Bridget R. Southwell

Localisation of NK1 receptor immunoreactivity to neurons and interstitial cells of the guinea-pig gastrointestinal tract.

Tachykinins, including substance P, neurokinin A, and neuropeptides K and γ, are expressed widely in the peripheral nervous system where they affect smooth muscle contraction, exocrine gland secretion, vascular permeability, and neurotransmission. Substance P, the preferred ligand for the NK1 receptor, is found in high concentrations in the enteric nervous system. In the present study, the localisation and distribution of the NK1 receptor was studied throughout the gastrointestinal tract of the guinea‐pig by using a polyclonal antiserum raised against the C‐terminal 15 amino acids of the NK1 receptor. Co‐localisation with other neuronal markers was examined in the ileum. Nerve cell bodies reactive for the NK1 receptor were found in the myenteric plexus of all regions and the submucous plexus of the small and large intestines. In the small intestine, the interstitial cells of Cajal were also immunoreactive. Immunoreactivity was largely confined to cell surfaces. Almost all immunoreactive myenteric nerve cells had Dogiel type I morphology, and most of these were immunoreactive for nitric oxide synthase, a transmitter of inhibitory neurons to the muscle and of descending interneurons. Neuropeptide Y‐containing secretomotor neurons in the submucous and myenteric plexuses also exhibited NK1 receptor immunoreactivity. NK1 receptors were present on a minority of tachykinin immunoreactive neurons of submucous ganglia. The results suggest that receptors on the longitudinal muscle might not be conventional NK1 receptors, that excitation of the circular muscle of the ileum is indirect, perhaps via the interstitial cells of Cajal, and that enteric inhibitory neurons may be excited via NK1 receptors.

What is new in cryptorchidism and hypospadias—a critical review on the testicular dysgenesis hypothesis ☆

It has been hypothesized that poor semen quality, testis cancer, undescended testis, and hypospadias are symptoms of one underlying entity--the testicular dysgenesis syndrome--leading to increasing male fertility impairment. Though testicular cancer has increased in many Western countries during the past 40 years, hypospadias rates have not changed with certainty over the same period. Also, recent studies demonstrate that sperm output may have declined in certain areas of Europe but is probably not declining across the globe as indicated by American studies. However, at the same time, there is increasing recognition of male infertility related to obesity and smoking. There is no certain evidence that the rates of undescended testes have been increasing with time during the last 50 years. In more than 95% of the cases, hypospadias is not associated with cryptorchidism, suggesting major differences in pathogenesis. Placental abnormality may occasionally cause both cryptorchidism and hypospadias, as it is also the case in many other congenital malformations. The findings of early orchidopexy lowering the risk of both infertility and testicular cancer suggest that the abnormal location exposes the cryptorchid testis to infertility and malignant transformation, rather than there being a primary abnormality. Statistically, 5% of testicular cancers only are caused by cryptorchidism. These data point to the complexity of pathogenic and epidemiologic features of each component and the difficulties in ascribing them to a single unifying process, such as testicular dysgenesis syndrome, particularly when so little is known of the actual mechanisms of disease.

Localisation of neurokinin 3 (NK3) receptor immunoreactivity in the rat gastrointestinal tract.

Abstract.The localisation of the neurokinin 3 receptor (NK3r) in the rat gastrointestinal tract has been studied by using a polyclonal antiserum against the C-terminal portion (amino acids 388–452) of the rat NK3r. In the oesophagus, immunoreactivity for the NK3r was found on smooth muscle cells of the muscularis mucosae. NK3r immunoreactivity was not present on muscle cells of other regions. Nerve cell bodies immunoreactive for NK3r were seen in the myenteric and submucous plexuses of the small and large intestine, but not in the stomach or oesophagus. Immunoreactivity was largely confined to nerve cell surfaces. The reaction product was on the cell soma and initial parts of axons. Reactivity was not seen on nerve terminals. Immunoreactive nerve cells had Dogiel Type II morphology. Patterns of co-localisation of NK3r and immunoreactivity for other markers were examined in the ileum, to provide a basis from which to deduce the functional identity of NK3r-immunoreactive nerve cells. Most of the NK3r-immunoreactive nerve cells were also immunoreactive for the calcium-binding proteins, calretinin and calbindin, and all were immunoreactive for the NK1 receptor (NK1r). Nerve cells that were immunoreactive for nitric oxide synthase were not immunoreactive for either NK3r or NK1r. The projections of the calbindin and calretinin neurons were determined by nerve lesion studies. Their morphology, projections to the mucosa and other ganglia and immunoreactivity for the calcium-binding proteins suggest that the NK3r-immunoreactive neurons are intrinsic sensory neurons.

Pilot study using transcutaneous electrical stimulation (interferential current) to treat chronic treatment-resistant constipation and soiling in children.

Background:  Chronic constipation in children may have organic or behavioral causes. The purpose of the present study was to investigate the effect of treatment with transcutaneous electrical stimulation (using interferential current) in children with chronic treatment‐resistant constipation with proven organic disorders.

Distribution of neurokinin-2 receptors in the guinea-pig gastrointestinal tract

Abstract.The distribution of neurokinin-2 (NK2) tachykinin receptors was investigated by immunohistochemistry in the guinea-pig oesophagus, stomach, small and large intestine. Receptor immunoreactivity occurred at the surfaces of smooth muscle cells throughout the digestive tract. Nerve fibre varicosities in enteric ganglia were also immunoreactive. In myenteric ganglia, these varicosities were most numerous in the ileum, frequent, but less dense, in the proximal colon and caecum, rare in the distal colon, extremely infrequent in the rectum and duodenum, and absent from the stomach and oesophagus. Reactive varicosities were rare in the submucous ganglia. Reactive nerve fibres in the mucosa were only found in the caecum and proximal colon. Strong NK2 receptor immunoreactivity was also found on the surfaces of enterocytes at the bases of mucosal glands in the proximal colon. Receptors were not detectable on the surfaces of nerve cells or on non-terminal axons. Reactivity did not occur on nerve fibres innervating the muscle. Denervation studies showed that the immunoreactive varicosities in the myenteric plexus of the ileum were the terminals of descending interneurons. Immunoreactivity for nitric oxide synthase was colocalised with NK2 receptor (NK-R) immunoreactivity in about 70% of the myenteric varicosities in the small intestine. Bombesin immunoreactivity occurred in about 30% of NK2-R immunoreactive varicosities in the small intestine.

Choline acetyltransferase immunoreactivity of putative intrinsic primary afferent neurons in the rat ileum

Abstract The colocalisation of choline acetyltransferase (ChAT) with markers of putative intrinsic primary afferent neurons was determined in whole-mount preparations of the myenteric and submucosal plexuses of the rat ileum. In the myenteric plexus, prepared for the simultaneous localisation of ChAT and nitric oxide synthase (NOS), all nerve cells were immunoreactive (IR) for ChAT or NOS, but seldom for both; only 1.6±1.8% of ChAT-IR neurons displayed NOS-IR and, conversely, 2.8±3.3% of NOS-IR neurons were ChAT-IR. In preparations double labelled for NOS-IR and the general nerve cell marker, neuron-specific enolase, 24% of all nerve cells were immunoreactive for NOS, indicating that about 75% of all nerve cells have ChAT-IR. All putative intrinsic primary afferent neurons in the myenteric plexus, identified by immunoreactivity for the neurokinin 1 (NK1) receptor and the neurokinin 3 (NK3) receptor, were ChAT-IR. Conversely, of the ChAT-IR nerve cells, about 45% were putative intrinsic primary afferent neurons (this represents 34% of all nerve cells). The cell bodies of putative intrinsic primary afferent neurons had Dogiel type II morphology and were also immunoreactive for calbindin. All, or nearly all, nerve cells in the submucosal plexus were immunoreactive for ChAT. About 46% of all submucosal nerve cells were immunoreactive for both neuropeptide Y (NPY) and calbindin; 91.8±10.5% of NPY/calbindin cells were also ChAT-IR and 99.1±0.7% were NK3 receptor-IR. Of the nerve cells with immunoreactivity for ChAT, 44.3±3.8% were NPY-IR, indicating that about 55% of submucosal nerve cells had ChAT but not NPY-IR. Only small proportions of the ChAT-IR, non-NPY, nerve cells had NK3 receptor or calbindin-IR. It is concluded that about 45% of submucosal nerve cells are ChAT/calbindin/NPY/VIP/NK3 receptor-IR and are likely to be secretomotor neurons. Most of the remaining submucosal nerve cells are immunoreactive for ChAT, but their functions were not deduced. They may include the cell bodies of intrinsic primary afferent neurons.

Decreased colonic transit time after transcutaneous interferential electrical stimulation in children with slow transit constipation

PURPOSE Idiopathic slow transit constipation (STC) describes a clinical syndrome characterised by intractable constipation. It is diagnosed by demonstrating delayed colonic transit on nuclear transit studies (NTS). A possible new treatment is interferential therapy (IFT), which is a form of electrical stimulation that involves the transcutaneous application of electrical current. This study aimed to ascertain the effect of IFT on colonic transit time. METHODS Children with STC diagnosed by NTS were randomised to receive either 12 real or placebo IFT sessions for a 4-week period. After a 2-month break, they all received 12 real IFT sessions-again for a 4-week period. A NTS was repeated 6 to 8 weeks after cessation of each treatment period where able. Geometric centres (GCs) of activity were calculated for all studies at 6, 24, 30, and 48 hours. Pretreatment and posttreatment GCs were compared by statistical parametric analysis (paired t test). RESULTS Thirty-one pretreatment, 22 postreal IFT, and 8 postplacebo IFT studies were identified in 26 children (mean age, 12.7 years; 16 male). Colonic transit was significantly faster in children given real treatment when compared to their pretreatment NTS at 24 (mean CG, 2.39 vs 3.04; P < or = .0001), 30 (mean GC, 2.79 vs 3.47; P = .0039), and 48 (mean GC, 3.34 vs 4.32; P = .0001) hours. By contrast, those children who received placebo IFT had no significant change in colonic transit. CONCLUSIONS Transcutaneous electrical stimulation with interferential therapy can significantly speed up colonic transit in children with slow transit constipation.

Quality of life in children with slow transit constipation

BACKGROUND Slow transit constipation (STC) causes intractable symptoms not readily responsive to laxatives, diet, or life-style changes. Children with STC have irregular bowel motions associated with colicky abdominal pain and frequent uncontrollable soiling. This study assessed the physical and psychosocial quality of life (QOL) in children with long-standing (> or =2 years) STC vs healthy controls. METHODS Children (aged 8-18) were recruited from gastrointestinal and surgical clinics and a Scout Jamboree. After informed consent was obtained, the questionnaire (Pediatric Quality of Life Inventory) was administered. This consists of parallel child and parent self-report scales encompassing physical functioning, emotional functioning, social functioning, and school functioning. Higher scores indicate better QOL. P value less than .05 was considered statistically significant. RESULTS In 51 children with STC (mean, 11.5 years; male/female, 2:1) and 79 controls (mean, 12.1 years; male/female, 1.9:1), Pediatric Quality of Life Inventory QOL score was significantly lower in the STC group (72.90 vs 85.99; P < .0001). In addition, parents of children with STC reported a significantly lower QOL score than their child compared with the childs own report (64.43 vs 72.90; P = .0034). Parents of controls did not (84.25 vs 85.99; P = .12). CONCLUSIONS Slow transit constipation is a debilitating condition affecting both physical and emotional functioning in children. Parental perception of QOL is significantly worse, highlighting the considerable family impact of constipation and uncontrollable soiling.

Glycosylphosphatidylinositol biosynthetic enzymes are localized to a stable tubular subcompartment of the endoplasmic reticulum in Leishmania mexicana

Glycosylphosphatidylinositols (GPI) are essential components in the plasma membrane of the protozoan parasite Leishmania mexicana, both as membrane anchors for the major surface macromolecules and as the sole class of free glycolipids. We provide evidence that L.mexicana dolichol‐phosphate‐mannose synthase (DPMS), a key enzyme in GPI biosynthesis, is localized to a distinct tubular subdomain of the endoplasmic reticulum (ER), based on the localization of a green fluorescent protein (GFP)–DPMS chimera and subcellular fractionation experiments. This tubular membrane (termed the DPMS tubule) is also enriched in other enzymes involved in GPI biosynthesis, can be specifically stained with the fluorescent lipid, BODIPY‐C5‐ceramide, and appears to be connected to specific subpellicular microtubules that underlie the plasma membrane. Perturbation of microtubules and DPMS tubule structure in vivo results in the selective accumulation of GPI anchor precursors, but not free GPIs. The DPMS tubule is closely associated morphologically with the single Golgi apparatus in non‐dividing and dividing cells, appears to exclude luminal ER resident proteins and is labeled, together with the Golgi apparatus, with another GFP chimera containing the heterologous human Golgi marker β1,2‐N‐acetylglucosaminyltransferase‐I. The possibility that the DPMS‐tubule is a stable transitional ER is discussed.

MIGRATION OF ENTERIC NEURAL CREST CELLS IN RELATION TO GROWTH OF THE GUT IN AVIAN EMBRYOS

Neural crest cell migration in the gut and the growth of the mid- and hindgut of avian embryos was investigated by a combination of whole-mount immunofluorescence of the HNK-1 neural crest marker epitope, chorioallantoic membrane grafting and morphometry. HNK-1-labelled cells advanced rostrocaudally in the gut of quail embryos (to the duodenum by stage HH 21, to the umbilicus by HH 25, to the ceca by HH 27, to the cloaca by HH 33). The timetable in chick embryos appeared to be slightly slower, but neural cells were obscured by background fluorescence in this species. More rostral regions of the gut commenced rapid growth earlier than more caudal regions (preumbilical small intestine after HH 26, postumbilical small intestine after HH 27 and colorectum after HH 28), and the small intestine and ceca grew most rapidly in length while the colorectum grew most rapidly in diameter. The rates of growth of the gut were low prior to the stage when HNK-1-labelled cells normally arrive in the small intestine, ceca and rostral colorectum, but increased dramatically after arrival. In the caudal colorectum rapid growth had commenced at the time of arrival of these cells. These data are consistent with the idea that a delay in arrival of vagal neural crest cells at any point in the intestine could jeopardize the ability of the cells to fully populate the remainder of the gut, due to the normal growth spurt causing the migration end-point to recede faster than the rate of neural crest cell migration. Thus, a mismatch in timing of neural crest cell migration and gut growth could play a role in the etiology of some forms of Hirschsprungs disease.