Interstitial cells of Cajal and telocytes in the gut: twins, related or simply neighbor cells?

AbstractIn the interstitium of the connective tissue several types of cells occur. The fibroblasts, responsible for matrix formation, the mast cells, involved in local response to inflammatory stimuli, resident macrophages, plasma cells, lymphocytes, granulocytes and monocytes, all engaged in immunity responses. Recently, another type of interstitial cell, found in all organs so far examined, has been added to the previous ones, the telocytes (TC). In the gut, in addition to the cells listed above, there are also the interstitial cells of Cajal (ICC), a peculiar type of cell exclusively detected in the alimentary tract with multiple functions including pace-maker activity. The possibility that TC and ICC could correspond to a unique cell type, where the former would represent an ICC variant outside the gut, was initially considered, however, further studies have clearly shown that ICC and TC are two distinct types of cells. In the gut, while the features and the roles of the ICC are established, part of the scientific community is still disputing these 'new' interstitial cells to which several names such as fibroblast-like cells (FLCs), interstitial Cajal-like cells or, most recently, PDGFRα+cells have been attributed. This review will detail the main features and roles of the TC and ICC with the aim to establish their relationships and hopefully define the identity of the TC in the gut

Neurokinin receptors in the gastrointestinal muscle wall: cell distribution and possible roles.

AbstractThe neurokinin receptors are G-protein-linked receptors; three distinct molecules, called neurokinin-1, neurokinin-2, and neurokinin-3 receptors, have been identified. Their physiological ligands are the tachykinins, which, in the mammalian gut, correspond to substance P, neurokinin A, and neurokinin B. In this apparatus, the main source of tachykinins is represented by intrinsic neurons located either in the myenteric plexus and projecting mainly to the muscle coat, or in the submucous plexus and projecting to the mucosa and submucosal blood vessels. The availability of specific antibodies has allowed identifying the sites of distribution of the neurokinin receptors in the gut, and important differences have been found among cell types and animal species. The complexity of the receptor distribution, either intraspecies or interspecies, is in agreement with the variegated picture coming out from physiological and pharmacological experiments. Interestingly, most of the knowledge on the tachykinin systems has been obtained from pathological conditions. Here, we tried to collect the main information available on the cellular distribution of the neurokinin receptors in the gut wall in the attempt to correlate their cell location with the several roles the tachykinins seem to play in the gastrointestinal apparatus

NK-receptors, Substance P, Ano1 expression and the ultrastructural features of the muscle coat are modified in the Cav-1-/- mouse ileum

Caveolin (Cav)-1 is an integral membrane protein of caveolae playing a crucial role in various signal transduction pathways. Caveolae represent the sites for calcium entry and storage especially in smooth muscle cells (SMC) and interstitial cells of Cajal (ICC). Cav-1-/- mice lack caveolae and show abnormalities in pacing and contractile activity of the small intestine. In particular, the absence of caveolae in ICC compromised their ability to maintain frequencies of contraction. Presently, we investigated, by transmission electron microscopy (TEM) and immunohistochemistry, whether the absence of Cav-1 in Cav-1-/- mouse small intestine affects ICC, SMC and neuronal morphology, the expression of NK1 and NK2 receptors, and of Ano1 (also called Dog1 or TMEM16A) an essential molecule for slow wave activity in gastrointestinal muscles. ICC were also labeled with c-Kit and tachykinergic neurons with Substance P (SP). Immunohistochemical results showed that in Cav-1-/- mice: i) ICC were Ano1-negative but maintained c-Kit expression, ii) NK1 and NK2 receptor immunoreactivity was increased and, in the SMC, mainly intracytoplasmatic, iii) SP-immunoreactivity was significantly reduced. Under TEM: i) ICC and SMC lacked typical caveolae but had few and large flask-shaped vesicles we called large-sized caveolae; ii) SMC and ICC contained an extraordinary high number of mitochondria; iii) neurons were unchanged. In conclusion, the present study shows important changes in SMC, ICC and neurons of the Cav-1-/- mice. Loss of Ano1 expression in the ICC and rearrangement of NK receptors in the SMC are interpretable as consequence of Cav-1/caveolae loss and possibly responsible for the impaired contractile activity. However, the impressive richness in mitochondria and the decrease in SP content might represent the ways to compensate the reduced calcium availability and the increased expression of NKr, allowing the maintenance of a certain cell function

Repeated administration of the spasmolytic otilonium bromide counteracts functional and neurotransmitters’ changes in the colon of rats underwent to wrap restraint stress

Otilonium bromide (OB) is a quaternary ammonium derivative successfully used for the treatment of irritable bowel syndrome (IBS). Several in vitro experiments in human and rat colon demonstrated its spasmolytic capability due to the block of muscarinic and tachykinin receptors and L-type Ca2+ channels. Moreover, in vivo OB administrations showed interesting interaction with the enteric nervous system in healthy rats (1). The wrap restrain stress (WRS) is considered an adequate model of psychosocial stressor, able to induce most of the IBS signs and symptoms. WRS leads to important changes in the enteric neurotransmitters of rat colon, as recently demonstrated (2). Consequently, we chose this animal model to investigate whether a repeated, oral treatment with OB prevented the functional and neurotransmitters’ changes reported in rats underwent to WRS. The results obtained by using multiple experimental approaches (in vivo colonic functional evaluations, routine histology, immunohistochemistry and western blot) showed that OB is able to counteract most of the morphological changes caused by WRS in the colonic wall. In particular, the drug prevents the decrease in SP-, NK1r-, nNOS-, VIP- and S100β-immunoreactivity (IR) and the increase in CGRP- and CRF1r-IR detected in WRS rats. On the contrary, OB does not interfere with the mild mucosal inflammation and does not affect the increase in CRF2r-immunoreactive neurons observed in WRS rats. Moreover, OB per se increases the muscarinic receptor 2 expression in the muscle wall and decreases the number of the myenteric ChAT-immunoreactive neurons. Functional findings show a significantly reduction in the number of spontaneous abdominal contraction in OB treated rats. The ability of OB to block L-type Ca2+ channels, also expressed by enteric neurons, might explain the drug efficacy in preventing excessive neuronal response to stress. This work was supported by grants from Menarini I.F.R

Relaxin increases contractile activity and neuronal Nitric Oxide Synthase (nNOS) expression in the muscle coat of mouse colon

Since the first observations, many actions have been attributed to Relaxin (RLX), so that it has been defined a pleiotropic hormone. This peptide of 6000 Da is synthesized by reproductive organs both in females and males. In particular, in females RLX is produced by corpus luteum (30-150 pg/ml) and its highest plasma levels (900 pg/ml) are reached during pregnancy, when RLX is also released by the decidua and placenta. In pregnancy, RLX plays its traditional role as inhibitor of spontaneous myometrial contractions and maintains uterine quiescence. Recently, RLX has shown to affect the smooth muscle of mouse gastric fundus and small intestine, depressing its motility through a nitric oxide (NO)-mediated mechanism. In this study, we investigate the functional role of RLX in the muscle coat of female mouse colon. In the presence of 50 nmol/l RLX, continuous recordings of isometric tension showed a fast reduction of circular muscle tone, followed by a stable increase of contraction amplitude. This double effect was mimicked by the application of 40 μmol/l NaNO2, a NO donor, and completely blocked by 1 μmol/l ODQ, an inhibitor of cyclic GMP which represents the main intracellular mediator of NO actions. The treatment of the tissue with 1 μmol/l TTX prevented the decrease of muscle tone caused by RLX, indicating that this specific effect was neuronally triggered. To clarify the underling cellular mechanism, the neuronal Nitric Oxide Synthase (nNOS) expression was evaluated in colonic segments treated with (50 nmol/l) RLX for 10 min or 40 min. In particular, two different antibodies able to recognize respectively the neurogenic and the myogenic nNOS were used. Interestingly, at both times of treatment, we observed an increase of neurogenic nNOS immunoreactivity (IR) and a decrease in myogenic nNOS-IR. These two opposite effects result statistically significant. Present functional and morphological findings demonstrate that RLX produces a paradoxical effect in the colonic muscle coat which likely represents the final result of an interaction between the two splice variants of the nNOS enzyme

The wrap partial restrain stress, an animal model of the irritable bowel syndrome: immunohistochemical and functional characterization

Several animal models have been proposed to mimic the human irritable bowel syndrome (IBS) all based on two etio-pathogenic hypotheses: infection and stress, both responsible for the development of a local inflammation. We investigated the wrap partial restrain stress (WRS) animal model with the aim to evaluate its validity in understanding the human IBS. Male Wistar rats were used and WRS was maintained for 2h. Abdominal contractions (AC) were recorded by a distension balloon in the colon-rectum. The number of faecal pellets and their total weight were determined. Colonic specimens from both groups were examined by routine histology, immunohistochemistry and western blot (WB). WRS animals were characterized by: 1) a statistically significant increase in the number of AC and in the mean number and mean weight of faecal pellets; 2) the presence of large clusters of mononucleated cells and a significant increase in eosinophilic granulocytes and mast cells in the mucosa; 3) reduction of GLP1r-immunoreactivity (IR) located at the basolat- eral periphery and the Golgi level of the cells of the glandular funds; 4) an increase in CGRP-IR in the lamina propria; 5) no significant difference in the muscle wall for Cav1, L- type Ca+2-channels, Mr2, NK1r and NK2r; 6) a significant decrease in the myenteric and a significant increase in the submucous NK1-IR neuron number; 7) a significant decrease in Substance P-IR in the myenteric plexus and muscle coat; 8) a significant decrease in myenteric and submucous nNOS-IR neuron number; 9) no difference in ChAT-IR neurons of both enteric plexuses; 10) a reduction in S-100-IR in the entire colonic wall; 11) no difference in the total number of neurons evaluated by the pan-neuronal marker PGP 9.5; 12) no change of all the ICC populations. The functional data are in favor of a lowering in the colonic wall distention threshold; the morphological results obtained in the lamina propria demonstrate the presence of a local inflammation, particularly intense at the level of the mucosa. Both of these findings agree with the hypothesis that inflammation might have a main role in the insurgence and maintenance of the typical IBS symptoms and support the validity of our WRS model. Moreover, while the smooth muscle cells do not show any significant variation, numerous and consistent changes in the excitatory, inhibitory and NK1r-IR neurons are detected

Telocytes: New Connecting Devices in the Stromal Space of Organs

Telocytes (TCs) represent a new type of interstitial cells, and were discovered by Prof. Popescu and his collaborators from Bucharest in 2005, and described as Interstitial Cajal-Like Cells (ICLCs). In 2010, Prof. Popescu and Prof. Faussone-Pellegrini from Florence, based on their expertise in morphology, agreed that in fact ICLCs were a brand-new entity and they renamed them telocytes. TCs are characterized by specific veil- or ribbon-like extensions called telopodes. Telopodes aid TCs in forming homo- or hetero-cellular contacts; thus, assembling three-dimensional networks that organizes the stromal and the parenchymal components of the organs. TCs can transfer information to neighbor cells ensuring a short-distance communication, and remotely by the release a wide variety of extracellular vesicles: exosomes, ectosomes, and multivesicular bodies. Here, we reviewed the evolution of the interest regarding TCs in different organs, in normal and pathological conditions. The main focus was on the role of TCs in gastrointestinal tract, urinary bladder, reproductive tract, and heart. This chapter sums up information about the possibilities that TCs are capable to behave as sensors/mediators in nervous activity, to represent mesenchymal stem cell precursors in adulthood, and to control and determine the differentiation/maturation of other cell types either during development or in postnatal life

Chronic treatment with otilonium bromide affects the tachykinergic and nitrergic systems in the rat colon

Otilonium bromide (OB), a quaternary ammonium derivative used for the treatment of intestinal motility disorders such as the irritable bowel syndrome (IBS). It exerts several actions, among which the ability to bind to the neurokin-2 receptor (NK2r) inhibiting NK2r mediated contraction and, in the human colon, NK2r internalization in the smooth muscle cells (SMC) (Cipriani et al., 2011). Substance P (SP) is an excitatory neurotransmitter that, interacting mainly with the neurokinin-1 receptor (NK1r), can stimulate bowel motility by SMC direct activation or inhibit it by an indirect action through enteric neural circuits. In an IBS rat model, the increase in NK1rmediated colonic motor response was associated to a decrease in the nitrergic activity. On these basis, we tested whether OB modifies NK1r, NK2r, SP and neuronal nitric oxide synthase (nNOS) expression in rat colon after chronical administration of the drug (2 or 20mg/Kg/daily) for 10 or 30 days. At the end of the treatments, specimens of proximal colon were collected and the expression of NK1r, NK2r, SP and neurogenic and myogenic nNOS were evaluated by immunohistochemistry and Western blot. Our data show that SP expression was significantly decreased in 10 and 30 days treated rats in myenteric ganglia and, in 30 days treated rats, also in the intramuscular nerve fibres. No quantitative change of the two NKr was observed, whereas, after 30 days, the NK1r was concentrated in the SMC cytoplasm. In parallel, the neurogenic nNOS expression increased and reached the significance after 30 days of treatment; the myogenic nNOS expression increased, but these increase reached the significance only at 10 days. Our findings suggest that the main target of the OB chronically administered is the NO-mediated system that is stimulated earlier at the muscular level, later at the neuronal level. We interpret the systemic decrease in the SP expression as consequence of the potentiated NO availability in the ganglia and muscle coat. If true, the late concentration of NK1r in the cytoplasm could represent an attempt of the SMC to overcome the deficit of its main ligand SP

Adaptive changes of telocytes in the urinary bladder of patients affected by neurogenic detrusor overactivity

Urinary bladder activity involves central and autonomic nervous systems and bladder wall. Studies on the pathogenesis of voiding disorders such as the neurogenic detrusor overactivity (NDO) due to supra-sacral spinal cord lesions have emphasized the importance of an abnormal handling of the afferent signals from urothelium and lamina propria (LP). In the LP (and detrusor) three types of telocytes (TC) are present and form a 3D-network. TC are stromal cells able to form the scaffold that contains and organizes the connective components, to serve as guide for tissue (re)-modeling, to produce trophic and/or regulatory molecules, to share privileged contacts with the immune cells. Specimens of full thickness bladder wall from NDO patients were collected with the aim to investigate possible changes of the three TC types by using histology, immunohistochemistry and transmission electron microscopy. The results show that NDO causes several morphological TC changes without cell loss or network interruption. With the exception of those underlying the urothelium, all the TC display signs of activation (increase in Caveolin1 and caveolae, SMA and thin filaments, Calreticulin and amount of cisternae of the rough endoplasmic reticulum, CD34, euchromatic nuclei and large nucleoli). In all the specimens a cell infiltrate, mainly consisting in plasma cells located in the vicinity or taking contacts with the TC, is present. In conclusion, our findings show that NDO causes significant changes of all the TC. Notably, these changes can be interpreted as TC adaptability to the pathological condition likely preserving each of their peculiar functions