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Keywords:

  • constipation;
  • Hirschsprung’s;
  • megacolon;
  • neurotrophins;
  • pseudo-obstruction;
  • tyrosine kinase

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. ONTOGENY OF THE ENTERIC NERVOUS SYSTEM
  5. THE ROLE OF ENDOTHELINS
  6. NUCLEAR TRANSCRIPTION FACTORS
  7. CONGENITAL NEUROPATHIC MOTILITY DISORDERS
  8. INTERSTITIAL CELLS OF CAJAL IN MALDEVELOPMENT AND ACQUIRED DISEASES OF THE COLON
  9. NEUROTROPHIC FACTORS AND THEIR POTENTIAL ROLE IN THERAPY
  10. CONCLUSION
  11. ACKNOWLEDGMENTS
  12. References

The goals of this review are to summarize some of the novel observations on the genetic and molecular basis of enteric nervous system disorders, with particular emphasis on the relevance of these observations to the practicising neurogastroenterologist. In the last two decades, there has been a greater understanding of genetic loci involved in congenital forms of pseudo-obstruction and Hirschsprung’s disease; and the contribution of endothelins and nuclear transcription factors to the development of the enteric nervous system. In addition, clarification of the molecules involved in the activation of the peristaltic reflex, the disorders of the interstitial cells of Cajal, the clinical manifestations of mitochondrial cytopathies affecting the gut, and the application of neurotrophic factors for disorders of colonic function have impacted on practical management of patients with gut dysmotility.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. ONTOGENY OF THE ENTERIC NERVOUS SYSTEM
  5. THE ROLE OF ENDOTHELINS
  6. NUCLEAR TRANSCRIPTION FACTORS
  7. CONGENITAL NEUROPATHIC MOTILITY DISORDERS
  8. INTERSTITIAL CELLS OF CAJAL IN MALDEVELOPMENT AND ACQUIRED DISEASES OF THE COLON
  9. NEUROTROPHIC FACTORS AND THEIR POTENTIAL ROLE IN THERAPY
  10. CONCLUSION
  11. ACKNOWLEDGMENTS
  12. References

The enteric nervous system (ENS) is a vast network of ganglionated plexuses located in the wall of the gastrointestinal (GI) tract.1,2 Although several plexuses are identified anatomically, the most important, from a functional perspective, are the myenteric and submucosal plexuses. In association with the muscle layers, the networks of interstitial cells of Cajal are recognized as the likely pacemakers activating neuromuscular function. The ENS consists of approximately 100 million neurones in higher mammals, and this number is roughly equal to the number of neurones in the spinal cord (Fig. 1). This article reviews the development of ENS, examples of genetic or molecular disorders that manifest as clinical syndromes, and the potential therapeutic application of neurotrophins.

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Figure 1.  Extrinsic and enteric control of gut motility. The enteric nervous system controls stereotypic motor functions such as the migrating motor complex and the peristaltic reflex; enteric control is modulated by the extrinsic parasympathetic and sympathetic nerves, which, respectively, stimulate and inhibit nonsphincteric muscle. ACh, acetylcholine; ATP, adenosine triphosphate; CGRP, calcitonin gene-related peptide; ICC, interstitial cells of Cajal; IPAN, intrinsic primary afferent neurone; NOS, nitric oxide synthase; PACAP, pituitary adenylate cycle-activating peptide; SubP, substance P; VIP, vasoactive intestinal peptide.

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The enteric nervous system develops in utero by migration of neural crest cells to the developing alimentary canal. Migration of neural crest cells, as well as the sequence of innervation of different levels of the gut, are regulated by specific signalling molecules that include transcription factors (e.g. the mouse achaete-scute homologue-1, or mash-1, a mammalian homologue of a proneural gene in Drosophila that is essential for development of peripheral autonomic nerves and neural tube nerve cells in mammals), neurotrophic factors (e.g. the glial-derived neurotrophic factor, GDNF, and its receptor subunits) and the neuregulin signalling system. These facilitate the growth, differentiation and persistence of the migrating nerve cells once they arrive in the gut. Neuregulins are a large group of structurally related signalling proteins, which are likely to have important roles in the development, maintenance and repair of the nervous system and other selected tissues. Their receptors are the ErbB protein tyrosine kinases, which are important in cell signalling.

Histological and electrophysiological studies3–6 of the intestinal tract have characterized the properties of the neurones and transmitters mediating its functions, including the peristaltic reflex, and the neuroimmune interactions between neurones and inflammatory cells.7 Discussion of extrinsic neural control is beyond the scope of this review. It is important to note however (1) that the extrinsic parasympathetic and sympathetic nerves serve to modulate the preprogrammed functions controlled by the enteric nervous system (Fig. 1); and (2) that the peristaltic reflex8 involves an afferent component mediated by intrinsic primary afferent neurones (IPANS, e.g. [CGRP] gene-related peptide neurones), ascending contraction (e.g. cholinergic and tachykininergic neurones) and descending relaxation (nitrergic, or vasointestinal peptidergic [VIPergic] neurones) systems. The peristaltic reflex is discussed later on.

These vast neural networks involve several molecular mechanisms that may be deranged during development, disturbed after maturation and modulated by medications.

ONTOGENY OF THE ENTERIC NERVOUS SYSTEM

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. ONTOGENY OF THE ENTERIC NERVOUS SYSTEM
  5. THE ROLE OF ENDOTHELINS
  6. NUCLEAR TRANSCRIPTION FACTORS
  7. CONGENITAL NEUROPATHIC MOTILITY DISORDERS
  8. INTERSTITIAL CELLS OF CAJAL IN MALDEVELOPMENT AND ACQUIRED DISEASES OF THE COLON
  9. NEUROTROPHIC FACTORS AND THEIR POTENTIAL ROLE IN THERAPY
  10. CONCLUSION
  11. ACKNOWLEDGMENTS
  12. References

Migration

The ENS cells are derived from precursor cells from three axial levels of the neural crest.9 These include the vagal,10 rostral-truncal,11 and lumbo-sacral10,12 levels. The enteric neurones mainly arise from the vagal neural crest of the developing hindbrain and colonize the gut by migration in a rostro-caudal direction. Vagal crest cells are not restricted to a particular intestinal region. Some enteric neurones arrive in the hindgut from the lumbosacral level via a caudo-rostral wave of colonization. In rare cases, the migrating cells do not reach the entire gut; usually this affects the terminal portion of the bowel, as in classical forms of Hirschsprung’s disease. Even more rarely, there may be a zonal form of Hirschsprung’s disease (Fig. 2), which is postulated to result from incomplete caudo-rostral migration of neuroblasts during embyonal development.13 The neural crest cells that migrate and colonize the gut become neuroblasts or neuronal support cells, glioblasts. However, differentiation into neurones and glial cells seems not to take place until they have reached their final destinations in the gut. Movement through the gut mesenchyme, survival in the gut and differentiation into mature cells are influenced by contacts of precursor cells with the microenvironment.

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Figure 2.  Zonal adult Hirschsprung’s disease in an adult. Narrowed segment of the sigmoid colon on barium enema and gross pathological examination. Reproduced from Fu et al., Gut 1996; 39: 765–7.

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The microenvironment consists of other cells in the mesenchyme, neural crest-derived cells, and the extracellular matrix. The extracellular matrix components provide directional signals to migrating neural crest cells and, together with neighbouring cells, provide signals for neural crest cell differentiation. For example, the appearance of neural crest cells in the gut is preceded by expression of extracellular matrix molecules,14 and other factors, such as glial derived neurotropic factor (GDNF), ensure survival of committed neuroblasts.15

A subpopulation of sacral neural crest cells appears predetermined to function in the hindgut. They do not require the presence of vagal-derived enteric precursors in order to colonize the hindgut, nor are they capable of dramatically altering their proliferation or differentiation.16 On the other hand, the environment at the sacral level allows neural crest cells from other levels of the axial region of the developing nervous system to enter the mesentery and gut mesenchyme. At least two environmental conditions at the sacral level enhance ventral migration of the sacral neural crest cells. Firstly, sacral neural crest cells take a ventral rather than a medial-to-lateral path through the somites and arrive near the gut mesenchyme many hours earlier than their counterparts at the thoracic level. There is only a narrow window of opportunity to invade the mesenchyme of the mesentery and the gut, therefore, their earlier arrival assures the sacral neural crest cells of gaining access to the gut. Secondly, the gut endoderm is more dorsally situated (i.e. closer to the crest with its migrating cells) at the sacral level than at the thoracic level. As a result, sacral neural crest cells preferentially populate the colo-rectum. In addition, a barrier to migration at the thoracic level prevents neural crest cells at that axial level from migrating to the gut.17 The barrier also prevents lumbo-sacral crest neurones and glial cells from migrating to the nearby small intestinal tissue.18

Defects of the neural crest cells themselves or alterations of the microenvironment of the pathway through which the neural crest cells migrate may result in maldevelopment of the ENS. In humans, this disordered development results in congenital enteric neuromuscular diseases.

Differentiation of neurones

Migrating crest cells are multipotent.19 The gut wall is itself a critical site where terminal differentiation of enteric neurones and glia occurs, and determines what kind of nervous system arises within the bowel.20 Enteric growth factor–receptor combinations influence differentiation. Combinations that enhance differentiation include: glial cell line-derived neurotrophic factor (GFR)-1-Ret, neurotrophin-3 (NT-3)-TrkC, and serotonin (5-HT)-2B. ret is a proto-oncogene that encodes for a tyrosine kinase receptor necessary for the development of the ENS. Because ret is a proto-oncogene, a single ‘activating’ mutation on one allele is sufficient to cause neoplastic transformation. Receptor tyrosine kinases transduce diverse processes, including cell growth, cell differentiation, survival and programmed cell death (apoptosis). A receptor tyrosine kinase is a 28-amino-acid single peptide that has an extracellular domain (rich in cadherin and cysteine), a transmembrane domain and an intracellular domain. Binding of ligands (e.g. GDNF, neurotrophins) to the receptor results in its activation leading to auto-phosphorylation of tyrosine residues and signal transduction. Figure 3 shows examples of mutations in the tyrosine kinase receptor that are associated with specific genetic disorders.

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Figure 3.  Tyrosine kinase receptor: genetic disorders and dysmotility. Tyrosine kinase receptor with examples of mutations associated with specific genetic isorders. ([F]MTC , [familial] medullary carcinoma of the thyroid; MEN,multiple endocrine neoplasia). Drawn from Edery et al., Nature 1994; 367: 378–80, and Romeo et al., Nature 2000; 367: 377–8.

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A qualitatively different effect on enteric nervous system development is shown by the peptide–receptor combination of endothelin-3 and the endothelin B receptor, ET-3/ETB, which prevents the premature differentiation of enteric neurones before colonization of the GI tract has been completed.

The first molecule found to affect the development of enteric neurones and glia was neurotrophin-3 (NT-3).21 Crest-derived cells in the fetal bowel express TrkC, the high-affinity receptor for NT-3. Overexpression of NT-3 in transgenic mice causes an increase in the size of developing ganglia and neurones in the myenteric plexus.20 The ENS, however, is relatively normal in the bowel of mice following the knockout of NT-3,22 suggesting that NT-3 affects the development of only a subset of enteric neurones or glia.19,23

Stimulation by glial cell line-derived neurotrophic factor (GDNF) is absolutely necessary for the survival of the vagal and sacral crest-derived cells that colonize the gut. If either GDNF24,25 or its signalling receptor, Ret,26 are knocked out in developing mice, the gut becomes totally aganglionic in the vagal and sacral domains of the bowel and persists only in the small region of the gut that is colonized by cells from the truncal crest. GDNF potently promotes neuronal development in vitro27,28 and acts as a mitogen in early development,27 greatly expanding the numbers of enteric crest-derived neural precursors. The initial GDNF-dependent crest-derived precursor that colonizes the bowel gives rise to multiple cell lineages that require particular growth or transcription factors. For example, the truncal crest depends on GDNF, not on Ret,11 but it also needs Mash-1. From this GDNF- and Mash-1-dependent lineage, all of the serotonergic neurones (which develop early in ontogeny), and many excitatory and inhibitory motor neurones, will develop. Later, GDNF loses its ability to promote proliferation, and it acts only as a growth-differentiation factor for enteric neurones, not for glia. All enteric neurones that contain CGRP are derived from Mash-1-independent neurones and differentiate late in ontogeny, after the last serotonergic neurone has become postmitotic (i.e. terminally differentiated).

Both crest- and noncrest-derived cells of the enteric mesenchyme also contain GFR-1,27 a peripheral glycosylphospho-inositol-anchored molecule, which binds GDNF and is necessary for the activation of Ret.29 Neural crest cells anchor GFR-1 to their plasma membranes (perhaps in a complex with Ret), where GDNF can bind to it and ensure survival of the crest-derived cells that colonize most of the bowel.20

Enteric serotonergic neurones appear so early that they coexist in primordial enteric ganglia with still-dividing neural precursors. 5-HT may not only be a neurotransmitter; the 5-HT2B receptor in the fetal bowel is regulated, and optimal at specific times, thus, 5-HT strongly promotes development of neurones at specific times and affects the development of late-arising enteric neurones.30

These growth-differentiation factors affect virtually the entire bowel. The peptide ET-3, and its receptor, ETB, play a critical, localized role in ENS development.31,32

THE ROLE OF ENDOTHELINS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. ONTOGENY OF THE ENTERIC NERVOUS SYSTEM
  5. THE ROLE OF ENDOTHELINS
  6. NUCLEAR TRANSCRIPTION FACTORS
  7. CONGENITAL NEUROPATHIC MOTILITY DISORDERS
  8. INTERSTITIAL CELLS OF CAJAL IN MALDEVELOPMENT AND ACQUIRED DISEASES OF THE COLON
  9. NEUROTROPHIC FACTORS AND THEIR POTENTIAL ROLE IN THERAPY
  10. CONCLUSION
  11. ACKNOWLEDGMENTS
  12. References

The endothelins are a family of three peptides, endothelin-1, -2 and -3, which are coded for by distinct, but related, genes. Endothelins act on cells via two G protein-coupled receptors, ETR-A (or ET-A) and ETR-B (or ET-B). In Hirschsprung’s disease, aganglionosis results from defects in the ET-3/ETB receptor.33 As stated above, aganglionosis may also result from disturbances of a different cell signalling system, Ret/GDNF. Mice with targeted disruption of ET-B receptor and ET-331,34 have congenital distal intestinal aganglionosis.

The endothelins are synthesized as much larger proproteins, which are cleaved by an endothelin-converting enzyme (ECE-1) to produce the active 21-amino-acid peptide.35 Of the three endothelins, it appears that ET-3 is most important as it binds to ET-B on vagal neural crest cells, is required for neuroblast colonization of the hindgut and eventual formation of the hindgut ENS.

Mutations of either ET-B or ET-3 have been identified in several naturally occurring animal models of Hirschsprung’s disease, the piebald lethal mouse and the lethal spotted mouse36, respectively. A homozygous substitution/deletion mutation of ET-3 gene has been reported in a patient with Waardenburg–Shah–Hirschsprung syndrome, which is characterized by piebaldism, heterochromia iridis, neural deafness, and congenital megacolon.37

Recent studies have suggested that the role of ET-3/ETB is not to promote but to inhibit enteric neuronal development.27,38 ET-3 enhances the development of smooth muscle, downregulating muscle secretion of laminin, which is a powerful promoter of enteric neuronal development.39 ET-3 may prevent the premature differentiation of neurones, which can result if a brake, ET-3, is missing and an accelerator, laminin-1, is present in excess. Crest-derived cells are motile, migratory and capable of multiplying; neurones are not. Differentiation of neurones must be prevented in order to prevent the depletion of the precursor pool before the entire bowel has been colonized. The ET-3/ETB deficiency-induced premature differentiation of neurones would leave the terminal colon uncolonized and thus aganglionic.

NUCLEAR TRANSCRIPTION FACTORS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. ONTOGENY OF THE ENTERIC NERVOUS SYSTEM
  5. THE ROLE OF ENDOTHELINS
  6. NUCLEAR TRANSCRIPTION FACTORS
  7. CONGENITAL NEUROPATHIC MOTILITY DISORDERS
  8. INTERSTITIAL CELLS OF CAJAL IN MALDEVELOPMENT AND ACQUIRED DISEASES OF THE COLON
  9. NEUROTROPHIC FACTORS AND THEIR POTENTIAL ROLE IN THERAPY
  10. CONCLUSION
  11. ACKNOWLEDGMENTS
  12. References

Nuclear transcription factors are involved in the process of neuronal differentiation. Several families of transcription factors exist and some of these are being evaluated for their role in the development of the enteric nervous system.

All autonomic ganglia fail to form properly and degenerate in mice lacking the homeodomain transcription factor Phox2b, as do the three cranial sensory ganglia that are part of the autonomic reflex circuits. In the primordial enteric nervous system and sympathetic ganglia, Phox2b is needed for the expression of Ret, for maintaining Mash-1 expression and for the switching on of the genes that encode dopamine-beta-hydroxylase and tyrosine hydroxylase. Thus, Phox2b regulates the noradrenergic phenotype in vertebrates,40 as seen in Fig. 4.

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Figure 4.  Absence of sympathetic, parasympathetic and enteric ganglia in Phox homozygous knockout embryo; note the absence of superior cervical ganglion and stomach ENS precursors and apoptosis of nerve cells in primitive oesophagus and sympathetic chain. TUNEL (in situ terminal deoxy-nucleotidyl transferase assay) analysis (c, d, f, g) showing apoptosis in sympathetic chain and oesophagus; wholemount X-gal stain (e, g) on dissectied gut. da, Dorsal aorta; lb, left bronchus; rb, right bronchus; oe, oesophagus; sc, sympathetic chain; st, stomach. Reproduced from Pattyn et al., Nature 1999; 399: 366–370.

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Another example of the role of nuclear transcription factors is demonstrated by the Sox10 mutation in the Dom Hirschsprung mouse model. Sox 10 is a member of the SRY-like HMG box family of transcription factors that are essential for proper peripheral nervous system development.41 This mutation arose spontaneously. In Sox10(Dom)/Sox10(Dom) embryos, apoptosis was increased in sites of early neural crest cell development, before these cells entered the gut. The underlying problem is probably not the enteric microenvironment, because Sox10(Dom)/Sox10(Dom) intestine supports colonization and neuronal differen-tiation by wild-type neural crest cells. Instead, excessive cell death occurs in mutant neural crest cells early in their migratory pathway.42

The rostro-caudal specification of the GI tract is likely to involve a spatial, temporal and combinatorial pattern of expression of homeobox genes, the so-called enteric hox code. These are highly conserved developmental control genes that appear necessary for correct morphogenesis in the embryonal gut.43 Homeobox gene products have a conserved domain of 60 amino acids that binds to specific DNA sequences. There are four clusters of homeobox genes located on four different chromosomes. Each rearranges in linear array in the cluster, and the gene located on the more 3′ side of the cluster is expressed in the more anterior part of the body segments. Hence, these genes are important in segmentation and determination of the anterior–posterior axis in embryos.

A number of transgenic models provide evidence of the importance of homeobox genes in the control of morphogenesis of the gut. The models include the knockout of ENX, causing increased innervation of the hindgut44 and overexpression of hox A4, resulting in megacolon.45ncx is a hox 11-related gene that is expressed in tissues derived from the neural crest, such as adrenals, enteric nerve ganglia, and dorsal root ganglia. Megacolon results from a deficiency of the Ncx/Hox 11L.1 transcription factor (Fig. 5). This disorder is associated with flat and degenerate myenteric ganglia and thinning of the smooth muscle layer in the affected segments of the bowel. At the ultrastructural level, transmission electron microscopy shows degenerate ganglion cells with irregular nuclei and condensed chromatin, consistent with apoptosis, which is observed in both myenteric and submucous neurones. Endothelins are candidate downstream molecules for the developmental control (hox) genes. The role of hox genes in human enteric neuromuscular disease requires further exploration.

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Figure 5.  Megacolon associated with knockout of nuclear transcription factor. Knockout of the Ncx/Hox 1 1 L1 results in megacolon in mice and overexpression of NADPH diaphorase (nitric oxide-containing inhibitory) nerves. WT, wild-type, KO, knockout. Reproduced from Hatano et al., J Clin Invest 1997; 100: 795–801.

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CONGENITAL NEUROPATHIC MOTILITY DISORDERS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. ONTOGENY OF THE ENTERIC NERVOUS SYSTEM
  5. THE ROLE OF ENDOTHELINS
  6. NUCLEAR TRANSCRIPTION FACTORS
  7. CONGENITAL NEUROPATHIC MOTILITY DISORDERS
  8. INTERSTITIAL CELLS OF CAJAL IN MALDEVELOPMENT AND ACQUIRED DISEASES OF THE COLON
  9. NEUROTROPHIC FACTORS AND THEIR POTENTIAL ROLE IN THERAPY
  10. CONCLUSION
  11. ACKNOWLEDGMENTS
  12. References

These disorders may be broadly classified46 as: (1) disorders of colonization by migrating neural crest derived neurones as in Hirschsprung’s disease, related to abnormalities in the ret gene and GDNF, or with disorders of ET-3 and its receptor, ETB; (2) disorders of differentiation of enteric nerves, as in intestinal ganglioneuromatosis related to a specific germline point mutation in ret at codon 918 of exon 16 (M918T) or codon 883 (A883F) in multiple endocrine neoplasia (MEN) 2b syndrome; or (3) disorders of the survival or maintenance of enteric nerves, as in hypoganglionosis and possibly congenital achalasia, which can result from one of several mechanistic derangements: GFRα2/neurturin; GFRα3/artemin; tyrosine kinase C/NT-3, and 5HT2B/5HT.

The next section reviews some of the congenital neuropathic motility disorders. However, it is important to note that genetic defects account for only a minority of patients with motility disorders and that the genetic defects described may only be relevant to a minority of familial cases. Nevertheless, careful review of these basic and clinical insights provides a further understanding of the mechanisms that may become disordered and result in acquired motility disorders.

Hirschsprung’s disease

Hirschsprung’s disease, a relatively common condition affecting 1 in 5000 live births, is characterized by the absence of intestinal nerve plexuses, causing intestinal obstruction in neonates and megacolon in infants and adults. There are germline mutations in genes encoding GDNF/Ret47,48 or ET-3/ETB32,49 in up to 40% of patients. Mutations in Ret and ETB (i.e. the receptors) are much more frequently encountered than mutations in the two ligands. At least two different mechanisms can cause the terminal colon to become aganglionic: firstly, a deficiency of GDNF/Ret that is not so severe as to cause the entire bowel to become aganglionic; and secondly, a deficiency of ET-3/ETB, in which crest-derived cells differentiate prematurely, and precursors cease dividing and migrating before the gut has been entirely colonized, leaving the last segment uncolonized. The mutations associated with Hirschsprung’s are located at different sites along the length of the ret gene. The roles of endothelins and gene mutations in the maldevelopment of the enteric nervous system and their role in Hirschsprung’s disease are discussed below.

There are at least six separate genetic loci involved in the control of four different intracellular mechanisms that result in models of congenital dysmotility (Table 1). These include abnormalities of c-ret, the gene that encodes for the tyrosine kinase receptor; the endothelin B system; Sox10 transcription factor; and c-kit, which is a marker for the interstitial cells of Cajal. Disturbances in these mechanisms result in syndromic dysmotilities such as Hirschsprung’s disease, Waardenburg–Shah syndrome (piebaldism, neural deafness, megacolon) and idiopathic hypertrophic pyloric stenosis. Figure 3 demonstrates some of the mutations in the tyrosine kinase receptor that have been reported in dysmotilities associated with familial or sporadic medullary carcinoma of the thyroid, multiple endocrine neoplasia type 2 A or B, and Hirschsprung’s disease. Table 1 provides a summary of the genetic defects and their phenotypic manifestations in the gut and other tissues.

Table 1.   Genetic models of Hirschsprung’s disease Thumbnail image of

Multiple endocrine neoplasia type 2B syndrome

Multiple endocrine neoplasia syndromes are characterized by the occurrence of tumours in two or more endocrine glands in a single patient or in close relatives. The three syndromes encountered clinically are MEN 2A, MEN 2B and familial medullary carcinoma of the thyroid.

MEN 2B is a serious condition that presents with severe constipation, diarrhoea (when associated with enterocolitis), megacolon or obstruction, often in infancy.50,51 Other external stigmata of MEN 2B are a characteristic facies, ‘blubbery lips’ from mucosal neuromas, marfanoid habitus, medullated corneal nerve fibres and medullary thyroid carcinoma.50,52 The latter develops eventually in almost all patients.

MEN 2B is an autosomal, dominantly inherited disorder affecting one in 30 000 people, but at least half of all patients present with a de novo mutation because only few survive to reproductive age or are disabled by a variety of neurological symptoms impairing reproduction. The precise molecular abnormality is an intracellular tyrosine kinase domain in the ret proto-oncogene, which is located on chromosome 10q11.2 ret encodes a tyrosine kinase receptor that is expressed particularly in neural crest-derived cells including the enteric ganglia. The receptor is expressed in neural crest cell-derived enteric nervous system, adrenal medulla, parathyroid, and the C cells of the thyroid. Function mutations increase susceptibility for endocrine tumours (medullary carcinoma of the thyroid, phaeochromocytoma and parathyroid tumours). A specific germline point mutation (methionine to threomine) in ret in exon 16 at codon 918 (M918T) occurs in 95% of patients; the remainder have a point mutation at codon 883 (A883F).53,54 The M918T mutation is located in the region encoding the substrate recognition pocket of the tyrosine kinase catalytic core. This alters ret substrate specificity and seems to act in a ligand-independent fashion (a gain of function mutation).55–57 Receptor tyrosine kinases with these mutations are not only constitutively activated but also bind to and phosphorylate substrates preferred by nonreceptor (or cytoplasmic) tyrosine kinases. A883F ret has not been directly tested but, given its location, it is thought that kinase specificity may also be altered in this mutant.54

In MEN 2B, intestinal pathology shows transmural intestinal ganglioneuromatosis, that is, massive proliferations of neural tissue (neurones, supporting cells, and nerve fibres) appearing as thickened nerve trunks among mature nerve cells (Fig. 6).

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Figure 6.  Histopathological features of ganglioneuromatosis in MEN 2b; note the extensive ganglioneuromatosis filling the submucosa, and the giant ganglioneuroma of the myenteric plexus (original magnification: a, ×50; b, ×200) Reproduced from Smith et al., Gut 1999; 45: 143–6.

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As surgery is the only curative procedure for medullary carcinoma of the thyroid, Milla et al. recommended that patients who present with a clinical phenotype of Hirschsprung’s disease should have adequate full-thickness biopsies of the bowel at the time of the pullthrough operation, and those with transmural intestinal ganglioneuromatosis should undergo molecular diagnostic testing by ret mutation analysis.58 If germline M918T or A883F mutations are found, a prophylactic thyroidectomy is indicated, and adrenal gland surveillance with abdominal ultrasound scanning, and urinary fractionated catecholamines and metanephrines, or vanillyl-mandelic acid, are indicated, as ˜50% of patients may subsequently develop phaeochromocytoma.58 Mutation-negative members of such families with documented germline mutations can be reassured they do not have the disease.

Mitochondrial DNA and cytopathies

These are diverse diseases characterized by mitochondrial abnormalities in skeletal muscle, which show ‘ragged red fibres’ on a Gomori trichrome stain. While the condition is generally recognized by clinicians as a result of the skeletal myopathy, this metabolic abnormality involves cells in other tissues: central and peripheral nervous system, gut, heart, kidney, liver, thyroid, pancreas, and bone marrow. The enzymes in the respiratory chain of mitochondria are encoded by nuclear and mitochondrial DNA. The disorders are inherited as sporadic, autosomal dominant or recessive, or X-linked; maternal inheritance is more frequent.

Mitochondria are involved in the production of energy (adenosine triphosphate; ATP), the generation of reactive oxygen species, and the initiation of apoptosis through the activation of mitochondrial permeability transition pores.59 Mitochondrial DNA is made up of 2–10 double-stranded circular DNA molecules in the mitochondrial matrix. At least 37 genes have been discovered, some of which encode the respiratory chain enzymes. Transcription of mitochondrial DNA is not closely related to the cell cycle; mitochondrial DNA is more susceptible to mutation than nuclear DNA.

Mitochondrial disorders manifest organ disturbances that reflect the importance of mitochondria to normal function, as in skeletal muscle and the nervous system (Table 2), similarly, metabolic disorders result from deranged cellular mitochondrial functions. A recent publication documents the location for selected mutations in the mitochondrial 16 569 base-pair genome, resulting in pathological disorders and syndromes.59

Table 2.   Classical mitochondrial disorders Thumbnail image of

The mitochondrial disorder affecting the gut is called mitochondrial neurogastro-intestinal encephalomyopathy (MNGIE), but it is also referred to as mitochondrial encephalomyopathy with sensorimotor polyneuropathy opthalmoplegia and pseudo-obstruction (MEPOP), as oculogastrointestinal muscular dystrophy (OGIMD), or as familial visceral myopathy type II. This is an autosomal recessive condition with GI and hepatic manifestations that may present at any age; typically, with hepatomegaly or hepatic failure in the neonate, seizures or diarrhoea in infancy, and hepatic failure or chronic intestinal pseudo-obstruction in children or adults.

MNGIE is characterized clinically by the presence of severe GI dysmotility, external ophthalmoplegia, ptosis, peripheral neuropathy and leukoencephalopathy. The small intestine is dilated or has multiple diverticula, and the amplitude of contractions is typical of a myopathic disorder.60 Some patients have a combination of intestinal dysmotility with the Kearns–Sayre syndrome, or transfer dysphagia due to abnormal co-ordination and propagation of swallows through the pharynx and the skeletal muscle portion of the oesophagus. Clearly, this becomes even more devastating when the smooth muscle portion of the oesophagus is affected by the associated MNGIE.

Apart from the obvious external ophthalmoplegia, these patients manifest skeletal muscle pain and cramps, and systemic (lactic) acidosis. Circulating muscle enzyme levels (CPK, ALT, aldolase, etc.) are elevated and muscle biopsy shows characteristic ragged red fibres on modified Gomori stain. This appearance results from the hypertrophy of mitochondria in the subsarcolemmal position in a few muscle fibres and the lack of mitochondria in other muscle fibres. Special stains for the respiratory muscle enzymes can identify the precise functional defect. Thus, for example, succinate dehydrogenase-positive fibres appear ‘ragged’ blue and staining of the adjacent tissue section with cytochrome C oxidase demonstrates a deficiency in the latter enzyme and a gene defect in the control of the complex IV respiratory chain proteins (Fig. 7).60 In the intestine, there is hypertrophy of the circular muscle layer and atrophy of the longitudinal muscle, and megamitochondria in myenteric neurones and muscle cells (Fig. 8).61

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Figure 7.  Histological and histochemical studies of skeletal muscle biopsy from a patient with mitochondrial myopathy. (A) Modified Gomori stain: note the ragged red fibres characterized by the subsarcolemmal location of giant mitochondria in a few fibres, and the paucity of mitochondria in other fibres. On histochemical analysis, a few fibres are succinate dehydrogenase positive (ragged blue appearance; broken arrows in B) but the same fibres do not express cytochrome C oxidase (C, solid arrows), suggesting a defect in the respiratory enzyme chain that results in mitochondrial dysfunction and systemic acidosis. Reproduced from Mueller et al., Gastroenterology 1999; 116: 959–63.

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Figure 8.  Submucosal plexus neurones showing megamitochondria in rectal biopsy from a patient with mitochondrial myopathy (left panels, light microscopy; right panels, electron microscopy). Reproduced from Perez-Atayde et al., Am J Surg Path 1998; 22: 1141–7.

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Screening tests for MNGIE are measurement of serum lactic acid, muscle enzymes, and thymidine phosphorylase in circulating leucocytes.62 The latter test is based on mutations identified in the gene for thymidine phosphorylase in 21 probands with MNGIE.62

Molecular dysregulation of peristalsis and myenteric neuropathology

Thus far, this review has considered disturbances in the development of the enteric nervous system and the role of molecular events. However, it is likely that molecular dysregulation is more prevalent in acquired diseases. This principal is illustrated by the quantitative disturbances in enteric neural elements in patients with acquired disorders associated with slow colonic transit. The neural elements deranged in these diseases are chiefly nerves and transmitters associated with the peristaltic reflex.

IPANs are activated by neurotransmitters released from the enteroendocrine cells in the lining of the gut. These cells serve as chemical or mechanical transducers of luminal signals, and release the content of their secretory granules (e.g. serotonin) through the basal membrane to activate the intrinsic afferents in the wall of the bowel. In turn, this leads to the release of another transmitter (such as CGRP, acetylcholine and substance P, although the transmitter coding of IPANs very likely varies with species and regions within the gut) to activate the ascending contractile system (cholinergic and tachykininergic neurones) and descending relaxatory system (nitrergic and VIPergic neurones) that facilitate peristalsis (Fig. 1). Different types of stimuli that activate the peristaltic reflex may involve different sensory mechanisms, including enterochromaffin cells (which act as sensory or mechanical transducers), and IPANs in the submucous and myentericplexus that serve different functions. Thus, Kunze et al. have demonstrated that IPANs in the myenteric plexus may respond directly to mechanical stimuli without any involvement of enterochromaffin cells.63 Other evidence supports the hypothesis that IPANs code for wall tension rather than simple stretch or distension.64 These basic observations provide an explanation for the inability of the paralysed or atonic gut to initiate the peristaltic reflex even if distended by an intraluminal bolus, and hence the delays in transit that may result in gastroparesis, pseudoobstruction, constipation or megacolon. Further understanding of the receptors, receptor subtypes and transmitters involved in the reflex activation of peristalsis is crucial for the development of novel therapies for motility disorders.

Much work also needs to be accomplished to understand the disturbances of these neural mechanisms in sporadic, acquired dysmotilities. These studies have been difficult because they require quantitative measurements, neuropathological expertise, availability of special techniques to evaluate a potentially large number of neurotransmitters, and comparison with control tissues. In most centres, such expertise is not available, and histopathological studies of tissue have received limited attention in relatively small systematic studies. Table 3 summarizes information from a number of studies in the literature.65–74 In general, it appears that reduced substance P and increased nitrergic neurones are associated with constipation that leads to surgical resection of the colon. In Parkinson’s disease, there is evidence of a reduction in the dopamine content of the myenteric plexus, and a significant reduction in the dopamine positive neurones of whole mounts of the plexus.75 Ultrastructural quantitative studies have demonstrated abnormalities of the morphology and numbers of interstitial cells of Cajal.76

Table 3.   Colonic neuropathology in slow transit constipation Thumbnail image of

INTERSTITIAL CELLS OF CAJAL IN MALDEVELOPMENT AND ACQUIRED DISEASES OF THE COLON

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. ONTOGENY OF THE ENTERIC NERVOUS SYSTEM
  5. THE ROLE OF ENDOTHELINS
  6. NUCLEAR TRANSCRIPTION FACTORS
  7. CONGENITAL NEUROPATHIC MOTILITY DISORDERS
  8. INTERSTITIAL CELLS OF CAJAL IN MALDEVELOPMENT AND ACQUIRED DISEASES OF THE COLON
  9. NEUROTROPHIC FACTORS AND THEIR POTENTIAL ROLE IN THERAPY
  10. CONCLUSION
  11. ACKNOWLEDGMENTS
  12. References

The interstitial cells of Cajal (ICCs) rose to prominence in the last 25 years initially through the careful histological and electrophysiological observations of Faussone-Pellegrini and Thuneberg;3[4][5]–6 the latter established the candidacy of ICCs as the intestinal pacemaker cells. The proto-oncogene c-kit encodes for a tyrosine kinase receptor that facilitates the development of the interstitial cells of Cajal.77 Furthermore, mice with mutations in the dominant white spotting (W) locus, which have cellular defects in haematopoiesis, melanogenesis and gametogenesis as a result of mutations in the c-kit gene, also lack the network of ICCs and intestinal pacemaker activity.78 In the last decade, the role of ICCs has become more clearly understood through studies of a mutant strain of mice (W/Wv), which lack c-kit-positive cells and develop GI dilatation and failure of peristalsis.79–83

ICCs are typically surrounded by collagen fibres and form close contact (Fig. 9) with smooth muscle cells.79 These gap junctions formed with smooth muscle cells result in transmission of the pacemaking activity that reflects the spontaneous oscillation in resting membrane potentials, which are unaffected by 10–6 mol L–1 verapamil, an L-type calcium channel blocker.79 When studied in short-term primary culture, these cells take on a triangular shape with 3–4 branches that are in apposition with co-cultured smooth muscle cells. Ward, Sanders and colleagues80[81]–82 have characterized the expression profiles of pacemaker ICC isolated from the murine small intestine and ICC and smooth muscle cells involved in neurotransmission from the gastric fundus; all cell types express muscarinic receptor types M2 and M3, neurokinin receptors NK1 and NK3, and inhibitory receptor VIP182.

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Figure 9.  Gap junctions between interstitial cells of Cajal and smooth muscle cells. Note spontaneous electrical oscillations of the resting membrane potential of ICCs and lack of inhibition by the l-type calcium channel blocker, verapamil. Nu, Nucleus; SM, smooth muscle cell; m, mitochondria; sER, smooth endoplasmic reticulum; ce, centride; co, collagen fibres; small arrows, caveolae. Reproduced from Lee et al., Am J Physiol 1999; 277: G409–23.

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In clinical studies, a relative deficiency of c-kit-positive cells has been reported in Hirschsprung’s disease, chronic intestinal pseudo-obstruction,84,85 GI stromal tumours and multiple GI autonomic tumours.86,87

There is also a well-documented observation of delayed maturation or maldevelopment88 of ICCs. At 1 month of age, a child with colonic chronic pseudo-obstruction underwent a biopsy of the affected colon, which showed no peristaltic activity and no c-kit immunoreactivity within the circular or submuscular layers, but normal ICC population in the myenteric plexus. At 6 months of age, there was normal peristaltic activity and ICCs were fully developed in all layers of a full-thickness biopsy of colon. This careful case study illustrates several points: (1) the concept of postnatal maturation of ICCs; (2) the imperative to manage children with neonatal megacolon or pseudo-obstruction conservatively in the first instance; and (3) the importance of ICCs to the overall peristaltic function of the colon. Little is known about the transmitters expressed by ICCs in disease. However, NK1 immunoreactivity has been detected in ICCs, and might be used as a marker for ICCs at the deep muscular plexus; these cells may participate in the actions exerted by tachykinins on muscle cells.89

Megacolon in an adult (Fig. 10) has also been associated with abnormal morphology and ultrastructure of ICCs. Faussone-Pellegrini et al. demonstrated the presence of ICCs with several branches in the dilated transverse colon, but abnormal ICCs with paucity of mitochondria, filaments and caveolae in the nondilated descending colon.90

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Figure 10.  Histological and ultrastructural studies of the interstitial cells of Cajal in an adult with megacolon. Note the cells have multiple branches in the unaffected segment (A, B; semithin section, toluidine blue), and abnormal cells in the affected segment with cells showing a paucity of mitchondria, filaments and caveolae (C; electron microscopy ×25 000). Reproduced from Faussone-Pellegrini et al., Gut 1999; 45: 755–9.

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In patients with acquired slow transit constipation, unassociated with colonic dilatation, the number of ICCs in the different layers of the sigmoid colon is lower (Fig. 11 compared to controls (resection specimens for other indications such as diverticular stricure cancer), and in addition, the confocal images showed that the cells had irregular surface markings and a paucity of branches76 (Fig. 12). c-kit staining of myenteric plexus cells is reduced, consistent with reduced ICCs or reduced tyrosine kinase content in them.

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Figure 11.  Distribution of interstitial cells of Cajal in whole transverse mounts of the sigmoid colon in a normal-appearing disease control section of the sigmoid colon (A) and the sigmoid colon of a patient with slow transit constipation (B). Reproduced from He et al., Gastroenterology 2000; 118: 14–21.

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image

Figure 12.  High-magnification confocal microscopy of the interstitial cells of Cajal from human sigmoid colon. A and C are single slices; B and D are reconstructions of 20 consecutive single slices. A and B are from a healthy disease-control colon; note multiple fine processes and the network of interconnecting ICCs. C and D are from a patient with slow transit constipation; note the irregular markings and loss of fine processes (bar=10 micron) Reproduced from He et al., Gastroenterology 2000; 118: 14–21.

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NEUROTROPHIC FACTORS AND THEIR POTENTIAL ROLE IN THERAPY

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. ONTOGENY OF THE ENTERIC NERVOUS SYSTEM
  5. THE ROLE OF ENDOTHELINS
  6. NUCLEAR TRANSCRIPTION FACTORS
  7. CONGENITAL NEUROPATHIC MOTILITY DISORDERS
  8. INTERSTITIAL CELLS OF CAJAL IN MALDEVELOPMENT AND ACQUIRED DISEASES OF THE COLON
  9. NEUROTROPHIC FACTORS AND THEIR POTENTIAL ROLE IN THERAPY
  10. CONCLUSION
  11. ACKNOWLEDGMENTS
  12. References

The neurotrophins comprise a multigene family that includes nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4/5 (NT-4), and neurotrophin-6 (NT-6).91,92 These factors signal their effects through the Trk family of protein tyrosine kinases; NGF acts primarily via TrkA, BDNF and NT-4 via TrkB, and NT-3 via TrkC. However, all these neurotrophins bind with equal affinity to the low-affinity neurotrophin receptor p75LNR.93 Neurotrophins bind to plasma membrane bound molecules that signal activation of the tyrosine kinase gene, Ret. GDNF and NTN promote the survival of enteric neurones as well as the survival, proliferation and differentiation of multipotential ENS progenitors present in the gut of E12.5–13.5 rat embryos. However, the effects of these growth factors are stage-specific, and a subpopulation of enteric neural crest undergoes apoptotic cell death specifically in the foregut of embryos lacking the Ret receptor. Thus, Ret is a marker for early lineage development of the ENS,94 and normal function of the Ret tyrosine kinase receptor is required in vivo during early stages of ENS histogenesis for the survival of undifferentiated enteric neural crest cells.95

BDNF promotes survival and maturation of certain subpopulations of sensory neurones92 and modulates high-frequency synaptic transmission at developing neuromuscular junctions in Xenopus nerve muscle cultures. BDNF also induces long-term potentiation of hippocampal neurones.96[97]–98 NT-3 promotes survival and maturation of certain subpopulations of sensory neurones.91

In a clinical study of patients with amyotrophic lateral sclerosis who were treated with r-metHuNT-3, there was a dose-related tendency to develop an increased number of softer stools.99 The mechanism of the increased bowel frequency in humans treated with r-metHuNT-3 is unclear. The timing of onset of the effects on bowel movements with exogenous r-metHuBDNF and r-metHuNT-3 suggest direct actions on the neuromuscular apparatus, or a very rapid trophic or regenerative effect on gut neuromuscular function. These considerations led to the hypothesis that r-metHuBDNF and r-metHuNT-3 alter colonic motor function, leading to increased frequency of bowel movements. This hypothesis was formally tested in a scintigraphic study.100

r-met-HuBDNF accelerated overall and proximal colonic emptying in healthy subjects (Fig. 13). r-metHuNT-3 accelerated overall colonic transit in healthy subjects and in patients with constipation, and gastric and small bowel transit in healthy subjects. r-metHuBDNF tended to increase stool frequency compared with placebo in healthy subjects, and increased stool frequency and facilitated passage of stool in constipated patients.100 The effects on stool frequency started within 3 days of onset of neurotrophin administration, and lasted up to 5 days after treatment cessation. r-metHu neurotrophic factors were well tolerated, although half of the participants in the two studies developed injection site reactions or paresthesiae. Thus, exogenous neurotrophic factors stimulate human gut motility in healthy subjects and in patients with constipation.

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Figure 13.  Scintigaphic images of the colon in healthy volunteers before and after pretreatment with placebo (vehicle) subcutaneously, or r-metHu-BDNF. Note the acceleration of transit measured by the geometric centre method at 24 hours (weighted average location of isotopic counts in the colon). Reproduced from Coulie, Szarka et al., Gastroenterology 2000; 119: 41–50.

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Two mechanisms mediating the actions of neurotrophins on neuromuscular function were considered: (i) trophic effects; or (ii) a direct effect on neurotransmission. The neurotrophins have long-term trophic actions including the promotion of survival and phenotypic maturation of many types of neurones.92,101 These functions are mediated by the tyrosine kinase receptors.102 Direct modulation of neurotransmission is suggested by acute or short-lived effects of neurotrophins. For example, BDNF modulates neurotransmitter synthesis, increases neuronal excitability, and provides long-term synaptic potentiation of neurones.103[104][105]–106 The rapidity of onset of diarrhoea in clinical trials with these neurotrophins and the acceleration of transit and increased frequency of bowel movements in our study argue in favour of a direct effect on neurotransmission. In vitro studies suggest that chronic parenteral administration of NT-3 to guinea pigs over 7 days results in increased electrical field stimulation (EFS)-induced contractions in colonic longitudinal muscle strips, which were partially blocked by atropine 10−7 mol L−1 and completely blocked by tetrodotoxin 10−6 mol L−1. Under nonadrenergic noncholinergic conditions, the EFS-induced relaxation was almost abolished by the rHuNT-3. Nitric oxide synthase-immunoreactive neurones per ganglion were significantly decreased in the longitudinal muscle-myenteric plexus preparations of rHuNT-3-treated guinea-pigs compared to placebo, thus the neurotrophin appears to increase sensitivity to excitatory transmitters and to reduce inhibitory innervation.107

CONCLUSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. ONTOGENY OF THE ENTERIC NERVOUS SYSTEM
  5. THE ROLE OF ENDOTHELINS
  6. NUCLEAR TRANSCRIPTION FACTORS
  7. CONGENITAL NEUROPATHIC MOTILITY DISORDERS
  8. INTERSTITIAL CELLS OF CAJAL IN MALDEVELOPMENT AND ACQUIRED DISEASES OF THE COLON
  9. NEUROTROPHIC FACTORS AND THEIR POTENTIAL ROLE IN THERAPY
  10. CONCLUSION
  11. ACKNOWLEDGMENTS
  12. References

Knockout models and mutant strains have provided interesting insights into the development of the enteric nervous system, and underscored the role of genetic derangements in relatively rare dysmotilities such as Hirschsprung’s disease. However it is important to keep this information in perspective because the identified genetic defects account for only a minority of familial cases, and the precise mechanism for the majority of sporadic Hirschsprung’s and pseudo-obstruction cases is still unclear. Nevertheless, studies of the molecular and genetic mechanisms of enteric neurones provide the foundation to characterize acquired diseases and, potentially, to develop new diagnostic and therapeutic strategies in clinical neurogastroenterology.

ACKNOWLEDGMENTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. ONTOGENY OF THE ENTERIC NERVOUS SYSTEM
  5. THE ROLE OF ENDOTHELINS
  6. NUCLEAR TRANSCRIPTION FACTORS
  7. CONGENITAL NEUROPATHIC MOTILITY DISORDERS
  8. INTERSTITIAL CELLS OF CAJAL IN MALDEVELOPMENT AND ACQUIRED DISEASES OF THE COLON
  9. NEUROTROPHIC FACTORS AND THEIR POTENTIAL ROLE IN THERAPY
  10. CONCLUSION
  11. ACKNOWLEDGMENTS
  12. References

This study was supported in part by grants RO1-DK54681-03 and K24-DK02638-03 (Dr M. Camilleri) and by General Clinical Research Center grant (RR00585) from the National Institutes of Health. Mrs Cindy Stanislav is thanked for excellent secretarial assistance.

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  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. ONTOGENY OF THE ENTERIC NERVOUS SYSTEM
  5. THE ROLE OF ENDOTHELINS
  6. NUCLEAR TRANSCRIPTION FACTORS
  7. CONGENITAL NEUROPATHIC MOTILITY DISORDERS
  8. INTERSTITIAL CELLS OF CAJAL IN MALDEVELOPMENT AND ACQUIRED DISEASES OF THE COLON
  9. NEUROTROPHIC FACTORS AND THEIR POTENTIAL ROLE IN THERAPY
  10. CONCLUSION
  11. ACKNOWLEDGMENTS
  12. References
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