'Rapid transit' constipation in children: a possible genesis for irritable bowel syndrome.

Children with chronic idiopathic constipation (CIC) often end up at the surgeon when medical treatments have failed. This opinion piece discusses a recently described pattern of CIC called 'Rapid transit constipation (RTC)' first identified in 2011 as part of surgical workup. RTC was identified using a nuclear medicine gastrointestinal transit study (NMGIT or nuclear transit study) to determine the site of slowing within the bowel and to inform surgical treatment. Unexpectedly, we found that RTC occured in 29% of 1000 transit studies in a retrospective audit. Irritable bowel syndrome (IBS) occurs in 7-21% of the population, with a higher prevalence in young children and with constipation type dominating in the young. While 60% improve with time, 40% continue with symptoms. First-line therapy for IBS in adults is a diet low in fermentable oligosaccharides, disaccharides, monosaccharides and polyols which reduces symptoms in > 70% of patients. In children with functional gastrointestinal disorders, fructose intolerance occurs in 35-55%. Reducing fructose produced significant improvement in 77-82% of intolerant patients. In children with RTC and a positive breath test upon fructose challenge, we found that exclusion of fructose significantly improved constipation, abdominal pain, stool consistency and decreased laxative use. We hypothesise that positive breath tests and improvement of pain and bowel frequency with sugar exclusion diets in RTC suggest these children have IBS-C. These observations raise the possibility that many children with CIC could be treated by reducing fructose early in their diet and this might prevent the development of IBS in later life.

The effect of food withdrawal in children with rapid-transit constipation.

BACKGROUND: Rapid proximal colonic transit with anorectal holdup is a subtype of chronic constipation linked to food intolerance. We aimed to determine the effectiveness of dietary exclusion as a treatment for constipated children with rapid-transit constipation by scintigraphy. METHODS: Questionnaires on diet and symptoms were mailed out to 125 children with chronic constipation and rapid proximal colonic transit on nuclear transit study at our institute between 1998 and 2014 years. Patients were given instructions and encouraged to undertake a six-food elimination diet targeting common protein allergens (dairy, wheat, soy, eggs, nuts, seafood). Answers were completed by circling an option or on visual analogue scale. Results were evaluated statistically using GraphPad Prism 6 by a Wilcoxon matched-pairs rank test. P < 0.05 was considered significant. RESULTS: We received 44/125 responses, 26 patients [mean age 11 years (5-21)] had attempted elimination diet and 18 had not. Dairy and wheat were the most common foods eliminated and symptomatic improvement was greater for patients who had completely eliminated foods. Constipation, abdominal pain and pain on defecation were reduced (p < 0.01). Laxative usage decreased, although this was not statistically significant. Families encountered problems with dietary exclusion, particularly expense. Assistance from a dietician or nutritionist was sought by >50 % of families. CONCLUSION: Dietary exclusion is a promising strategy to treat constipation in children with rapid proximal colonic transit. However, it was hard for many families, demonstrating the need for identifying the cause more specifically and a better set of instructions for the family and/or dietitian to follow.

Vaginal agenesis, the hymen, and associated anomalies.

STUDY OBJECTIVES: Review anomalies in patients with vaginal agenesis. In particular, to clarify the impact of an absent hymen on the presence of other anomalies; on the success of creating a vagina with dilators; and on sexual function outcomes. DESIGN: Retrospective medical record review; questionnaire on sexual function. SETTING: Gynecology service at a children's hospital and the practice of 1 gynecologist. PARTICIPANTS: All patients with vaginal agenesis were identified from the databases, as well as the subgroup in which hymenal status was known. OUTCOME MEASURES: Data regarding hymen, renal, skeletal, cardiac, and other anomalies; for women who had a neovagina, the technique used to create a functional vagina. RESULTS: Of 69 females (age range 2-70 years), renal tract anomalies (43.3%), vertebral anomalies (29%), cardiac anomalies (14.5%), and syndromes including Klippel-Feil (7%) and MURCS association (7%) were identified. Where hymenal status was known (n = 47), 31 were normal, and 16 had an absent hymen. Where the hymen was absent, renal agenesis was increased (odds ratio = 13.5, P < .001). There was no association between other anomalies and an absent hymen, or between the various anomalies. For women without a hymen, the likelihood of failing dilation therapy was increased (odds ratio = 21.7; P < .01]. CONCLUSION: An absent hymen makes renal agenesis more likely and increases the likelihood that dilator techniques will fail. This condition appears to be associated with reports of long-term problems with poor lubrication that are potentially related to the absence of the peri-hymenal Bartholin's glands.

Development of the gubernaculum during testicular descent in the rat.

Gubernacular elongation during inguinoscrotal testicular descent and cremaster muscle development remains poorly described in mammals. The role of the genitofemoral nerve (GFN) remains elusive. We performed detailed histological analysis of testicular descent in normal rats to provide a comprehensive anatomical description for molecular studies. Fetuses and neonatal male offspring (5-10 per group) from time-mated Sprague-Dawley dams (embryonic days 15, 16, and 19; postnatal days 0, 2, and 8) were prepared for histology. Immunohistochemistry was performed for nerves (Class III tubulin, Tuj1) and muscle (desmin). At embryonic days 15 and 16, the gubernaculum and breast bud are adjacent and both supplied by the GFN. By embryonic day 19, the breast bud has regressed and the gubernacular swelling reaction is completed. Postnatally, the gubernacular core regresses, except for a cranial proliferative zone. The cremaster is continuous with internal oblique and transversus abdominis. By postnatal day 2 (P2), the gubernaculum has everted, locating the proliferative zone caudally and the residual mesenchymal core externally. Eversion creates the processus vaginalis, with the everted gubernaculum loose in subcutaneous tissue but still remote from the scrotum. By P8, the gubernaculum has nearly reached the scrotum with fibrous connections attaching the gubernaculum to the scrotal skin. A direct link between GFN, gubernaculum, and breast bud suggests that the latter may be involved in gubernacular development. Second, the cremaster muscle is continuous with abdominal wall muscles, but most of its growth occurs in the distal gubernacular tip. Finally, gubernacular eversion at birth brings the cranial proliferative zone to the external distal tip, enabling gubernacular elongation similar to a limb bud.

Neuromodulation for constipation: sacral and transcutaneous stimulation.

Constipation is a frequently occurring digestive ailment that is usually treated conservatively. Neuromodulation is altering function of an organ by altering neural activity. This paper reviews methods of neuromodulation used to treat constipation. This includes direct stimulation of sacral nerves and stimulation across the skin. Direct stimulation of sacral nerves is the most well developed method and is presented in detail. It is generally accepted that the mechanism of action is modulation rather than stimulation so it is called sacral neuromodulation (SNM). SNM involves percutaneous placement of an electrode in the third sacral foramen and implanting a stimulating device under the skin in the buttocks. SNM is founded on the physiological principle that activity in one neural pathway modulates pre-existing activity in another through synaptic interaction. The mechanism of action in constipation may be neuromodulation of the extrinsic neural control of the large bowel or modulation of reflexes inhibiting large bowel function. Limited evidence is available to assess the outcome of SNM in constipation. Results in the medium term seem promising for selected patients with idiopathic slow and normal transit constipation not responding to optimal conservative treatment. Adverse events include electrode migration and infection. The availability of a testing phase provides a predictor of treatment outcome. In addition, transcutaneous stimulation using sticky pad electrodes over the lumbosacral region or acupuncture points has been reported to improve constipation symptoms. In general, the level of evidence is low and further studies are needed.

Substance P and vasoactive intestinal peptide are reduced in right transverse colon in pediatric slow-transit constipation.

BACKGROUND: Slow-transit constipation (STC) is recognized in children but the etiology is unknown. Abnormalities in substance P (SP), vasoactive intestinal peptide (VIP) and nitric oxide (NO) have been implicated. The density of nerve fibers in circular muscle containing these transmitters was examined in colon from children with STC and compared to other pediatric and adult samples. METHODS: Fluorescence immunohistochemistry using antibodies to NO synthase (NOS), VIP and SP was performed on colonic biopsies (transverse and sigmoid colon) from 33 adults with colorectal cancer, 11 children with normal colonic transit and anorectal retention (NAR) and 51 with chronic constipation and slow motility in the proximal colon (STC). The percentage area of nerve fibers in circular muscle containing each transmitter was quantified in confocal images. KEY RESULTS: In colon circular muscle, the percentage area of nerve fibers containing NOS > VIP > SP (6 : 2 : 1). Pediatric groups had a higher density of nerve fibers than adults. In pediatric samples, there were no regional differences in NOS and VIP, while SP nerve fiber density was higher in sigmoid than proximal colon. STC children had lower SP and VIP nerve fiber density in the proximal colon than NAR children. Twenty-three percent of STC children had low SP nerve fiber density. CONCLUSIONS & INFERENCES: There are age-related reductions in nerve fiber density in human colon circular muscle. NOS and VIP do not show regional variations, while SP nerve fiber density is higher in distal colon. 1/3 of pediatric STC patients have low SP or VIP nerve fiber density in proximal colon.

Localization of muscarinic receptors M1R, M2R and M3R in the human colon.

BACKGROUND: Muscarinic acetylcholine receptors (MR) are involved in multiple intestinal reflexes. The cellular localization of subtypes of MRs within enteric circuits mediating muscle and mucosal reflexes remains to be demonstrated. This study aimed to localize the three functionally significant subtypes of MRs in human colon. METHODS: Reverse transcriptase-PCR was used to determine expression levels of muscarinic receptor subtype (MRs) M1Rs, M2Rs and M3Rs in human colon. Indirect immunofluorescence and confocal microscopy was used to localize MRs in cryostat-cut sections of human colon. Sections were double labeled for multiple cellular and neurochemical markers. Western blotting was used to confirm specificity of the muscarinic antisera used. KEY RESULTS: All three MR subtypes were expressed in human colon. Immunoreactivity (IR) for M2Rs and M3Rs was most abundant in circular and longitudinal muscle. M1R-IR was most abundant on myenteric and submucosal nerve cells, both cholinergic and nitrergic. M3R-IR was also present on populations on myenteric nerve cell bodies. Immunoreactivity for all three receptors was present on nerve fibers in the circular muscle. CONCLUSIONS & INFERENCES: In the human colon, subtypes of MRs were present on multiple cell types within the enteric circuits underlying motility, secretory and vasoactive reflexes. The cellular distribution for MRs found in this study agrees with data from functional studies, providing insight into the role MRs have in mediating enteric cholinergic neurotransmission.

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.

The Kelly technique of bladder exstrophy repair: continence, cosmesis and pelvic organ prolapse outcomes.

PURPOSE: The Kelly technique of radical soft tissue mobilization, an alternative to osteotomy and modern staged repair, has been used extensively at our tertiary referral center for bladder exstrophy in the last 2 decades. We present what is to our knowledge the first long-term followup of the Kelly technique in 31 patients treated at our institution. MATERIALS AND METHODS: Patients admitted for bladder exstrophy at our institution since 1980 were identified and the medical charts were reviewed. Continence questionnaires were completed during followup appointments or by mail. Continence was defined as complete-dry greater than 3 hours during the day and night with 2 or fewer night wets per month and partial-dry 2 hours or more during the day and 3 or greater night wets per month, and/or stress incontinence. The degree of pelvic organ prolapse was assessed in females older than 12 years. RESULTS: Data were available on 31 Kelly patients, including 14 females, 4 to 25 years old and 13 patients, including 4 females, 2 to 29 years old treated with another staged technique. Of 30 Kelly patients without urinary diversion 21 (70%) were completely or partially continent. Of the 30 patients 17 voided spontaneously without clean intermittent catheterization or augmentation, of whom 12 (71%) were continent. Lower abdominal appearance was graded as abnormal in 11 of 12 male Kelly patients vs in 2 of 7 nonKelly males with pubic approximation (p = 0.01). Of the 12 females assessed none of 9 Kelly patients had prolapse, whereas 2 of 3 nonKelly patients had prolapse (p <0.05). CONCLUSIONS: The continence rate after the Kelly operation compares favorably with that in recent series. The abnormal appearance of the lower abdomen and bony pelvis in Kelly males may result from a lack of pubic approximation. Importantly pelvic organ prolapse may be decreased in women after the Kelly technique.

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

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.

The role of the gubernaculum in the descent and undescent of the testis.

Testicular descent to the scrotum involves complex anatomical rearrangements and hormonal regulation. The gubernaculum remains the key structure, undergoing the 'swelling reaction' in the transabdominal phase, and actively migrating out of the abdominal wall to the scrotum in the inguinoscrotal phase. Insulin-like hormone 3 (Insl3) is the primary regulator of the first phase, possibly augmented by Müllerian inhibiting substance/anitmüllerian hormone (MIS/AMH), and regression of the cranial suspensory ligament by testosterone. The inguinoscrotal phase is controlled by androgens acting both directly on the gubernaculum and indirectly via the genitofemoral nerve, and release of calcitonin gene-related peptide from its sensory fibres. Outgrowth of the gubernaculum and elongation to the scrotum has many similarities to an embryonic limb bud.Cryptorchidism occurs because of both failure of migration congenitally, and failure of elongation of the spermatic cord postnatally. Germ cell development postnatally is disturbed in congenital cryptorchidism, but our current understanding of germ cell biology suggests that early orchidopexy, around 6 months of age, should provide a significant improvement in prognosis compared with a previous generation. Hormone treatment is not currently recommended. Acquired cryptorchid testes may need orchidopexy once they no longer reach the scrotum, although this remains controversial.

Immunohistochemical localisation of pre-synaptic muscarinic receptor subtype-2 (M2r) in the enteric nervous system of guinea-pig ileum.

The cholinergic muscarinic 2 receptor (M2r) is known to be present on smooth muscle cells in the intestine. Pharmacological studies also suggest that M2rs regulate transmitter release from nerves in the enteric nervous system. This study localised M2rs in the guinea-pig ileum using different antibodies and fluorescence immunohistochemistry. Double labelling with antibodies against neurochemical markers was used to identify the type of nerves bearing M2r. Guinea-pig ileum were fixed, prepared for sections and wholemounts and incubated with antisera against the M2r sequence. Tissue was double labelled with antibodies against neuronal nitric oxide synthase (nNOS), common choline acetyltransferase (cChAT), substance P (SP), synaptophysin and vesicular acetylcholine transporter (VAChT). Immunofluorescence was viewed using confocal microscopy. Abundant M2r-immunoreactivity (IR) was present on the surface of circular and longitudinal smooth muscle cells. M2r-IR was present in many but not all nerve fibres in the circular muscle and ganglia. M2r-IR was present in VAChT-IR and cChAT-IR cholinergic nerve fibres and SP-IR nerve fibres in the myenteric ganglia and submucosal ganglia. M2r-IR was present on a few nNOS-IR nerve fibres and around nNOS-IR neurons in the myenteric ganglia. In the circular muscle and deep muscular plexus, M2r-IR was present in many VAChT-IR and SP-IR nerve fibres and in few nNOS-IR nerves. M2rs are not only present on muscle cells in the intestine, but also on nerve fibres. M2rs may mediate cholinergic reflexes via their location on muscle and also via neural transmission. The pre-synaptic location supports pharmacological studies suggesting M2rs mediate neurotransmitter release from nerve fibres. The presence of M2rs on VAChT-IR, SP-IR and nNOS-IR-containing nerve fibres suggests M2rs may regulate ACh, SP and nitric oxide release.

Immunohistochemical localisation of cholinergic muscarinic receptor subtype 1 (M1r) in the guinea pig and human enteric nervous system.

Little is known regarding the location of cholinergic muscarinic receptor 1 (M1r) in the ENS, even though physiological data suggest that M1rs are central to cholinergic neurotransmission. This study localised M1rs in the ENS of the guinea pig ileum and human colon using fluorescence immunohistochemistry and RT-PCR in human colon. Double labelling using antibodies against neurochemical markers was used to identify neuron subytpes bearing M1r. M1r immunoreactivity (IR) was present on neurons in the myenteric and submucosal ganglia. The two antibodies gave similar M1r-IR patterns and M1r-IR was abolished upon antibody preabsorption. M1r-IR was present on cholinergic and nNOS-IR nerve cell bodies in both guinea pig and human myenteric neurons. Presynaptic M1r-IR was present on NOS-IR and VAChT-IR nerve fibres in the circular muscle in the human colon. In the submucosal ganglia, M1r-IR was present on a population of neurons that contained cChAT-IR, but did not contain NPY-IR or calretinin-IR. M1r-IR was present on endothelial cells of blood vessels in the submucosal plexus. The localisation of M1r-IR in the guinea pig and human ENS shown in this study agrees with physiological studies. M1r-IR in cholinergic and nitrergic neurons and nerve fibres indicate that M1rs have a role in both cholinergic and nitrergic transmission. M1r-IR present in submucosal neurons suggests a role in mediating acetylcholine's effect on submucosal sensory and secretomotor/vasodilator neurons. M1r-IR present on blood vessel endothelial cells suggests that M1rs may also mediate acetylcholine's direct effect on vasoactivation.

Immunohistochemical localization of substance P NK1 receptor in guinea pig distal colon.

BACKGROUND: Neurokinin receptors facilitate tachykinin mediated intestinal motility and secretion. Distribution of Substance P (SP) neurokinin 1 receptor (NK1r) immunoreactivity (IR) has been previously characterized in guinea pig ileum, but not colon. This study localizes NK1rs in guinea pig distal colon. METHODS: Neurons were double labelled for NK1r and either acetylcholine transferase (ChAT), calbindin (calb), neuropeptide Y (NPY), nitric oxide synthase (NOS) or SP. The NK1r endocytosis was induced by 10(-5) mol L(-1) SP, septide, [SarMet] SP or neurokinin A. RESULTS: In guinea pig distal colon, NK1r-IR was present on 70% of submucosal neurons. Sixty-threepercent of the NK1r-IR submucosal neurons were ChAT-IR, 16% calb/SP-IR, 19% NPY-IR and 14% NOS-IR neurons. The NK1r-IR was present on 5% of myenteric neurons. Of these 63% were ChAT-IR, 16% calb-IR neurons and 25% NOS-IR. The NK1rs were also on myenteric plexus interstitial cells of Cajal and on circular muscle. CONCLUSION: In guinea pig distal colon, NK1rs were on 70% of submucosal neurons including all three secretomotor neuron subtypes and sensory neurons, suggesting NK1rs have a major role in neuronal control of mucosal reflexes. The NK1rs were on few myenteric neurons but were dense on muscle cells, suggesting NK1rs affect motility through neuro-muscular rather than neuro-neuronal transmission.

Chronic constipation in children: organic disorders are a major cause.

Diagnostic tools for paediatric chronic constipation have been limited, leading to over 90% of patients with treatment-resistant constipation being diagnosed with chronic idiopathic constipation, with no discernible organic cause. Work in our institution suggests that a number of children with intractable symptoms actually have slow colonic transit leading to slow transit constipation. This paper reviews recent data suggesting that a significant number of the children with chronic treatment-resistant constipation may have organic causes (slow colonic transit and outlet obstruction) and suggests new approaches to the management of children with chronic treatment-resistant constipation.

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

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 micromol L-1) significantly depressed responses to EFS in all preparations. Blockade of NK2 receptors (SR 48968, 2 micromol 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.

Localization of protein kinase C theta immunoreactivity to interstitial cells of Cajal in guinea-pig gastrointestinal tract.

In the gastrointestinal tract, interstitial cells of Cajal (ICC) are located between nerve fibres and muscle cells and have a role in neuromuscular transmission and muscle contractility. Protein kinase C (PKC) is involved in modulation of muscle contractility by neurotransmitters, but it is not known if PKC has a role in ICC. There are 11 different PKC isoforms. The presence of PKC isoforms in ICC in guinea-pig gastrointestinal tract was examined using fluorescence immunohistochemistry and confocal microscopy. Segments of guinea-pig stomach, duodenum, ileum, proximal and distal colon were fixed in zambonis fixative. Frozen sections and wholemounts were incubated with anti-PKC antibodies (alpha, beta, delta, epsilon, gamma, iota, lambda, mu, theta) followed by fluorescent secondary antibody. Only PKC theta (theta) immunoreactivity was found in ICC. None of the other PKC isoforms (alpha, beta, delta, epsilon, gamma, iota, lambda, mu) localized to the ICC. PKC theta immunoreactivity was prominent in ICC located between the circular and longitudinal muscle layers (ICC-MY) in all regions except stomach and within the circular muscle (ICC-IM) in the large intestine. PKC theta was not present in ICC in the deep muscular plexus in either duodenum or ileum. PKC theta immunoreactivity was present in the cell body and proximal processes of the ICC. The cells containing PKC theta also contained cKit confirming the cells were ICC. ICC-MY in the ileum also contained the neurokinin (NK) 1 receptor. In conclusion, PKC theta is present in pacemaker ICC, but its function is not yet known. Functional studies will be needed to determine the role of this kinase in ICC. Knowing the second messenger cascades and being able to manipulate subpopulations of ICC will add to our understanding of the molecular and cell biology of ICC networks within the gastrointestinal tract and may ultimately help in understanding the aetiology of some gastrointestinal motor pathologies.

Immunohistochemical demonstration of the NK(1) tachykinin receptor on muscle and epithelia in guinea pig intestine.

BACKGROUND AND AIMS: Previous immunohistochemical studies failed to reveal neurokinin (NK)(1) tachykinin receptors on intestinal muscle, despite convincing pharmacologic data indicating their presence. This study aimed to apply optimal immunohistochemical methods to reveal the receptors. METHODS: NK(1)-receptor immunoreactivity was examined by confocal microscopy in tissue incubated with or without 10(-7) mol/L substance P (SP), 10(-7) mol/L SP plus 10(-6) mol/L NK(1) receptor antagonist (CP99994), or with fluorescent cyanine 3.18 (Cy3) SP. RESULTS: Without incubation, NK(1)-receptor immunoreactivity was strong on muscle of the rectum and distal colon and weak in proximal colon and small intestine. NK(1) receptor was located on the surface of muscle cells in all gut regions. Exposure to SP increased the intensity of immunoreactivity, and the receptor moved into the cytoplasm. Mobilization of the receptor by SP was blocked by the NK(1)-receptor antagonist CP99994. Cy3-SP was internalized by muscle cells and colocalized with the receptor. NK(1)-receptor immunoreactivity occurred on crypt epithelial cells in the small intestine and the base of glands in the proximal colon. CONCLUSIONS: The NK(1) receptor occurs on the external muscle throughout the small and large intestines. SP binds and triggers NK(1)-receptor aggregation and internalization in the muscle.

Independent endocytosis of the NK(1) and NK(3) tachykinin receptors in neurons of the rat myenteric plexus.

In the myenteric plexus of rat ileum, NK(1) and NK(3) receptors are co-located almost exclusively on neurons of a single population. This study compares endocytosis of NK(1) and NK(3) receptors in these neurons. In the absence of agonist, 26.2+/-2.8% of NK(1) receptor and 29.1+/-1.1% of NK(3) receptor was located in the cytoplasm of the neurons; the remaining receptor was on the surface. The tachykinin neurotransmitters, substance P (10 pM-10 microM) and neurokinin A (10 pM-100 microM), both induced concentration-dependent endocytosis of NK(1) and NK(3) receptors. The selective NK(1) receptor agonist, [Sar(9),Met(O(2))(11)]-substance P (1 microM), induced endocytosis of NK(1) receptor (64.2+/-1.5% in cytoplasm) but not NK(3) receptor (32.9+/-5.0%). The NK(1) receptor endocytosis was reduced by the selective NK(1) receptor antagonist, CP-99994 (100 nM), but not by the selective NK(3) receptor antagonist, SR-142801 (1 microM). The selective NK(3) receptor agonist, senktide (10 nM), induced endocytosis of NK(3) receptor (61.2+/-5.4%) but not NK(1) receptor (34.0+/-4.5%). The NK(3) receptor endocytosis was blocked by SR-142801 but not by CP-99994. We also investigated the effects of monensin, which generally blocks recycling of endocytosed receptor. In the absence or presence of exogenous agonist, monensin caused a build-up of NK(1) receptor, but not NK(3) receptor, in the cytoplasm of neurons.The results demonstrate independent, agonist-induced endocytosis of NK(1) and NK(3) receptors in neurons of the myenteric plexus of rat ileum and suggest that the mechanisms of recycling of NK(1) and NK(3) receptors differ.

The rat visceral yolk sac internalizes maternal transferrin and secretes hydrolyzed products towards the fetus.

The uptake of transferrin by the rat visceral yolk sac membranes, and the fate of this protein, were measured in a two-chambered system which allowed access to both surfaces of these membranes, i.e. that facing the maternal compartment and that facing the fetal compartment. 125I-labeled transferrin was internalized by the maternal surface of the visceral yolk sac but not by the fetal surface. Following internalization, this transferrin was degraded and the amino acids were secreted exclusively towards the fetal compartment. Transcytosis of intact transferrin was not detected in either direction. These results suggest that transport across the rat visceral yolk sac bound to maternally derived transferrin is not a major mechanism of iron transport in vivo. These results support a role for the visceral yolk sac in fetal metabolism, or supplying the fetus with amino acids derived from degradation of specific maternal plasma proteins, in this case, transferrin.