Effect of Calcium-Sensitive Receptor Agonist R568 on Gastric Motility and the Underlying Mechanism.

INTRODUCTION: Calcium-sensitive receptor (CaSR) is expressed in the enteric nervous system of gastrointestinal tract. However, its role in the regulation of gastrointestinal motility has not yet been fully elucidated. We aimed to investigate the effect of the CaSR agonist - R568 on gastric motility and its potential mechanism. METHODS: In vivo, R568 was given by gavage to explore gastric emptying with or without capsaicin which specifically blocks the function of vagal afferents; neurotransmitters synthetized in the myenteric plexus of the gastric corpus and antrum were analysed by ELISA and immunofluorescence staining; gastric muscle strips contraction recording and intracellular single unit firing recording were used to study the effect of R568 on muscle strips and myenteric interstitial cells of Cajal (ICCs) ex vitro. RESULTS: Gastric emptying was inhibited by R568 in Kunming male mice, and capsaicin weakened this effect. The expression of c-fos-positive neurons increased in the nucleus tractus solitarius when R568 was treated. R568 decreased the expression of cholinergic neurons and reduced the synthesis of acetylcholine. Conversely, R568 increased the expression of nitrogenic neurons and enhanced the synthesis of nitric oxide in the myenteric plexus. Ex vitro results showed that R568 inhibited the contraction of the gastric antral muscle strip and suppressed the spontaneous firing activity of pacemaker ICCs. CONCLUSION: Activation of the gastrointestinal CaSR inhibited gastric motility in vivo and ex vitro. Transmitting nutrient signals to the brain through the vagal afferent nerve, modulating the cholinergic and nitrergic system in the enteric nervous system, and inhibiting activity of pacemaker ICCs in the myenteric plexus are involved in the mechanism of CaSR in gastric motility suppression.

Relation between Cajal Cell Density and Radiological and Scintigraphic Outcomes in Patients with Ureteropelvic Junction Obstruction.

INTRODUCTION: In this study, we aimed to investigate the correlation between Cajal cell density and preoperative and postoperative radiological and scintigraphic parameters in ureteropelvic junction obstruction (UPJO). METHODS: The study group consisted of 41 renal units (38 consecutive patients; 13 female and 25 male) surgically treated for UPJO. UPJ specimens from patients were immuno-stained with CD117 (c-kit) antibody for interstitial Cajal cells (ICCs). The relation between Cajal cell density and preoperative and postoperative radiological and scintigraphic parameters was evaluated. RESULTS: The mean age of the patients was 8.52 ± 8.86 (0-35) years. The density of Cajal cells was defined in 2 groups for convenient analysis as 0-5 cells (low) in 19 (46.3%) patients and >5 cells (moderate-high) in 22 (53.6%). There was significant difference between the preoperative and postoperative anteroposterior diameters of the related kidneys in both Cajal groups (p = 0.001-low, p = 0.000-moderate-high) independent of Cajal cell density. Regression in hydronephrosis postoperatively was determined in both Cajal groups (77.8%-low, 64.7%-moderate-high); however, there was no difference between them (p = 0.39). Preoperative T1/2 was significantly longer in the low Cajal group (p = 0.02). Postoperative T1/2 decreased in both low (p = 0.000) and moderate-high (p = 0.001) Cajal groups, but no difference was found between them (p = 0.24). There was significant improvement in the kidney differential function after surgery in the low Cajal density group (p = 0.015) while there was no correlation between the scintigraphic success or improvement and Cajal cell density (p = 0.51). DISCUSSION/CONCLUSION: ICC deficiency/density could not be shown as a predictive factor for the determination of success rate of pyeloplasty. Despite the lack of any evidence for the degree of deficiency as an indicator for the severity of obstruction and prediction of surgical success, further studies are needed for confirmation.

The impact of acute stress disorder on gallbladder interstitial cells of Cajal.

Physical and psychological stress exerts a substantial effect on gastrointestinal motility disorders, where trauma enhances symptoms of digestive dysfunction. Interstitial cells of Cajal (ICCs) act as pacemakers for gastrointestinal motility regulation and are likely important in stress-associated gastrointestinal motility disorders. This study explored the mechanisms underlying gallbladder ICCs function under acute stress conditions using a rabbit chest puncture and cholecystectomy model. The stem cell factor (SCF)/c-kit pathway is essential for the development of ICCs, and gene expression was investigated to identify stress-induced transcriptional alterations. Immunohistochemistry, terminal deoxynucleotidyl transferase dUTP nick end labeling assays were used to determine ICCs apoptosis, whereas western blot analysis and reverse-transcription polymerase chain reaction were used to detect changes in the SCF/c-kit signaling pathway. These methods revealed a reduction in ICCs via apoptosis following stress, and ICCs increased over time after stressor removal. Therefore, this study demonstrates the impact of stress on ICCs development and survival and further confirms the link between stress and gastrointestinal motility.

Acute Cholecystitis Reduces Interstitial Cells of Cajal in Porcine Gallbladder Through Decreased mRNA Synthesis.

BACKGROUND/AIMS: Acute cholecystitis is a common gastrointestinal disorder, often characterized by acute cholecystitis with gallbladder motility disorder. Interstitial cells of Cajal (ICCs) are the pacemaker cells of gut motility in the gastrointestinal tract. Disruption of ICC function is related to motility disorders. The aim of this study was to explore the cellular and molecular mechanisms of ICCs in acute cholecystitis and after the resolution of acute inflammation. MATERIALS AND METHODS: Fifty adult guinea pigs were randomly divided into five groups: a sham-administered group (control group); two groups that were intraperitoneally administered an anti-polyclonal neutrophil (PMN) antibody 24 h before common bile duct ligation (CBDL); and two groups of guinea pigs that were subjected to CBDL without receiving the PMN antibody. Guinea pigs that underwent CBDL were held for 24 h or 48 h after surgery before being subjected to laparotomy and cholecystectomy. Immunohistochemistry, TUNEL assays, western blotting, and real-time PCR were performed to determine ICC morphology and density, to detect ICC apoptosis, and to examine stem cell factor (SCF) and c-kit protein expression and SCF and c-kit mRNA levels, respectively. RESULTS: Both hematoxylin-eosin staining and histological inflammation scores in the PMN groups were lower than those in the control groups (P < 0.01). No differences were observed in ICC morphology between groups. During acute cholecystitis, ICCs numbers were reduced. Conversely, the density of ICCs increased after inflammation was relieved (P < 0.01). In addition, SCF and c-kit protein and mRNA expression levels decreased during acute cholecystitis (P < 0.05) and increased after inflammation was relieved (P < 0.05). Furthermore, ICC apoptosis increased during acute cholecystitis and decreased after resolution of acute cholecystitis (P < 0.01). CONCLUSIONS: In acute cholecystitis, ICC injury may be related to gallbladder motility disorder.

Lineage Tracing of FOXL1+ Cells in the Tunica Muscularis Suggests Mutual Origin for Telocytes and Smooth Muscle Cells.

We recently identified a FOXL1+ intestinal subepithelial network of telocytes (TCs) without which epithelial stem and progenitor cells cannot proliferate and support regeneration. In addition to FOXL1 lineage cell distribution along the intestinal epithelium, we also observed their presence within the muscle layers. Here, we characterized FOXL1+ lineage cells along the muscle layers of the duodenum in order to understand their progeny and relation to interstitial Cajal cells (ICCs), smooth muscle cells (SMCs) and the previously reported PDGFRa+ TCs. Using a FOXL1-Cre transgenic line in conjunction with genetic lineage labeling using the Rosa26-mTmG allele, in which Cre-marked cells produce a membrane-targeted version of green fluorescent protein (GFP), we found that within the muscle layers FOXL1 lineage GFP+ cells had two main progeny; (i) elongated multinucleated SMA+ SMCs, intermingled in parallel or perpendicular to muscle fibers. (ii) TCs displaying small cell body with multiple cell processes, expressing PDGFRa and CD34. These findings may suggest a mutual origin for TCs and SMCs.

The complex involvement of the digestive tract in human defense behavior - structural and functional arguments.

The digestive system has an innate monitoring and defense capacity, which allows the recognition and elimination of different dangerous substances. The complex analysis of the intestinal content comprises the cross-interactions between the epithelial cells, the enteroendocrine cells, the neural tissue and the cellular defense mechanisms. The enteric nervous system, also called "the enteric brain" or "the second brain" is the only neuronal network outside the central nervous system capable of autonomous reflex activity. The enteric nervous system activity is mostly independent of the central nervous system, but not in all aspects. In fact, even the enteral reflexes are a consequence of the bidirectional intestine-brain relation. The central nervous and enteric nervous systems are coupled through the sympathetic and parasympathetic branches of the autonomic nervous system. The gastrointestinal functions are regulated due to the interaction between the intrinsic neurons within the gastrointestinal wall and the extrinsic neurons outside the gastrointestinal tract. Here we provide an overview of the important role of the enteric brain in defensive behavior, as well as its structural and functional particularities that make it a special organ.

Telocytes and Lymphatics of the Human Colon.

BACKGROUND: Telocytes (TCs) are a peculiar morphological type of stromal cells. They project long and moniliform telopodes, visible on various bidimensional sections. Originally regarded as "interstitial Cajal-like cells", gastrointestinal TCs were CD34+. Further double-labelling studies found that colon TCs are negative for the expressions of the PDGFR-α and α-SMA. However, the TCs in colon were not distinguished specifically from endothelial cells (ECs), vascular or lymphatic. A combinational approach is important for accurate TC identification. Hence, we designed an immunohistochemical study of human colon to check whether ECs and CD34+ TCs express different markers. METHODS: Immunohistochemistry was performed on archived paraffin-embedded samples of human colon (nine cases) for the following markers: CD31, CD34, CD117/c-kit and D2-40 (podoplanin). RESULTS: A distinctive population of CD34+ TCs was found coating the myenteric ganglia. However, also perivascular cells and vascular ECs were CD34+. c-kit expression was equally found in interstitial Cajal cells (ICCs) and perivascular cells. The CD34 TCs did not express c-kit. As they were equally CD31- and D2-40- they were assessed as different from ECs. CONCLUSIONS: Testing specific markers of ECs, vascular and lymphatic, in the same tissues in which CD34+ TCs are found, is much more relevant than to identify TCs by transmission electron microscopy alone.

Therapeutic effect of protease-activated receptor 2 agonist SLIGRL-NH2 on loperamide-induced Sprague-Dawley rat constipation model and the related mechanism.

PURPOSE: To investigate the therapeutic effects of protease-activated receptor 2 (PAR-2) agonist SLIGRL-NH2 on loperamide-induced Sprague-Dawley (SD) rat constipation animal models. MATERIALS AND METHODS: Loperamide was injected subcutaneously to induce constipation twice a day for 3 days. SD rats (n = 30) were randomly divided into five groups: non-constipation group (control, n = 6), constipation group (constipation, n = 6), constipation + SLIGRL-NH2 low-dosage group (SLIGRL-NH2 low, n=6), constipation + SLIGRL-NH2 high-dosage group (SLIGRL-NH2 high, n = 6), and constipation + prucalopride (positive control, n = 6). The SLIGRL-NH2 low group and SLIGRL-NH2 high group were administered with 2.5 µmol/kg and 5 µmol/kg SLIGRL-NH2, respectively, and the prucalopride group received 2 mg/kg prucalopride. The control and constipation group received 1× PBS under the same pattern. SLIGRL-NH2 and prucalopride were orally administrated once daily for 7 days. On the final day of oral administration, food intake, water intake, the number of stool pellets, weight, and fecal water content was calculated; moreover, the colons of rats in different groups were collected and histological features were examined by hematoxylin and eosin staining; furthermore, the expression of anoctamin-1 was determined by Immunohistochemical methods, and the expressions of c-kit and PAR-2 were examined using real-time quantitative polymerase chain reaction and Western blot methods; finally, the expressions of neurotransmitter vasoactive intestinal peptide (VIP) and substance P (SP) were examined using enzyme-linked immuno-sorbent assay methods. RESULTS: The feeding and excretion behaviors, intestinal transit ratio, and the histological feature of the colon in the constipated rats were all improved by SLIGRL-NH2 treatment; moreover, SLIGRL-NH2 treatment induced significant increase in the expression of PAR-2 and also increased number of interstitial Cajal cells. Furthermore, SLIGRL-NH2 also decreased the contents of the inhibitory neurotransmitter VIP and increased the expression of the excitatory neurotransmitter SP. High dose of SLIGRL-NH2 has shown similar anti-constipation effects as prucalopride. CONCLUSION: These results suggested that SLIGRL-NH2 can enhance gastrointestinal transit and alleviate in rats with loperamide-induced constipation.

Network of telocytes in the temporomandibular joint disc of rats.

The phenotypes of the temporomandibular joint (TMJ) disc cells range from fibroblasts to chondrocytes. There are relatively few reported studies using transmission electron microscopy (TEM) to determine the ultrastructural features of these cells. It was hypothesized that at least a subpopulation of TMJ stromal cells could be represented by the telocytes, cells with telopodes. In this regard a TEM study was performed on rat TMJ samples. Collagen-embedded networks were found built-up by cells with telopodes with subplasmalemmal caveolae, moderate content in matrix secretory organelles and well-represented intermediate filaments. Appositions of cell bodies were found. Prolongations of such cells were closely related to nerves and microvessels. Our study indicates that the TMJ disc attachment seems equipped with telocytes capable of stromal signaling. However, further studies are needed to assess whether the telocytes belong to a renewed cell population derived from circulating precursors.

Histological and functional organization in human testicle: expression of receptors c-kit and androgens / Organización histológica y funcional en el testículo humano: expresión de los receptores c-kit y andrógenos

The objective of this work was identify the presence of interstitial cells of Cajal, muscle cells, nerves and androgen receptor positive cells in adult human testicle, using immunohistochemical detection for c-kit/CD-117, actin smooth muscle specific (ASMS), neurofilament (N) and androgen receptor (AR), respectively. The samples were obtained from patients (n= 10) with diagnosis of prostate cancer, with surgery of orchiectomy. Subsequently were processed by histology and for immunohistochemistry using specific antibodies. It showed the presence of cells c-kit/CD-117, with diverse degrees of positivity, distributed mainly in the interstitial peritubular area of the human testicle. The peritubular myoides cells were positive to the presence of the actin smooth muscle and androgen receptor. The neurofilaments elements (+) only were observed in the vascular tunic. The specific immunohistochemistry describe the presence of the interstitial cells of Cajal in human testicular interstitium, opening a new perspective for the functional interpretation of the testicular cellularity and tubular motility. Possibly associated functionally to peribubulars cells of smooth muscle to regulate the mobility of the seminiferous tabules, whose integration and function would be androgen dependent. The cells that express the c-kit receptor, were found exclusively in the interstitial compartment. This cellular type in addition of the muscular cells of peritubules and the absence of nervous fibers to the interior of the testicle, could be responsible for the regulation of tubular mobility, as it happens in the gastrointestinal apparatus.

El objetivo de este trabajo fue identificar la presencia de células interticiales de Cajal, células musculares lisas, células nerviosas y células que expresan receptores de andrógeno en el testículo de humano adulto, usando inmunohistoquímica específica para: c-kit/CD-117, músculo liso actina específico (ASMS), neurofilamentos (N) y para receptores de andrógenos (AR). Las muestras fueron obtenidas de pacientes (n=10) con diagnóstico de cáncer prostático sometidos a cirugía de orquiectomía. Las biopsias se procesaron para histología e inmunohistoquímica usando anticuerpos específicos. Se muestra la presencia de células c-kit/CD-117, con diversos grados de positividad y distribuidas en el compartimento interticial del testículo. Las células peritubulares mioides fueron positivas para la presencia de músculo liso actina específico y para receptor de andrógenos. La marcación de neurofilamentos positivos, sólo fueron observados en la túnica vascular. Conclusiones: La inmunohistoquímica específica describe la presencia de células interticiales de Cajal en los interticios testiculares humanos, abriendo una nueva visión en la interpretación funcional de la celularidad testicular y la motilidad tubular. Lo anterior asociado a la funcionalidad de las células peritubulares (músculo liso) regularían la motilidad de los túbulos seminíferos. Este proceso posiblemente es andrógeno dependiente. Las células que expresan receptores c-kit se encuentran exclusivamente en los compartimentos interticiales, estas células en conjunto con las células musculares peritubulares agregado a la ausencia de fibras nerviosas al interior del testículo, podrían ser los responsables de la regulación de la motilidad tubular, similar a como se informa para el tracto gastrointestinal.