Decrease in nerve fibre density in human sigmoid colon circular muscle occurs with growth but not aging

Background Studies in animals suggest that enteric neurons decrease in density or number with increasing age. Neurons containing nitric oxide (NO), vasoactive intestinal peptide (VIP) and Substance P (SP) have been implicated. In human large intestine, NO‐utilizing neurons decrease during childhood or early adulthood but it is not known if the innervation of the muscle changes. This study examined the density of nerve fibres containing these transmitters in sigmoid colon circular muscle from children and adults. Methods Fluorescence immunohistochemistry using antibodies to neuronal NO synthase (nNOS), VIP and SP was performed on sigmoid colon from 18 adults with colorectal cancer, two children with familial adenomatous polyposis, and normal colon from nine children with Hirschsprung’s disease. The percentage area of immunoreactive (IR) nerve fibres containing each transmitter in circular muscle was quantified in confocal images. Key Results In the adult sigmoid colon circular muscle, the percentage area of nerve fibres containing nNOS>VIP>SP (6 : 2 : 1). Paediatric groups had significantly higher percentage area of nerve fibres containing nNOS, VIP or SP‐IR than adults, with the decrease in nerve fibre density occurring from birth to 30 years. Circular muscle thickness increased between 12 and 30 years. Total nerve fibre area remained constant, while the muscle increased in thickness. Conclusions & Inferences In human sigmoid colon circular muscle, there are reductions in nNOS‐, VIP‐ and SP‐IR nerve fibre density with growth from newborn to late adolescence but little further change with aging. The reduction in nerve density is due to an increase in circular muscle thickness rather than a loss of nerve fibres.     

Cholinergic transmission to colonic circular muscle of children with slow-transit constipation is unimpaired, but transmission via NK2 receptors is lacking

Abstract  Tachykinins (TKs) colocalize with acetylcholine in excitatory motor neurones supplying human colonic circular muscle (CCM). Some children with slow-transit constipation (STC) have reduced TK-immunoreactivity in nerve terminals in CCM suggesting a deficit in neuromuscular transmission. This study aimed to test this possibility. Seromuscular biopsies of transverse colon were obtained laparoscopically from STC children (37, 17 with low density of TK-immunoreactivity). Specimens of transverse (17) and sigmoid colon (20) were obtained from adults undergoing colonic resection for cancer. CCM contractions were measured isotonically and responses to carbachol, neurokinin A (NKA) and electrical field stimulation (EFS) recorded. Carbachol and NKA-evoked contractions in adult and STC colon. Hyoscine (2  μ mol L⁻¹) significantly depressed responses to EFS in all preparations. Blockade of NK₂ receptors (SR 48968, 2  μ mol L⁻¹) significantly depressed EFS-evoked contractions of adult transverse CCM, but had no effect on STC preparations. Thus, neuromuscular transmission in both adults and STC children is predominantly cholinergic and this component is unimpaired in the latter, indicating that reduced TK-immunoreactivity is not a marker for depressed cholinergic responses. Although pharmacologically responsive TK receptors are present in STC colon, we did not detect neuromuscular transmission mediated by release of TKs in these preparations.     

Regional variation in the neurochemical coding of the myenteric plexus of the human colon and changes in patients with slow transit constipation

Abstract There are differences in the structure and function between regions of the colon. In patients with slow transit constipation the activity of all regions is markedly slowed. Counts of colonic neurones in slow transit constipation have been semiquantitative and led to varying results. We have applied new methods of quantification of markers in whole mounts of the colonic myenteric plexus to compare density of innervation between regions and between normal patients and those undergoing resection for severe constipation. Whole mounts of colonic myenteric plexus were made from specimens removed for cancer treatment (controls) and cases of severe constipation. All neurones were labelled by anti‐human neuronal protein antibodies. Neurones synthesizing acetyl choline were labelled for choline acetyltransferase (ChAT) and those for nitric oxide by antisera to nitric oxide synthase (NOS). Four populations of neurones were distinguished and quantified according to the two selective markers, ChAT and NOS. In the normal major populations were NOS alone (51% of ascending colon neurones and 44% of descending colon neurones) and ChAT alone (41% ascending colon, 48% descending colon). Nitric oxide synthase/ChAT and NOS‐/ChAT‐comprised only small populations. In all regions in severe constipation, the percentage of NOS‐only colonic myenteric neurones was raised (54% ascending colon, 49% descending colon) and ChAT only was reduced (36% ascending colon, 42% descending colon). The other populations were not changed. Accurate quantification of neuronal populations in whole mounts of human colon reveals inter‐regional differences in innervation and marked changes in innervation in cases of very severe constipation.     

The effect of mosapride citrate on proximal and distal colonic motor function in the guinea-pig in vitro.

Mosapride citrate (mosapride), a substituted benzamide, is a selective 5-HT(4) receptor agonist, and is known to have prokinetic properties on the stomach. However, it is unclear whether mosapride also has a prokinetic effect on the colon. We previously found that mosapride significantly shortened colonic transit time in the guinea-pig, an animal with a distribution of colonic 5-HT(4) receptors similar to that of a human. So, we aimed to separately evaluate the effect of mosapride on proximal and distal colonic motor function in the guinea-pig. Proximal (approximately 8 cm from the ileocolic junction) and distal colon (approximately 8 cm from the anus) were removed. Both ends of the colon were connected to a chamber containing a Krebs-Henseleit solution. To measure colonic transit time, artificial faeces were inserted into the oral side of the lumen and moved towards the anal side by intraluminal perfusion via a peristaltic pump. A total of 6 cm of transit was observed and time was measured in 2 cm increments. A tissue bath study, using electrical stimulation, was performed to estimate the contractile activity of the circular musculature of the colon. Immunohistochemical staining for 5-HT(4) receptors was performed in the myenteric plexus and circular muscle in both proximal and distal colon, and the stained area was measured using a microscope and computer software. Mosapride enhanced contraction at 10(-9) to 10(-7) mol L(-1), coinciding with rapid transit both in proximal and distal colon. This pattern was more prominent in proximal colon. At the high dose (10(-6) mol L(-1)) mosapride had little or no effect on colonic contraction. This stimulatory effect was attenuated by GR113808, atropine and tetrodotoxin. In the myenteric plexus, the density of 5-HT(4) receptors was significantly greater in the proximal colon than in the distal colon, but in circular muscle the density was greater in the distal colon. Thus, mosapride accelerates transit through increased contraction in the proximal colon more than distal colon. The different distribution of neuronal and muscular 5-HT(4) receptors may support these findings. Therefore, mosapride may be a useful alternative to tegaserod and cisapride for constipation.     

Pancolonic spatiotemporal mapping reveals regional deficiencies in,and disorganization of colonic propagating pressure waves in severe constipation

Background The morphology, motor responses and spatiotemporal organization among colonic propagating sequences (PS) have never been defined throughout the entire colon of patients with slow transit constipation (STC). Utilizing the technique of spatiotemporal mapping, we aimed to demonstrate ‘manometric signatures’ that may serve as biomarkers of the disorder. Methods In 14 female patients with scintigraphically confirmed STC, and eight healthy female controls, a silicone catheter with 16 recording sites spanning the colon at 7.5 cm intervals was positioned colonoscopically with the tip clipped to the cecum. Intraluminal pressures were recorded for 24 h. Key Results Pan‐colonic, 24 h, spatiotemporal mapping identified for the first time in STC patients: a marked paucity of propagating pressure waves in the mid‐colon (P = 0.01), as a consequence of a significant (P < 0.0001) decrease in extent of propagation of PS originating in the proximal colon; an increase in frequency of retrograde PS in the proximal colon; a significant reduction in the spatiotemporal organization among PS (P < 0.001); absence of the normal nocturnal suppression of PS. Conclusions & Inferences Pan‐colonic, 24 h, spatiotemporal pressure mapping readily identifies characteristic disorganization among consecutive PS, regions of diminished activity and absent or deficient fundamental motor patterns and responses to physiological stimuli. These features are all likely to be important in the pathophysiology of slow transit constipation.     

Neuropeptides in idiopathic chronic constipation (slow transit constipation)

Tissue specimens from the large bowel of 18 patients with long-standing slow transit constipation were investigated to determine the distribution and density of several neuropeptides and amines in the enteric nerve system, and also of endocrine cells in comparison to normal individuals. CGRP (calcitonin gene-related peptide), galanin, glucagon, GRP (gastrin-releasing peptide), metenkephalin, motilin, neuropeptide Y (NPY), PACAP, peptide YY (PYY), serotonin, somatostatin, substance P and VIP were studied by immunohistochemistry. Tissue concentrations of VIP, substance P and galanin were also measured by radioimmunoassay. Significantly increased VIP, SP and galanin contents were found in specimens from the ascending colon. Levels of VIP and galanin were also increased in the transverse colon. Immunohistochemistry revealed only marginal changes with an increased density of PACAP nerve fibres in the smooth muscle and of VIP and PACAP nerves in the myenteric plexus of the transverse colon. In the descending colon substance P and NPY immunoreactivity were also increased in the myenteric plexus while the density of VIP nerve fibres was reduced in the mucosa/submucosa. The frequency of PYY-containing cells and the 5-HT-containing cells in the ascending colon was significantly increased in the constipated patients.     

High‐resolution colonic manometry accurately predicts colonic neuromuscular pathological phenotype in pediatric slow transit constipation

Background Severe pediatric slow transit constipation (STC) is commonly due to intrinsic colonic neuromuscular disease. We sought to correlate neuromuscular histological phenotypes in pediatric STC with colonic manometric phenotypes using high‐resolution manometry (HRM). We tested the hypothesis that failure of motor quiescence (FQ) between bisacodyl‐induced high amplitude propagating sequences (HAPSs) might predict neuromuscular pathology. Methods Eighteen children (10 males, median age: 7.5 years) with refractory STC underwent stationary colonic HRM before segmental colonic resection. Six age‐matched constipated children with normal colonic transit served as controls. Colonic resection specimens underwent histopathological analysis. Conventional manometric parameters and area under the curve (AUC) during a 1‐min period following bisacodyl‐induced HAPSs [PBAUC₁], as measure of FQ, were calculated. Key Results Numbers of postbisacodyl HAPSs in descending and sigmoid segments were lower in patients than controls (P < 0.01, respectively). Low amplitude propagating sequences (LAPSs) were common prebisacodyl in controls and rare in STC (P < 0.001), whereas postbisacodyl LAPS were more common in STC (P < 0.001). Postbisacodyl, both retrograde propagating contractions and bursts of contractions were present in STC patients only (P < 0.001 and P < 0.01). Postbisacodyl simultaneous pressurization was seen only in STC (P < 0.05 and P < 0.001, in descending and rectosigmoid segments). Histological abnormalities were present in 17/18. Fourteen were neurogenic, one neuro‐myogenic, and two myogenic. In segments with HAPS, PBAUC₁ was predictive of colonic neuropathy using a cutoff of 205 mmHg.s^‐1 (Sensitivity 100%, specificity 86%, PPV92%, NPV100%). Conclusions & Inferences PBAUC₁ is increased in multiple colonic segments in neuropathic pediatric STC and constitutes a sensitive and specific biomarker of neuropathy.     

Distribution and actions of galanin and vasoactive intestinal peptide in the human colon.

Responses to galanin (GAL) and vasoactive intestinal peptide (VIP) and their distribution were investigated in muscularis externa from the human sigmoid colon. Immunohistochemical studies revealed GAL-like immunoreactivity (GAL-IR) and VIP-IR in nerve fibres supplying both muscle layers as well as the myenteric ganglia. Additionally GAL-IR was shown to co-exist with VIP-IR in many nerve fibres innervating human circular and longitudinal (taenial) colonic muscle layers. Circular and longitudinal muscle strips were contracted by GAL and relaxed by VIP. There was a marked difference in sensitivity between muscle layers to both peptides; circular muscle being less sensitive to GAL, longitudinal muscle less sensitive to VIP. When given simultaneously GAL attenuated responses to VIP on longitudinal muscle, thus in the same neurone there are substances having opposing actions on smooth muscle motility.     

Distribution of vasoactive intestinal peptide- and substance P-containing nerves originating from neurons of airway ganglia in cat bronchi.

This study examined the possibility that vasoactive intestinal peptide (VIP)- and substance P (SP)-containing nerve fibers in bronchial smooth muscle, glands, epithelium, and blood vessels originate from neurons of airway ganglia. Explants of airway walls were maintained in culture with the expectation that nerve fibers from neurons of airway ganglia would remain viable, whereas fibers originating from neurons not present in the airway walls would degenerate. Airways were dissected and placed into culture dishes containing CMRL 1066 medium for 3, 5, and 7 days. In controls (noncultured), VIP- and SP-like immunoreactivity was observed in nerve fibers associated with bronchial smooth muscle, glands, and blood vessel walls and in nerve cell bodies of airway ganglia. Nerve fibers containing SP were also observed within the bronchial epithelium. After 3, 5, and 7 days in culture, VIP- and SP-containing fibers were identified in all of the same locations except in the airway epithelium where SP-containing fibers could not be demonstrated. VIP and SP were frequently colocalized in the same nerve fibers of bronchial smooth muscle and glands in controls and cultured airways. There were no statistically significant differences in nerve fiber density for either VIP- or SP-containing fibers in bronchial smooth muscle between controlled and cultured airways. VIP concentrations in cultured airways were significantly less than in controls. The results suggest that a large proportion of VIP- and SP-containing nerve fibers supplying bronchial smooth muscle, glands, and blood vessels in the airways originate from neurons of airway ganglia.     

Morphological and neurochemical differences in peptidergic nerve fibers of the mouse vagina

The vagina is innervated by a complex arrangement of sensory, sympathetic, and parasympathetic nerve fibers that contain classical transmitters plus an array of neuropeptides and enzymes known to regulate diverse processes including blood flow and nociception. The neurochemical characteristics and distributions of peptide‐containing nerves in the mouse vagina are unknown. This study used multiple labeling immunohistochemistry, confocal maging and analysis to investigate the presence and colocalization of the peptides vasoactive intestinal polypeptide (VIP), calcitonin‐gene related peptide (CGRP), substance P (SP), neuropeptide tyrosine (NPY), and the nitric oxide synthesizing enzyme neuronal nitric oxide synthase (nNOS) in nerve fibers of the murine vaginal wall. We compared cervical and vulvar areas of the vagina in young nullipara and older multipara C57Bl/6 mice, and identified differences including that small ganglia were restricted to cervical segments, epithelial fibers were mainly present in vulvar segments and most nerve fibers were found in the lamina propria of the cervical region of the vagina, where a higher number of fibers containing immunoreactivity for VIP, CGRP, SP, or nNOS were found. Two populations of VIP‐containing fibers were identified: fibers containing CGRP and fibers containing VIP but not CGRP. Differences between young and older mice were present in multiple layers of the vaginal wall, with older mice showing overall loss of innervation of epithelium of the proximal vagina and reduced proportions of VIP, CGRP, and SP containing nerve fibers in the distal epithelium. The distal vagina also showed increased vascularization and perivascular fibers containing NPY. Immunolabeling of ganglia associated with the vagina indicated the likely origin of some peptidergic fibers. Our results reveal regional differences and age‐ or parity‐related changes in innervation of the mouse vagina, effecting the distribution of neuropeptides with diverse roles in function of the female genital tract.     

Distribution of calcitonin gene-related peptide, somatostatin, substance P and vasoactive intestinal polypeptide in experimental colitis in rats

Immunohistochemistry was used to examine the distribution of calcitonin gene-related peptide (CGRP), substance P, somatostatin and vasoactive intestinal polypeptide (VIP) in experimental colitis induced with trinitrobenzene sulphonic acid (TNBS) in rats. CGRP immunoreactivity was observed throughout the colonic wall. A significant reduction of CGRP-immunoreactive (IR) nerve fibres was observed in the mucosa after the induction of colitis. After TNBS treatment substance P immunoreactivity was reduced throughout the colon; however, after 7 days there was a marked re-innervation of the circular muscle. Somatostatin immunoreactivity was distributed sparsely within the colonic wall, and was comparatively less affected by colitis. VIP immuno- reactivity was abundantly distributed in the colonic wall and underwent an immediate reduction in the mucosa after TNBS treatment. After 2 days, there was a consistent and progressive increase in the number and density of VIP-IR nerve fibres in the inflamed colon, particularly the circular muscle. This change was associated with a proliferation of nerve fibres within the muscle layers. It was concluded that the early decrease in these neuropeptides was consistent with release from peripheral nerve terminals or the loss of nerves during the initial stages of colonic inflammation, which may be an essential condition for the development of colitis in this model. The observation that the intensity and density of substance P and VIP-IR nerve fibres increased in the circular muscle 7 days after the induction of colitis suggests their possible involvement in tissue repair.     

Myogenic regional responsiveness to cholinergic and vipergic stimulation in human colon

Background Differences in the actions of enteric neurotransmitters on colonic circular and longitudinal muscle layers have not been clearly determined, nor the possible existence of intrinsic myogenic phenotypes that might contribute to regional differences in human colon motor activity. The aim of this study was to analyze the direct pharmaco‐mechanical coupling of carbachol (CCh) and vasoactive intestinal polypeptide (VIP) on human colonic smooth muscle strips and cells. Methods Circular and longitudinal muscle strips and cells were obtained from 15 human specimens of ascending and sigmoid colon. Both isometric tension on muscle strips and contraction and relaxation on cells were measured in response to increasing CCh and VIP concentrations. Key Results Circular muscle strips of ascending colon were more sensitive to the effect of CCh than that of sigmoid colon, EC₅₀ values being, respectively, 4.15 μmol L^?1 and 8.47 μmol L^?1 (P < 0.05), although there were no differences in maximal responses. No regional differences were observed in longitudinal muscle strips or in smooth muscle cells. Maximal responses to CCh were higher on circular than longitudinal muscle strips and cells throughout the colon. A greater sensitivity to VIP was observed in ascending colon compared with sigmoid colon, both in circular (EC_50: 0.041 and 0.15 μmol L^?1, respectively, P < 0.01) and longitudinal (EC_50: 0.043 and 0.09 μmol L^?1, respectively, P < 0.05) strips, and similar differences were observed in longitudinal smooth muscle cells (EC_50: 44.85 and 75.24 nmol L^?1, respectively, P < 0.05). Conclusions & Inferences Regional myogenic differences in pharmaco‐mechanical coupling between the enteric neurotransmitters and smooth muscle contribute to the complex regional motor patterns of human colon.     

Anatomical registration and three‐dimensional visualization of low and high‐resolution pan‐colonic manometry recordings

Background Colonic propagating sequences (PS) are important for the movement of colonic content and defecation, and aberrant PS patterning has been associated with slow transit constipation. However, because these motor patterns are typically recorded over long periods (24 h +), the visualization of PS spatiotemporal patterning is difficult. Here, we develop a novel method for displaying pan‐colonic motility patterns. Methods A 3D mesh representing the geometry of the human colon was created as follows: (i) Human colon images from the Visible Human Dataset were digitized to create a 3D data cloud, and (ii) A surface mesh was fitted to the cloud using a least‐squares minimization technique. Colonic manometry catheters were placed in the ascending colon of healthy controls and patients with slow transit constipation (STC), with the aid of a colonoscope. The colonic manometry data were interpolated and mapped to the model according to the following anatomical landmarks: cecum, hepatic flexure, splenic flexure, sigmoid‐descending junction, and anus. Key Results These 3D images clearly and intuitively communicate characteristics of normal and abnormal colonic motility. Specifically we have shown the reduced amplitude of the antegrade propagating pressure waves (PPW) throughout the colon and reduced frequency of PPWs at the mid‐colon in patients with STC. Conclusions and Inferences A novel method for the 3D visualization of PS is presented, providing an intuitive method for representing a large volume of physiological data. These techniques can be used to display frequency, amplitude or velocity data, and will help to convey regions of abnormally in patient populations.     

Peptidergic innervation in the cerebral blood vessels of the guinea pig: an immunohistochemical study.

The distribution of peptidergic nerve fibers containing substance P (SP), calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP), and neuropeptide Y (NPY) in the cerebral arteries and veins of the guinea pig was studied using immunohistochemical techniques. The ultrastructure of these immunoreactive nerve terminals was also compared. The cerebral arteries were innervated by abundant peptidergic nerve fibers with characteristic running patterns, i.e., SP fibers in a meshwork, VIP and NPY fibers in a spiral fashion. Only CGRP fibers showed both meshwork and spiral patterns. In the cerebral veins, the abundant SP fibers innervated the cortical veins, deep cerebral veins, and dural sinuses. However, CGRP, VIP, and NPY fibers in extremely low density were noted merely in the cortical veins. Electron microscopic observations demonstrated that SP-immunoreactive nerve terminals existed apart from the arterial smooth muscle cells, while VIP- and NPY-immunoreactive nerve terminals adjoined them. As for CGRP nerve terminals, some existed close to the arterial smooth muscle cells, and others were found some distance from them. These morphological characteristics observed by light and electron microscopy suggest that SP fibers are not related directly to the vasomotor function, but VIP and NPY fibers are, and that CGRP fibers have a more complicated function. The distribution patterns of the peptidergic nerve fibers are consistent with the suggestion that vasomotor peptidergic fibers may function actively on cerebral arteries and passively on cerebral veins and that SP fibers regarded as sensory fibers may provide information regarding cerebral vascular conditions, innervating every part of both cerebral arteries and veins.     

Fall in density, but not number of myenteric neurons and circular muscle nerve fibres in guinea-pig colon with ageing

Abstract  In guinea-pig ileum, ageing has been associated with a decrease in enteric neurons. This study examined guinea-pig colon and measured changes in gut dimensions, neuron size, density and ganglionic area. Changes in motor nerve fibres in the circular muscle were also measured. Myenteric neurons in whole-mount preparations of mid-colon from 2-week, 6-month, and 2-year-old guinea-pigs were labelled immunohistochemically with the neuronal marker human neuronal protein HuC/HuD, and numbers of neurons mm⁻², neuronal size, ganglionic area mm⁻², gut length, circumference and muscle thickness were measured. Corrected numbers of neurons mm⁻² and ganglionic area mm⁻² accounting for growth of the colon were calculated. Additionally, nerve fibres in circular muscle cross-sections were labelled with antibodies against nitric oxide synthase (NOS) and substance P (SP) and the density of nerve fibres in circular muscle was measured. The numbers of neurons mm⁻² decreased by 56% (from 2 weeks to 2 years) with no change in neuron size. Total neuron numbers decreased by 19% ( P  = 0.14) when adjusted for changes in length and circumference with age. The percentage area of NOS- and SP-immunoreactive (IR) nerve fibres in the circular muscle decreased ( P  < 0.001), but the total area of NOS and SP-IR nerve fibres increased ( P  < 0.01) due to an age-related increase in muscle thickness. The density of myenteric neurons in guinea-pig mid-colon halved from 2 weeks to 2 years, but when the increase in colon dimensions was considered, the number of neurons decreased by only 19%. The percentage area of motor nerve fibres in the circular muscle decreased with no change in total volume of nerve fibres.     

Morphology and projections of myenteric neurons to colonic fiber bundles of the cat

Regulation of colon function depends on the location of nerve cell bodies and the distribution of intrinsic nerve fibers in the myenteric plexus. The morphology and projections of myenteric neurons through colonic fiber bundles in cat colon were determined using in vivo retrograde transport of HRP and Fast blue. Myenteric neurons were found to project from at least 5 to 59 mm orad (mean: 42 mm) or aborad (mean: 54 mm) through colonic fiber bundles. Approximately 73% of labelled cells were in ganglia within 2.8 mm of colonic fiber bundles in the axis of circular muscle fibers; none was beyond 7.7 mm. There were 2 soma morphologies. One type (Dogiel type I) had a mean soma diameter of 40.5 microns and had a rough somal surface. There were few if any short, broad dendrites, but its one long process extended to a branch point of an adjacent colonic fiber bundle. The other type (Dogiel type III) had a mean soma diameter of 26.4 microns, had a smooth somal surface and had few if any fine dendrites. It also projected a single long axon to colonic fiber bundles. There were twice as many Dogiel type III neurons. We conclude that myenteric neurons in the cat colon project both orad and aborad over relatively long distances through colonic fiber bundles where they form another intrinsic neuronal connection for the myenteric plexus.     

Response of Smooth Muscle from Proximal and Distal Human Colon

Regional differences in colonic motility may be responsible for the orderly transit of intraluminal contents through the colon. The aims of this study were to compare the effect of stretch on active and passive stress development in colonic muscle from the proximal and distal colon and to compare the responses of these tissues to KC1 or bethanechol stimulation. Strips of taenia or circular smooth muscle were obtained from the disease-free segment of the colon removed for adenocarcinoma. Passive, active, and total isometric stress were measured on full-thickness strips of circular or longitudinal taenial muscle stimulated with bethanechol (10⁻⁴ M) as the muscles were stretched to 120% of the length of optimum tension ( L _o.) The tissues then were stimulated with increasing concentrations of KCI and bethanechol while being stretched at L _o. The active stress in the proximal circular muscle was greater at all levels of stretch than in distal circular or longitudinal muscle ( p <.001). The resting and passive stress were greater in distal circular and longitudinal taenial muscle than in proximal circular muscle ( p < .05). There was a dose-dependent increase in stress development to bethanechol and KCl in each type of muscle. Proximal circular muscle had the greatest response. The EDSO was shifted to the right in distal circular muscle (2.6 ± 0.1 × 10⁻⁵ M) compared to proximal circular muscle (1.1 ± 0.1 × 10⁻⁵ M) ( p < .001). These studies suggest that muscle stress differs in different locations of the colon and the role of active and passive stress development must be considered in models explaining in vivo colonic motility disturbances.     

Internal anal sphincter relaxation associated with bisacodyl‐induced colonic high amplitude propagating contractions in children with constipation: a colo‐anal reflex?

Background Describe the association of internal anal sphincter (IAS) relaxation with colonic high‐ amplitude peristaltic contractions (HAPCs). Methods Retrospective review of colon manometry tracings of children with constipation to determine the IAS relaxation characteristics associated with HAPC’s (HAPC‐IASR) events and compare them to the those seen during the performance of the anorectal manometry (ARMRAIR) events. Key Results A total of 70 HAPC‐ IASRs were observed in 15 patients, 65 after bisacodyl, two during fasting and three after a meal. In 64% of events, the IAS relaxation started when the HAPC reached left colon and in 36% as proximal as the hepatic flexure. High‐ amplitude peristaltic contraction propagation seems to be important in HAPC‐IASR characteristics; those propagating distal to sigmoid colon demonstrated larger and longer IAS relaxation as well as lower residual pressure, but equivalent resting pressure compared with HAPC’s ending proximal to sigmoid colon. Although IAS resting pressure was comparable for ARM‐RAIRs and HAPC‐IASRs, the duration and magnitude of anal relaxation was higher, and the anal residual pressure was lower in HAPC‐IASRs. Conclusions & Inferences We demonstrated that IAS relaxation in constipated children is associated with HAPCs migrating in the proximal and distal colon; in most cases, starting when peristalsis is migrating through left colon and in an important proportion while migrating proximally. We also demonstrated that HAPC‐IASRs are different from ARM‐RAIRs suggesting a neurally mediated reflex. Finally, the IAS relaxation characteristics are highly dependent on the degree of propagation of HAPCs, which could have important implications in the understanding of defecation disorders.     

Qualitative and quantitative analysis of tachykinin NK2 receptors in chemically defined human colonic neuronal pathways

The involvement of NK2 receptors (NK2r) in the neuroregulation of human colonic motility has been mainly assessed by using pharmacological approaches. The aim of this study was to characterize the intramural neurons and nerve varicosities expressing NK2r in human colonic neuronal pathways. Neuronal coding in the myenteric plexus and external muscle layers was identified on the basis of the patterns of colocalization of tachykinins (TK), vesicular acetylcholine transporter (VAChT), nitric oxide synthase (NOS), glutamate decarboxylase (GAD), and vasoactive intestinal peptide (VIP) with NK2r immunoreactivity. The proportions of chemically defined synaptophysin-immunoreactive nerve varicosities were accurately determined by using specific software. NK2r immunoreactivity was detected in the soma of many myenteric neurons (71.8%). A large proportion of these neurons was immunoreactive to VAChT (39.3%), TK (30%), and GAD (23.5%) and, to a lesser extent, to NOS and VIP. The proportions of nerve varicosities expressing NK2r showed significant regional differences: the highest proportion (59.8%) was located in the myenteric plexus. High proportions of the myenteric nerve varicosities expressing NK2r were immunoreactive to VIP (80.9%) and NOS (77.9%), and lower proportions were recorded with VAChT, TK, and GAD. In the circular and longitudinal muscle layers, the proportions of nerve varicosities expressing NK2r were 49.6% and 45.3%, respectively. The chemically defined intramuscular varicosities were closely apposed to smooth muscle cells expressing NK2r. In conclusion, the data obtained in this study, in which the expression of NK2r was mapped in the human colonic neuronal pathways, confirm that these receptors are involved in the neuroneuronal and neuromuscular processes regulating human colonic motility.     

Impaired propulsive motility in the distal but not proximal colon of BK channel β1‐subunit knockout mice

Background Large‐conductance Ca²⁺‐activated K⁺ (BK) channels regulate smooth muscle tone. The BK channel β1‐subunit increases Ca²⁺ sensitivity of the α‐subunit in smooth muscle. We studied β1‐subunit knockout (KO) mice to determine if gastrointestinal (GI) motility was altered. Methods Colonic and intestinal longitudinal muscle reactivity to bethanechol and colonic migrating motor complexes (CMMCs) were measured in vitro. Gastric emptying and small intestinal transit were measured in vivo. Colonic motility was assessed in vivo by measuring fecal output and glass bead expulsion time. Myoelectric activity of distal colon smooth muscle was measured in vitro using intracellular microelectrodes. Key Results Bethanechol‐induced contractions were larger in the distal colon of β1‐subunit KO compared to wild type (WT) mice; there were no differences in bethanechol reactivity in the duodenum, ileum, or proximal colon of WT vsβ1‐subunit KO mice. There were more retrogradely propagated CMMCs in the distal colon of β1‐subunit KO compared to WT mice. Gastrointestinal transit was unaffected by β1‐subunit KO. Fecal output was decreased and glass bead expulsion times were increased in β1‐subunit KO mice. Membrane potential of distal colon smooth muscle cells from β1‐subunit KO mice was depolarized with higher action potential frequency compared to WT mice. Paxilline (BK channel blocker) depolarized smooth muscle cells and increased action potential frequency in WT distal colon. Conclusions & Inferences BK channels play a prominent role in smooth muscle function only in the distal colon of mice. Defects in smooth muscle BK channel function disrupt colonic motility causing constipation.