Neurotrophin signal transduction by the Trk receptor

The initial event in the neuronal differentiation of PC12 cells is the binding of the neurotrophin nerve growth factor (NGF) to the Trk receptor. This interaction stimulates the intrinsic tyrosine kinase activity of TRk, initiating a signalling cascade involving the phosphorylation of intracellular proteins on tyrosine, serine, and threonine residues. These signals are then in turn propagated to other messengers, ultimately leading to differentiation, neurotrophin-dependent survival and the loss of proliferative capacity. To transmit NGF signals, NGF-activated Trk rapidly associated with the cytoplasmic proteins, SHC, PI-3 kinase, and PLC-γ1. These proteins are involved in stimulating the formation of various second messenger molecules and activating the Ras signal transduction pathway. Studies with Trk mutants indicate that the acivation of the Ras pathway is necessary for complete differentiation of PC12-derived cells and for the maintenance of the differentiated phenotype. Trk also induces the tyrosine phosphorylation of SNT, a specific target of neurotrophic factor activity in neuronal cells. This review will discuss the potential roles of Trk and the proteins of the Trk signalling pathways in NGF function, and summarize our attempts to understand the mechanisms used by Trk to generate dthe many phenotypic responses of PC12 cells to NGF. 1994 John Wiley & Sons, Inc.     

Bombesin-like immunoreactivity in human gastrointestinal tract

In the present study the distribution and molecular characteristics of bombesin-like immunoreactivity (BLI) were studied in acid extracts of human gastrointestinal tract. The highest levels were found in the fundus, antrum, pylorus and pancreas with lower levels in the duodenum, jejunum, terminal ileum and colon. BLI was also detected in both the muscle and mucosal layers of the antrum and colon. Sephadex G-50 gel chromatography under acid dissociating conditions revealed two peaks of immunoreactivity, one in the position of synthetic porcine gastrin releasing peptide (GRP) and the second eluting with synthetic amphibian bombesin. Variations in the proportions of the two molecular forms were seen in different regions of the gut. In the stomach and pancreas greater than 70% of the BLI eluted with the GRP marker while in pylorus, jejunum and terminal ileum only 20% was present in this form. Reverse-phase ODS silica HPLC of the major antral BLI peak, utilising a methanol/trifluoroacetic acid gradient indicated that this peptide was similar to porcine GRP. We have therefore (1) demonstrated the presence and heterogeneity of bombesin-like immunoreactivity throughout the human gastrointestinal tract and (2) shown for the first time that a proportion of this BLI closely resembles porcine GRP.     

Distribution of substance p-like immunoreactivity in nerves of the gastrointestinal tract of the frog Rana esculenta L.

Summary Substance P-like immunoreactivity in the alimentary canal of the frogRana esculenta L. was studied by means of the indirect immunoperoxidase method. In all segments of the gastrointestinal tract, immunoreactivity was revealed in both the myenteric and the submucosa plexus. Stained nerve cells were observed in the myenteric plexus but not in the submucous plexus. The proximal part of the oesophagus and hindgut were free of immunoreactive perkarya. The stained nerve cells were of the Dogiel type I category in the foregut, and type II in the midgut. Both the musculature and gastrointestinal glands were supplied with immune-positive fibres. These results indicate that substance P may play similar roles in the frog gut, as described previously in mammals and fish.     

Trk neurotrophin receptor-like proteins in the teleost Dicentrarchus labrax

In recent years, data have accumulated suggesting that the role of neurotrophins and Trk receptors may not be limited to the nervous system, and the presence of these substances has been detected in a variety of vertebrate and invertebrate non-nervous tissues. This study was designed to map the expression of immunoreactivity (IR) for Trk-like proteins in alevins of the teleost Dicentrarchus labrax, with particular emphasis on non-nervous structures. We used antibodies against specific epitopes of the intracellular domain of these proteins, a region that is highly conserved in phylogeny. Trk-like IR was seen in segregate cell populations of the nervous system, and non-nervous tissues. In the central nervous system TrkA-like and TrkC-like IR was abundant, whereas TrkB-like IR was restricted to a low number of brain areas. Expression of Trk-like protein IR was observed in the peripheral nervous system and sensory organs, with the exception of the lateral line organ. Outside the nervous system, TrkA-like IR was mainly found in different epithelia, TrkB-like IR in the endocrine and digestive system, and TrkC-like IR in the cardiovascular and immune systems. The gills showed IR for all three Trk-like proteins, whereas they were absent from the gonads. Furthermore, scattered cells positive for Trk-like proteins were found in most of the investigated tissues. The distribution of Trk-like IR in this teleost is compared with that of mammals and birds, which it often paralleled, and the possible role of neurotrophins and Trk-like receptor proteins in different non-neuronal tissues is discussed.     

Distribution of ACTH-like immunoreactivity in rat brain and gastrointestinal tract

Summary Immunocytochemistry reveals ACTH-like immunoreactivity to reside not only in the pituitary but also in central nerves and in antral gastrin cells. In all probability, the central nerves store a peptide identical with or closely resembling true ACTH. Their pattern of distribution is, in some regions, similar to that of enkephalin-immunoreactive nerves. The antiserum demonstrating ACTH-like immunoreactivity in central nerves and in antral gastrin cells is directed towards the COOH-terminal part of the hormone. A peptide corresponding to this part, the corticotrophin-like intermediate peptide (CLIP) is manufactured by the pars intermedia of the pituitary. CLIP is devoid of adrenocortical activity but has recently been shown to possess insulin-releasing activity. The occurrence of CLIP-like peptides in antral gastrin cells may indicate a role for such peptides in the gastrointestinal regulation of insulin release. The simultaneous occurrence of enkephalin-like and ACTH-like immunoreactivity in the antral gastrin cells is of particular interest since a large precursor molecule, containing both the enkephalin and the ACTH sequence has recently been identified.     

Somatostatin-immunoreactive cells in the gastrointestinal tract of the frog Rana esculenta

The morphology and topographic distribution of somatostatin-immunoreactive cells in the stomach and small intestine of the frog Rana esculenta were studied at the light-microscopic level by the use of the peroxidase-antiperoxidase method. Scattered immunostained cells occurred in all regions of the gastrointestinal tract investigated. In the small intestine, the number of these cells decreased gradually in the oral to anal direction, i.e. from the pyloric (antral) stomach to the entrance into the colon. Most of the immunostained cells possessed thick, short cytoplasmic processes, which did not display a preferential spatial orientation. Other somatostatin-immunoreactive cells, which were exclusively located in the small intestine, gave rise to a single long extension oriented toward the lumen. In both stomach and small intestine, a complete penetration of the epithelial surface by these processes of somatostatin-immunoreactive cells was observed only occasionally. The morphological features of the somatostatin-immunostained cells speak in favor of endocrine, paracrine, and possibly also intraluminal secretory functions of the enteroendocrine somatostatin system in frogs.Fellow of the Alexander von Humboldt Foundation, Bonn, Germany     

Distribution of ACTH-like immunoreactivity in rat brain and gastrointestinal tract.

Immunocytochemistry reveals ACTH-like immunoreactivity to reside not only in the pituitary but also in central nerves and in central nerves and in antral gastrin cells. In all probability, the central nerves store a peptide identical with or closely resembling true ACTH. Their pattern of distribution is, in some regions, similar to that of enkephalin-immunoreactive nerves. The antiserum demonstrating ACTH-like immunoreactivity in central nerves and in antral gastrin cells is directed towards the COOH-terminal part of the hormone. A peptide corresponding to this part, the corticotrophin-like intermediate peptide (CLIP) is manufactured by the pars intermedia of the pituitary. CLIP is devoid of adrenocortical activity, but has recently been shown to possess insulin-releasing activity. The occurrence of CLIP-like peptides in antral gastrin cells may indicate a role for such peptides in the gastrointestinal regulation of insulin release. The simultaneous occurrence of enkephalin-like and ACTH-like immunoreactivity in the antral gastrin cells is of particular interest since a large precursor molecule, containing both the enkephalin and the ACTH sequence has recently been identified.     

Effect of cisapride on the concentrations of beta-endorphinlike immunoreactivity and substance P-like immunoreactivity in the rat gastrointestinal tract

Cisapride is a gastrointestinal prokinetic agent reported to be devoid of direct cholinergic effect from the myenteric plexus of the gut. The effect of cisapride (0.125, 0.5, 2mg/kg, i.p.) on the concentration beta-endorphin and substance P in rat gastrointestinal tract was studied. beta-Endorphinlike immunoreactivity contents were significantly increased in both mucosal and muscular layers of the entire gastrointestinal tract (from gastric body to rectum) of the rats treated with 2 mg/kg of cisapride. beta-Endorphinlike immunoreactivity contents were also increased in a part of the gastrointestinal tract of the rats treated with 0.125 or 0.5 mg/kg of cisapride. Substance P like immunoreactivity contents were significantly decreased in muscular layers of the rectosigmoid colon of the rats treated with 2 mg/kg of cisapride. This study suggests that the prokinetic effects of cisapride may relate to the contents of beta-endorphinlike immunoreactivity and substance P like immunoreactivity in gastrointestinal tract.     

Trk-neurotrophin receptor-like immunoreactivity in the gut of teleost species

Neurotrophins, acting through their high-affinity signal-transducing Trk receptors, are involved in the development, differentiation and maintenance of discrete neuron populations in the higher vertebrates. Furthermore, the presence of Trk receptors in some non-neuronal tissues, including the endocrine cells of the gut, could indicate an involvement of neurotrophins also in these tissues. Recently, neurotrophins and neurotrophin receptor proteins have been identified in the lower vertebrates and invertebrates, whose amino acid sequences are highly homologous with those found in mammals. The present study investigates the occurrence and distribution of Trk-like proteins in the neurons and gut endocrine cells in five species of teleost. Single and double immunolabeling was carried out on fresh and paraffin-embedded tissue using commercially available antibodies against sequences of the intracytoplasmic domain of the mammalian Trk. Western-blot analysis, carried out on samples of stomach and intestine of bass, identified proteins whose estimated molecular masses (140 kDa, 145 kDa and 143–145 kDa) were similar to those reported for full-length TrkA, TrkB and TrkC in the higher vertebrates. TrkA-like immunoreactivity was found in the enteric nervous system plexuses of three fish species. Trk-like immunoreactivity was observed in the endocrine cells as follows: sparse TrkA-like immunoreactive endocrine cells were detected only in the intestine; TrkB-like immunoreactive cells were detected only in the stomach; and TrkC-like immunoreactive cells were found both in the intestine of the carp and in the stomach of the bass, where they also showed TrkB-like immunoreactivity. These findings confirm the occurrence and distribution of Trk-like proteins in teleosts. These proteins are closely related to the Trk neurotrophin receptors of mammals. The functional significance of Trk-like proteins in both neuronal and non-neuronal cells of teleosts is still not clear. Received: 3 August 1998 / Accepted: 2 October 1998     

Peptide hormone-like immunoreactivity in the gastrointestinal tract and endocrine pancreas of eleven teleost species

Summary The distribution of peptide hormone-like immunostaining in the gastrointestinal tract of 11 teleost species was investigated by immunofluorescence.Cells immunoreactive for somatostatin were found in the glandular epithelium of the stomach of four species and in the epithelium of the pyloric appendage of one species. The mid-gut epithelium contained cells reactive with antibodies to glucagon (three species), gastrin (five species), pancreatic polypeptide (five species), and substance P (two species). Cells immunoreactive for met-enkephalin were found in the epithelium of both the mid-gut and the stomach of six species.In six species in which the endocrine pancreas was investigated, insulin-, glucagon-, and somatostatin-like immunoreactivity was observed. Pancreatic polypeptide was definitely localised by immunostaining in cells of the endocrine pancreas of only one out of three species examined.Vasoactive intestinal polypeptide-, neurotensin-, bombesin-, and enkephalin-like immunoreactivity was identified in the gastrointestinal nerve fibres in various species.In view of the considerable species variation found, caution should be exercised in generalising about the peptides present in the gastrointestinal tract of fish.     

CD34 immunoreactivity and interstitial cells of Cajal in the human and mouse gastrointestinal tract

Immunoreactivity for the tyrosine kinase receptor Kit (Kit-ir) is an established marker for the interstitial cells of Cajal (ICC) of the gut. Recently, the presence of CD34 immunoreactivity (CD34-ir) has been reported in Kit-ir ICC around the myenteric plexus in human small intestine. Conversely, we observed that CD34-ir labeled Kit-negative fibroblast-like cells, closely adjacent to, but distinct from, the Kit-ir ICC. The existence of cells expressing both CD34-ir and Kit-ir remains controversial. CD34-ir and Kit-ir were studied by high-resolution confocal microscopy on cryostat sections of human and murine gut as well as murine whole-mounts, using specific antibodies raised to human and murine CD34, respectively. CD34-ir labeled numerous cells in all parts of the gut, in man and in mouse. CD34-ir was consistently observed in Kit-negative cells, distinct from the closely adjacent Kit-ir ICC. Thin processes of both cell types intermingled extensively, often at the limit of resolution for light microscopy. CD34-ir was also observed in Kit-negative mesenchymal cells in the submucosa, in capillaries and in mesothelial cells. CD34-ir is not a marker for Kit-ir ICC in the human and murine gut. No CD34-ir, Kit-ir-expressing cells were encountered. Conversely, CD34-ir cells, closely adjacent to, but distinct from, Kit-ir ICC were consistently identified. The intimate relationship between these cells may offer an alternative explanation for reports of CD34 and Kit co-localization. The ontogeny and function of CD34-ir cells in the gut, as well as the origin of gastrointestinal stromal tumors, remain unclear.     

Distribution of bombesin- and cholecystokinin-like immunoreactivity in rat and dog brain and gastrointestinal tract

Using a specific bombesin radioimmunoassay and an immunoassay for cholecystokinin which sees all C-terminal fractions, the distribution of bombesin-like (BLI) and cholecystokinin-like (CCK-LI) immunoreactivity in the brain and gastrointestinal tract of the rat and dog has been studied. Both peptides are found in the brain and gut but the rat contains more CCK and BLI than the dog; this is particularly noted in the stomach, colon and cerebral cortex whereas the small intestine of both species contains equivalent amounts of peptides. This contrasts with other comparative studies, mainly on nervous system CCK, which find no major distribution differences in man, monkey, pig and rat. This finding suggests that CCK-LI and BLI peptides may have a more predominant role in the rat than in the dog.     

Distribution and characterization of cyclo(His-Pro)-like immunoreactivity in the rat gastrointestinal tract

The distribution of cyclo(His-Pro), thyrotropin-releasing hormone (TRH) and $\text{pyroglutamate aminopeptidase}$ activity was examined in the rat gastrointestinal (GI) tract. Cyclo(His-Pro)-like immunoreactivity was present in the following order of distribution (fmoles/mg protein): caecum > colon = jejunum = ileum > stomach = duodenum = rectum, and was immunologically and chromatographically identical with the authentic cyclo(His-Pro). Cyclo(His-Pro) concentrations showed significantly positive correlations with TRH concentrations, but not with $\text{pyroglutamate aminopeptidase}$ activities, in most tissues of the GI tract, suggesting a precursor role of TRH for gut cyclo(His-Pro). These data suggest that cyclo(His-Pro) may be involved in regulating rat GI functions.     

Localisation of somatostatin- and gastrin-like immunoreactivity in the gastrointestinal tract of Ciona intestinalis L.

Summary Somatostatin- and gastrin-like immunoreactivity has been found by immunofluorescence in cells of the stomach and intestinal epithelia of Ciona intestinalis L. The cells containing the peptide immunoreactive to mammalian anti-gastrin can be restained with the Grimelius' technique for argyrophilia.     

In situ visualization of o-phthalate esters in gastrointestinal tract of the frog Rana esculenta

The regional distribution and relative occurrence of phthalates were studied immunohistochemically by confocal laser scanning microscopy in the alimentary tract of the green frog, Rana esculenta, using an antibody against o-phthalate esters. Many positive sites indicating the basal presence of phthalate esters were identified. The immunoreactive cells were located in the gastric glands of the stomach and in the intestinal epithelium regions with variable frequencies. The regional distribution of phathalate-accumulating cells resembled that of fish and demonstrated that these endocrine disruptors not only enter via the alimentary canal, but also bioaccumulate inside cells specialized in secretion as well as absorption functions.     

Immunocytochemical localization of neuropeptide Y-like immunoreactivity in adrenergic and non-adrenergic neurons of the rat gastrointestinal tract

The immunocytochemical location of neuropeptide Y (NPY)-like immunoreactivity (LI) within the neuronal structures of the rat gastrointestinal (GI) tract was investigated with the indirect immunofluorescence method. NPY immunoreactive neurons were found throughout all regions of the GI tract with the largest number in the duodenum. NPY immunoreactive perikarya were mainly located in the submucosal ganglia. NPY labeled processes were extensively seen in the submucosal and myenteric plexuses, smooth muscles, muscularis mucosa, mucosa and surrounding blood vessels. Following 6-hydroxydopamine (6-OHDA) treatment, NPY immunoreactive nerve fibers around blood vessels disappeared completely and the reactive fibers in other regions were reduced in number. NPY immunoreactive nerve cell bodies in the ganglionic plexuses, however, were not affected by 6-OHDA treatment. Serial sections of the coeliac ganglion showed that NPY-LI was present in cell bodies which also displayed tyrosine hydroxylase (TH) immunoreactivity. Our results suggest that NPY is abundantly contained in both adrenergic and non-adrenergic neurons of the gut and may play an important role in the regulation of the GI tract.     

Neuronal nitric oxide synthase immunoreactivity in the respiratory tract of the frog,Rana temporaria

Summary Physiological and histochemical studies have recently supported the notion that nitric oxide (NO) is the transduction signal responsible for the non-adrenergic, non-cholinergic relaxation of the vasculature as well as the airways of the mammalian lung. We report the presence of immunoreactivity to NO synthase (NOS) in nerve cell bodies and nerve fibres in the neural plexus of the buccal cavity and lungs of the frog, Rana temporaria, using the indirect immunocytochemical technique of avidin-biotin and the NADPH-diaphorase technique. The neural ganglia located next to the muscle layer and within the connective tissue of the buccal cavity were partially immunoreactive for NOS. In the lungs, NOS immunoreactivity occurred in nerve cell bodies, as well as in both myelinated and unmyelinated nerve fibres. Fine nerve fibres immunoreactive to NOS were observed within the muscle fibre bundles and next to the respiratory epithelium. Both the presence of NOS immunoreactivity and the positive histochemical reaction for NADPH-diaphorase in the neural plexus of amphibian respiratory tract suggests a broad evolutionary role for NO as a peripheral neurotransmitter.