Tim Robson

Neurochemical coding in the small intestine of patients with Crohn's disease.

BACKGROUND: There have been conflicting results regarding the effect of Crohns disease on the neurochemical composition of the enteric nervous system. AIMS: To examine the effect of Crohns disease on the neurochemical composition of enteric nerve fibres and cell bodies using whole mount preparations of human ileum. METHODS: Whole wall ileum from seven normal subjects and nine patients with Crohns disease was used to investigate the neurochemical composition of neurones and nerve fibres in the myenteric plexus, circular muscle, and serosa layer of ileum using immunohistochemical techniques. RESULTS: Increased tyrosine hydroxylase, 5-hydroxytryptamine, and neuropeptide Y immunoreactivity was exclusively seen in the myenteric plexus. There was increased neurofilament immunoreactivity in the myenteric plexus and nerve fibres of the circular muscle layer, and thick bundles of immunoreactive nerve fibres in the serosa layer. Increased vasoactive intestinal polypeptide, nitric oxide synthase, and pituitary adenylate cyclase activating peptide immunoreactivity was seen in the myenteric plexus and nerve fibres of the circular muscle layer, and aggregates of inflammatory cells in the serosa layer of the afflicted segment of Crohns ileum. In addition, there was a chaotic display of nerve fibres containing some of the neuroactive substances with a high frequency of enlarged varicosities in the myenteric ganglia and/or nerve fibres of the circular muscle layer of Crohns ileum. CONCLUSION: Results show quantitative as well as qualitative changes in the neurochemical composition of enteric nerve fibres and nerve cell bodies of Crohns ileum. These changes and the presence of nitric oxide synthase and peptides immunoreactive inflammatory cells in the serosa layer suggest that nerve-immune interactions may have a significant role in the process of the inflammatory changes seen in Crohns ileitis.

Localisation of P2X5 and P2X7 receptors by immunohistochemistry in rat stratified squamous epithelia.

Abstract In order to investigate whether purinoceptors are involved in the physiological renewal and regeneration of epithelia, we used immunohistochemical techniques on fresh frozen sections of various stratified squamous epithelial tissues (cornea, tongue, soft palate, oesophagus, vagina and footpad) of the rat and specific polyclonal antibodies to unique peptide sequences of P2X1–7 receptors. Only two of the antibodies, anti-P2X5 and anti-P2X7, reacted with epithelial structures. P2X5 immunoreactivity was mainly associated with the membranes of the proliferating and differentiating cell layers (spinous and granular layer) in both keratinised and non-keratinised epithelia and growing hair follicles. In contrast, P2X7 immunoreactivity was clearly associated with the keratinisation process, the staining being most intense in the upper keratinised and the exfoliated layers. These findings suggest, for the first time, that P2X5 and P2X7 receptors play an important role in the physiological turnover of continuously regenerating cells, and further, raise the possibility that they represent novel targets for the development of pharmacological tools of potential benefit for diseases of epithelial dysfunction.

P2X receptors in the rat duodenal villus

Abstract Immunohistochemical techniques were performed on freshly frozen sections of the duodenum of the rat using specific polyclonal antibodies to unique peptide sequences of P2X1–7 receptors. Of the antibodies to the seven known P2X receptor subtypes that mediate extracellular signalling by nucleotides, three reacted with discrete structures in the duodenal villus of the rat. Anti-P2X1 reacted with the capillary plexus in the intestinal villus, which did not extend to the crypt region, suggesting that nucleotides may be involved in the uptake and transport of metabolites. Anti-P2X5 immunostained the membranes of the narrow ”stem” of villus goblet cells, where the nucleus and cell organelles reside, possibly influencing synthesis and release of mucins. P2X7 receptor immunoreactivity was only seen in the membranes of enterocytes and goblet cells at the tip of the villus, where cells are exfoliated into the lumen, consistent with earlier findings that P2X7 is involved in apoptotic events. Thus, in complex structures such as the intestinal villus, purinoceptors appear to participate in several and diverse signalling functions.

Expression of two ATP-gated ion channels, P2X5 and P2X6, in developing chick skeletal muscle

Physiological and pharmacological studies have shown that ATP has potent effects on developing chick skeletal muscle. These effects have previously been shown to be developmentally regulated, and the responses were characteristic of activation of the P2X ligand‐gated ion‐channel family of ATP receptors. Here, using immunohistochemistry, we describe the expression patterns of two members of the P2X receptor family, P2X5 and P2X6, during development of skeletal muscle in the chick embryo. These receptors were first expressed at early stages of skeletal muscle development, and expression disappeared immediately before the stage at which fusion of myoblasts to form myotubes occurs. P2X5 was also demonstrated in nerves supplying developing skeletal muscle, in some dorsal root ganglion cells, and in dorsal and ventral spinal cord. No expression of the other five members of the P2X family were demonstrated in developing skeletal muscle. Dev Dyn 1999;216:442–449. ©1999 Wiley‐Liss, Inc.

P2X and P2Y purinoceptor expression in pancreas from streptozotocin-diabetic rats

The expression of the nucleotide receptors P2X1, P2X2, P2X7, P2Y1, P2Y2 and P2Y4, in the pancreas of the streptozotocin-induced diabetic rat was investigated using immunohistochemistry. In diabetic animals, P2X7 receptor expression, normally located in the outer periphery of the islet, was increased and located inside the islet. Double-labelling experiments, using antibodies raised against insulin, somatostatin and glucagon, showed, for the first time, an increase in immunostaining for P2X7 receptors on islet glucagon-containing alpha cells (which had migrated to the interior), while no P2X7 receptors were found in beta and delta cells. P2Y1 receptors were present in intra-islet capillaries, while P2Y4 receptors were found on both alpha and beta cells. P2Y1 and P2Y2 receptor expression was also found in pancreatic duct cells and P2X1, P2X2, P2Y1 and P2Y2 receptors were identified in small blood vessels.

Changes in expression of P2 receptors in rat and mouse pancreas during development and ageing

In view of the evidence for a role for extracellular ATP in both pancreatic endocrine and exocrine functions, we have investigated the expression of P2X and P2Y receptors in this tissue in neonate and aged rat and mouse. Using immunohistochemistry it was shown that P2X1, P2X4, P2X7, P2Y1 and P2Y2 receptors were present in different regions of the rat and mouse pancreas; P2X3 and P2X6 receptors were not found, and P2X5 immunolabelling was only found in some nerves. The pancreatic vasculature of both rat and mouse expressed P2X1, P2X2, P2Y1 and P2Y2 receptors in the smooth muscle. P2X1 and P2X4 receptors were absent in the islets of the neonate pancreas, but were progressively upregulated with age after birth. In contrast, the greatest expression of P2Y1 in cells from the duct system was in neonate pancreas, while there was no P2Y1 expression in aged rat pancreas. P2X7 receptors had a consistent pattern of distribution in all of the groups examined, being located in the outer periphery of the islet. Using antibodies raised against insulin, somatostatin and glucagon, double-labelling immunofluorescence was used to identify P2X7-positive cells in different islet of Langerhans cell populations. Our results demonstrated a clear immunoreaction to P2X7 receptors in islet α cells, while no P2X7 was expressed in β and δ cells. The significance of the differential expression of P2 receptors in the pancreas during development and ageing, and a possible role for the proliferation and death of the islet cell population are discussed.

An Increase of Neuropeptide Y But Not Nitric Oxide Synthase-Immunoreactive Nerves in the Bladder Neck from Male Patients with Bladder Neck Dyssynergia

PURPOSE To determine the distribution of neuropeptides in male patients with bladder neck dyssynergia and benign prostatic hyperplasia. MATERIALS AND METHODS Bladder neck tissue, obtained from male patients with bladder neck dyssynergia (BND) and control patients with benign prostatic hyperplasia (BPH), was studied immunohistochemically for protein gene product 9.5 (a general neuronal marker), vasoactive intestinal polypeptide, neuropeptide Y, calcitonin gene-related peptide, substance P, growth associated protein 43 and nitric oxide synthase. RESULTS In the bladder neck from control patients, the greatest density of nerves contained protein gene product 9.5, followed in decreasing order by neuropeptide Y; vasoactive intestinal polypeptide; calcitonin gene-related peptide; nitric oxide synthase; substance P and serotonin. The neuropeptides were found in the smooth muscle and were also associated with blood vessels. In patients with BND there was a statistically significant increase (P < 0.05) in the density of protein gene product 9.5- and neuropeptide Y-immunoreactive nerves in the smooth muscle and the base of the mucosa but not in blood vessels in the bladder neck, while the density of the other neuropeptides studied, nitric oxide synthase and serotonin did not significantly change from that of control tissue. Growth associated protein 43-immunoreactive nerves were absent from the bladder neck from both groups of patients. CONCLUSION It is suggested that the increase in density of protein gene product 9.5- and neuropeptide Y-immunoreactive nerves, part of the sympathetic contractile system of the bladder neck, may exacerbate bladder outlet obstruction and thus play a role in the pathogenesis of BND.

Expression of Nucleotide P2X Receptor Subtypes during Spermatogenesis in the Adult Rat Testis

The expression of ATP-gated ion channels (P2X receptors) was investigated in testes of adult rats by immunohistochemistry and Western blotting with antibodies against all seven P2X receptor subtypes. Immunoreactive cells were identified and monitored during germ cell maturation. Results of immunohistochemical and Western blotting experiments showed the expression of P2X₁, P2X₂, P2X₃, P2X₅ and P2X₇ receptors, while P2X₄ and P2X₆ receptors were absent from the testis. Blood vessels displayed immunostaining for P2X₁ and P2X₂ receptors; the P2X₁ receptors were present exclusively in blood vessels. P2X₂, P2X₃ and P2X₅ receptors were found to be expressed differentially in the various germ cell types throughout the different stages of the cycle of the seminiferous epithelium; P2X₂ and P2X₃ receptors were always observed together in the same cell types and at the same stages. Sertoli cells also showed differential staining for P2X₂ and P2X₃ receptors during the cycle of the seminiferous epithelium, whereas P2X₇ receptor expression was present throughout all stages. No immunostaining for P2X receptors was detected on Leydig cells. The possible roles of purinergic signalling in the control of germ cell maturation are discussed. In particular, it is suggested that purinergic signalling may play a role in controlling the maturation of germ cell subsets of different developmental ages that exist alongside each other in the adult testis.

Changes in expression of P2X7 receptors in NOD mouse pancreas during the development of diabetes.

This study examined the expression of P2X7 receptors in pancreatic islets of the non-obese diabetic (NOD) mouse model of human autoimmune insulin-dependent diabetes mellitus, to determine whether they are involved in islet cell destruction during early- and late-developing diabetes. Pancreatic cells containing glucagon (α-cells), insulin (β-cells) and somatostatin (δ-cells) were co-localized with P2X7 receptors. We examined P2X7 receptor expression in normal and diabetic spleens using flow cytometry. In non-diabetic NOD controls, P2X7 receptors were expressed in glucagon-containing cells at the periphery of islets, being consistent with previous studies. In early NOD diabetes (12 weeks), there was migration of peripheral P2X7 receptor positive, glucagon-containing cells into the center of islets. In late NOD diabetes (34 weeks), P2X7 receptor- and glucagon-stained α-cells were gone from islets. Migration of macrophages and dendritic cells into islets took place, but they lacked P2X7 immunoreactivity. There was no significant difference in the percentage of splenic macrophages stained for P2X7 receptors from control and diabetic spleens. In conclusion, in the development of early to late diabetes, there is a down-regulation of P2X7 receptors on islet cells and a loss of α- and β-cell populations. P2X7 receptor signalling might be involved in α-cell clearance from late diabetic islets.

Radiation-induced changes in neuropeptides in the rat urinary bladder

PURPOSE To determine whether there was a change in innervation in the rat urinary bladder following x-ray irradiation. MATERIALS AND METHODS The urinary bladders were obtained from rats irradiated 6 months previously with single doses of 15 Gy and 25 Gy x-radiation, and from nonirradiated (control) animals. They were examined immunohistochemically to localize neuropeptide Y (NPY), substance P (SP), vasoactive intestinal polypeptide (VIP), calcitonin gene-related peptide (CGRP), met-enkephalin (m-ENK), leu-enkephalin (l-ENK), somatostatin (SOM) and the enzyme, tyrosine-hydroxylase (TH). Computer assisted image analysis was used to assess the density of immunoreactive nerve fibres. RESULTS The greatest density of nerves observed in the bladder from control animals contained NPY, followed (in decreasing order) by CGRP, VIP, SP and TH. The nerves appeared to run predominantly along the longitudinal axis of the circular and longitudinal muscle fibres. SP-, CGRP-, TH- and occasionally VIP-immunoreactive nerves were observed in the lamina propria, at the base of the urothelium. Perivascular nerves containing neuropeptides and TH were observed throughout the bladder wall. There was an absence of m-ENK-, l-ENK- and SOM-immunoreactive nerves in the control and irradiated rat urinary bladders. In the rat urinary bladder irradiated with 25 Gy x-radiation, there was a significant increase (P < 0.05) in the density of NPY-, TH- and SP- but not CGRP- and VIP-immunoreactive nerves. There were regional differences within the bladder, that is, there was an increase in VIP-, CGRP- and SP-immunoreactive nerves around and within the urothelium. NPY-immunoreactive nerves were seen in the connective tissue and elastic fibres of the lamina propria for the first time. An increase in the density or fluorescence intensity of perivascular TH- but not neuropeptide-containing nerves was observed. CONCLUSIONS The increase in the density of NPY-, SP- and TH-immunoreactive nerves in the irradiated bladders may be due to axonal sprouting which contributes to the symptoms of radiation injury.