Ohlsson et al.indd Drug Target Insights 2007:2 229–237 229 REVIEW Correspondence: Bodil Ohlsson, Entrance 35, 205 02 Malmö, Sweden. Tel: +46 40 33 10 00; Fax: +46 40 33 62 08; Email: bodil.ohlsson@med.lu.se Copyright in this article, its metadata, and any supplementary data is held by its author or authors. It is published under the Creative Commons Attribution By licence. For further information go to: http://creativecommons.org/licenses/by/3.0/. New Insights into the Understanding of Gastrointestinal Dysmotility Bodil Ohlsson1 and Sabina Janciauskiene2 Department of Clinical Sciences, Gastroenterology Division1, Wallenberg Laboratory2, Entrance 46, 2nd floor, University Hospital Malmö, Lund University, 20502 Malmö, Sweden. Abstract: Our understanding of the physiology of digestion, absorption, secretion, and motility in the gastrointestinal tract has improved immensely. Today it is well established that the gross functions of the gastrointestinal tract depend on the coordination between the muscles, nerves and hormones. The enteric nervous system (ENS) is involved in most of the physiological and pathophysiological processes in the gastrointestinal tract. Therefore, clinical and experimental studies on the ENS provide the basis for a better understanding of the mechanisms involved in gastrointestinal disorders and promote the development of therapeutic options. This review outlines some of the current views on the role of the ENS and its related hormones in gastrointestinal motility. Keywords: gonadotropin-releasing hormone (GnRH), oxytocin, chronic intestinal pseudo-obstruction (CIPO), apoptosis Introduction Gastrointestinal motility is a term used to describe the contraction of the muscles in the gastrointestinal tract. The normal motility of the gastrointestinal tract is dependent on the function of the enteric nervous system (ENS), the smooth muscle layers, and the interstitial cells of Cajal (ICCs) (Fig. 1) (Goyal and Hirano, 1996; Wood, 2000). Diseases characterised by gastrointestinal dysmotility are highly prevalent conditions. For instance, the mild form of dysmotility, also called irritable bowel syndrome (IBS), is assumed to affect almost 15%–20% of the population (Gwee, 2005). Chronic intestinal pseudo-obstruction (CIPO) is the most diffi cult of these clinical challenges, characterised by the presence of chronic dys- motility and intestinal dilatation in the absence of mechanical obstruction (De Giorgio et al. 2004a). The pathogenesis of IBS and CIPO remains unclear, although putative mechanisms, including infl ammation, altered calcium signalling, mitochondrial dysfunction, free radical production, and others, may contrib- ute to the degeneration and loss of enteric neurons (Hall and Wiley, 1998; Spiller, 2003). Over recent years much effort has been made to try to improve the motility and decrease the abdom- inal pain occasioned by these conditions by targeting enteric peptides and their receptors. One example is motilin, an important peptide in digestive motility whose receptor is the site of action for erythromy- cin in the treatment of gastro paresis (Galligan and Vanner, 2005). The peptide ghrelin, related to moti- lin, which originates primarily in the stomach (Möller et al. 2003), has recently also been shown to enhance gastric emptying in idiopathic gastro paresis (Tack et al. 2005). Serotonin and its receptor have been the goal for an intensive effort to develop different agonists and antagonists (Johanson, 2004; Cash and Chey, 2005; Wessinger et al. 2005). So far, these attempts have not been very successful. Recent fi ndings support a new concept that alterations in intracellular mechanisms of neuronal survival might play a crucial role in the degeneration of the enteric nervous system (De Giorgio et al. 2000; Bassotti et al. 2006). Furthermore, two newly discovered peptides localized in the human gastrointestinal tract, oxytocin and gonadotropin releasing hormone (GnRH) seem to play crucial roles in the regulation of gastrointestinal dysmotility (Monstein et al. 2004; Ohlsson et al. 2006a; Ohlsson et al. 2007). Oxytocin is known to enhance gastric emptying (Hashmonai et al. 1979; Petring, 1989). Our own studies have shown that oxytocin is expressed in the myenteric and submucous ganglia and nerve fi bres along the entire human gastrointestinal tract (Monstein et al. 2004; Ohlsson et al. 2006b), and that it increases colonic peristalsis while the receptor antagonist delays the gastric emptying rate (Ohlsson et al. 2004; Ohlsson et al. 2006a). Similarly, GnRH has also been shown to stimulate intestinal motor http://creativecommons.org/licenses/by/3.0/. 230 Ohlsson and Janciauskiene Drug Target Insights 2007:2 activity in rat (Khanna et al. 1992; Ducker et al. 1996), and a loss of GnRH-containing neurons in the ENS has been related to CIPO development (Ohlsson et al. 2007). The Enteric Nervous System and Gastrointestinal Dysmotility Enteric neuron apoptosis The ENS is a highly integrated neural system which consists of distinct subclasses of enteric neurons localized within the wall of the alimentary tract throughout its entire length. The ENS closely resembles the central nervous system (Gershon et al. 1994), and has a unique ability to control virtually all gut functions, including motility, inde- pendently of the central nervous system (CNS) (Furness et al. 2005). Remarkably, in response to different types of stimuli/conditions, enteric neurons are able to change their structural, functional and chemical phenotype (Lomax et al. 2005). These changes in the functional and/or structural integrity of the ENS may occur as a consequence of normal aging or due to pathologies ranging from enteric neuropathies Enteric Nervous System Neurones ICCs Glial cells Hormones and neurotransmitters TRH GnRH Oxytocin GLP - 1 CCK GIP Secreto neurin CRH Ghrelin Motilin PP VIP Serotonin Figure 1. A schematic overview over the enteric nervous system and its most important peptides according to motility. CCK = cholecystoki- nin; CRH = corticotrophin-releasing hormone; ICCs = Interstitiasl cells of Cajal; GIP = gastric inhibitory polypeptide; GLP-1 = glucagon-like peptide 1; GnRH = gonadotropin-releasing hormone; PP = pancreatic polypeptide; TRH = thyrotropin-releasing hormone; VIP = vasoactive intestinal peptide. 231 Gastroinestinal motility Drug Target Insights 2007:2 (i.e. Hirschsprung’s disease) to intestinal or extra intestinal diseases (i.e. ulcerative colitis and Crohn`s disease, amyloidosis, scleroderma and etc) (Di Lorenzo, 1999; De Giorgio and Camilleri, 2004b; De Giorgio et al. 2004c). It has been suggested that some motility disor- ders originate from developmental defects, i.e. Hirschsprung’s disease (Kim et al. 2006), whereas others are due to neurodegeneration. In fact, pathological changes of the ENS are often accom- panied by nerve process degeneration and necrosis (Dvorak et al. 1993). Ultra structural evaluation of tissue specimens from patients with Crohn’s dis- ease and ulcerative colitis have shown swollen, empty axons, fi lled with large vacuoles, swollen mitochondria and concentrated neurofi brils (Vasina et al. 2006). The B-cell leukaemia/lymphoma-2 (bcl-2) protein has the functional role of blocking apop- tosis, i.e. programmed cell death. This protein is widely expressed in the developing central and peripheral nervous systems. The expression of bcl-2 is also displayed in enteric neurons (Wester et al. 1999). The reduced expression of bcl-2 has been demonstrated in degenerative disorders of both the central nervous system and ENS (Merry and Korsmeyer, 1997). Thus, the current state of knowledge allows speculations that alterations in the intracellular mechanisms involved in neuronal survival may play a critical role in various gastro- intestinal motility disorders. To support of this postulation, the decreased expression of the Bcl-2 protein in enteric neurons has been demonstrated in patients with severe forms of CIPO (De Giorgio et al. 2000; De Giorgio et al. 2004a; De Giorgio and Camilleri, 2004b). We found further support when, examining full thickness biopsies, we noticed signifi cantly lower bcl-2 expression in a patient with CIPO than in controls. In parallel, histological examination revealed the presence of swollen or shrunken neu- rons of the myenteric plexus with or without vacuolisation of the cytoplasm (Ohlsson et al. 2007). The increased number of apoptotic enteric neurons and decreased expression of bcl-2 have also been found in patients with slow-transit con- stipation (Bassotti et al. 2006) and in patients with Hirschsprung’s disease (Song et al. 2002). Thus, the improved knowledge on the changes and regulation in proteins associated with apoptosis in the cells of ENS may be crucial for regulating the apoptosis program. A number of animal models such as a rat hemispheric ischemia/reperfusion model (Gabryel et al. 2006) and an apoptosis- dependent emphysema mouse model (Petrache et al. 2006), as well as age-related macular degen- eration (Glotin et al. 2006) have demonstrated that substances controlling intracellular pathways of cell apoptosis can reduce disease processes character- ised by excessive cell apoptosis. Therefore, we believe that clinical and experimental studies on the role of enteric neuronal apoptosis are of funda- mental importance because they may improve our understanding regarding the pathophysiology of gastrointestinal dysmotility and may also provide the basis for new therapeutic approaches. Enteric glial cells The ENS is composed of both neurons and enteric glial cells, which play a central role in sustaining the structural and functional integrity of enteric neurons. Enteric glial cells were fi rst described by Dogiel in 1899 as nucleated satellite cells accom- panying enteric neuronal cells. Dogiel assumed that enteric glia represented a kind of connective tissue, and consequently very little research was conducted to reveal their functions (Dogiel, 1889). Today, there is evidence from transgenic animal models that enteric glial cells are essential for gastrointestinal integrity and function, but still little is known about the underlying mechanisms. For example, in genetically modifi ed animals, loss of enteric glia results in neuronal degeneration and changes in the neurochemical coding of enteric neurons (Bush et al. 1998; Aube et al. 2003). New data suggest that enteric glial cells have an impor- tant role in maintaining the integrity of the muco- sal barrier of the gut, and may also serve as a link between the nervous and immune systems of the gut as indicated by their potential to synthesize cytokines, present antigens and respond to infl am- matory insults. The role of enteric glia in human disease has not yet been systematically studied, but, based on the evidence available it can be pre- dicted that enteric glia are involved in the aetio- pathogenesis of various pathological processes in the gut, particularly those with neuroinfl ammatory or neurodegenerative components (Ruhl, 2005). The number of glia cells has been shown to increase in response to pro-infl ammatory cytokines, such as interleukin-1 (IL-1) and tumour necrose factor alpha (TNFα), or lipopolysaccharide (von Boyen et al. 2004). Notably, several studies have described 232 Ohlsson and Janciauskiene Drug Target Insights 2007:2 an association between increased glial cell proliferation and neuronal disintegration in patients with infl ammatory bowel diseases (Cabarrocas et al. 2003; Lomax et al. 2005). In another study, examination of non-involved intestinal tissue from patients with Crohn’s disease, ulcerative colitis, or histological normal controls demonstrated that the enteric glia cell network was signifi cantly disrupted in Crohn’s disease, but not in ulcerative colitis (Cornet et al. 2001). In patients with slow transit constipation a remarkable decrease both in the number of enteric neurons and interstitial cells of Cajal (ICCs), and also in the number of glial cells has been found. These patients had signifi cantly more apoptotic enteric neurons than controls (Bassotti et al. 2006). It is likely that a dynamic equilibrium between enteric neurons and glia plays an important role in vivo. Therefore, the insufficient support of enteric neurons by glial cells may lead to enhanced neuronal apoptosis and neurodegeneration, char- acteristic features of gastrointestinal dysmotility disorders such as idiopathic chronic constipation, IBS and CIPO (Törnblom et al. 2002; De Giorgio et al. 2004a; De Giorgio and Camilleri, 2004b; De Giorgio et al. 2004c; Bassotti et al. 2006). Interstitial Cells of Cajal (ICCs) Interstitial cells of Cajal (ICCs) were originally described in the gut more than a century ago by Ramóny Cajal (He et al. 2001). ICCs are a unique class of mesenchymal cells found in the gastroin- testinal tract of mammals. In the region of the gastric corpus and antrum, multipolar ICCs form two-dimensional networks, and have been mis- taken for neurons, glial cells, smooth muscle cells, macrophages and fi broblasts. ICCs are the pace- maker cells responsible both for initiating slow wave activity in gastrointestinal muscles and for the active propagation of the electrical slow waves (Thomsen et al. 1998). ICCs can be recognised either by their characteristic ultra structure by electron microscopy or by the immunohistochem- ical demonstration of their surface receptor tyro- sine kinase Kit. Recent studies demonstrated that the c-Kit receptor is essential for the development of ICCs. Mesenchymal ICC precursors that carry the c-Kit receptor require the kit ligand, which can be provided by neuronal cells or smooth muscle cells. Accordingly the ICCs develop as either myenteric or muscular ICCs (Wu et al. 2000). The evidence from experimental models and human diseases increasingly point to a central role of ICCs in the aetiology of human gastrointestinal dysmotility. Many gastrointestinal motor disorders like gastro paresis, abnormal small bowel motility, infl ammatory bowel disease, CIPO, gastrointesti- nal stromal and multiple autonomic tumours, achalasia and Hirschsprung’s disease show a changed number and/or structure of ICCs (He et al. 2000; Sanders et al. 1999; Hagger et al. 2000). Gastro paresis is associated with electrical abnormalities, and deviations from normal slow- wave rhythm (dysrhythmias) have been reported to result in delayed gastric emptying. In a diabetic rat model it has been demonstrated that degenera- tion of ICCs is responsible for these gastro- electrical dysrhythmias (Ordög et al. 2000). Therefore, the identifi cation of abnormalities in ICCs which are linked to specifi c gastrointestinal motor disorders should be taken more into focus in the future. Newly Discovered Peptides in the Enteric Nervous System (ENS) A wide range of peptides are described as having a decreased expression in dysmotility. It is not known whether these down-regulated peptides are primary or secondary to development of the disease (Krischnamurthy and Schuffl er, 1993; De Giorgio and Camilleri, 2004b). Gut peptides exert diverse effects, regulating gastrointestinal motility and acid secretion, epithelial integrity, and both nutrient absorption and disposal. These actions are initiated by the activation of specifi c G protein-coupled receptors and may be mediated by direct or indirect effects on target cells (Kutchai, 2004). More recent evidence demonstrates that gut peptides, such as glucagon-like peptides-1 and 2, also directly regu- late signalling pathways coupled to cell prolifera- tion and apoptosis (Drucker, 2003). A number of signalling pathways between mesenchymal and neural crest cells are required for the development of the ENS (Natarajan et al. 2002). These signalling pathways involve peptides secreted by intestinal mesenchymal cells such as endothelin-3, glial cell line-derived neurotrophic factor (GDNF), neuroturin, neurotrophin-3 (NT-3), and netrin-1 (Chalazonitis et al. 1998; Young et al. 2004; Nagy and Goldstein, 2006). The presence of both motilin and ghrelin in guinea-pig myen- teric neurons is suggested to play a role in the 233 Gastroinestinal motility Drug Target Insights 2007:2 activation of the ENS and hence in the regulation of gastrointestinal motility (Xu et al. 2006), which is further supported by a close relationship between Ghrelin and gastric motility in rats (Masuda et al. 2000). The fi ndings that patients with functional dyspepsia (FD) have altered plasma profi le of ghrelin suggest a possible role for this peptide in the pathophysiology of FD (Takamori, 2006). Obestatin, a newly discovered ghrelin-associated peptide, was initially suggested to decrease gastric emptying (Zhang, 2005). Unfortunately, recent studies have not been able to confi rm these results, and existing reports do not support obestatin as a regulator of digestive motility (Gourcerol and Taché, 2007). Serotonin is a biochemical neurotransmitter, found primarily in the CNS, gastrointestinal tract, and blood platelets (Vialli, 1966). The bowel exhib- its refl exes in the absence of CNS input. To do so, epithelial sensory transducers, such as enterochro- maffi n (EC) cells, activate the mucosal processes of intrinsic and extrinsic primary afferent (sensory) neurons by secretion of serotonin (5-HT) in response to mucosal stimuli (Gershon, 2005). The enteric serotonin reuptake transporter has been proposed to play a critical role in serotonergic neu- rotransmision and in the initiation of peristaltic and secretory refl exes (Chen et al. 2001). The current knowledge suggests that serotonin initiates peristal- tic and secretory refl exes because of its ability to stimulate secretion of acetylcholine (Ach) and cal- citonin gene related peptide (CGRP) (Pan et al. 1994; Sidhu et al. 1995; Grider, 1994, 2003). These afferent refl ex pathways also lead to perceptions of nausea, and discomfort and pain from the gastroin- testinal tract (Grundy, 2002). Serotonin is thus implicated in the pathology of irritable bowel syn- drome (IBS), which is characterised by visceral hypersensitivity and altered motility (Simrén et al. 2003; Costedio et al. 2006). Multiple receptor families explain the broad physiological actions and distribution of serotonin, therefore, many agonists and antagonists to the serotonin receptors have been developed and clinically used. So far, no one has given successful results in the treatment of IBS (McLean et al. 2006). The neuropeptide vasoactive intestinal peptide (VIP) is the most important peptidergic transmitter in intestinal relaxation, which regulates smooth muscle- and epithelial function. For the fi rst time, VIP/pituitary adenylate cyclase activating peptide (PACAP) receptors have been detected in the human gastrointestinal tract by the use of specifi c antibodies (Rettenbacher and Reubi, 2001). Observed correlation between delayed gastrointes- tinal transit and an increase of VIP neurons in a rat ischemia/reperfusion model suggests that changes in enteric transmitters might contribute to gastro- intestinal dysmotility (Calcina et al. 2005). Secretoneurin is a functional neuropeptide derived from secretogranin II (chromogranin C). Both in the myenteric and submucous plexuses, nerve fi bres and the majority of ganglion cells were found to be secretoneurin-immunoreactive. Thus, secretoneurin is a new major peptide within the human enteric neuroendocrine system. Its abundant presence in myenteric ganglion cells may imply a role in the modulation of gastroin- testinal motility. The chemotactic properties of secretoneurin and its possible localization in sensory fi bres suggest that this peptide may be involved in the genesis of intestinal infl ammation (Schurmann et al. 1995). Oxytocin Oxytocin is a hormone with its most well-known effects on myoepithelial cells of the breast during lactation and the uterine contractions during par- turition. Oxytocin is detected not only in plasma but also in almost all segments of the gastrointes- tinal tract (Monstein et al. 2004). The indirect immunofl uorescence approach has shown that oxytocin is expressed in myenteric and submucous ganglia, suggesting that it is important for both gastrointestinal sensitivity and motility (Ohlsson et al. 2006b). Oxytocin is released into plasma in response to a meal (Ohlsson et al. 2002), and has been shown to stimulate gastric emptying (Hashmoni et al. 1979; Petring, 1989) and colonic peristalsis (Ohlsson et al. 2004). In addition, inhi- bition of the binding of endogenous oxytocin by the receptor antagonist atosiban delayed gastric emptying (Ohlsson et al. 2006a). The prokinetic effect of oxytocin on the gastrointestinal tract is speculated to be similar to the one in uterine myo- metrium and mammary myoepitheal cells; intracel- lular release of Ca2+ which leads to muscle contraction via myosin light kinase activity (Gimpl and Fahrenholz, 2001). A woman with chronic gastro paresis demand- ing continuous treatment with prokinetic drugs, was completely out of symptoms during pregnancy and breast feeding, and could stop medicamentation 234 Ohlsson and Janciauskiene Drug Target Insights 2007:2 every time she was pregnant (Ohlsson, 2006c). Although the mechanism behind this phenomenon is not proven, these states are characterised by elevated oxytocin levels in plasma (Chiodera et al. 1991, Silber et al. 1991), and together with other observations mentioned above, one may speculate whether oxytocin defi ciency may be the aetiology to the gastro paresis in this woman (Ohlsson, 2006c). Despite the stimulatory effect of oxytocin on peristalsis, treatment with nasally administered oxytocin did not improve the stool habits in women with refractory constipation (Ohlsson et al. 2005). Oxytocin is also known to have analgesic effects (Petersson et al. 1996), and its plasma levels are found to be decreased in patients suffering from dyspepsia and IBS, conditions characterised by abdominal pain and discomfort (Uvnäs-Moberg et al. 1991). Furthermore, children suffering from recurrent abdominal pain exhibit lower plasma levels of oxytocin than healthy controls (Alfven, 2004). Interestingly, both depression and fi bromy- lagia are associated with IBS (Lydiard et al. 1993; Sperber et al. 1999), and both of these conditions are also characterised by low plasma levels of oxytocin (Frash et al. 1995; Anderberg and Uvnäs- Moberg, 2000). Accordingly, treatment of IBS patients with intravenously (Louvel et al. 1996) or nasally (Ohlsson et al. 2005) administered oxyto- cin resulted in the reduction of abdominal pain and reduced depression. The questions remain as to whether oxytocin could be used clinically to improve the suffering of patients with IBS and CIPO by reducing their pain and their depressive mood rather than by attempting to improve motil- ity. Further randomised clinical trials are needed to answer these questions. Gonadotropin releasing hormone (GnRH) The central core of the hypothalamic-pituitary- gonadal axis, in all vertebrate species, is the group of neurons that produce and secrete gonadotropin- releasing hormone (GnRH). Over the past 20 years, techniques have become available to identify the GnRH-producing neurons and measure both GnRH content and levels of GnRH mRNA in brain tissue. Indeed, several types of differentiated lym- phocytes, including spleenocytes, thymocytes, peripheral T- and B-lymphocytes, and mast cells have been demonstrated to produce GnRH or a GnRH-like peptide (Marchetti et al. 1996). Although it is not known whether different forms of GnRH might have different receptor types, GnRH receptors have been found throughout the human body (Fekete et al. 1989; Kakar and Jennes, 1995), but have not been studied in the gastroin- testinal tract. In rats, GnRH mRNA has been found in parietal cells of gastric glands, the epithelium of the small and large intestine, and in parasym- pathetic ganglion cells of the myenteric plexus. In addition, the GnRH receptor has been found in the epithelium of gastric pits (Huang et al. 2001) and GnRH receptor mRNA in the myenteric neurons in the rat (Ho et al. 1996). GnRH has also been detected in rat pancreas (Wang et al. 2001). In the dog, GnRH has been shown to inhibit the release of gastric secretions and gastrin release (Soldani et al. 1982), possibly due to diminished vagal activity. Apart from its effects on reproduction, these fi ndings suggest a role for GnRH also in the regulation of the gastrointestinal tract. Accord- ingly, the GnRH analogue leuprolide (pGlu-His- Trp-Ser-Tyr-DLeu-Arg-Pro-EtNH2) has been shown to stimulate intestinal motor activity in rats (Khanna et al. 1992; Ducker et al. 1996). Further- more, symptom resolution and alleviation of intestinal motility abnormality after treatment with leuprolide have been reported in a patient with CIPO (Mathias et al. 1992). In a study of the effect of leuprolide in the treatment of IBS, the overall symptom score was improved, but the greatest therapeutic effect was seen on abdominal pain and nausea (Mathias et al. 1994a; Mathias et al. 1998). This effect persisted when the treatment was con- tinued for up to 6–12 months (Mathias et al. 1994b; Palomba et al. 2005). Other GnRH analogues, such as buserelin, are used in the treatment of in vitro fertilization (IVF), endometriosis, polycystic ovary syndrome, pros- tate cancer, uterine leiomyoma and precocious puberty. Gastrointestinal side effects are considered infrequent (WHO) but nausea and abdominal pain have been reported in 7%–17% of women treated with buserelin for endometriosis and uterine leiomyoma (FASS, Micromedex). The aetiology to these side effects is not known. Recently we demonstrated for the fi rst time GnRH positive neurons in the human gastrointes- tinal tract and have shown a decreased number of GnRH positive neurons in a CIPO patient. The patient, who had been treated with buserelin, 235 Gastroinestinal motility Drug Target Insights 2007:2 developed CIPO with pronounced abdominal pain and nausea/vomiting. Remarkably, immunohisto- chemical analysis of intestinal resects revealed that in the patient only 3% of myenteric neurons are GnRH positive as compared to 53% in controls (Ohlsson et al. 2007). The patient had high plasma titres of anti-GnRH antibodies that correlated with the occasions of the treatment with buserelin. The latter led us to the hypothesis that the patient devel- oped CIPO due to buserelin-induced formation of anti-GnRH antibodies which destroyed GnRH- producing neurons of the myenteric plexus. We believe that GnRH plays a pivotal role not only in the regulation of different hormones involved in repro- duction, but also in the regulation of the motor activity of the gastrointestinal tract. Degeneration of GnRH neurons might be of central importance for the patho- physiology of different forms of IBS and CIPO. Concluding Remarks Over recent decades, a number of peptides have been characterised, which led to an explosion in our understanding of their biological action and function in the central and enteric nervous system. Gut hormones, including cholecystokinin, cortico- trophin-releasing hormone, gastrin, gastric inhibi- tory polypeptide, ghrelin, glucagon-like peptide-1, motilin, neurotensin, pancreatic polypeptide, secre- toneurin, serotonin, thyrotrophic-releasing hormone and VIP have been shown to play a role in modu- lating gastrointestinal motility. New experimental and clinical data point to GnRH and oxytocin, two other peptide candidates, as being involved in controlling gastrointestinal motility. These fi ndings open new, fascinating perspectives for research and the therapeutic potential of the peptidal role in gastrointestinal diseases. Furthermore, the role of neuronal apoptosis and agents which improve neuronal survival deserves further attention concerning their function in pre- venting neuronal degeneration which results in dysmotility. References Alfven, G. 2004. Plasma oxytocin in children with recurrent abdominal pain. J. Pediatr. Gastroenterol. Nutr., 38:513–7. Anderberg, U.M. and Uvnäs-Moberg, K. 2000. Plasma oxytocin levels in female fi bromyalgia syndrome patients. Z. Rheumatol., 59:373–9. Aube, A.C., Cabarrocas, J., Bauer, J., et al. 2006. Changes in enteric neurone phenotype and intestinal functions in a transgenic mouse model of enteric glia disruption. Gut., 55:630–7. Bassotti, G., Villanacci, V., Maurer, C.A., et al. 2006. The role of glial cells and apoptosis of enteric neurons in the neuropathology of intractable slow transit constipation. Gut., 55:41–6. Bush, T.G., Savidge, T.C., Freeman, T.C., et al. 1998. Fulminant jejuno- ileitis following ablation of enteric glia in adult transgenic mice. Cell, 93:189–201. Cabarrocas, J., Savidge, T.C. and Liblau, R.S. 2003. Role of enteric glial cells in infl ammatory bowel disease. Glia., 41:81–93. Calcina, F., Barocelli, E., Bertoni, S., et al. 2005. Effect of N-methyl-d-aspartate receptor blockade on neuronal plasticity and gastrointestinal transit delay induced by ischemia/reperfusion in rats. Neuroscience, 134:39–49. Cash, B.D., Chey, W.D. 2005. Review article: The role of serotonergic agents in the treatment of patients with primary chronic constipation. Aliment. Pharmacol. Ther., 22:1047–60. Chalazonitis, A., Rothman, T.P., Chen, J., et al. 1998. Age-dependent dif- ferences in the effects of GDNF and NT-3 on the development of neurons and glia from neural crest-derived precursors immunose- lected from the fetal rat gut: expression of GFRalpha-1 in vitro and vivo. Dev. Biol., 204:385–406. Chen, J.J., Zhishan, L., Pan, H., Murphy, D.L., Tamir, H., Koepsell, H. and Gershon, M.D. 2001. Maintenance of serotonin in the intestinal mucosa and ganglia of mice that lack the high-affi nity serotonin transporter (SERT): abnormal intestinal motility and the expression of cation transporters. J. Neurosci., 21:6348–61. Chiodera, P., Salvarani, C., Bacchi-Modena, A., et al. 1991. Relationship between plasma profi les of oxytocin and adrenocorticotropic hormone during suckling or breast stimulation in women. Horm. Res., 35:119–23. Cornet, A., Savidge, T.C., Cabarrocas, J., et al. 2001. Enterocolitis induced by autoimmune targeting of enteric glial cells: a possible mechanism in Crohn’s disease? Proc. Natl. Acad. Sci. U.S.A., 98:13306–11. Costedio, M.M., Hyman, N. and Mawe, G.M. 2006. Serotonin and its role in colonic function and in gastrointestinal disorders. Dis. Colon. Rectum, 50:376–88. De Giorgio, R., Barbara, G. and Stanghellini, V. 2000. Reduced Bcl-2 expression in the enteric nervous system as a marker for neuronal degeneration in patients with gastrointestinal motor disorders. Gastroenterology, 118:A867 (abstract). De Giorgio, R., Sarnelli, G., Corinaldesi, R., et al. 2004a. Advances in our understanding of the pathology of chronic intestinal pseudo- obstruction. Gut, 53:1549–52. De Giorgio, R. and Camilleri, M. 2004b. Human enteric neuropathies: morphology and molecular pathology. Neurogastroenterol. Motil., 16:515–31. De Giorgio, R., Guerrini, S., Barbara, G., et al. 2004c. New insights into human enteric neuropathies. Neurogastroenterol Motil., 16:143–7. Di Lorenzo, C. 1999. Pseudo-obstruction: Current approaches. Gastroenterology, 116:980–7. Dogiel, A.S. 1889. Ueber den Bau der Ganglien in den Gefl echten des Darmes und der Gallenblase des Menschen und der Säugethiere. Z. Naturforsch. B., 5:130–58. Drucker, D.J. 2003. Glucagon-like peptides: regulators of cell proliferation, differentiation, and apoptosis. Mol. Endocrinol., 17:161–71. Ducker, T.E., Boss, J.W., Altug, S.A., et al. 1996. Luteinizing hormone and human chorionic gonadotropin fragment the migrating myoelectrical complex in rat small intestine. Neurogastroenterol Motil., 8:95–100. Dvorak, A.M., Onderdonk, A.B., McLeod, R.S., et al. 1993. Axonal necro- sis of enteric autonomic nerves in continent ileal pouches. Possible implications for pathogenesis of Crohn’s disease. Ann. Surg., 217:260–71. FASSR, the Swedish register of drugs. Fekete, M., Redding, T.W. and Comaru-Schallu, A.M. 1989. Receptors for luteinizing hormone-releasing hormone, somatostatin, prolactin, and epidermal growth factor in rat and human prostate cancers and in benign prostate hyperplasia. Prostate, 14:191–208. 236 Ohlsson and Janciauskiene Drug Target Insights 2007:2 Frasch, A., Zetzsche, T., Steiger, A., et al. 1995. Reduction of plasma oxytocin levels in patients suffering from major depression. Adv. Exp. Med. Biol., 395:257–8. Furness 2005, Goyal, R.K. and Hirano, I. 1996. The enteric nervous system. N. Engl. J. Med., 334:1106–15. Gabryel, B., Chalimoniuk, M., Stolecka, A., et al. 2006. Inhibition of arachi- donic acid release by cytosolic phospholipase a (2) is involved in the antiapoptotic effect of FK506 and cyclosporine a on astrocytes exposed to simulated ischemia in vitro. J. Pharmacol. Sci., 102:77–87. Galligan, J.J. and Vanner, S. 2005. Basic and clinical pharmacology of new motility promoting agents. Neurogastroenterol Motil., 17:643–53. Gershon, M.D., Kirchgessner, A.L. and Wade, P.R. 1994. Functional anatomy of the enteric nervous system. In: Johnson, L.R., ed. Physiology of the gastrointestinal tract. 3rd ed. New York: Raven Press, p381–422. Gershon, M.D. 2005. Review article: Nerves, refl exes, and the enteric nervous system. Pathogenesis of the irritable bowel syndrome. J. Clin. Gastroenterol., 39:S184–S193. Gimpl, G. and Fahrenholz, F. 2001. The oxytocin receptor system: structure, function, and regulation. Physiol. Rev., 81:629–83. Glotin, A.L., Calipel, A., Brossas, J.Y., et al. 2006. Sustained versus transient ERK1/2 signaling underlines the anti- and proapoptotic effects of oxidative stress in human RPE cells. Invest. Ophthalmol. Vis. Sci., 47:4614–23. Gourcerol, G. and Taché, Y. 2007. Obestatin- a ghrelin-associated peptide that does not hold its promise to suppress food intake and motility. Neurogastroenterol Motil., 19:161–5. Goyal, R.K. and Hirano, I. 1996. The enteric nervous system. N. Engl. J. Med., 334:1106–15. Grider, J.R. 1994. CGRP as a transmitter in the sensory pathway mediating peristaltic refl ex. Am. J. Physiol., 26:G1139-G1145. Grider, J.R. 2003. Neurotransmitters mediating the intestinal peristaltic refl ex in the mouse. J. Pharmacol. Exp. Ther., 307:460–7. Grundy, D. 2002. Towards a reduction of rectal pain? Neurogastroenterol Motil., 14:217–9. Gwee, K.A. 2005. Irritable bowel syndrome in developing countries-a disorder of civilization or colonization? Neurogastroenterol Motil., 17:317–24. Haggers, R., Finlayson, C., Kahn, F., et al. 2000. A defi ciency of intersti- tial cells of Cajal in Chagasic megacolon. J. Auton. Nerv. Syst., 80:108–11. Hall, K.E. and Wiley, J.W. 1998. Neural injury, repair and adaptation in the GI tract. I. New insights into neuronal injury: a cautionary tale. Am. J. Physiol., 274:G978–83. Hashmonai, M., Torem, S., Argov, S., et al. 1979. Prolonged post-vagotomy gastric atony treated by oxytocin. Br. J. Surg., 66:550–51. He, C.L., Burgart, L., Wang, L., et al. 2000. Decreased intestitial cell of cajal volume in patients with slow-transit constipation. Gastroenter- ology, 118:14–21. He, C.L., Soffer, E.E., Ferris, C.D., et al. 2001. Loss of interstitial cells of cajal and inhibitory innervation in insulin-dependent diabetes. Gas- troenterology, 121:427–34. Ho, J.S., Nagle, G.T., Mathias, J.R., et al. 1996. Presence of gonadotropin- releasing hormone (GnRH) receptor mRNA in rat myenteric plexus cells. Comp. Biochem. Physiol., 113:817–21. Huang, W., Yao, B., Sun, L., et al. 2001. Immunohistochemical and in situ hybridization studies of gonadotropin releasing hormone (GnRH) and its receptor in rat digestive tract. Life. Sci., 68:1727–34. Johanson, J.F. 2004. Options for patients with irritable bowel syndrome: contrasting traditional and novel serotonergic therapies. Neurogas- troenterol Motil., 16:701–11. Kakar, S.S. and Jennes, L. 1995. Expression of gonadotropin-releasing hormone and gonadotropin-releasing hormone receptor mRNAs in various non-reproductive human tissues. Cancer Lett., 98:57–62. Khanna, R., Browne, R.M., Heiner, A.D., et al. 1992. Leuprolide acetate affects intestinal motility in female rats before and after ovariectomy. Am. J. Physiol., 262:G185–G190. Kim, J.H., Yoon, K.O., Kim, J.K., et al. 2006. Novel mutations of RET gene in Korean patients with sporadic Hirschsprung’s disease. J. Pediatr. Surg., 41:1250–54. Krishnamurthy, S. and Schuffl er, M.D. 1993. Pathology of neuromuscular disorders of the small intestine and colon. Gastroenterology, 104:1398–1408. Kutchai, H.C. 2004. The gastrointestinal system. In: Berne, R.M., Levy, M.N., Koeppen, B.M., Stanton, B.A., edr. Physology. Fifth edition. Mosby, St Louis, Missouri. Lomax, A.E., Fernandez, E. and Sharkey, K.A. 2005. Plasticity of the enteric nervous system during intestinal infl ammation. Neurogastroenterol Motil., 17:4–15. Louvel, D., Delvaux, M., Felez, A., et al. 1996. Oxytocin increases thresh- olds of colonic visceral perception in patients with irritable bowel syndrome. Gut., 39:741–7. Lydiard, R.B., Fossey, M.D., Marsh, W., et al. 1993. Prevalence of psychi- atric disorders in patients with irritable bowel syndrome. Psychoso- matics, 34:229–34. Masuda, Y., Tanaka, T., Inomata, N., et al. 2000. Ghrelin stimulates gastric acid secretion and motility in rats. Biochem. Biophys. Res. Commun., 276:905–8. Marchetti, B., Gallo, F., Farinella, Z., et al. 1996. Luteinizing hormone- releasing hormone (LHRH) receptors in the neuroendocrine-immune network. Biochemical bases and implications for reproductive phys- iopathology. Ann. N. Y. Acad. Sci., 784:209–36. Mathias, J.R., Baskin, G.S., Reeves-Darby, V.G., et al. 1992. Chronic intestinal pseudoobstruction in a patient with heart-lung transplant. Therapeutic effect of leuprolide acetate. Dig. Dis. Sci., 37:1761–8. Mathias, J.R., Clench, M.H., Reeves-Darby, V.G., et al. 1994a. Effect of leuprolide acetate in patients with moderate to severe functional bowel disease. Double-blind, placebo-controlled study. Dig. Dis. Sci., 39:1155–62. Mathias, J.R., Clench, M.H., Roberts, P.H., et al. 1994b. Effect of leuprolide acetate in patients with functional bowel disease. Long-term follow-up after double-blind, placebo-controlled study. Dig. Dis. Sci., 39:1163–70. Mathias, J.R., Clench, M.H., Abell, T.L., et al. 1998. Effect of leuprolide acetate in treatment of abdominal pain and nausea in premenopausal women with functional bowel disease: a double-blind, placebo- controlled, randomized study. Dig. Dis. Sci., 43:1347–55. McLean, P.G., Borman, R.A. and Lee, K. 2006. 5-HT in the enteric nervous system: gut function and neuropharmacology. Trends in Neurosci- ences, 30:9–13. Merry, D.E., Korsmeyer, S.J. 1997. Bcl-2 gene family in the nervous system. Annu. Rev. Neurosci., 20:245–67. Micromedex, Drugdex, Drug Evaluation. Monstein, H-J., Grahn, N., Truedsson, M., et al. 2004. Oxytocin and oxy- tocin receptor mRNA expression in the human gastrointestinal tract: A polymerase chain reaction study. Regul. Pept., 119:39–44. Möller, N., Nygren, J., Hansen, T.K., et al. 2003. Splanchnic release of ghrelin in humans. J. Clin. Endocrinol. Metabol., 88:850–52. Nagy, N. and Goldstein, A.M. 2006. Endothelin-3 regulates neural crest cell proliferation and differentiation in the hindgut enteric nervous system. Dev. Biol., 293:203–17. Natarajan, D., Marcos-Gutierrez, C., Pachnis, V., et al. 2002. Requirement of signalling by receptor tyrosine kinase RET for the directed migra- tion of enteric nervous system progenitor cells during mammalian embryogenesis. Development, 129:5151–60. Ohlsson, B., Forsling, M.L., Rehfeld, J.F., et al. 2002. Cholecystokinin leads to increased oxytocin secretion in healthy women. Eur. J. Surg., 168:114–18. Ohlsson, B., Ringström, G., Abrahamsson, H., et al. 2004. Oxytocin stimu- lates colonic motor activity in healthy women. Neurogastroenterol Mot., 16:33–40. Ohlsson, B., Björgell, O., Ekberg, O., et al. 2006a. The oxytocin/vasopres- sin receptor antagonist atosiban delays the gastric emptying of a semisolid meal compared to saline in human. BMC. Gastroenterology, 6:11 (16 Mars 2006). 237 Gastroinestinal motility Drug Target Insights 2007:2 Ohlsson, B., Truedsson, M., Djerf, P., et al. 2006b. Oxytocin is present throughout the human gastrointestinal tract. Reg. Pept., 135:7–11. Ohlsson, B. 2006c. A case report on a patient suffering from recurrent vomiting episodes, whose condition improved remarkedly during pregnancy and breast feeding. BMC. Gastroenterology, 6:28. Ohlsson, B., Veréss, B., Janciauskiene, S., et al. 2007. Chronic intestinal pseudo-obstruction due to buserelin-induced formation of anti-GnRH antibodies. Gastroenterology, 132:45–51. Ordög, T., Takayama, I., Cheung, W.K.T., et al. 2000. Remodeling of net- works of interstitial cells of Cajal in a murine model of diabetic gastroparesis. Diabetes, 49:1731–9. Palomba, S., Orio, F., Manguso, F., et al. 2005. Leuprolide acetate treatment with and without coadministration of tibolone in premenopausal women with menstrual cycle-related irritable bowel syndrome. Fertility and Sterility, 83:1012–20. Pan, H. and Galligan, J.J. 1994. 5-HT1A and 5-HT4 receptors mediate inhi- bition and facilitation of fast synaptic transmission in enteric neurons. Am. J. Physiol., 266:G230–G238. Petersson, M., Alster, P., Lundeberg, T., et al. 1996. Oxytocin increases nociceptive thresholds in a long-term perspective in female and male rats. Neurosci. Lett., 212:87–90. Petrache, I., Fijalkowska, I., Medler, T.R., et al. 2006. [alpha]-1 antitrypsin inhibits caspase-3 activitiy, preventing lung endothelial cell apopto- sis. Am. J. Pathol., 169:1155–66. Petring, O.U. 1989. The effect of oxytocin on basal and pethidine-induced delayed gastric emptying. Br. J. Clin. Pharmacol., 28:329–32. Rettenbacher, M. and Reubi, J.C. 2001. Localization and characterization of neuropeptide receptors in human colon. Naunyn. Schmiedebergs. Arch. Pharmacol., 364:291–304. Ruhl, A. 2005. Glial cells in the gut. Neurogastroenterol Motil., 17:777–90. Sanders, K.M., Ordög, T., Koh, S.D., et al. 1999. Development and plasticity of interstitial cells of Cajal. Neurogastroenterol Motil, 11:311–38. Schurmann, G., Bishop, A.E., Facer, P., et al. 1995. Secretoneurin: a new peptide in the human enteric nervous system. Histochem. Cell Biol., 104:11–9. Sidhu, M. and Cooke, H.J. 1995. Role for 5-HT and Ach in submucosal refl exes mediating colonic secretion. Am. J. Physiol. Gatsrointest. Liver. Physiol., 269:G346–G351. Silber, M., Larsson, B. and Uvnäs-Moberg, K. 1991. Oxytocin, somatosta- tin, insulin and gastrin concentrations vis-à-vis late pregnancy, breastfeeding and oral contraceptives. Acta. Obstet. Gynecol. Scand., 70:283–9. Simren, M., Simms, L., D’Souza, D., Abrahamsson, H., Bjornsson, E.S. 2003. Lipid-induced colonic hypersensitivity in irritable bowel syndrome: the role of 5-HT3 receptors. Aliment. Pharmacol. Ther., 17:279–87. Soldani, G., Del Tacca, M., Bambini, G., et al. 1982. Effects of gonadotropin- releasing hormone (GnRH) on gastric secretion and gastrin release in the dog. J. Endocrinol. Invest., 5:393–6. Song, Y., Li, J.C., Li, M.J. 2002. Bcl-2 expression in enteric neurons of Hirschsprung’s disease and its signifi cance. Shi Yan Sheng Wu Xue Bao, 35:155–8. Sperber, A.D., Atzmon, Y., Neumann, L., et al. 1999. Fibromyalgia in the irritable bowel syndrome: Studies of prevalence and clinical implica- tions. Am. J. Gastroenterol, 94:3541–6. Spiller, R.C. 2003. Postinfectious irritable bowel syndrome. Gastroenterol- ogy, 124:1662–71. Tack, J., Depoortere 1, Bisschops, R., et al. 2005. Infl uence of ghrelin on gastric emptying and meal-related symptoms in idiopathic gastropa- resis. Aliment. Pharmacol. Ther., 22:847–53. Takamori, K.I., Mizuta, Y., Takshima, F., et al. 2007. Relation among plasma ghrelin level, gastric emptying and psychologic condition in patients with functional dyspepsia. J. Clin. Gastroenterol., 41:477–83. Thomsen, L., Robinson, T.L., Lee, J.C.F., et al. 1998. Interstitial cells of Cajal generate a rhythmic pacemaker current. Nat. Med., 4:848–50. Törnblom, H., Lindberg, G., Nyberg, B., et al. 2002. Full-thickness biopsy of the jejunum reveals infl ammation and enteric neuropathy in irri- table bowel syndrome. Gastroenterology, 123:1972–79. Uvnäs-Moberg, K., Arn, I., Theorell, I., et al. 1991. Gastrin, somatostatin and oxytocin levels in the patients with functional disorders of the gastrointestinal tract and their response to feeding and interaction. J. Psychosom. Res., 35:525–33. Vasina, V., Barbara, G., Talamonti, L., et al. 2006. Enteric neuroplasticity evoked by infl ammation. Auton. Neurosci., 126–127:264–72. Vialli, M. 1966. Histology of the enterochromaffin cell system. In: ERspamer, V., ed. Handbook of experimental pharmacology: 5-hydroxytrypramine and related indolealkylamines. Vol 19. New York: Springer-Verlag, p 1–65. Von Boyen, G.B., Steinkamp, M., Reinshagen, M., et al. 2004. Proinfl am- matory cytokines increase glial fi brillary acidic protein expression in enteric glia. Gut., 53:222–8. Wang, L., Xie, L.P., Huang, W.Q., et al. 2001. Presence of gonadotropin- releasing hormone (GnRH) and its mRNA in rat pancreas. Mol. Cell. Endocrinol., 14:172–85. Wessinger, S., Jones, M.P. and Crowell, M.D. 2005. Editorial overview: serotonergic agents in functional GI disorders: targeting the brain-gut axis. Curr. Opin. Invest. Drugs., 6:663–6. Wester, T., Olsson, Y. and Olsen, L. 1999. Expression of bcl-2 in enteric neurons in normal human bowel and hirschsprung disease. Arch. Pathol. Lab. Med., 123:1264–8. Wood, J.D. 2000. Neuropathy in the brain-in-gut. Eur. J. Gastroenterol Hepatol., 12:597–600. Wu, J.J., Rothman, T.P., Gershon, M.D. 2000. Development of the intersti- tial cell of Cajal: origin, kit dependence and neuronal and nonneuro- nal sources of kit ligand. J. Neurosci. Res., 59:384–401. Xu, L., Depoortere, I., Tomasetto, C., et al. 2005. Evidence for the presence of motilin, ghrelin, and the motilin and ghrelin receptor in neurons of the myenteric plexus. Regul. Pept., 124:119–25. Young, H.M., Anderson, R.B. and Anderson, C.R. 2004. Guidance cues involved in the development of the peripheral autonomic nervous system. Auton. Neurosci., 112:1–14. Zhang, J.V., Ren, P.G., Avsian-Kretchmer, O., et al. 2005. Obestatin, a peptide encoded by the ghrelin gene, opposes ghrelin’s effects on food intake. Science, 310:996–9. << /ASCII85EncodePages false /AllowTransparency false /AutoPositionEPSFiles true /AutoRotatePages /None /Binding /Left /CalGrayProfile (Dot Gain 20%) /CalRGBProfile (sRGB IEC61966-2.1) /CalCMYKProfile (U.S. Web Coated \050SWOP\051 v2) /sRGBProfile (sRGB IEC61966-2.1) /CannotEmbedFontPolicy /Error /CompatibilityLevel 1.4 /CompressObjects /Tags /CompressPages true /ConvertImagesToIndexed true /PassThroughJPEGImages true /CreateJDFFile false /CreateJobTicket false /DefaultRenderingIntent /Default /DetectBlends true /ColorConversionStrategy /LeaveColorUnchanged /DoThumbnails false /EmbedAllFonts true /EmbedJobOptions true /DSCReportingLevel 0 /EmitDSCWarnings false /EndPage -1 /ImageMemory 1048576 /LockDistillerParams false /MaxSubsetPct 100 /Optimize true /OPM 1 /ParseDSCComments true /ParseDSCCommentsForDocInfo true /PreserveCopyPage true /PreserveEPSInfo true /PreserveHalftoneInfo false /PreserveOPIComments false /PreserveOverprintSettings true /StartPage 1 /SubsetFonts true /TransferFunctionInfo /Apply /UCRandBGInfo /Preserve /UsePrologue false /ColorSettingsFile () /AlwaysEmbed [ true ] /NeverEmbed [ true ] /AntiAliasColorImages false /DownsampleColorImages true /ColorImageDownsampleType /Bicubic /ColorImageResolution 300 /ColorImageDepth -1 /ColorImageDownsampleThreshold 1.50000 /EncodeColorImages true /ColorImageFilter /DCTEncode /AutoFilterColorImages true /ColorImageAutoFilterStrategy /JPEG /ColorACSImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /ColorImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /JPEG2000ColorACSImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /JPEG2000ColorImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /AntiAliasGrayImages false /DownsampleGrayImages true /GrayImageDownsampleType /Bicubic /GrayImageResolution 300 /GrayImageDepth -1 /GrayImageDownsampleThreshold 1.50000 /EncodeGrayImages true /GrayImageFilter /DCTEncode /AutoFilterGrayImages true /GrayImageAutoFilterStrategy /JPEG /GrayACSImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /GrayImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /JPEG2000GrayACSImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /JPEG2000GrayImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /AntiAliasMonoImages false /DownsampleMonoImages true /MonoImageDownsampleType /Bicubic /MonoImageResolution 1200 /MonoImageDepth -1 /MonoImageDownsampleThreshold 1.50000 /EncodeMonoImages true /MonoImageFilter /CCITTFaxEncode /MonoImageDict << /K -1 >> /AllowPSXObjects false /PDFX1aCheck false /PDFX3Check false /PDFXCompliantPDFOnly false /PDFXNoTrimBoxError true /PDFXTrimBoxToMediaBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXSetBleedBoxToMediaBox true /PDFXBleedBoxToTrimBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXOutputIntentProfile () /PDFXOutputCondition () /PDFXRegistryName (http://www.color.org) /PDFXTrapped /Unknown /Description << /JPN /FRA /DEU /PTB /DAN /NLD /ESP /SUO /ITA /NOR /SVE /ENU >> >> setdistillerparams << /HWResolution [2400 2400] /PageSize [612.000 792.000] >> setpagedevice