convergence of Lemniscal and Local Excitatory Inputs on Large GABAergic Tectothalamic Neurons.

Large GABAergic (LG) neurons form a distinct cell type in the inferior colliculus (IC), identified by the presence of dense VGLUT2‐containing axosomatic terminals. Although some of the axosomatic terminals originate from local and commissural IC neurons, it has been unclear whether LG neurons also receive axosomatic inputs from the lower auditory brainstem nuclei, i.e., cochlear nuclei (CN), superior olivary complex (SOC), and nuclei of the lateral lemniscus (NLL). In this study we injected recombinant viral tracers that force infected cells to express GFP in a Golgi‐like manner into the lower auditory brainstem nuclei to determine whether these nuclei directly innervate LG cell somata. Labeled axons from CN, SOC, and NLL terminated as excitatory axosomatic endings, identified by colabeling of GFP and VGLUT2, on single LG neurons in the IC. Each excitatory axon made only a few axosomatic contacts on each LG neuron. Inputs to a single LG cell are unlikely to be from a single brainstem nucleus, since lesions of individual nuclei failed to eliminate most VGLUT2‐positive terminals on the LG neurons. The estimated number of inputs on a single LG cell body was almost proportional to the surface area of the cell body. Double injections of different viruses into IC and a brainstem nucleus showed that LG neurons received inputs from both. These results demonstrated that both ascending and intrinsic sources converge on the LG somata to control inhibitory tectothalamic projections. J. Comp. Neurol. 523:2277–2296, 2015.