Positive Interstitial Cells Of Cajal Research Articles

The Dome Wall of Bladder Acts as a Pacemaker Site in Detrusor Instability in Rats.

BackgroundThe aim of this study was to confirm that the interstitial cells of Cajal (ICCs) in the dome wall of the bladder are pacemaker cells, and that the dome wall of the bladder acts as a pacemaker site in the detrusor instability (DI) rat model.Material/MethodsThe model of DI in Wistar rats was established and urodynamic studies measuring the bladder volume and pressure were performed. The detrusor excitability was investigated using the amplitude and frequency of phasic contraction of strips. The localization and quantity of ICCs was identified by immunohistochemistry and c-KIT protein expression in the rat bladder. PCR assay and Western blot were used to assess the expression of HCN2 and Cx43.ResultsThe bladder capacity, residual volume, voiding volume, and maximum voiding pressure were significantly increased in the DI group. The contraction frequency and amplitude of the strips from the dome of the bladder in the DI group were higher than the triangle, body, and base parts. Both the concentration of c-KIT positive ICCs cells and expression of the c-KIT protein in the dome wall were higher than in other parts of the bladder. The expression of HCN2 and Cx43 in each part of the DI rat group were obviously higher than each part in the control group. Compared to the body, base, and triangle parts, the expression of HCN2 and Cx43 in the dome wall were obviously higher in the DI group.ConclusionsThe quantity of ICCs was higher in the dome wall and the dome wall of bladder acts as a pacemaker site in the DI rat model.

ATP-sensitive K+ channels maintain resting membrane potential in interstitial cells of Cajal from the mouse colon

To investigate the role of ATP-sensitive K+(KATP) channels on pacemaker activity in interstitial cells of Cajal (ICC), whole-cell patch clamping, RT-PCR, and intracellular Ca2+([Ca2+]i) imaging were performed in cultured colonic ICC. Pinacidil (a K+ channel opener) hyperpolarized the membrane and inhibited the generation of pacemaker potential, and this effect was reversed by glibenclamide (a KATP channel blocker). RT-PCR showed that Kir 6.1 and SUR2B were expressed in Ano-1 positive colonic ICC. Glibenclamide depolarized the membrane and increased pacemaker potential frequency. However, 5-hydroxydecanoic acid (a mitochondrial KATP channel blocker) had no effects on pacemaker potentials. Phorbol 12-myristate 13-acetate (PMA; a protein kinase C activator) blocked the pinacidil-induced effects, and PMA alone depolarized the membrane and increased pacemaker potential frequency. Cell-permeable 8-bromo-cyclic AMP also increased pacemaker potential frequency. Recordings of spontaneous intracellular Ca2+([Ca2+]i) oscillations showed that glibenclamide increased the frequency of [Ca2+]i oscillations. In small intestinal ICC, glibenclamide alone did not alter the generation of pacemaker potentials, and Kir 6.2 and SUR2B were expressed in Ano-1 positive ICC. Therefore, KATP channels in colonic ICC are activated in resting state and play an important role in maintaining resting membrane potential.

Effect of acupuncture at points selected from different regions on SCF-kit signaling pathway in diabetic gastroparesis rats

To observe the effect of acupuncture at points selected from different regions on the positive expression of interstitial cells of Cajal (ICC) and the stem cell factor (SCF) in gastric antrum tissues in diabetic gastroparesis (DGP) rats, and to explore the influence of region-based point selection on the acupoint combination efficacy. Sixty Sprague-Dawley (SD) rats were randomly divided into a normal group (group A), a model group (group B), a group of Zusanli (ST 36) plus Zhongwan (CV 12) (group C), a group of Zusanli (ST 36) plus Neiguan (PC 6) (group D), and a group of Zusanli (ST 36) plus non-meridian non-acupoint points (group E), based on the random number table (n=12). DGP rat model was established by single intraperitoneal injection of 2% streptozotocin and common diet. After successful modeling, the rats were treated once a day for 4 weeks. Positive ICC and SCF expressions were measured by immunohistochemistry. Compared with group A, the gastrointestinal propulsion rate of group B showed a statistically significant decrease (P<0.05). Compared with group B, the gastrointestinal propulsion rate and the expression of ICC in the gastric antrum were significantly higher in group C, group D and group E, and the between-group differences were statistically significant (P<0.05); the expression of C-kit protein in group C was statistically significantly higher than that in group D and group E (P<0.05). The expression of SCF protein was significantly increased in group C than in group B, and the difference was statistically significant (P<0.05). Acupuncture can improve the symptoms of delayed gastric emptying in DGP model rats, and regulate the expression of ICC and SCF in gastric antrum tissues. The effect of Zusanli (ST 36) plus Zhongwan (CV 12) in the gastric region is superior to that of the Zusanli (ST 36) plus distal Neiguan (PC 6) or non-meridian non-acupoint point, indicating that region-based point selection is the key factor affecting the effect of acupoint combination.

Regulation of the Pacemaker Activity of Colonic Interstitial Cells of Cajal by Protease-Activated Receptors: Involvement of Hyperpolarization-Activated Cyclic Nucleotide Channels

Background and Purpose: The exact mechanism of protease-activated receptors (PARs) on pacemaker activity of interstitial cells of Cajal (ICCs) has not been reported. We investigated the effects on pacemaker activity by the activation of PARs and its signal mechanisms in colonic ICCs. Methods: The whole-cell patch-clamp technique, RT-PCR and Ca²⁺ imaging were used in cultured ICCs from mouse colon. Results: PAR-1 and PAR-2 were expressed in Ano-1 positive ICCs. TFLLR-NH₂ (a PAR-1 agonist) and trypsin (a PAR-2 agonist) depolarized the membrane and increased the pacemaker potential frequency. U-73122 (a phospholipase C (PLC) inhibitor) and thapsigargin (a Ca²⁺ ATPase inhibitor) suppressed the TFLLR-NH₂- and trypsin-induced effects on pacemaker potential. TFLLR-NH₂ and trypsin also increased intracellular Ca²⁺ ([Ca²⁺]_i) intensity with increasing of Ca²⁺ oscillations. Genistein (a tyrosine kinase inhibitor), SP600125 (a JNK inhibitor), CsCl, ZD7288, clonidine (hyperpolarization-activated cyclic nucleotide (HCN) channel blockers), SQ-22536 and dideoxyadenosine (adenylate cyclase inhibitors) suppressed the increased pacemaker potential frequency without effects on depolarization of the membrane induced by TFLLR-NH₂ and trypsin. Conclusion: These results suggest that activation of PAR-1 and PAR-2 modulates the pacemaker activity of colonic ICCs through the PLC-dependent [Ca²⁺]_i release pathway. The increased pacemaker potential frequency by PAR-1 and PAR-2 was also dependent on tyrosine kinase, JNK, and HCN activation.

Effect of intestinal resection on hydrogen sulfide biosynthesis and the damage of Cajal interstitial cells

To investigate the effect of intestinal resection on hydrogen sulfide (H2S) biosynthesis and interstitial cells of Cajal(ICC) in mice. After intestinal resection mouse model was established, the activity of MPO in the proximal anastomosis intestinal tissue were detected. Sensitive sulphur electrode assay was applied to measure the H2S level. RT-PCR technique was employed to investigate the mRNA expression of the endogenous H2S biosynthesis enzymes, cystathionine-b-synthase (CBS) and cystathionine-c-lyase (CSE). Immunofluorescence staining was used to detect the expression of c-kit in order to calculate the area of ICC. The mRNA expression of CSE was detected in the small intestine tissue of mice, while no CBS mRNA was found. The mRNA expression of CSE in proximal anastomotic stoma increased in time-dependent manner in the model group. CSE mRNA expression began to increase 1 hour after operation, reached the peak at 6th hour, then decreased gradually, and was similar to the control group at postoperative 24th hour. Compared to the model group, in the intestinal tissues of proximal 3 cm to anastomotic stoma, the mRNA expression of CSE (1.16 ± 0.18 vs. 1.63 ± 0.13, P<0.05), the activity of MPO [(0.54 ± 0.07) U/g vs. (0.83 ± 0.09) U/g, P<0.05], the H2S level [(36.1 ± 6.1) nmol/mg vs. (5.3 ± 5.6) nmol/mg, P<0.05] were significantly reduced in the PPG group. Meanwhile, average percentage of positive ICC area in the PPG groups was significantly higher [(2.26 ± 0.19)% vs. (1.65 ± 0.24)%, P<0.05]. Inflammatory reaction in muscular layer induced by intestinal resection up-regulates the mRNA expression of CSE proximal to anastomotic stoma, generates excess H2S to damage ICC leading to intestinal motor dysfunction. Preoperative inhibition of endogenous H2S generation may protect the ICC.

Existence of c-Kit negative cells with ultrastructural features of interstitial cells of Cajal in the subserosal layer of the W/W(v) mutant mouse colon.

Interstitial cells of Cajal (ICC) are mesenchymal cells that are distributed along the gastrointestinal tract and function as pacemaker cells or intermediary cells between nerves and smooth muscle cells. ICC express a receptor tyrosine kinase c-Kit, which is an established marker for ICC. The c-kit gene is allelic with the murine white-spotting locus (W), and some ICC subsets were reported to be missing in heterozygous mutant W/Wv mice carrying W and Wv mutated alleles. In this study, the characterization of interstitial cells in the subserosal layer of W/Wv mice was analyzed by immunohistochemistry and electron microscopy. In the proximal and distal colon of W/Wv mutant mice, no c-Kit-positive cells were detected in the subserosal layer by immunohistochemistry. By electron microscopy, the interstitial cells, which were characterized by the existence of caveolae, abundant mitochondria and gap junctions, were observed in the W/Wv mutant colon. The morphological characteristics were comparable to those of the multipolar c-Kit positive ICC seen in the subserosa of proximal and distal colon of wild-type mice. Fibroblasts were also located in the same layers, but the morphology of the fibroblasts was distinguishable from that of ICC in wild type mice or of ICC-like cells in W/Wv mutant mice. Collectively, it is concluded that c-Kit-negative interstitial cells showing a typical ICC ultrastructure exist in the proximal and distal colon of W/Wv mutant mice.

The KIT gene is associated with the english spotting coat color locus and congenital megacolon in Checkered Giant rabbits (Oryctolagus cuniculus).

The English spotting coat color locus in rabbits, also known as Dominant white spotting locus, is determined by an incompletely dominant allele (En). Rabbits homozygous for the recessive wild-type allele (en/en) are self-colored, heterozygous En/en rabbits are normally spotted, and homozygous En/En animals are almost completely white. Compared to vital en/en and En/en rabbits, En/En animals are subvital because of a dilated (“mega”) cecum and ascending colon. In this study, we investigated the role of the KIT gene as a candidate for the English spotting locus in Checkered Giant rabbits and characterized the abnormalities affecting enteric neurons and c-kit positive interstitial cells of Cajal (ICC) in the megacolon of En/En rabbits. Twenty-one litters were obtained by crossing three Checkered Giant bucks (En/en) with nine Checkered Giant (En/en) and two en/en does, producing a total of 138 F1 and backcrossed rabbits. Resequencing all coding exons and portions of non-coding regions of the KIT gene in 28 rabbits of different breeds identified 98 polymorphisms. A single nucleotide polymorphism genotyped in all F1 families showed complete cosegregation with the English spotting coat color phenotype (θ = 0.00 LOD = 75.56). KIT gene expression in cecum and colon specimens of En/En (pathological) rabbits was 5–10% of that of en/en (control) rabbits. En/En rabbits showed reduced and altered c-kit immunolabelled ICC compared to en/en controls. Morphometric data on whole mounts of the ascending colon showed a significant decrease of HuC/D (P<0.05) and substance P (P<0.01) immunoreactive neurons in En/En vs. en/en. Electron microscopy analysis showed neuronal and ICC abnormalities in En/En tissues. The En/En rabbit model shows neuro-ICC changes reminiscent of the human non-aganglionic megacolon. This rabbit model may provide a better understanding of the molecular abnormalities underlying conditions associated with non-aganglionic megacolon.

Distribution of interstitial cells of Cajal in the neurogenic urinary bladder of children with myelomeningocele

C-kit positive interstitial cells of Cajal (ICCs) play an important role in the regulation of the smooth muscle motility, acting as pacemakers to provide the slow wave activity in various organs. Recent studies have shown that c-kit positive ICCs are widely distributed in the urinary tract of animals and humans. The aim of our study was to examine the distribution of ICCs in the children's neurogenic bladder. An immunohistochemical study of specimens obtained from neurogenic urinary bladder (from the trigonum and the corpus) of children with meningomyelocele and during autopsy was performed using antibody against c-kit (CD 117). Histological morphometry of immunoexpression of c-kit positive ICCs was performed by means of an image analyzing system. Our investigation demonstrated ICCs located in the vesical muscle layers. The distribution of those cells is different in the trigonum and the corpus of the urinary bladder. No remarkable differences were observed in c-kit immunoexpression between the neurogenic and the control group. There was no difference in the distribution of ICCs in the urinary bladder of healthy children as compared to children with myelomeningocele. Biopsy revealed different distribution of ICCs in particular parts of the bladder (trigonum/ corpus) in both groups of children.

The possible roles of hyperpolarization-activated cyclic nucleotide channels in regulating pacemaker activity in colonic interstitial cells of Cajal

BackgroundHyperpolarization-activated cyclic nucleotide (HCN) channels are pacemaker channels that regulate heart rate and neuronal rhythm in spontaneously active cardiac and neuronal cells. Interstitial cells of Cajal (ICCs) are also spontaneously active pacemaker cells in the gastrointestinal tract. Here, we investigated the existence of HCN channel and its role on pacemaker activity in colonic ICCs.MethodsWe performed whole-cell patch clamp, RT-PCR, and Ca2+-imaging in cultured ICCs from mouse mid colon.ResultsSQ-22536 and dideoxyadenosine (adenylate cyclase inhibitors) decreased the frequency of pacemaker potentials, whereas both rolipram (cAMP-specific phosphodiesterase inhibitor) and cell-permeable 8-bromo-cAMP increased the frequency of pacemaker potentials. CsCl, ZD7288, zatebradine, clonidine (HCN channel blockers), and genistein (a tyrosine kinase inhibitor) suppressed the pacemaker activity. RT-PCR revealed expression of HCN1 and HCN3 channels in c-kit and Ano1 positive colonic ICCs. In recordings of spontaneous intracellular Ca2+ [Ca2+]i oscillations, rolipram and 8-bromo-cAMP increased [Ca2+]i oscillations, whereas SQ-22536, CsCl, ZD7288, and genistein decreased [Ca2+]i oscillations.ConclusionsHCN channels in colonic ICCs are tonically activated by basal cAMP production and participate in regulation of pacemaking activity.

Comparison of different methods of intestinal obstruction in a rat model

To investigate different methods of creating incomplete intestinal obstruction in a rat model and to compare their electrophysiologic, morphologic and histologic characteristics. Rat ileum was partially obstructed by the respective application of: braided silk (penetrated the mesentery and surrounded intestine); half ligation (penetrated directly and ligated 1/2 cross-section of the intestine); wide pipe (6 mm in width, surrounded the intestine); narrow pipe (2 mm in width, surrounded the intestine). A control was also included (no obstruction). Various behavioral and electrophysiologic variables, as well as morphologic and immunohistochemical observations were recorded by blinded investigators at different time points (12, 24, 48, 72 h), including daily general condition, ileal wet weight and circumference, macromorphous and micromorphous intestine, bowel movement capability in vivo and in vitro, slow wave and neural electrical activity, and the number of c-Kit positive interstitial cells of Cajal (ICC). Despite being of a similar general condition, these methods resulted in different levels of obstruction in each group compared with the control at different time points (12, 24, 48, 72 h). However, these fields of the wide pipe rat showed significantly differences when compared with the other three obstructed groups at 12 to 72 h, including macroscopic and histological presentation, intestinal transit ratio and contractility, circumference and wet weight, amplitude and frequency of nerve electrical discharge and slow wave, and ICC numbers (all P < 0.01). The wide pipe rat method is significantly more reliable and stable than the other methods of obstruction, demonstrating that use of the wide pipe method can be a useful model of incomplete intestinal obstruction.

Neurotensin Receptor 1 Is Expressed in Gastrointestinal Stromal Tumors but Not in Interstitial Cells of Cajal

Gastrointestinal stromal tumors (GIST) are thought to derive from the interstitial cells of Cajal (ICC) or an ICC precursor. Oncogenic mutations of the KIT or PDGFRA receptor tyrosine kinases are present in the majority of GIST, leading to ligand-independent activation of the intracellular signal transduction pathways. We previously investigated the gene expression profile in the murine Kit K641E GIST model and identified Ntsr1 mRNA, encoding the Neurotensin receptor 1, amongst the upregulated genes. Here we characterized Ntsr1 mRNA and protein expression in the murine Kit K641E GIST model and in tissue microarrays of human GIST. Ntsr1 mRNA upregulation in Kit K641E animals was confirmed by quantitative PCR. Ntsr1 immunoreactivity was not detected in the Kit positive ICC of WT mice, but was present in the Kit positive hyperplasia of Kit K641E mice. In the normal human gut, NTSR1 immunoreactivity was detected in myenteric neurons but not in KIT positive ICC. Two independent tissue microarrays, including a total of 97 GIST, revealed NTSR1 immunoreactivity in all specimens, including the KIT negative GIST with PDGFRA mutation. NTSR1 immunoreactivity exhibited nuclear, cytoplasmic or mixed patterns, which might relate to variable levels of NTSR1 activation. As studies using radio-labeled NTSR1 ligand analogues for whole body tumor imaging and for targeted therapeutic interventions have already been reported, this study opens new perspectives for similar approaches in GIST.

Effects of Mechanical Stretch on Interstitial Cells of Cajal in Guinea Pig Bladder

C-kit positive interstitial cells of Cajal (ICCs) play an important role in the regulation of the smooth muscle motility. In this study, we investigated the mechanical sensitivity of ICCs in guinea pig bladder and their possible relationship with detrusor overactivity (DO). Stretch load was performed in vivo in DO models produced by 4 wk of partial bladder outlet obstruction (PBOO). Number and morphology of ICCs were examined using a specific ICC marker, c-kit, immunochemistry staining. The spontaneous and stretch-induced calcium transients (SICT) of ICCs were investigated in cells cultured onto flexible silicone membranes preloaded with the Ca(2+) indicator fluo-4AM in vitro. C-kit positive ICCs were mainly located along and between bladder smooth muscle bundles. ICCs in DO bladders displayed more lateral branching with mutual connections. The number of c-kit positive bladder ICCs was increased in the DO group compared with the control group (n = 50, P < 0.05). Cultured ICCs from DO bladders showed spontaneous calcium waves with higher frequency and lower amplitude than those from control bladders (n = 15, P < 0.05). Significant SICT were detected in cultured bladder ICCs. SICT generated in ICCs from DO were more likely to transfer to adjacent smooth muscle cells through cell membrane connection than ICCs from control bladders. Long-term overload tension following PBOO caused changes in morphology, quantity and spontaneous calcium transients of ICCs in guinea pig bladder. Mechanical sensitivity and interaction with SMC of ICCs may contribute to the mechanosensitive conductances in bladder regulation, and may play a role in the pathogenesis of DO.

Morphological Expression of KIT Positive Interstitial Cells of Cajal in Human Bladder

PurposeWe investigated the 3-dimensional morphological arrangement of KIT positive interstitial cells of Cajal in the human bladder and explored their structural interactions with neighboring cells.Materials and MethodsHuman bladder biopsy samples were prepared for immunohistochemistry/confocal or transmission electron microscopy.ResultsWhole mount, flat sheet preparations labeled with anti-KIT (Merck, Darmstadt, Germany) contained several immunopositive interstitial cell of Cajal populations. A network of stellate interstitial cells of Cajal in the lamina propria made structural connections with a cholinergic nerve plexus. Vimentin positive cells of several morphologies were present in the lamina propria, presumably including fibroblasts, interstitial cells of Cajal and other cells of mesenchymal origin. Microvessels were abundant in this region and branched, elongated KIT positive interstitial cells of Cajal were found discretely along the vessel axis with each perivascular interstitial cell of Cajal associated with at least 6 vascular smooth muscle cells. Detrusor interstitial cells of Cajal were spindle-shaped, branched cells tracking the smooth muscle bundles, closely associated with smooth muscle cells and vesicular acetylcholine transferase nerves. Rounded, nonbranched KIT positive cells were more numerous in the lamina propria than in the detrusor and were immunopositive for anti-mast cell tryptase. Transmission electron microscopy revealed cells with the ultrastructural characteristics of interstitial cells of Cajal throughout the human bladder wall.ConclusionsThe human bladder contains a network of KIT positive interstitial cells of Cajal in the lamina propria, which make frequent connections with a cholinergic nerve plexus. Novel perivascular interstitial cells of Cajal were discovered close to vascular smooth muscle cells, suggesting interstitial cells of Cajal-vascular coupling in the bladder. KIT positive detrusor interstitial cells of Cajal tracked smooth muscle bundles and were associated with nerves, perhaps showing a functional tri-unit controlling bladder contractility.

Regional Distribution of Interstitial Cells of Cajal (ICC) in Human Stomach

We elucidated the distribution of interstitial cells of Cajal (ICC) in human stomach, using cryosection and c-Kit immunohistochemistry to identify c-Kit positive ICC. Before c-Kit staining, we routinely used hematoxylin and eosin (HE) staining to identify every structure of human stomach, from mucosa to longitudinal muscle. HE staining revealed that the fundus greater curvature (GC) had prominent oblique muscle layer, and c-Kit immunostaining c-Kit positive ICC cells were found to have typical morphology of dense fusiform cell body with multiple processes protruding from the central cell body. In particular, we could observe dense processes and ramifications of ICC in myenteric area and longitudinal muscle layer of corpus GC. Interestingly, c-Kit positive ICC-like cells which had morphology very similar to ICC were found in gastric mucosa. We could not find any significant difference in the distribution of ICC between fundus and corpus, except for submucosa where the density of ICC was much higher in gastric fundus than corpus. Furthermore, there was no significant difference in the density of ICC between each area of fundus and corpus, except for muscularis mucosa. Finally, we also found similar distribution of ICC in normal and cancerous tissue obtained from a patient who underwent pancreotomy and gastrectomy. In conclusion, ICC was found ubiquitously in human stomach and the density of ICC was significantly lower in the muscularis mucosa of both fundus/corpus and higher in the submucosa of gastric fundus than corpus.

Interstitial cells of Cajal reduce in number in recto-sigmoid Hirschsprung's disease and total colonic aganglionosis

Interstitial cells of Cajal (ICCs) play a key role in regulating gastrointestinal tract motility. The pathophysiological basis of colonic aperistalsis in Hirschsprung's disease (HD) is still not fully understood. Many studies reported that decreased numbers or disrupted networks of ICCs were associated with HD. Little information is available on the distribution of different subtypes of ICCs in HD. The aim of this study was to determine the alterations in density of different subtypes of ICC in colonic specimens of patients with total colonic and recto-sigmoid HD. Full thickness colonic specimens were obtained from five children with total colonic aganglionosis (TCA), sixteen with recto-sigmoid HD and seven controls. ICCs were visualized in frozen sections by c-Kit (CD117) fluorescent staining. In the control colon, c-Kit positive ICCs formed a dense network surrounding the myenteric plexus (IC-MY), along the submucosal surface of the circular muscle layer (IC-SM) and in the circular and longitudinal muscle layer (IC-IM). In the aganglionic region of the colon of the patients affected by HD, the number of ICCs (especially IC-IM and IC-SM) was markedly reduced and IC-MY networks were disrupted. Nearly total lack of three subtypes of ICCs was observed in the TCA specimens. This study demonstrated the altered distribution of different subtypes of ICCs in the resected colon of patients with recto-sigmoid HD and TCA. These findings suggest that the reduction of each subtype of ICCs may play an important role in the etiology of HD.

Distribution of interstitial cells of Cajal and nitrergic neurons in normal and diabetic human appendix

The objective of this study was to determine the distribution of enteric nerves and interstitial cells of Cajal (ICC) in the normal human appendix and in type 1 diabetes. Appendixes were collected from patients with type 1 diabetes and from non-diabetic controls. Volumes of nerves and ICC were determined using 3-D reconstruction and neuronal nitric oxide synthase (nNOS) expressing neurons were counted. Enteric ganglia were found in the myenteric plexus region and within the longitudinal muscle. ICC were found throughout the muscle layers. In diabetes, c-Kit positive ICC volumes were significantly reduced as were nNOS expressing neurons. In conclusion, we describe the distribution of ICC and enteric nerves in health and in diabetes. The data also suggest that the human appendix, a readily available source of human tissue, may be useful model for the study of motility disorders.

C-Kit receptor (CD117) in the porcine urinary tract

C-Kit positive interstitial cells of Cajal (ICC) play an important role in the regulation of the smooth muscle motility, acting as internal pacemakers to provide the slow wave activity within various luminal organs. Recently c-Kit-(CD117)-positive interstitial cells (IC) have been shown in the genitourinary tract, but systematic studies on the distribution and density of IC within the urinary tract are still lacking. Therefore the aim of the present study was to analyze systematically the localization and distribution of the c-Kit receptor in the urinary tract of the pig using immunohistochemical and molecular methods. Tissue samples were harvested from the porcine urinary tract including renal calices and pelvis, ureteropelvic junction, proximal, middle and distal ureter, ureteral orifice, fundus, and corpus of the bladder and the internal urethral orifice. Small and large intestine specimen served as controls. Immunohistochemistry (APAAP, IF) was applied on serial frozen sections using four monoclonal and polyclonal antibodies recognizing CD117. Whole mounts of the porcine upper urinary tract were prepared and investigated using conventional and confocal fluorescence microscopy followed by three-dimensional reconstruction. UV-laser microdissection and RT-PCR were applied to confirm the immunohistochemical results. CD117-immunoreactivity labeled bipolar IC and round-shaped mast cells (MC) throughout the adventitia, tunica muscularis and submucosa within the whole porcine urinary tract. While MC were found continuously in all investigated segments, a gradient of bipolar IC was evident. The whole mount preparations gave a detailed cytomorphology of IC within the various layers of the porcine urinary tract. Whole mount preparations revealed closed apposition of bi- and tripolar c-Kit positive IC parallel to the smooth muscle bundles and to veins of the tunica muscularis and adventitia. In the urothelium single CD117-positive interepithelial cells were found. The highest density of CD117-positive cells was found at the ureteropelvic junction, however the differences in between the segments were minimal. Microdissection and RT-PCR confirmed the results uncovered by immunohistochemistry. The ubiquitous distribution of IC and their close relationship to smooth muscle provides strong evidence that IC could contribute to the intrinsic pacemaker activity within the porcine (upper and lower) urinary tract. The role of the interepithelial CD117-positive cells as mechanosensors or as a precursor cell in the regeneration of the urothelium, is conceivable.

LOSS OF INTERSTITIAL CELLS OF CAJAL AND GAP JUNCTION PROTEIN CONNEXIN 43 AT THE VESICOURETERAL JUNCTION IN CHILDREN WITH VESICOURETERAL REFLUX

Symmetrical muscular contractions and unidirectional peristalsis are prerequisites for competent valve function at the ureterovesical junction. Interstitial cells of Cajal (ICCs) are pacemaker cells that create and coordinate peristaltic motility. We investigated ureteral endings immunohistochemically to elucidate the presence of c-kit positive ICCs as well as the occurrence of gap junction protein connexin 43 in children with vesicoureteral reflux (VUR) to identify a possible pathogenic factor of an insufficient antireflux mechanism. Distal ureteral specimens were obtained from 27 children undergoing antireflux surgery. Routine histological paraffin embedded sections were immunostained detecting the c-kit proto-oncogene to study the presence of ICCs as well as connexin 43 positive cells to determine the gap junction density within the ureteral wall. Age matched nonrefluxing ureteral endings served as controls. All investigations were done using high power field magnification for semiquantitative analysis and statistically analyzed. ICCs were present in refluxing and nonrefluxing ureteral endings. Healthy individuals displayed significantly more ICCs than children with VUR. Connexin 43 immunoreactivity was significantly decreased in all refluxing ureteral specimens, whereas it was homogeneously distributed in normal controls. C-kit positive ICCs are found at the ureterovesical junction. Refluxing ureteral endings significantly lack these pacemaker cells, implying a malfunctioning valve mechanism permitting VUR. A substantial decrease in gap junctions in this region adversely affects intercellular signaling, aggravating coordinated peristalsis, which is essential for a competent anti-reflux mechanism.

Interstitial cells of cajal in the human normal urinary bladder and in the bladder of patients with megacystis‐microcolon intestinal hypoperistalsis syndrome

OBJECTIVE To investigate the distribution of c-kit-positive interstitial cells of Cajal (ICCs) in normal bladder and bladders from patients with megacystis-microcolon-intestinal peristalsis syndrome (MMIHS, a rare congenital and generally fatal cause of functional intestinal obstruction in the newborn), the most characteristic feature of which is abdominal distension caused by a distended unobstructed urinary bladder. PATIENTS AND METHODS Full-thickness bladder specimens were obtained from four infants with MMIHS and four controls, and processed as paraffin-wax and frozen sections. Sections were assessed using single immunohistochemistry with monoclonal and polyclonal anti-c-kit antibodies. Anti-α-smooth muscle actin (SMA) antibody was used to investigate the contractile apparatus in smooth muscle cells of the urinary bladder. Specimens were examined using light and confocal scanning microscopy. RESULTS There were many c-kit positive ICCs in the normal urinary bladder, appearing as small, long, bipolar cells with only two long and several short processes. In contrast, ICCs were absent in the MMIHS bladder. α-SMA immunoreactivity was lower in MMIHS urinary bladder than in control sections. CONCLUSION This study shows for the first time the presence of c-kit-positive ICCs in the normal human urinary bladder. The lack of ICCs in the MMIHS bladder may contribute to the voiding dysfunction in this disease.

Motility patterns and distribution of interstitial cells of Cajal and nitrergic neurons in the proximal, mid‐ and distal‐colon of the rat

The aim of this work was to study the patterns of spontaneous motility in the circular and longitudinal muscle strips and to characterize the distribution of c-kit positive interstitial cells of Cajal (ICCs) and nitrergic neurons (nNOS) in the proximal, mid- and distal-colon of Sprague-Dawley rats. We described two types of spontaneous contractions: high frequency (HF) and low frequency (LF) contractions, which were recorded in the presence of tetrodotoxin, suggesting a non-neurogenic origin. Regional differences were found in the motility patterns depending on the muscle layer and on the part of the colon studied. Muscle strips without submuscular plexus (SMP) showed only LF contractions. The density of ICCs was of the same magnitude along the extent of the colon: about 90-120 cells mm(-2) at Auerbach's plexus (AP) and 50-60 cells mm(-2) at the SMP. nNOS positive cells were found at the level of the AP and the major density was found in the mid-colon. Electrical field stimulation abolished LF but did not affect HF contractions. Our results indicate that HF contractions are due to the ICC network found associated with the submuscular plexus (ICC-SMP). The origin of LF contractions is still unknown.