Chapter 10 The role of nitric oxide in motility of the developing gastrointestinal tract

10 The role of nitric oxide in motility of the developing gastrointestinal tract M. Ceregrzyn and A. Kuwahara Laboratory of Environmental Physiology, Institute for Environmental Sciences, University of Shizuoka, 52-1 Yada, Shizuoka, Shizuoka 422-8526, Japan

Nitric oxide (NO) is produced by nitric oxide synthase (NOS) and by nonenzymatic pathways. In the gastrointestinal tract (GIT) of adults the majority of NO-producing neurons are localized in the myenteric plexus where they act as inhibitory inter and motoneurons. The distribution and density of NOproducing neurons change during development. These developmental variations are observed in the central nervous and respiratory systems as well as in the GIT. The first NO-positive cells in the GIT are seen at an early stage of pregnancy. The number of NO-positive neurons increase until the end of pregnancy, but after birth rapidly decrease in the submucosal plexus, while the density of these neurons in the myenteric plexus remains unchanged. NO production is important for the development of normal GI motor function, especially pyloric contractility. Animals with insufficient production of NO (due either to inhibition of NOS or to genetically induced lack of the enzyme) present signs of pyloric stenosis. Decreased production of NO during pregnancy induces significant growth retardation as a result of malnutrition caused by a diminution in blood flow in both the placenta and foetal circulation. In conclusion, NO plays an important role in the maturation of the GIT in terms of development of normal motility but the precise mechanism of its action remains a subject for further research.

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1.

INTRODUCTION

The biological functions of nitric oxide (NO) can be divided into two main categories: involvement in physiological regulation, and contributions to pathological processes. NO is a molecule that acts as an intercellular messenger regulating numerous physiological functions, e.g. vascular tone, platelet aggregation, immune response, and neurotransmission, both in the central and the peripheral nervous system. Nitrergic transmission in the peripheral nervous system takes place in non-adrenergic, non-cholinergic (NANC) nerves (Moncada and Higgs, 1995). In particular, NO takes part in the regulation of motility, blood flow, water and electrolyte transport, and acid secretion in the gastrointestinal tract (GIT). In addition, NO is involved in mucosal inflammation (Salzman, 1995). In pathological conditions, NO is responsible for neurotoxicity during (brain) stroke (Snyder and Bredt, 1992). These multiple functions involve in part the enteric nervous system (ENS). NO is one of the neurotransmitters produced by neurons of the ENS that has an important role in regulation of gastrointestinal motility. In this chapter, the authors would like to provide an overview of the functions of NO in the GIT in adult and developing animals and humans. In particular, we will discuss the distribution of nitrergic neurons in the GIT in foetuses, growing and adult animals and humans. Moreover, the actions of NO and the distribution of nitrergic neurons in the GIT of growing animals will be presented, and some other aspects of the role of NO in the development of the GIT will be discussed.

2.

2.1.

NITRIC OXIDE SYNTHASE AND NITRIC OXIDE PRODUCTION Detection of nitric oxide synthase activity and presence

Methods for investigation of nitric oxide synthase (NOS) activity and distribution are summarized in table 1. NOS synthesizes NO from L-arginine with subsequent formation of citrulline. Nitrite and nitrate are formed as endproducts of NO oxygenation (fig. 1). The activity of NOS can therefore be expressed as changes in concentration of citrulline (Misko et al., 1993; Kuemmerle, 1998; Cullen et al., 1999). Moreover, levels of nitrite and nitrate can also be used as indicators of NOS activity. The most common method for detection of nitrate and nitrite is the Griess reaction, which has been used for measurements in culture media or body fluids (Miller et al., 1993; Guevara et al., 1998; Iizuka et al., 1999).

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Table I Summary of methods used for investigation of distribution and function of nitric oxide synthase in the gastrointestinal tract (for details see text) Type of determination Activity of NOS

Compound Nitrate, nitrite

Griess method

Nitric oxide L-citrulline Visualization of NOS

Methods Liquid chromatography

UV detector EPR Liquid Radioassay chromatography Fluorescence microscopy

NADPHdiaphorase histochemistry NOS immuno Fluorescence histochemistry microscopy

Confocal microscopy

Electron microscopy

Protein degradation

/

cAl)NH£

Arginine

.-:

~'r::\

\~

~~

Arginine

~line

/

O2 O2 O 2

O2

O2

<, N0 2 /

O2

/

\)

\

°

N0 3 Fig. I.

Nitric oxide (NO) syntehsis in nonhepatic cells. NOS - nitric oxide synthase, ASargininosuccinate synthetase, AL-argininosuccinate lyase.

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Another method for measuring nitrite and nitrate, is liquid chromatography using UV absorbance (Wiklund et al., 1993c). Conversions of nitrite or nitrate to NO in body fluids and organ preparations can be detected using chemiluminescence (Hobbs et al. 1991; Aneman et al., 1996; Iizuka et al., 1999; Tangphao et al., 1999). Another method that allows measurement of NO uses electron paramagnetic resonance (EPR). In this method, NO formation is determined by the increase in intensity of the characteristic triplet hyperfine EPR spectrum of NO and spin-trapping agent. One of the spintrapping agents is iron (11)- diethyldithiocarbamate (FeDETC2). NO is attached to the spin-trapping compound previously administered to the body (Mikoyan et al., 1994; Bune et al., 1995) or cell culture (Corbett etai.,1991). Studies concerning NO production involve determination of NOS in animal tissues. There are few approaches to investigate NOS distribution in tissues. This enzyme is expressed in the genome of certain cells and thus it can be detected using biochemical methods such as he polymerase chain reaction ~ and the products can be detected by Western blotting. NOS has been detected and quantified by this method in studies considering the distribution and action of NOS in the GIT (Bandyopadhyay et al., 1997; Hoffman et al., 1997; Vos et al., 1997; Closs et al., 1998; Cullen et al., 1999; Fischer et al., 1999). NOS can also be detected using histochemical methods that allow the distribution of this enzyme in all parts of the tissues to be demonstrated. The method which allows the visualization of all three isoforms of NOS, neuronal (nNOS), endothelial (eNOS), and inducible (iNOS) in cells and tissues uses reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry, immunocytochemistry and in situ hybridization (Beesley, 1995). It has been reported that in amphibian (Li et al., 1992), guinea pig (Young et al., 1992), dog (Ward et al., 1992), pig (Timmermans et al., 1994b) and human intestine (Timmermans et al., 1994a) NADPH-d and NOS are co-localized in the ENS. This provides morphological support that the same protein has two different substrate sites that are involved in two enzymatic actions (Dawson et al. 1991; Hope et al., 1991). Almost all the NOS-positive myenteric neurons also stain for NADPH-d in all regions of the rat small intestine, caecum, and distal colon. In contrast, in the stomach, duodenum, and ileum, only a few of the NOS-positive nerve fibres in the tertiary and secondary plexuses and in the circular muscle layer are positive for NADPH-d (Belai et al., 1992). In a further study, the pattern of distribution and co-localization of NADPH-d activity and NOS-immunoreactivity in the myenteric plexus of human foetal (6-17 weeks of gestation) stomach and small intestine have been investigated (Belai and Burnstock, 1999). Only 15% of the NADPH-d-positive myenteric neurons

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were NOS-immunoreactive in foetuses at 6-10 weeks of gestation, whereas a 100% co-localization was found in samples at 12-17 weeks of gestation. This finding suggests that NADPH-d-reactivity does not always represent NOS activity (Belai and Burnstock, 2000). In spite of this inconsistency, the NADPH-d staining method has been widely used to visualize NOS. Furthermore, this method is not selective, and it stains all isoforms of NOS equally. Since the function of each isoform of NOS is different this method is not precise enough for detailed studies. Thus, immunohistochemical staining using specific antibodies against each isoform of NOS is the best method to precisely localize all NOS isoforms. Using such antibodies, neural NOS has been found in the rat gut (Bredt et al., 1990; Belai et al., 1992), in the canine colon (Ward et al., 1992), the guinea pig gastric fundus (Miller et al., 1991), in the guinea pig small intestine (Costa et al., 1991; Furness et al., 1991; Costa et al., 1992; Young et al., 1992), the guinea pig colon (Young et al., 1992; McConalogue and Furness, 1993), the guinea pig caecum (Shuttleworth et al., 1991), and cultured myenteric neurons of the caecum (Saffrey et al., 1992). NOS stained immuohistochemically can be detected by several methods: conventional fluorescence, confocal, and electron microscopy (Mann et al., 1997). Electron microscopy provides precise determination of NOS localization within cells (Llewellyn-Smith et al., 1992; Beesley, 1995; O'Brien et al., 1995; Matini and Faussone-Pellegrini, 1997; Sang and Young, 1997; Li and Furness, 2000). In conclusion, there are two approaches to investigate the effects of NO produced by NOS in the GIT. One is the functional determination of NOS activity, which can be expressed as changes in the concentrations of reaction substrates, such as L-arginine, used by NOS to produce NO, or as changes in the concentration of reaction products such as L-citrulline, NO, nitrite, or nitrate. The other way is visualization of NOS. Using light or electron microscopy methods NOS can be visualized both at tissue and subcellular levels. The localization of NOS by NADPH-d histochemistry is non-selective, only immunohistochemical methods allow the various isoforms to be visualized separately.

2.2.

Nitric oxide production

There are two general pathways of NO production: the first involves the NOS pathway and can be called an internal source of NO since it involves activity of body cells; the second is NOS independent, and does not involve activity of body cells, but depends instead upon external sources, such as bacterial denitrification and reduction of dietary nitrate in the acidic environment of the stomach (table 2).

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Table 2 Sources of NO in the gastrointestinal tract Body cells source

Biosynthesis

Neurons

Neuronal NOS (Huang et aI., 1993; Nichols et aI., 1993; Berezin et aI., 1994; Lin et aI., 1998; Fischer et al., 1999) Inducible NOS (Miampamba and Sharkey, 1999; Torihashi et aI., 2000) Constitutive and inducible NOS (M'Rabet-Touil et aI., 1993; Laszlo et al., 1994; Chen et aI., 1998; Chen et aI., 1999; Takada et al., 1999; Hsu et aI., 2000) Constitutive NOS (Nichols et aI., 1993; O'Brien et aI., 1995; Fischer et al., 1999) Inducible NOS (Boughton-Smith et aI., 1993)

Monocytes/macrophages Enterocytes

Endothelial cells

Granulocytes External sources Dietary/intraluminal nitrate and luminal bacteria

Reduction in acidic conditions and bacterial denitrification (McKnight et aI., 1997; Weitzberg and Lundberg, 1998; Iizuka et al., 1999)

2.2.1. Nitric oxide produced by different isoforms of nitric oxide synthase NOS produces NO from L-arginine in various cells. The main cell types producing NO are endothelial cells, macrophages and neuronal cells (for reviews see: (Moncada and Higgs, 1991; Moncada et al., 1991; Snyder and Bredt, 1992). Several differences are seen among the three distinct isoforms of NOS (nNOS, eNOS, and iNOS), including differences in tissue distribution, regulation of transcription, post-translational modification, and cofactor demand. Neuronal NOS is present in the brain, in the innervation of the posterior pituitary, in autonomic nerve fibres of the retina, in cell bodies and nerve fibres in the myenteric plexus of the intestine, in the adrenal medulla, and in vascular endothelial cells (Bredt et al., 1990). Endothelial NOS is found in vascular endothelial cells and in the GIT, eNOS is involved in regulation of blood flow (Stenson, 1999). Both nNOS and eNOS are constitutive isoforms of the enzyme. In contrast, the isoform iNOS is induced by proinflammatory cytokines (e.g. IL-l, TNF-a, and IFN-g) and by bacteriallipopolysaccharide. Inducible NOS produces NO in amounts that greatly exceed those which are produced under physiological conditions for neurotransmission or vasodilatation. In pathological conditions NO is used for killing bacteria or tumour cells (Stenson, 1999). However, NO produced after activation of iNOS may also influence functions that are mediated by eNOS and nNOS.

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2.2.2. Non-enzymatic sources of nitric oxide Dietary nitrite is reduced in the acidic environment of the stomach to NO and other oxides of nitrogen. Nitrate in the diet, may also be an important source of gastric production of reactive nitrogen oxide species (McKnight et al., 1997). Various microorganisms reduce nitrate to nitrite, and as the result of subsequent acidification in the stomach nitrite is further reduced to NO (Benjamin et al., 1994). NO is synthesized in the human and rat mammary gland (lizuka et al., 1997; lizuka et aI., 1998). Moreover, human breast milk containing high concentrations of nitrite leads to an increased NO level in the stomach gas of neonates fed only breast milk. As a result, the maximum concentration of NO in stomach gas is observed 2 to 5 days after birth. In addition to this, NO is also present on the milk surface in vitro. Thus, breast milk and its content of nitrate/nitrite or NO may be an important factor in regulating the mucosal blood flow and gastric motility and in achieving bacteriostasis in the neonatal stomach (Iizuka et al., 1999). In any event, it has been shown that nitrite can directly induce changes in gastrointestinal motility (Ceregrzyn et aI., 1998). Nonenzymatic production of NO has been demonstrated, in the stomach, on the surface of the skin, in the ischaemic heart, and in infected nitritecontaining urine (Weitzberg and Lundberg, 1998). This non-enzymatic NO may playa role in an effective host defence mechanism against gastrointestinal pathogens, as a modulator of platelet activity and may possibly take part in the regulation of gastrointestinal motility and microcirculation (McKnight et aI., 1999).

2.3. Distribution of nitric oxide synthase within the gastrointestinal wall In the GIT, NOS is expressed both in enteric neurons and in blood vessels. Extensive NOS activity is present in submucosal arterioles and staining for NOS is uniform in the small and large intestine of guinea pig and rat (Nichols et al., 1992; Nichols et aI., 1993). Non-neuronal cells such as mucosal epithelium, enteroglia, mast cells, endothelial cells and muscle fibres are not NADPH-d positive, whereas neuronal cells show NADPHd-positive reaction (Aimi et al., 1993). The presence of NOS in neuronal cells has been confirmed by immunohistochemical methods. Thus, it appears that the majority of NOS in the intestinal wall is present in the ENS. NOS activity has also been shown to be present in myenteric neurons and their fibres to the circular muscle in rat intestine (Nichols et aI., 1993). The ileum posesses the largest relative number of NOS-positive cells, and

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Type I Type II

Type III

t .

Type IV

Type VI

Type V

Type VII Fig. 2.

\

Giant neurones

Types of enetric neurones. Shapes of neurones observed in the human and pig intestine.

the colon the smallest. NOS-positive neuronal elements are also present within the submucosa throughout the intestine. These cells, in the myenteric as well as the submucosal nerve plexuse, are of Dogiel type I and type II morphologies (Nichols et al., 1993) (fig. 2). Finally, in submucosal arterioles, there is a regular pattern of NOS-positive patches that are unrelated to any perivascular innervation (Nichols et al., 1992; Nichols et al., 1993).

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2.3.1. Morphology of the enteric nervous system Enteric neurons were first classified by Dogiel, with the aid of methylene blue staining (Dogiel, 1899) The first division proposed by Dogiel included three types of neurons present in the gastrointestinal wall. These classical types are still the basis for the morphological classification of neurons in the ENS (Furness et aI., 1999; Stach et aI., 2000). Type I neurons are uniaxonal and multidendritic with spine-shaped structures. Type II neurons are multidendritic. They appear to have a few axons (2 to 10), but they may be pseudo-uniaxonal. Type III neurons are uniaxonal and multidendritic. These cells have two types of dendrites, branched dendrites or short, tapering ones. In addition to these classical Dogiel types of enteric neurons, some other types have been recently found in pig and human intestine (Furness et aI., 1999; Stach et aI., 2000), i.e. types IV, V, VI, VII, and giant neurons (table 3, fig. 2). The network of ENS inside the gastrointestinal wall is presented in figs. 3 and 4.

2.3.2.

Nitrergic neurons in the enteric nervous system

The ENS regulates many functions of the GIT. The neural network of the ENS involves a large number of neurotransmitters, which are released by Table 3 Types of neurons in the enteric nervous system (Furness et aI., 1999; Stach et aI., 2000) Neuron type Type I Type II Type

Type

Type

Type Type Giant

Dendrites number

Characteristics

multidendritic short multidendritic! short adendritic III multi dendritic long branched and short tapering IV multidendritic polardendritic, short or medium length V multi dendritic polardendritic, stem-like process emanation VI multi dendritic branched and tapering VII multidendritic axonopolar main dendrites neurons multidendritic long branched and tapering

Axons number

Characteristics

uniaxonal unipolar poliaxonal (2-10) pseudounipolar, multipolar uniaxonal unipolar

uniaxonal

unipolar

uniaxonal

emanating from stem-like process

uniaxonal

axonal dendrites

uniaxonal uniaxonal

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Fig. 3.

The enteric nervous system in transverse section (scheme).

relatively few kinds of neurons. Among these, nitrergic neurons have been shown to play an important role in the regulation of gastrointestinal motility. Histochemical and immunohistochemical methods allow one to investigate the distribution and projections of neurons expressing NOS and these methods have been useful in investigating the function of nitrergic neurons in the regulation of gastrointestinal motility and secretion.

2.3.2.1.

Rat

In the rat, NOSjNADPH-d-positive nerve cell bodies and nerve fibres are present throughout the digestive tract, from the oesophagus to the rectum. They are very common in the myenteric ganglia (fig. 5). Moreover, dense positive fibres are also present in internodal strands, the secondary plexus, and the tertiary plexus, and they are particularly abundant in the deep muscular plexus (fig. 6). On the other hand, very few NADPH-d-positive neurons are observed in the submucosal ganglia. The density of NADPH-d-positive structures is higher in the small and large intestine than in the oesophagus and stomach.

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Fig. 4.

281

The three components of the myenteric plexus in the guinea pig distal colon.

However, even though the density of NADPH-d-positive structures varies from one region to another of the GIT, the pattern of distribution appears to be similar in all parts of the GIT (Aimi et a!., 1993). In the rat distal ileum the NADPH-d-positive myenteric neurons comprise about 27% of the nerve cell bodies per ganglion and the morphologies of most ofthese neurons are of Dogiel type I (Aimi et a!., 1993; Cracco and Filogamo, 1994). Similarly, in the colon approximately one third of the NADPH-d-positive cells have Dogie! type I morphology (Aimi et a!., 1993). The expression ofnNOS differs between distinct parts of the GIT in the rat. Quantitative determination ofnNOS (brain isoform) expression in the rat GIT reveals that the highest content of this enzyme is present in the oesophagus, while levels in the colon, stomach, and ileum are significantly lower (Fischer et a!., 1999). There is both intrinsic and (extrinsic) vagal innervation of NOS-positive neurons in the myenteric and submucosal plexuses of the rat gastric corpus. These neurons are distinct from those containing vasoactive intestinal peptide (VIP). In addition, it has been shown that NOS-positive fibres innervate the circular muscle layer of the rat gastric corpus (Forster and Southam, 1993). The density of NOS-positive neurons is significantly higher in rat gastric corpus than in forestomach or antrum. Nevertheless, the proportion

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Fig. 5.

NOS immunostaining in myenteric plexus of the rat esophagus.

of NOS-positive cells observed in myenteric ganglia of the stomach (20%) was shown to be lower than that of these cells in the duodenum. Furthermore, there is a tendency for the proportion of NOS-positive neurons to become lower when passing in an aboral direction along the duodenum, e.g. the density of NOS-positive neurons in the first 0.5 em from pylorus is higher than in the next 3 em, and the proportion of NOS-positive neurons in the postpyloric region of the duodenum is higher than in the following part of the duodenum (Jarvinen et aI., 1999). Sixty four percent of neurons in the rat oesophagus are NOS-positive, 40% are choline acetyltransferase-positive while only 4% are positive for both. NOS-positive nerve fibres are present also in motor endplates, muscularis mucosa, and the submucosal layer. In addition, NOS neurons are descending while the choline acetyltransferase neurons are ascending in the rat oesophagus (Kuramoto and Brookes, 2000). On the other hand, double staining for NADPH-d/acetylcholinesterase and NADPH-d/substance P (SP) has shown that there is no co-localization of either acetylcholinesterase or SP with

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reactivity in the pig small intestine. In another study (Brehmer et al., 1998), the presence of nitrergic neurons has been demonstrated in outer and inner submucosal plexus of pig upper small intestine, suggesting that nitric oxide may playa role in the regulation of mucosal function in the duodenum. Similar co-localization of NOS immunohistochemistry and NADPH-d histochemistry has been shown in the pig large intestine, where the distribution and pattern of NOS and NADPH-d-positive nerve cells and fibres are apparently the same (Barbiers et al., 1993). Moreover, in this study it was shown that NOS/NADPH-d-positive neurons are present in large numbers in the myenteric plexus (45 cells/ganglion). In the outer submucosal plexus, NOS-positive nerve cells are distributed over all ganglia with an average density of 15 cells/ganglion. In the inner submucosal plexus, NOS-positive neurons are present in only a few ganglia and the density of positive neurons is lower (5 cells/ganglion). Another study performed by the same group (Barbiers et al., 1995) has shown that in porcine colon, nitrergic nerve fibres and cell bodies are numerous in the myenteric and outer submucosal plexuses, but that these fibres are present to a lesser extent in the inner submucosal plexus. It could also be shown that large numbers of NOS/NADPH-d positive fibres are present in the circular muscle, and that in the pig colon the nitrergic neurons project aborally within the myenteric plexus.

2.3.2.3.

Birds

The distribution of NOS, NADPH-d, and VIP in enteric neurons of the newly hatched chicken gut is similar to that of mammalian gut (Balaskas et al., 1995). Therefore, neurons expressing NADPH-d activity, NOS, and VIP are present in both the myenteric and submucosal ganglia in all regions of the GIT. Moreover, NADPH-d and NOS-positive neurons are completely co-localized in the chicken gut. NOS immunoreactivity is in part co-localized in the same neurons with VIP immunoreactivity. Moreover, VIP immunopositive neurons that are NADPH-d negative increase anally and such neurons are more prominent in the submucosal ganglia than in the myenteric ganglia. NADPH-d-, NOS-, and VIP-positive nerve fibres are present in the circular muscle, but very few, if any, are present in the longitudinal muscle. In contrast to the situation in the mammalian gut, VIP-immunoreactivity, but not NOS or NADPH-d activity, is present in mucosal fibres, (Balaskas et al., 1995). It has been shown that in the adult quail intestine only a minority of the NADPH-d-positive neurons stain for VIP. VIP-immunoreactive cell bodies are frequent in the myenteric plexus but not in the submucosal plexus, whereas there are considerable numbers of NADPH-d-positive neurons in both these plexuses. Nitrergic fibres are also observed in the outer muscle

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layer, but they are almost absent in the lamina muscularis mucosa and lamina propria, in contrast to the numerous VIP-ergic fibres encircling basal parts of the intestinal crypts (Boros et al., 1994).

2.3.2.4.

Guinea pig

NADPH-d reactivity in the guinea pig is found in a spectrum of enteric nerves along the intestine. This observation shows that NO can be released at multiple gastrointestinal sites. The density ofNADPH-d-positive cells is different for different regions of guinea pig intestine. In the duodenum the mean number of cells per ganglion is 5±1, 8±2 in the jejunum, 10±2 in the ileum, and 9± 1 in the colon (Nichols et al., 1992). Another study has shown that NOS containing neurons innervate several targets in guinea pig colon, including both longitudinal and circular muscles, and nerve cells in the myenteric and submucosal ganglia (McConalogue and Furness, 1993). NOS-immunoreactivity is present in many neurons of the myenteric ganglia of the guinea pig small intestine. The great majority of these neurons are Dogiel type I. On the other hand, in the submucosal ganglia NOS immunoreactivity is only observed in occasional neurons, while interstitial cells of Cajal, and the endothelium of submucosal or myenteric blood vessels do not show any NOS-immunoreactivity. NOSimmunoreactivity has been localized in varicose nerve fibres that are numerous in myenteric ganglia and internodal strands. In addition, NOS-positive neurons have been shown to project preferentially in the anal direction. All NOS-immunoreactive cell bodies are also immunoreactive for VIP, while most, but not all, VIP-immunoreactive cells contain NOS-immunoreactivity (Costa et al., 1992). In the guinea pig small intestine there are three classes of interneurons that project anally. One of these types is NOS and choline acetyltransferase immunoreactive (Costa et al., 1992, Li and Furness, 1998). The other types are choline acetyltransferase and somatostatin immunopositive (Portbury et al., 1995; Song et al., 1997) or choline acetyltransferase and 5-hydroxytryptamine immunopositive (Furness and Costa, 1982; Young and Furness, 1995). It has been proposed (Li and Furness, 2000) that these neurons may be involved in the control of various functions (motility, secretion and blood flow). In the case of motility, these three types of neurons may be involved in the coordination of three characteristic patterns of motility: migrating myoelectric complex, local propulsive (peristaltic) reflexes and mixing movements. However, the definite functional involvement of each class remains to be established. Furthermore, the same authors have shown that in the guinea pig ileum,

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all NOS-immunoreactive neurons appear to have Dogiel type I morphology. Only approximately 16% of neurons in the myenteric plexus are immunoreactive for both NOS and choline acetyltransferase (Li and Furness, 1998; Li and Furness, 2000). On the other hand, NOS-positive neurons do not show immunoreactivity for calbindin, however calbindinpositive varicosities are closely related to NOS-positive cells, forming identifiable synapses (approximately 80 close appositions) (Li and Furness, 2000). It was concluded in this study that choline acetyltransferaseand NOS-immunoreactive neurons are interneurons that are involved in local reflexes and are interposed between intrinsic primary afferent neurons and NOS-inhibitory neurons (Li and Furness, 2000). Another study (Clerc et al., 1998), succeeded in identifying several major neuron populations in the guinea pig duodenum. Calbindin immunoreactivity occurs in a population of myenteric nerve cells with Dogiel type II morphology (Clerc et al., 1998; Li and Furness, 2000), which have axons that project to other myenteric ganglia, the circular muscle, and the mucosa. All these neurons are immunoreactive for choline acetyltransferase (the enzyme synthesizing acetylcholine) and some are also immunoreactive for calretinin. Myenteric neurons with NOS-immunoreactivity project anally to the circular muscle. In addition, these neurons are also immunoreactive for VIP, and some of them have enkephalin and/or neuropeptide Y (NPY) immunoreactivity. A very few neurons (about 2%) are both NOS- and choline acetyltransferase immunoreactive. In general, the innervation of the duodenum closely resembles that of the ileum, both in the chemistries and the projections of the neurons. However, there are differences, which include the almost complete absence of NOSimmunoreactivity from VIP-immunoreactive interneurons in the duodenum, the projection of calbindin-immunoreactive Dogiel type II neurons to the circular muscle and the absence of tachykinin-immunoreactivity from these neurons (Clerc et al., 1998). In the guinea pig small intestine there are three classes of descending and one class of ascending interneuron (Costa et al., 1996; Furness et al., 2000). In the guinea pig colon there are four classes of descending neurons and three classes of ascending ones (Lomax and Furness, 2000; Lomax et al., 2000). NOS-immunoreactivity is present in 39% of the total population of myenteric neurons in the guinea pig distal colon (Lomax and Furness, 2000). The significant difference observed between the small and large intestine in the guinea pig is that VIP immunoreactive, non-cholinergic secretomotoneurons are NOS-positive only in the colon (Lomax and Furness, 2000). On the other hand, in the small intestine myenteric plexus all NOS-immunoreactive nerve cell bodies are also immunoreactive for VIP, while most VIP- immunoreactive neurons also contain NOS-immunoreactivity (Costa et al., 1992).

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Double staining for protein gene product 9.5 (PGP 9.5) and NADPH-d has shown that the myenteric plexus has different network patterns along the entire course of the oesophagus (Morikawa and Komuro, 1998). In addition, NADPH-d positive neurons make up on average 69% of the total number of myenteric neurons. Moreover, motor endplates of the oesophageal striated muscles (stained by acetylcholinesterase) are often associated with NADPH-d positive varicose fibres, which come from the myenteric ganglia, but it is uncertain if they are in direct continuity with the myenteric neuronal cell bodies. It was concluded that the myenteric NADPH-d positive neurons in the guinea pig oesophagus contribute to the innervation of both the striated muscle and smooth muscle of the lower oesophageal sphincter (Morikawa and Komuro, 1998).

2.3.2.5.

Human

The myenteric plexus, as well as the outer submucosal plexus, in both the porcine and the human ileocaecal junction contain NOS-positive neurons, which vary widely in size and shape. Compared to the myenteric plexus, significantly fewer NOS-containing neurons are present in the outer submucosal plexus. A large number of NOS- immunoreactive nerve fibres are observed in the enlarged circular muscle of the ileocaecal junction and in the circular muscle of the adjacent ileum. No NOS-immunoreactivity is present in the smooth muscle cells of the outer circular or longitudinal muscle layer (Bogers et al., 1994). Another study has shown that there are significant differences in the density of nitrergic neurons among various parts of human ileocaecal region. The highest percentage of NOS-positive neurons in the myenteric plexus is seen in the caecocolonic junction and that in the submucosal plexus is observed in the post-junctional (ascending) colon (Faussone-Pellegrini et al., 1994). Moreover, immuno-histochemistry of human stomach fundus wall has shown that only neural elements are immunoreactive for nNOS, while nerve endings or nerves supplying the vessels are negative. In human stomach wall, immunoreactivity for constitutive eNOS is found mainly in endothelialcells of arteries and veins (Fischer et al., 1999). Single NADPH-d-reactive or NOS-immunoreactive neurons are present in the submucosal plexus ganglia, while the myenteric plexus ganglia include several NADPH-d/NOS-positive neurons (often arranged in clusters) and nerve fibres. However, the distribution of these neurons is not uniform. The number of NADPH-d-positive nerves in the plexus is greater than that in the longitudinal muscle layer. Moreover, NADPH-d activity and VIP immunoreactivity is co-localized in some neurons of the myenteric ganglia (Keranen et al., 1995). Investigation of neuron

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density and distribution of the NADPH-d positive neurons in the fundus, corpus and antrum of adult human stomach has also shown that the submucosal plexus contains significantly less neurons than the myenteric plexus (Manneschi et aI., 1998). NADPH-d-positive neurons in the submucosal plexus are mostly located in ganglia close to the circular muscle layer. In the myenteric ganglia NADPH-d-positive neurons represent 50--60% of the neurons in the fundus, corpus and antrum, but their density is significantly lower in the fundus than in the corpus or antrum. NADPH-d positive fibres form a rich plexus in the innermost portion of the circular muscle layer of the corpus (Manneschi et aI., 1998).

2.3.2.6.

Opossum

A double-labelling study (Lynn et aI., 1995) demonstrated the coexistence of NOS and VIP in the opossum internal anal sphincter. VIP-immunoreactive and NADPH-d-positive neurons are present in ganglia of the myenteric and submucosal plexuses, and most of the VIP-immunoreactive neurons are also NADPH-d positive.

2.3.2.7.

Mouse

Immunohistochemical techniques have demonstrated the presence and colocalization of a range of putative neurotransmitters and other neuronal markers in the myenteric plexus of the small and large intestine of the mouse (Sang and Young, 1996). In the small intestine, there are two major classes of motor neurons in the circular muscle: one class is NOS/VIP/NPY positive and the other class contains calretinin and SP. There are seven classes of neurons that innervate myenteric ganglia. Two of them contain NOS. One of them is only NOS-positive and the other is both NOS and VIP immunoreactive. The other classes are VIP, NPY, calretin/calbindin, SP or 5-hydroxytryptamine positive. In the large intestine, there are five major classes of motor neurons that contain NOS, NOS/VIP, gamma amino butyric acid (GABA), SP, or calretin/Sl'. There are seven major classes of neurons that innervate myenteric ganglia in the large intestine, and these contain NOS, VIP, calretin/calbindin, calretin, SP, GABA or 5-hydroxytryptamine (Sang and Young, 1996). Continuation of this study by the same group demonstrated the projections of the neurons in both the small and large intestine of the mouse (Sang et aI., 1997). In this study myotomy was used to examine the polarity of myenteric neurons projecting to the myenteric ganglia, whereas myectomy was used to examine the polarity of myenteric neurons projecting to the circular muscle or to the myenteric

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0.4

0.3

0.1

o Oesophagus

Fig. 7.

Stomach

Ileum

Colon

Schematic presentation of projecton of ecitataory and inhibitory circular muscle motoneurons and intemeurons in the mouse small and large intestine.

ganglia. Those inhibitory, circular muscle motor neurons that have been determined in the small and large intestine arise from cells in the myenteric plexus and run anally to supply the muscle. Intemeurons positive for NOS, VIP, calbindin and 5-hydroxytryptamine project anally in both the small and large intestine. On the other hand, SP-immunoreactive intemeurons and excitatory motor neurons mainly project in an oral direction, however some SP-immunoreactive neurons may also project locally and/or a short distance anally (fig. 7) (Sang et aI., 1997).

2.3.2.8.

Hamster

NOS-immunoreactivity has been found in nerve fibres and cell bodies in the myenteric plexus of the proventriculus, stomach and small and large intestines of the golden hamster (Toole et aI., 1998). Neurons which are NOSpositive are also NADPH-d-positive. Moreover, the density of distribution of NADPH-d-positive neurons expressed as a percentage of the total number of cells visualized by PGP 9.5 is similar in the small and large intestine. The percentage of VIP-positive cells is markedly lower than NADPH-d positive ones. However, the percentage of VIP-immunoreactive cells is higher in the distal colon (fig. 8). The co-localization of NADPH-d expression with VIP immunoreactivity has been found in the myenteric plexus of all parts of the GIT of hamster, however the population of VIP /NADPH-d-positive neurons is small. The majority of NADPH-d positive neurons are not VIP immunoreactive.

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.NADPH-d DVIP

40

...

35

:::l

30

t:

0

u

..!!! 25 Qj e iii 20

...

....s 0

~ 0

15 10 5 0 Ileum

Caecum

Proximal colon

Distal colon

Fig. 8. The mean percentage of total cell count showing positive staining for NADPH-d (black colums) and VIP (white columns) in the intestinal tract of golden hamster. Results are expressed as a percentage of the total count of PGP 9.5 positive cells taken as 100% (Based on data from Toole, et al., 1998).

VIP/NADPH-d-positive fibres are also very scanty in tertiary components of the ENS. Although, there is a low occurrence of cells expressing both VIP and NADPH-d immunoreactivity, neurons which are only NADPH-d-positive are surrounded by VIP-positive fibres, which suggests there is a close connection between these two kinds of neurons (Toole et al., 1998). Thus, the overall pattern of distribution of NOS-positive neurons is similar between species, but differs in that the co-localization of NOS and VIP-positive neurons is much less frequent in the hamster intestine than in that of guinea pig or rat (Costa et al., 1992; Aimi et al., 1993).

2.3.2.9.

Nitrergic neurons in adults-summary

In conclusion, based on the available data, our conclusion is that there are no major differences among species in the pattern of distribution of nitrergic neurons along the GlT. Thus, the pattern ofnitrergic innervation is probably common for all mammals and for all vertebrates. However, there are a few small differences in certain parts of the GIT. For example, the highest occurrence of NOS-positive neurons is observed in the myenteric plexus of the oesophagus and the lowest is in the colon. In adult animals, many NOS-positive neurons are present in the myenteric plexus, while in the submucosal plexus the number of neurons positive for NOS is significantly smaller. This distribution difference of NOS-positive neurons in the gastro-

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intestinal wall is common for all parts of the GIT. A small number of nitrergic neurons is seen in the submucosal plexus of the stomach, and small and large intestine of diverse species. On the other hand, NOS-positive fibes are frequently seen in the submucosal plexus, which suggests the involvement of nitrergic transmission between the myenteric and submucosal plexuses. Two other common features of nitrergic neurons are that they preferentially project in the anal direction and innervate circular muscle. These features are present in both the small and large intestine. Since NO is not the only neurotransmitter involved in NANC neurotransmission, the co-localization of NO with other neurotransmitters in neurons has been widely studied. However, the relationship of NOS and VIP (one of the other NANC neurotransmitters) in neurons is not yet clear. There are data showing that considerable populations of neurons express both VIP and NO (Costa et al., 1992; Aimi et al., 1993) but conversely, other data show that the populations releasing these neurotransmitters are mainly separated (Toole et al., 1998). In spite of differences among species in the density of the population of neurons expressing both NOS and VIP, co-localization of these two neurotransmitters can be found in all species. Thus, one could conclude that, at least in part, VIP- and NO-dependent neurotransmission overlaps in neurons of the ENS. On the other hand, SP-positive neurons do not express NOS, indicating that NO-dependent is separate from SPdependent neurotransmission in the ENS. In like manner, there is also no common expression of NOS and choline acetyltransferase or acetylcholine esterase (both enzymes mark cholinergic neurons). This suggests that neurotransmission that depends on acetylcholine is not associated with nitrergic neurotransmission. In general, nitrergic fibres seem to be different from ones that are positive for neurotransmitters such as acetylcholine or SP, that activate gastrointestinal motility. However, a connection between nitrergic and SP transmission seems to exist, since some NADPH-d-positive neurons are surrounded by SP-positive varicose fibres (Aimi et al., 1993). Furthermore, a clear connection between nitrergic and cholinergic transmission can be seen in the oesophagus (Morikawa and Komuro, 1998; Kuramoto and Brookes, 2000).

2.3.3. Distribution of nitric oxide synthase inside enteric neurons NOS-immunoreactivity is patchily distributed in myenteric neurons of the guinea pig ileum and is not specifically associated with any subcellular organelle or with the plasma membrane. Moreover, NOS-immunoreactive fibres are close to smooth muscle cells in the circular muscle layer and some

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of them make synaptic contacts with NOS-immunoreactive and non-immunoreactive enteric neurons (Llewellyn-Smith et al., 1992). In comparison with enteric nerves, both NOS-immunoreactivity and NADPH-d activity of intracardiac neurons in the rat and guinea pig exhibit a light and patchy distribution of NOS-immunoreactive material at the ultrastructural level. However, some NOS-containing intracardiac neurons are very heavily labelled throughout the neuronal cell bodies and their processes (Sosunov et al., 1996). In enteric neurons of the dog ileum, NOS and VIP immunoreactivities are present in the same nerve varicosities, but not in the same organelles. NOS is localized in electron-dense material of undetermined nature, whereas VIP is associated with large granular vesicles. Moreover, SP and NOS are not present in the same nerves (Berezin et al., 1994). NOS-immunoreactivity in the guinea pig small and large intestine is present both in cell bodies and internodal strands. On the other hand, nerve fibres in the tertiary plexus do not show NOS-immunoreactivity (Costa et al., 1992), (fig. 4). In contrast to guinea pig, two forms ofNADPH-d-positive neurons can be distinguished in the rat duodenal myenteric plexus. The first are thick fibres without varicosities, that emanate from intrinsic positive neurons which can be traced to the internodal strands. The others are fine fibres with varicosities, that are seen in the secondary and tertiary components of the myenteric plexus (Aimi et al., 1993).

2.4.

Interstitial cells of Cajal and enteric nerves

In this section the review of ICC will refer to the connection of ICC with nitrergic neurons, the involvement of ICC in NO production, and neurotransmission involving nitrergic neurons.

2.4.1.

Definition of interstitial cells of Cajal

ICC were difficult to recognize for a long time, because of their similarities in structure to other cells present in the gastrointestinal wall. However, the problem of definition of ICC has now been overcome since these cells have been shown to express c-kit, the proto-oncogene which encodes the receptor tyrosine kinase (Sanders et al., 1999). Thus, ICC can be recognized by receptor expression, which is one of the most used tools to identify these cells under the light microscope (Komuro et al., 1996; Faussone-Pellegrini and Thuneberg, 1999). ICC, especially those cells distributed at the deep muscular plexus, also express tachykinin receptor NK1, a subtype of somatostatin receptors, and contain NOS (Vannucchi, 1999).

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2.4.2. Distribution of interstitial cells of Cajal within the GIT and their correlation with nitrergic neurons The number of contacts between ICC, smooth muscle cells, and nerve endings varies according to the location and morphology of ICC. The way from nerve endings through ICC to smooth muscle cells is not morphologically identical at each gastrointestinal level, suggesting that the roles played by the ICC in gastrointestinal motility may differ in different parts of the GIT (Faussone-Pellegrini, 1987a). In the human small intestine, ICC are located primarily in close relationship to the myenteric plexus, as well as between circular muscle layers. In the large intestine, ICC are also present in close affinity with the myenteric plexus, while there are great numbers in the circular muscle layer and in the taeniae of the longitudinal muscle layer (Romert and Mikkelsen, 1998). Neuronal NOS-immunoreactivity has been shown in several neurons, nerve fibres, and nerve endings in the myenteric plexus, and in nerve endings within the muscle layers in the rat ileum. These nerve endings are especially numerous in the deep muscular plexus and they are much closer to ICC than to smooth muscle cells. Some of the ICC in the myenteric plexus are also NOS-immunoreactive indicating that ICC are apparently able to produce NO. Moreover, in the deep muscular plexus, ICC are the ileal type that are very richly innervated by NOS-positive nerves (Matini and Faussone-Pellegrini, 1997). It has been shown that the contacts between ICC and neuronal cells are synapse-like and that ICC in the deep muscular plexus are closely associated with enteric neurons that are NOS or SP immunopositive (Wang, et al., 1999). In the guinea pig, the correlation between interstitial cells and NOS-positive neurons is similar to that in the rat. Another study has shown that c-kit-immunoreactive cells constitute dense reticular networks in the deep muscular plexus and the myenteric plexus of the guinea pig jejunum. Furthermore, the NOS-immunoreactivity occurs in the circular muscle layer, most densely at the deep muscular plexus, as well as within the ganglion strands or connecting strands of the myenteric plexus. In addition, close association between c-kit- and NOSimmunoreactive cells has been shown in the deep muscular plexus (Toma et aI., 1999). Moreover, axon profiles with NOS-immunoreactivity lie closely adjacent to the interstitial cells in the deep muscular plexus, but also close to smooth muscle cells ofthe circular muscle layer. However, the distance between the interstitial cells and NOS-positive axons in the myenteric plexus is considerable. NOS-immunoreactivity has been shown in nerve fibres in the myenteric plexus, circular muscle layer, and occasionally in smooth muscle cells and ICC (Berezin et aI., 1994).

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2.4.3. Functions of interstitial cells of Cajal The ICC have two functions. One is that these cells act as the pacemakers of rhythmic activity (Berezin et aI., 1990; Sanders et aI., 1991; Sanders, 1996; Huizinga et aI., 1998; Thomsen et aI., 1998). The other is to act as intermediary for neural inputs to the muscle. ICC may be involved in mediation of NANC neurotransmission (Huizinga et aI., 1990; Burns et aI., 1996). ICC may exclusively conduct inhibitory neurotransmission, or there may be parallel innervation to both ICC and smooth muscle cells. However, functional studies have shown that ICC can react to neurotransmitters or may produce NO and amplify inhibitory neurotransmission (Sanders, 1996). In the W/Wv mutant mouse ICC are absent in the lower oesophageal sphincter and pyloric sphincter. In these mutants, it has been shown that NO-dependent inhibitory neurotransmission is reduced in animals lacking ICC (Ward et al., 1998). Moreover, hyperpolarization to sodium nitroprusside is also attenuated in W/Wv animals. This study led to the conclusion that ICC play an important role in NO-dependent neurotransmission in the mouse lower oesophageal sphincter and pyloric sphincter by transducing NO signals into hyperpolarizing responses. Similar results have been shown in another study performed by the same group in the mouse stomach wall (Burns, et aI., 1996). Inhibitory nerves consitutively express NOS (Belai et aI., 1992; Ward et aI., 1992; Young et aI., 1992). In the stomach of W/Wv mutant mouse the distribution of inhibitory nerves is normal, but NO-dependent inhibitory neuroregulation is greatly reduced (Burns et aI., 1996). In addition, it has been shown that neurons with NOS and SP immunoreactivities are both closely associated (axonal varicosities) with cell bodies of ICC in the guinea pig small intestine, and ICC may be innervated together with smooth muscle cells. Thus, it is possible that ICC are not exlusive mediators of inhibitory neurotransmission from enteric neurons (Wang et aI., 1999). A study supporting parallel mediation shows that opossum oesophageal neurons are in close proximity with both ICC and smooth muscle cells (Daniel and Posey-Daniel, 1984). Even though the concept that ICC are involved in the generation of electrical rythmicity in the developing GIT is not yet fully accepted, there is some data that suggests that ICC in the deep myenteric plexus are involved in the development of inhibitory enteric transmission (Sanders et aI., 1999). In summary, ICC are important for generating spontaneous activity of the GIT. I) Slow waves originate from specific sites that are formed by networks of interstitial cells of CajaI. 2) Destruction or removal of these cells affects rythmicity of slow wave generation and propagation. 3) Isolated ICC show spontaneous electrical activity which

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may be linked to several voltage-dependent ion channels. 4) ICC are innervated by enteric neurons, and respond to neurotransmitters. In addition, these cells may produce NO and amplify inhibitory neurotransmission, but the close association of SP neurons with ICC suggest that transmission or propagation of excitatory nervous impulses may also be one of the functions of ICC.

2.4.4.

The significance of interstitial cells of Cajal during development of the GIT

The importance of ICC for normal motor function during the development of the GIT has been presented in the case of hypertrophic pyloric stenosis in humans. In the normal pylorus, ICC are present in large numbers, while in cases of pyloric stenosis, the immunoreactivity for c-kit is absent in the major part of the tissue. Moreover, ICC are present only in the inner part of the musculature near the submucosal edge and in the antrum (Vanderwinden et al., 1996). In mouse foetuses, ICC and their precursor cells (fibroblast-like cells rich in mitochondria) are not identifiable among cells present in the colonic submucosal area facing the circular muscle layer. Small numbers of cells regarded as precursors of ICC, are present in unfed neonates. On the other hand, large numbers of the precursor cells are present in suckling mice. Differentiation of the precursor cells into ICC undoubtedly starts during the second week of postnatal life. Some of them have mixed fibroblastic- and interstitial-cell features and some have many interstitial-cell features. During the weaning period, ICC are quite well differentiated. However, they have the same morphology as in adult mice after 30 days of age (Faussone-Pellegrini, 1987b). A study using the gene knockout mouse has shown that enteric neurons are not required for the development of functional ICC. The circular smooth muscle layer, which develops before ICC, is probably the source of stem cell factor required for ICC development (Ward et al., 1999). 3.

FUNCTIONS OF NITRIC OXIDE IN THE ENTERIC NERVOUS SYSTEM IN ADULT ANIMALS

There is a wide consensus that NO plays a role as a neurotransmitter in the ENS. This role has been recently investigated, and it was found that NO is mainly involved in NANC inhibitory neurotransmission in the GIT. However, NANC neurotransmission is not necessarily mediated only by NO. Indeed, there is evidence that NANC neurotransmission is mediated by at

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least four neurotransmitters: adenosine 5' -triphosphate (ATP), VIP, pituitary adenylate cyclase activating peptide (PACAP), and NO (Belai and Burnstock, 2000). It has been reported that NO acts as a neuromodulator in guinea pig ileum (Gustafsson et al., 1990) inhibiting SP transmission, but cholinergic transmission seems to be independent of NO modulation (Gustafsson et al., 1990). NO is released during stimulation of autonomic neurons in the longitudinal and circular muscle of the guinea pig caecum and the taenia caeci via activation of NOS (Wiklund et al., 1993c). NG-monomethyl-L-arginine (L-NMMA) enhances the contractile response to nerve stimulation in longitudinal muscle of the guinea pig ileum (Wiklund et al., 1993a; Wiklund et al., 1993b). Similar results have been reported using longitudinal muscle lying between the taenia caecum isolated from the guinea pig. Nw-nitro-L-arginine and haemoglobin significantly reduced electrically-induced relaxation, showing that this effect is related to NO production (Shuttleworth et al., 1991). Another line of evidence concerning the involvement of NO in inhibitory neurotransmission has been presented in experiments performed on isolated preparations of guinea pig ileum longitudinal muscle. The inhibitory effect of NO (in the form of acidic NaN0 2 ) has been observed in the case of acetylcholine-, SPand neurokinin A-induced contractions (Wiklund et al., 1993a). NOS inhibition modifies contractile responses to application of acetylcholine, SP, or direct muscle stimulation, indicating that endogenous NO has a mainly prejunctional inhibitory action on cholinergic and SP-like neurotransmission. This suggests that inhibition of NO synthesis enhances prejunctional neurotransmission (Wiklund et al., 1993a). Other actions of NO reported in the GIT are: regulation of gastrointestinal motor function such as sphincter opening (Tottrup et al., 1991a; Tottrup et al., 1991b; Rattan and Chakder, 1992; Rattan et al., 1992; Preiksaitis et al., 1994; Grisoni et al., 1996), limiting intestinal contractile activity e.g. of the circular muscle in the guinea pig ileum (Suzuki et al., 1994), gastric receptive and adaptive relaxation (Desai et al., 1991a; Desai et al., 1991b), accommodation process of the circular muscle in the small (Waterman et al., 1994a) and large (Ciccocioppo et al., 1994) intestine. NO is also involved in regulation of peristalsis (Waterman and Costa, 1994; Waterman et al., 1994b). In the rat and ferret gastric fundus, NO is mainly involved in short-lasting relaxations and initiating sustained relaxations (Li and Rand, 1990; D' Amato et al., 1992; Grundy et al., 1993). In circular muscle strips of the guinea pig gastric fundus, NO acts as a neurotransmitter during sustained relaxation (Lefebvre et al., 1992; Desai et al., 1994). However, in the longitudinal muscles of the rat ileum (Bartho et al., 1992; Bartho and Lefebvre, 1994a; Bartho and Lefebvre, I994b; Bartho and Holzer, 1995)

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and opossum oesophagus (Saha et a1., 1993), it has been reported that the first reaction to NO is excitatory. Certainly, NO is capable of dual excitatory and inhibitory effects on intestinal motility. In vitro studies show that sodium nitroprusside induces a prompt, concentration-dependent stimulation of peristalsis of isolated guinea pig intestine and has biphasic effects on longitudinal muscle contractions. The contractions are sensitive to atropine, which suggests that the excitatory effect involves cholinergic motor neurons, while relaxation is induced by a direct influence on the smooth muscle (Holzer et a1., 1997). In summary, studies of gastrointestinal function in adult animals show that NO plays an important role as a neurotransmitter in NANC nerves in the ENS. Nitrergic neurons are involved in the relaxation of sphincters, adaptive relaxation of the stomach and intestine, and in regulation of peristalsis. However, the role of inhibitory neurotransmitter in the ENS is not restricted only to NO because VIP, PACAP, and ATP are also involved (Hata et a1., 1990; D' Amato et a1., 1992; Smits and Lefebvre, 1996; Bartho et a1., 1998; Fernandez et a1., 1998; Glasgow et al., 1998; Krantis et a1., 1998; Belai and Burnstock, 2000).

4. NITRERGIC NEURONS IN THE DEVELOPING GASTROINTESTINAL TRACT 4.1.

Development of the enteric nervous system

The ENS of mammals and birds is a part of the peripheral nervous system. The characteristic feature of this system is that it can work independently, without any input from the central nervous system (CNS). Local ENS reflexes thus control many functions of the GlT, the ENS integrates information from the lumen, adjusting muscle contractility and secretion accordingly. This ability makes the ENS similar to the CNS. Despite this feature that differentiates the ENS from the rest of the peripheral nervous system in vertebrates, nevertheless the ENS originates from the same tissue. Detailed studies of the migration of neuroblasts and the formation of the ENS are possible because of several specific markers that allow cells to be labelled and their movements observed within the embryo. For example, in avian embryos HNK-1 was found to be a unique marker for neural crest derived cells (Newgreen et al., 1996; Mogi et al., 2000), while in the case of mammalian embryos several markers can be used: DbH-nLacZ-transgene, which is composed of b-galactosidase and dopamine b-hydroxylase (Kapur et al., 1992); C-RET-proto-oncogene of receptor tyrosine kinase (Pachnis et al., 1993; Young et a1., 1998); Phox2b--one of the homeodomain transcription

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factors--{Pattyn et al., 1997; Young et al., 1998); HNK-l-earbohydrate epitope expressed by neural crest cells (Chalazonitis et al., 1994, Young et al., 1998); SOX-IG-gene expressed in normal neural crest cells, but expression of which is disrupted in the mouse model of Hirschsprung disease (Southard-Smith et al., 1998); MASH-l--{mammalian achaete-scute homolog 1) gene required for normal differentiation of neuroblasts (Guillemot et al., 1993; Lo and Anderson, 1995); and p75-low affinity nerve growth factor receptor (Young et al., 1998). This information allows a general model of the interactions occurring during development of ENS to be drawn. The source of all branches of the peripheral nervous system is the neural crest-a transient structure of vertebrate embryos, which is formed on the edges of the neural folds as they fuse in the dorsal midline to form the neural tube. The ENS originates from cells that emerge from the posterior hindbrain and anterior spinal cord. These cells migrate ventrally to the cervical branches of the dorsal aorta and dorsally to the foregut. The neural crest-derived cells proliferate and colonize the entire bowel. The growth and maturation of the ENS is under the control of growth factors such as glial-derived, neurotrophic factor endothelin-3 (Natarajan and Pachnis, 2000).

4.2.

4.2.1.

Changes in NOS expression during foetal and postnatal development

General changes

There is much data showing that NOS expression changes during development. These changes are observed in the CNS, the respiratory system and in the ENS. Knowledge about NO has mainly focused on its action on vascular smooth muscles and on NANC neurotransmission in the ENS. In addition, NO is a potent cytotoxic compound. It can also act as a second messenger in the cell cytoplasm. This multipotentiality of the molecule and the fact that during development the production of NO, expression of NOS and distribution of nitrergic neurons change substantially in some tissues may reflect important roles of NO. This raises the question of what role NO plays in the whole mechanism during embryogenesis and postnatal development of the ENS. In rats, the cerebral cortical plate and hippocampus nNOS is transiently expressed during embryonic and early postnatal development. In adult animals the expression of NOS is absent in the olfactory epithelium, (Verma et al., 1993) but during the embryonic and early postnatal stages NOS is evidently expressed in this structure (Bredt and Snyder, 1994). Transient

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expression of NOS has also been observed in dorsal root ganglia. In rats in the embryonic state most cells express NOS, with the most intensive staining at E14 and E15. At E19, the pattern of staining resembles that in adult animals (Wetts and Vaughn, 1993). Similarly, NOS is expressed in the dorsal root ganglion in embryonic chicks (Ward et al., 1994). In the CNS, NO may participate in synaptic plasticity and neuronal death. These processes are prominent during development of the nervous system. The influence of NO on synaptic plasticity has been shown in studies of the effects of NOS inhibitors on the expression of long-term potentiation (Bohme et al., 1991; Schuman and Madison, 1991; Haley et aI., 1992). Another action of NO has been shown following damage of the spinal cord - i.e. following damage to the ventral roots NOS is expressed in spinal motoneurons which were previously NOS-negative (Wu, 1993). Thus, it is possible that NO plays a role in regeneration of damaged neurons. Another function of NO during development is postulated to be the mediation of the cytostatic effects of Nerve Growth Factor (Peunova and Enikolopov, 1995). These authors have shown that Nerve Growth Factor induces different forms of NOS in neuronal cells and that NO acts as a cytostatic agent in these cells. Thus, inhibition of NOS leads to inhibition of Nerve Growth Factor-induced cytostasis and thereby prevents full differentiation. The data suggests that induction of NOS initiates cytostasis during differentiation (Peunova and Enikolopov, 1995). The successful transition from foetal to neonatal life involves a marked decline in pulmonary vascular resistance that is modulated in part by endothelium-derived NO. During foetal development NOS expression is found in rat lung (North et al., 1994). Constitutive eNOS and nNOS proteins are detectable at E16, however maximal levels are observed at E20. During postnatal 1-5 days (PI-P5) the levels of both constitutive eNOS and nNOS decrease. A report supporting the previous data, showed that in the pig NOS-containing nerves supplying the airway smooth muscle are most abundant in the neonate, the density of NOS-positive nerves decreasingwith age (newborn, 38%; 16 weeks old, 18%) (Buttery et aI., 1995). Similarly, in the foetal rat NOS protein becomes more abundant during development, to reach maximal levels in perinatal life (North et al., 1994). The distribution of nNOS in foetal, neonatal, and adult mouse lung have been investigated with NADPH-d histochemistry and nNOS immunochemistry. NADPH-d positive cells can be detected at E13, while NOS-positive cells first appear at £15 in a small population of neurons. However, comparison of NOS and NADPH-d staining shows no apparent differences in terms of location and frequency during the rest of development. NADPH-d positive labelling is present both in cell bodies and in the terminal nerve fibres. NADPHd/NOS-positive neuronal cell bodies are present in the hilar region and

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bronchiolar wall, as well as in neuronal processes during all stages of foetal and postnatal development. However, NOS-positive terminal nerve fibres with varicosities are more frequent in pulmonary blood vessels than in the airways. NOS-positive nerve plexuses are also present in tracheae. However, there is no perinatal increase in the number or intensity of staining of nNOS-positive nerve structures (Guembe and Villaro, 1999). These studies suggest that in the CNS and respiratory system NO has its own specific role during foetal and postnatal development. Interestingly, in both the CNS and respiratory system the expression of NOS changes significantly during the early postnatal development. These facts suggest the involvement of NO in developmental mechanisms in these tissues.

4.2.2. 4.2.2.1.

Gastrointestinal tract Mouse

In the mouse, (also in rat, guinea pig, hamster and cat) NOS-containing neurons (visualized by NOS immunochemistry and NADPH-d histochemistry) are found in the muscular and submucosal layer throughout the gut. The population of myenteric neurons is prominent (Ekblad et al., 1994). However, during development of the intestine the frequency of NOS-positive neurons changes with the stage of development. Immunoreactivities of NOS and neuron specific enolase (NSE) have been shown in organ culture of embryonic mouse (Young et al., 1998). In this preparation, the distribution of NOS significantly differs between midgut and hindgut during development. In this study NOS and NSE were expressed from E 1 to E11.5 showing that both NOS and NSE in the midgut are present earlier than in the hindgut By way of comparison, Phox2b-positive cells are present in the mesenchyme adjacent to the foregut at E9.5. By EI0, Phox2b positive cells are present in the foregut mesenchyme. At ElLS the positive cells reach the caecal swelling, and by E12 they colonize the caecum and one-third of the hindgut. Finally, by E14 Phox2b positive cells are present throughout the entire gut of the embryonic mouse (Young et al., 1998). This data shows that NOS in the gut is expressed in the early developing ENS. However, there are no data about the production of NO and its function during development in the mouse. In the myenteric plexus of adult mice, NOS-positive neurons are abundant (Grozdanovic et al., 1992; Sang and Young, 1996). The number of NOS-positive neurons comprises 31.8% of all myenteric neurons. On the other hand, only 3% of submucosal neurons show NOS-immunoreactivity (Young and Ciampoli, 1998) (fig. 9).

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Fig. 9. Percentage of NOS-immunoreactive cells in developing intestine in mouse. Data expressed as percentage of total myenteric and submucosal neurons protein gene product 9.5 (PGP9.5)-stained in mouse at the day of birth and in adult animals (Young and Ciampoli, 1998).

During foetal development the expression of NOS changes in the submucosal plexus. Detailed study of the expression of NOS during foetal and postnatal development of the GIT of the mouse has shown that the first NOS-immunoreactive neurons are present in outer mesenchyme of the small intestine at E12 (Young and Ciampoli, 1998). These neurons are present in the presumptive region of the myenteric plexus. Another study has shown that NADPH-d activity in submucosal and myenteric neurons is first found on day Ell in clusters of cells in the dorsal mesogastrium. Moreover, these cells express neurofilament immunoreactivity and thus are developing along a neuronal lineage. Enteric neurons that express NADPH-d activity appear in the stomach on day E12. At this time, NADPH-d-containing cells are no longer present in the dorsal mesogastrium (Branchek and Gershon, 1989).

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At E16, the number of NOS-positive neurons increases and the majority of NOS-positive neurons in the myenteric plexus are present in ganglia. At this stage, in the region of the presumptive submucosal plexus, NOSpositive nerve fibres develop between the external smooth muscle and the mucosa but there are no NOS-positive cell bodies. At E 18, there are NOS-positive cell bodies in the submucosal plexus and most of them show weak immunoreactivity, but some are strongly immunoreactive. On the day of birth (PO), NOS-immunoreactivity is observed in the myenteric plexus (myenteric ganglia and nerve terminals) and in the circular muscle. In the submucosal plexus many neurons show strong NOS-immunoreactivity. In contrast to the submucosal plexus, the percentage of neurons that show NOS-immunoreactivity in the myenteric plexus does not change significantly during development (Young and Ciampoli, 1998). In the submucosal plexus at P7-P9, large variation of NOS-immunostaining is exhibited. An increase in the ratio of NOS-positive cells is observed at P14-P22 and by P42 the pattern of NOS-positive cells cannot be distinguished from that in adult animals (Young and Ciampoli, 1998). The results of these studies question the importance of NO production during the development of the ENS. The functional significance of changes of NOS expression in the submucosal plexus may indicate that NO is involved in regulation of mucosal function in the early postnatal stage. There are two possible explanations of the significance of transient NOS expression during the development of the ENS. The first, is the possibility that NO may act as a neurotransmitter or neuromodulator only in the foetus and the early postnatal period, while after this time it loses this function in the gut. Unfortunately there is not enough data to discuss this problem in detail. The fact that the myenteric plexus achieves its NOS expression level almost at the same time as the submucosal plexus, while after birth the percentage of NOS-positive neurons in the submucosal plexus dramatically decreases, may suggest that regulation of secretory functions and neurotransmission between the mucosa and ENS do not require much NO in adult life. On the other hand, in the large intestine of many adult animals, there are significant numbers of NOS-positive neurons in the submucosal plexus (Grozdanovic et aI., 1992; Ward et aI., 1992; Young et aI., 1992; Barbiers et aI., 1993; Furness et al., 1994; Timmermans et aI., 1994b). However, in the large intestine of adult animals, NOS-positive neurons innervate the circular muscle and other submucosal ganglia, while in the small intestine the submucosal NOS-positive neurons appear to innervate the mucosa (Young and Ciampoli, 1998). Thus, the changes in NOS expression during foetal and early postnatal life may reflect the functional involvement of NO in the regulation of neurotransmission in the intestine.

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The second explanation of the transient expression of NOS in the ENS during foetal and postnatal development is that NO may playa role in neuronal development by establishing synaptic connections and/or influencing cell survival (Bredt and Snyder, 1994; Roskams et al., 1994). We presented above evidence that NOS expression changes in the CNS, particularly in the dorsal root ganglia, the cerebral cortical plate, the hippocampus, and the olfactory epithelium (Wetts and Vaughn, 1993; Bredt and Snyder, 1994; Ward et al., 1994). Moreover, the time course of expression of NOS in the cerebral cortical plate (Bredt and Snyder, 1994) is similar to that in the submucosal plexus in the small intestine (Young and Ciampoli, 1998). It has been suggested that NO plays a role in the use-dependent modification of synaptic efficacy in the developing nervous system, because in adult life the extracortical non-transient-NOS-positive neurons express NOS when they stop dividing and begin to extend processes (Bredt and Snyder, 1994). A similar situation has been observed in developing olfactory neurons that express NOS during process extension. Olfactory neurons also become NOS-positive after bulbectomy, as they replace processes which were lost (Bredt and Snyder, 1994). Thus in the olfactory system, transient NOS expression is a feature of both ontogeny and regenerative response to injury.

4.2.2.2.

Guinea pig

In the guinea pig model it has been shown that NO may playa role during adaptation, after severance of extrinsic nerves. The number of NADPH-d positive cells in the myenteric plexus increases I week after (surgical) extrinsic denervation of an isolated loop of guinea pig ileum. On the other hand, NADPH-d staining in submucosal ganglia is not affected by the denervation. Similar data obtained after systemic capsaicin administration suggest that primary afferent denervation also increases NADPH-d staining (Yunker and Galligan, 1994). A further study, (Yunker and Galligan, 1998) confirmed that the number of NOS-positive neurons increases in the myenteric plexus but not in the submucosal plexus after extrinsic denervation or systemic capsaicin treatment in the guinea pig. Moreover, it has been shown that the increase in number of NOS-positive neurons is associated with potentiation of inhibitory transmission to longitudinal muscle in denervated intestine. The increase in number of NOS-positive neurons seems to be permanent, since twenty four weeks after the denervation the number of NOS-positive cells is still elevated (Yunker and Galligan, 1998). A developmental study performed by another group of researchers demonstrated age-related changes in the localization of NADPH-d in the ganglionated plexus of the guinea

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pig gallbladder. At embryonic day E52, all the PGP 9.5-immunoreactive neurons show positive reaction for NADPH-d. At 2-4 days after birth the relative number of nitrergic neurons decreases, reaching a minimum of 26.7% of the total number of neurons. The rate rises slightly to 33.6% at 6 months. Finally, the value returns close to 100% at 2 years of age (Siou et al., 1994). Thus, a similar situation can be observed as presented in mouse CNS and ENS - the transient expression of NOS with a significant decrease occurring after birth. Moreover, there is a similarity with the results observed in the regenerative response to injury of the olfactory bulb (Bredt and Snyder, 1994). Thus, we speculate that NOS expression in the myenteric plexus of the denervated intestine may be a manifestation of the adaptational process occurring in the ENS. The results of experiments performed with this model in comparison with foetal and early postnatal changes of NOS expression suggest that NO is an important factor during formation of the ENS connections. However, the differences between the myenteric and submucosal plexuses give some doubts about a unified hypothesis. Thus, further studies of the involvement of NO in the development of the ENS are necessary to elucidate the insight of the mechanism. In summary, based on up-to-date data, obtained by comparing developing animals with the denervated intestine model, one can suggest that NO plays an important role in the adaptation of intestinal function during foetal and postnatal development.

4.2.2.3.

Pig

Nitrergic neurons are present in the outer and inner submucosal plexus of the pig upper small intestine (Brehmer et al., 1998). NOS-expressing neurons develop together with the structures of the plexuses in the pig duodenum, from 50 days of pregnancy to 6-8 weeks (weaning at 5 weeks) after birth. Although the myenteric and outer submucosal plexuses, are already composed of ganglia and interconnecting strands at the beginning of the second half of the pregnancy, the inner submucosal plexus has not at this time been differentiated from these structures. A gradual development of ganglia and interconnecting strands is then observed. Consequently, at the time of birth, every enteric plexus possesses its own typical arrangement and shape of ganglia and nerve strands. Furthermore, the study shows that the density and number of NOS-expressing neurons change during development. In the outer submucosal and myenteric plexuses, the density of NOS-expressing neurons decreases constantly from 50--69 days of gestation to weaning, whereas the number of these neurons increases significantly. On the other hand, in the inner submucosal plexus, the density of these neurons increases markedly up to the end of

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pregnancy. In neonates, the density of NOS-expressing neurons in the inner submucosal plexus is markedly lower and the level is unchanged in weaned piglets. However, the number of these neurons significantly increases after weaning, while the density remains at the same level. In contrast, in spite of a significant increase in the number of NOS-positive neurons in the myenteric plexus, their density constantly decreases during development. The pattern of NOS-positive neurons is similar to that observed in other species (Ekblad et al., 1994). Morphological studies of neuronal projections (Costa et al., 1992; Ekblad et al., 1994; Timmermans et al., 1994b) suggest that NOS-positive neurons in the pig small intestine serve as interneurons and inhibitory motorneurons. These data are supported by findings that NO can modulate intestinal reflexes in addition to its inhibitory action on neuromuscular transmission (Yuan et al., 1995). Since the function of NOS-positive neurons has been investigated only in 6-8 week old pigs, the function of NO during foetal and early postnatal period of life may be different from that in older pigs. The pattern of changes of NOS-expression in the submucosal plexus is similar to that observed in the CNS and the gall bladder of other species (Wetts and Vaughn, 1993; Bredt and Snyder, 1994; Siou et al., 1994). Moreover, there is similarity to the mouse small intestine during foetal and postnatal development (Young and Ciampoli, 1998). The involvement of NO in the maturation of neurons was shown by the example of the ventral hom (Kalb and Agostini, 1993). In this study NOS antagonists blocked the molecular maturation of motor neurons and this effect is likely to be mediated by a subpopulation of the ventral motor neurons that transiently express NOS during early postnatal life. These results suggest that the local production of nitric oxide within the ventral hom may contribute to a late phase in the differentiation of the motor neuron.

4.2.2.4

Human

The developing human GLT is capable of producing NO, but the density of NOS-containing neurons differs between the foetus and neonate (Timmermans et al., I994a). This immunohistochemical study shows that the total number of NOS-containing neurons in the proximal jejunum and proximal colon of a 32 weeks old foetus is much higher than that in a 2 month old newborn. NOS-containing neurons are present in the distal ileum of the newborn, but not the foetus. The density of nitrergic neurons in adult humans is higher in the myenteric plexus than in the submucosal plexus. A study on the development of human oesophagus innervation has shown that maturation of the muscle layers and innervation occurs from 8 weeks

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of gestation, until foetal swallowing commences at 16 weeks. Immunoreactivities of the general nerve markers, PGP 9.5 and synaptophysin are present throughout the cytoplasm of immature neurons at 8 weeks of gestation, while from 10 weeks synaptophysin immunoreactivity is localized only at nerve terminals. A glial cell marker, SlOO immunoreactivity, is also detected from 8 weeks of gestation. Moreover, in this study the histochemistry of NADPH-d was used to visualize nerves in the developing oesophagus, but this method was used to demonstrate only myenteric neurons. A definite NADPH-d reaction first appears in foetal oesophagus at 8 weeks of pregnancy (Hitchcock et al., 1992). It has been reported that in human small intestine distribution of NOSpositive nerves changes with age (Belai and Bumstock, 2000). A subpopulation of myenteric neurons from preterm human foetal stomach and small intestine contain NOS and ATP as early as 6-17 weeks of gestation. The density of NADPH-d positive neurons is significantly higher in foetuses (14-19 weeks of gestation) compared to adults (42-71 years old). NADPH-d-positive neurons occupy 41% of all neurons (PGP 9.5-positive) in foetuses, while in young adults this number is two folds smaller - 20%. The level of NADPH-d positive neurons increases again in the elderly, such that the percentage of nitrergic neurons reaches 56% in 78-86 year old individuals, which is higher than in young adults or in foetuses. These findings show that in humans the density of NOS-positive neurons decreases after birth. These data are different than those in the mouse, where the density of NOS-positive neurons in the myenteric plexus does not change significantly after birth. However, the decrease in density of NOS-positive neurons after birth in humans seems to be a general rule for the development of the ENS. Thus, two functions can be postulated for NO in the development of human intestine: firstly, regulation of intestinal motility as an inhibitory neurotransmitter and secondly, involvement in the development and/or maturation of human ENS (Belai and Bumstock, 1999). The appearance, distribution and some histochemical features of nonneuronal cells associated with the myenteric plexus of human foetal small intestine have been studied in the 10th and 17th week of gestation (Fekete et al., 1999). It was shown that cells with immunoreactivity for glial proteins (S-IOO protein, glial fibrillary acidic protein (GFAP)) are already present in the 1Oth week of gestation. Cells with S-lOO protein immunoreactivity are located within the circular muscle layer, the myenteric, and submucosal plexuses. On the other hand, GFAP immunopositive cells are mainly restricted to the side of the myenteric plexus adjacent to the longitudinal muscle layer. In contrast to the dense network formed by S-IOO protein immunopositive structures, the GFAP immunopositive cells appear

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singly and do not connect into a network. NOS-immunoreactive varicosities are in close association with the surface of S-100-positive cells in both the circular muscle layer and in the tertiary plexus. Thus, the conclusion of the study was that two populations of non-neuronal cells with different locations and different immunohistochemical characters appear and develop together with the enteric ganglia in the human foetal intestine. Furthermore, the close morphological relation of NOS-immunoreactive fibres with the S-100 protein immunopositive cellular network indicates a functional relationship between S-IOO protein immunopositive cells and nitrergic nerves during the early development of human ENS (Fekete et aI., 1999).

4.2.2.5.

Rat

Increased biosynthesis of NO during pregnancy in rats has been demonstrated by the following data. First, levels of the stable NO metabolite, nitrate, are elevated in the plasma and the urine in pregnant rats. Moreover, urinary excretion of nitrate is also increased in pseudopregnant rats. Second, the cOMP level in the urine is also increased during pregnancy and pseudopregnancy, paralleling the rise in nitrate. In addition, chronic treatment with a NOS inhibitor limits the increase in urinary nitrate excretion. Finally, NO haemoglobin is detected in the blood of pregnant rats by electron paramagnetic resonance spectroscopy (Conrad et aI., 1993). Constitutive eNOS activity in rats, increases during the first 20 postnatal days and decreases after weaning at 25 days. In addition, lipopolysaccharide treatment results in a significant increase in iNOS activity. The maximal activity of iNOS occurs between 10 and 15 days of age. These data suggest that constitutive eNOS activity in the neonatal rat colon may be important during development and maturation of that tissue. Furthermore, the colon of the preweaned rat is more susceptible to the detrimental effects of lipopolysaccharide-induced NO production than that of postweaned animals (Brown and Tepperman, 1997).

4.2.2.6.

Birds

A study has been made of enteric nitrergic neurons from different regions of the digestive tract of the embryonic, neonatal and adult quail, using whole mounts and sections (Boros et aI., 1994). This study showed that NADPH-d is first found at embryonic days 4 and 5 in the mesenchyme of the gizzard primordium and at the caeco-colonic junction. Furthermore, at embryonic

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day 6, nitrergic neurons already begin to form myenteric nerve networks in the proventriculus, gizzard and proximal part of the large intestine. Finally, at embryonic day 9, myenteric networks can be visualized along the entire digestive tract. In addition, at the level of the stomach, this network is confined to the area covered by the intermediate muscles. NADPH-d-positive myenteric neurons in the distal parts of the blind-ending paired caeca also become organized into ganglia at embryonic days 12 and 13. From this developmental stage on, a submucosal nitrergic nerve network, sandwiched between the lamina muscularis mucosa and the luminal side of the outer muscle layer, becomes prominent in the proventriculus and the intestinal walls.

5.

THE INVOLVEMENT OF NITRIC OXIDE IN THE FOETAL AND POSTNATAL DEVELOPMENT OF THE GASTROINTESTINAL MUSCLE

The GIT changes rapidly during development. During this time, from the early foetal stage until weaning (in mammals), it develops all the functions of the adult organ. The mechanisms regulating this process involve the development of the ENS and are a part of the whole neuronal network - the nitrergic innervation. Early studies showed that the functional innervation during development of the GIT begins in sequential manner (Gershon and Thompson, 1973). The rat small intestine reacts to electrical field stimulation at E 15, but the reactions are observed in only 50% of the preparations, although both acetylcholine and excess potassium evoke a response in all preparations. These results suggest that in the rat intestine, functional cholinergic innervation is established at least by embryonic day 16 (Miyazaki et aI., 1982). Another study has shown the development of non-adrenergic responses in rat stomach (Ito et aI., 1988). These authors reported that electrical stimulation of the circular muscle layer of the gastric corpus of a foetus on the 18th day of pregnancy, induces a biphasic response consisting of a contraction followed by a long lasting relaxation. These responses are TTX-sensitive and guanethidine-resistant, suggesting a non-adrenergic origin. Thus, these data show that non-adrenergic innervation in the foetal rat stomach starts to function at day 18 (Ito et aI., 1988). Stomach motility changes during postnatal development, and in the dog gastric antral contractions increase from 0.2 contractions per minute at birth to a maximum 2.3 contractions per minute on the 11th day, before gradually declining (Malloy et aI., 1979). A study of the contractility of rabbit gastric muscles during postnatal development has shown that contractility of antral smooth muscle strips induced either by acetylcholine

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or potassium chloride is significantly smaller in newborn animals than adult ones. Moreover, the chemically skinned muscles of newborn rabbits showed significantly lower calcium-induced contractility (Zitterman and Ryan, 1990). Similarly, the bethanechol-induced maximal response in the smooth muscle of the rabbit proximal stomach is nearly 4-fold greater in weaning animals than in neonates. Maximum contractile response increases with age for high extracellular K+, and SP, but not for serotonin, cholecystokinin octapeptide, neurotensin, or bombesin. SP and serotonin are more potent in neonates than in weaning animals (Tomomasa et al., 1989). Similar differences have also been observed in muscle strips of gastric fundus from foetal and adult guinea pigs. Irrespective of the agonist, gastric muscle of adult animals develops more active force than that of foetuses, probably because of an increased influence of extracellular calcium (Paul et aI., 1994). The data presented here show that during foetal and postnatal development the gastrointestinal muscles change until eventually they can fulfill all functions of the adult organ. In general, these changes correspond with increase of contractility and the use of extracellular calcium, but the development of mechanical activity of gastrointestinal muscle is not only attributable to certain quantitative changes. It has been shown, that during the postnatal period the response of rat duodenum to purinergic agonists - ADP and ATP shifts from a contraction (1-7 days after birth) to a relaxation (around the third postnatal week). The ATP-induced contractions are resistant to hyoscine or TTX, suggesting that cholinergic neurons are not involved (Furukawa and Nomoto, 1989). Similarly, neurogenic contraction induced by nicotine in the neonatal rat duodenum changes to relaxation in adult animals (Irie et aI., 1994). This nicotineinduced relaxation involves NO production (Irie et aI., 1991) while relaxation induced by ATP or VIP is NO independent (Irie et aI., 1991; Irie et aI., 1992). Thus, the relaxant response of the GIT during development involves maturation of nitrergic, peptidergic, and purinergic transmission. Inhibition of NOS by non-selective inhibitors results in foetal growth retardation (Diket et aI., 1994). In particular, it has been observed that NG-nitro-L-arginine methyl ester (L-NAME) induces a dose-dependent reduction in placenta and pup size in addition to reduction of amniotic fluid levels of cyclic guanosine monophosphate. Moreover, haemorrhagic necrosis of foetal hind limbs also occurs due to treatment with L-NAME, while the pathological changes are prevented by coadministration of (the NO donor) sodium nitroprusside. In another study (Miller et aI., 1996) it was observed that L-NAME administration causes a paradoxical increase in NO synthesis in rats, indicating that a lack of NO may not be the cause of the foetal growth retardation. Indeed, in contrast to what was observed

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in the other study (Diket et al., 1994), supplemental L-arginine or NO donors failed to reverse the effects of L-NAME on foetal or placental size. The effect of endotoxin is similar to that of NOS inhibitor; the reductions in foetal and placental size and increased NO synthesis are comparable to those seen with L-NAME. In addition, it has been shown that iNOS is consitutively expressed in the pregnant uterus and placenta, while it is absent in the non-pregnant state. Interestingly, neither L-NAME nor endotoxin modifies the expression of iNOS. In rats treated with L-NAME or endotoxin, apoptosis is evident in the placenta and uterus and peroxynitrite formation is co-localized with apoptosis in the L-NAME and endotoxintreated rats. Thus, based on these data is has been concluded that inhibition of NOS induces retardation of foetal growth by a net reduction in cellular proliferation due to the induction of apoptosis, which is probably a response to peroxynitrite formation (Miller et al., 1996). In the face of these data we can conclude that NOS plays an important role in general development, however the precise involvement is still not clear. The increased production of NO after administration of NOS inhibitors is confusing, suggesting that NO production may not be the factor responsible for the observed effects on foetal growth. Moreover, these results suggest that the function of NOS during development is not only limited to production of NO, but may also be involved in lowering peroxynitrite formation. On the other hand, there is a strong line of evidence that NO formed by the constitutive isoforms of NOS, is a critical determinant of foetal and neonatal growth and maturation. A study of the effects of aminoguanidine, (predominantly inhibits iNOS) and L-NAME (non-selective NOS inhibitor) on development of rats shows that inhibition of constitutive NOS (cNOS) compromises foetal and placental growth. In addition, postnatal inhibition of cNOS compromises neonatal growth expressed as asymmetric retardation, with brain sparing, suggesting a nutritional origin. Furthermore, cNOS inhibition causes growth retardation. During the postnatal period inhibition of cNOS evokes gastrointestinal effects characterised by hypertrophic pyloric stenosis, and increases in stomach weight/pup weight ratio, and stomach volume/pup weight ratio. Moreover, a concomitant decrease in small intestine weight/length ratio, muscularis hypertrophy at the pyloric sphincter and elevated arterial blood pressure are seen in animals in which NOS was inhibited during postnatal development (Voelker et al., 1995). These findings have been supported by a study performed in mutant mice. It has been reported that in the genetically changed mouse it is possible to achieve low catalytic activity of NOS due to a defective nNOS isoform. The most evident effect of disrupting the neuronal NOS gene was the development of grossly enlarged stomachs, with hypertrophy of the pyloric sphincter and the circular muscle layer. This phenotype resembles infantile pyloric

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stenosis in humans, in which gastric outlet obstruction is associated with the lack of NADPH-d-positive neurons in the pylorus (Huang et al., 1993). These two lines of evidence show that it is most likely that NO plays an important role in development and maturation of the GIr. Thus, the results suggest that NO is a part of the regulatory system that controls motor function of the pyloric sphincter. The possible mechanism of hypertrophic pyloric stenosis induced by inhibition of cNOS involves most likely the inhibition of inhibitory nitrergic innervation to the pyloric muscle. It leads to constant constriction that evokes muscle hypertrophy. As a result, the stomach contents cannot pass to the small intestine, which causes malnutrition and growth retardation. In addition to the local effects of nitrergic regulation, sufficient production of NO during foetal development is necessary for the growth of the foetus as well as the placenta (Diket et al., 1994). It is unlikely that inhibition of placental growth results from decreased umbilical blood flow due to reduction of raised umbilical-placental vascular tone (Chang et al., 1992; Mule et al, 1997). During pregnancy NO contributes to maternal vasodilatation and uterine immune suppression of normal pregnancy, since the production of NO in the whole body has been shown to be elevated in pregnant animals (Conrad et al., 1993; Yang et al., 1996). Inhibition of NOS leads to vasoconstriction in the foetal circulation (Bustamante et al., 1996), placenta (Chang et al., 1992; Myatt et al., 1992), while maternal blood pressure is not affected (Diket et al., 1994). The importance of NO production for the foetus is supported by the fact that the concentration of nitrate in the foetus is 9-fold higher than in the mother sheep during pregnancy. This increased NO synthesis, may in part mediate the cardiovascular adaptations to normal pregnancy and the low systemic and umbilical vascular resistance in the foetus (Yang et al., 1996). NO production is crucial for the general development of the foetus. In addition to its involvement during development of the GIr, NO plays a role in regulation of cardiovascular function in the foetus. NO has been reported to be critical for maintaining the calibre of major arteries of the immature rat foetus. Moreover, the isoform responsible for production of NO during pregnancy, iNOS, most likely plays an important part in the regulation of foetal circulation. Significant constriction of the great vessels and ductus arteriosus was observed in foetuses receiving a selective iNOS inhibitor, whereas both lipopolysaccharide and sodium nitroprusside dilated these vessels. Moreover, the vasorelaxant effect of lipopolysaccharide was blocked by inhibition of iNOS. These results suggest that NO generated by iNOS plays a significant role in controlling the calibre of the major vessels and the ductus arteriosus in the rat foetus, whereas iNOS is not involved in the regulation of circulation in adults (Bustamante et al., 1996). Gastrointestinal blood flow and oxygen delivery decrease and vascular resistance increases after

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inhibition of NOS. Such changes are seen in the stomach, small intestine, and proximal colon and caecum, but not in the middle and distal colon and rectum. In addition, reduction of blood flow is greater in the gastrointestinal circulation than in the rest of the foetal circulation. Moreover, the blood flow to the mucosal-submucosal layer of the small intestine is also reduced by inhibiting NOS. On the other hand, the blood flow in the muscularis serosa of the small intestine remains at the same level. These results suggest that NO is an important regulator of vascular tone in the developing gastrointestinal circulation (Fan et aI., 1996; Fan et aI., 1998). The involvement of NO in the regulation of circulation in early ontogeny is supported by the fact that ischaemia-reperfusion has an age-dependent effect on endothelial production of NO in vitro by postnatal mesenteric artery. In addition, these changes mirror the effects of ischaemia-reperfusion on gut vascular resistance in vivo (Nowicki, 1996). Thus, NO plays dual role in the regulation ofthe developing GIT. One is the direct effect on gastrointestinal tissue while the second is a role as a regulator of blood flow.

6.

FUTURE PERSPECTIVES - CONCLUSIONS

In conclusion, NO production can have at least two important functions during the development of the GIT. The first function is a general effect of NO on either the placental or foetal circulation. A lack of appropriate NO actions on umbilical and placental blood vessels causes malnutrition of the foetus and consequently growth retardation which secondarily may influence development of the GIT. The second function of NO during maturation of the GIT is related to the development of the function of the pyloric sphincter muscle. The nitrergic innervation is very important for the growth, maturation, and proper function of the GIT since the inhibition of cNOS causes severe impairment of gastrointestinal function. Finally, the occurrence of NOS-positive cells in a variety of organs (CNS, respiratory system, and GIT) is significantly higher at the prenatal stage of development than at other times, which suggests that NO is also important for the development of these organs in addition to its importance for foetal and postnatal development of the mammalian GIT. ABBREVIATIONS USED IN THE TEXT ATP - adenosine 5'-triphosphate cNOS - constitutive nitric oxide synthase CNS - central nervous system

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eNOS - endothelial nitric oxide synthase ENS - enteric nervous system EPR - electron paramagnetic resonance GFAP - glial fibrillary acidic protein ICC - interstitial cells of Cajal iNOS - inducible nitric oxide synthase L-NAME - NG-nitro-L-arginine methyl ester L-NMMA - NG-monomethyl-L-arginine NADPH-d -- reduced nicotinamide adenine dinucleotide phosphate diaphorase NANC - non-adrenergic non-cholinergic nNOS - neural nitric oxide synthase NO - nitric oxide NOS - nitric oxide synthase NPY - neuropeptide Y NSE - neuron specific enolase PACAP - pituitary adenyl ate cyclase activating peptide PGP 9.5 - protein gene product 9.5 SP - substance P VIP - vasoactive intestinal peptide

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