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Abstract Orexins, or hypocretins, are peptides originally found in the hypothalamus, and have been shown to be involved in the stabilization and maintenance of sleep and wakefulness. In addition, these peptides are known for their actions on energy homeostasis by increased heat production or physical activity. Previous results suggest them to be also involved in peripheral actions on the regulation of intestinal secretion, depending on the subject’s nutritional status (fasted-fed). Orexin-A and Orexin-B peptides, are derived from the prepro-orexin precursor protein. These ligands bind to two G-protein-coupled receptors, orexin-1 and -2 -receptors. Despite intensive research, the role of orexins has not yet been clarified. The aim of the present study was to investigate the role of orexins and their receptors on sleep and wake patterns, energy homeostasis and intestinal secretion. The effects of orexins on sleep and wakefulness, and energy homeostasis were studied in a novel transgenic mouse line, overexpressing the human prepro-orexin gene. The overexpression of prepro-orexin and orexin-A was confirmed in the hypothalami of transgenic mice. The transgenic mice showed a significant reduction in their REM sleep during day and night time, and differences in their vigilance states in the light/dark transition periods. In addition, the mice demonstrated a significantly elevated day time food intake at room temperature, and an increased metabolic heat production independent of uncoupling protein 1 mediated thermogenesis in brown adipose tissue. Instead, transgenic mice showed increased levels of uncoupling protein 2 in white adipose tissue. Furthermore, transgenic mice significantly decreased their total locomotor activity during the first two nights in response to cold exposure (+4°C). The effect of orexins and their receptors on duodenal HCO3– secretion were studied after an overnight (16 h) food deprivation in an in situ perfused organ. Fasting decreased the expression of orexin receptors in rat duodenal mucosa and in acutely isolated duodenal enterocytes. Furthermore, food deprivation abolished OXA induced duodenal mucosal HCO3– secretion in rats, and intracellular calcium signalling in isolated rat and human duodenal enterocytes. In conclusion, the present thesis demonstrates that orexins inhibit REM sleep. In addition, peptides affect increasingly on metabolic heat production, independent of uncoupling protein 1 mediated thermogenesis. Furthermore, the orexin system has a significant role in duodenal bicarbonate secretion, which is regulated by the presence of food in the intestine.

The duodenal mucosa is regularly exposed to acid, digestive enzymes and ingested noxious agents. It is thus critical to maintain a protective barrier to prevent the development of mucosal injury and inflammation, which are often observed in situations when barrier function is impaired. The rate of mucosal bicarbonate secretion, the regulation of epithelial paracellular permeability and motility are each key components of duodenal barrier function. The hormone melatonin is present in high levels in the gastrointestinal tract and it has been hypothesized that melatonin exerts protective properties. This thesis aims to investigate the impact of exogenous melatonin on the regulation of duodenal barrier function and motility in anesthetized rats in vivo. In addition, duodenal tissue was examined histologically and the expression levels of tight junction proteins and melatonin receptors were assessed with qRT-PCR. It was found that melatonin stimulated mucosal bicarbonate secretion and decreased basal paracellular permeability. Exposing the duodenal mucosa to the well-characterized barrier breaker ethanol increased mucosal bicarbonate secretion, paracellular permeability and motility. Omission of luminal Clˉ abolished, while pretreatment with a nicotinic receptor antagonist reduced, the ethanol-induced bicarbonate secretion suggesting that the secretory response to ethanol is meditated via Clˉ/HCO3ˉexchangers and enteric neural pathways. Melatonin reduced the ethanol-induced increases in paracellular permeability and motility either when injected intravenously or when administered in drinking water for two weeks. The actions of melatonin were abolished by the melatonin receptor antagonist luzindole and by nicotinic acetylcholine receptor inhibition. Two weeks oral administration of melatonin up-regulated the expression levels of melatonin receptors, down-regulated the expression of ZO-3 while the expression of ZO-1, ZO-2, claudin 2-4, occludin and myosin light chain kinase were unaffected. Superficial epithelial changes in a few villi were seen in response to ethanol exposure, an effect that was histologically unchanged by melatonin pretreatment. In conclusion, the results suggest that melatonin plays an important role in the neurohumoral regulation of gastrointestinal mucosal barrier function and motility via receptor- and enteric neural-dependent pathways in vivo in rats. Melatonin might be a candidate for treatment of barrier dysfunction in humans.

In seawater, drinking is a fundamental part of the osmoregulatory strategy for teleost fish, and presents a unique challenge. The intestine has an established role in osmoregulation, and its ability to effectively absorb fluid from imbibed seawater is crucial to compensating for water losses to the surrounding hyperosmotic environment. Alongside solute-linked water transport (driven by NaCl cotransport), intestinal bicarbonate (HCO3-) secretion also benefits fluid absorption directly (via apical Cl-/HCO3- exchange), and indirectly through the formation of calcium carbonate (CaCO3) thus removing the osmotic influence of Ca2+ within the gut fluid. For the European flounder (Platichthys flesus), elevated luminal Ca2+ has proven to be a specific, potent stimulator of HCO3- secretion both in vitro and in vivo where these actions are presumably modulated by an extracellular Ca2+-sensing receptor (CaR). The focus of this work was to learn more about how intestinal HCO3- secretion is regulated, the role of Ca2+, and more specifically the CaR. To achieve this, in vitro ‘gut sac’ experiments investigated how luminal Ca2+ influenced HCO3- secretion, and associated ion and fluid transport. Contrary to expectation, increasing Ca2+ from 5 to 20 mM did not stimulate HCO3- secretion. In an attempt to elucidate the role of CaCO3 precipitation in fluid absorption, and further explore the physiological implications of HCO3- secretion, the intestine was perfused in vivo with salines containing varying concentrations of Ca2+ (10, 40 and 90 mM). The production and secretion of HCO3-, in addition to CaCO3 formation increased accordingly with Ca2+, and was associated with a dramatic 25 % rise in the fraction of fluid absorbed by the gut. Additional in vitro experiments, utilising the Ussing chamber, helped establish some of the characteristics of intestinal HCO3- secretion by the euryhaline killifish (Fundulus heteroclitus), but was unresponsive to elevated mucosal Ca2+. Further attempts to potentiate the activity of the CaR, and application of the receptor agonists gadolinium (Gd3+) and neomycin, failed to produce responses consistent with the effect of Ca2+ observed previously, either in vitro or in vivo. With no evidence supporting a direct role for an extracellular, intestinal CaR in HCO3- secretion it was argued that secretion would be principally regulated by two factors, the ability of the epithelia to generate high levels of intracellular HCO3- and the rate of CaCO3 formation.

The duodenal epithelium is regularly exposed to HCl, digestive enzymes, bacteria and toxins, and sometimes also to ethanol and drugs. The imbalance of aggressive factors in the intestinal lumen and mucosal barrier function increases the risk of tissue injury and inflammation. The key components of the duodenal barrier function include mucosal permeability, bicarbonate transport and the secretion or absorption of fluids. This thesis aims to elucidate the role of melatonin, neuropeptide S (NPS) and short chain fatty acids (SCFAs) in the regulation of intestinal mucosal barrier function and motility in the anesthetized rat in vivo and in tissues of human origin in vitro. Melatonin was found to reduce ethanol-induced increases in paracellular permeability and motility by a neural pathway within the enteric nervous system involving nicotinic receptors. In response to luminal exposure of ethanol, signs of mild mucosal edema and beginning of desquamation were observed in a few villi only, an effect that was not influenced by melatonin. Melatonin did not modify increases in paracellular permeability in response to luminal acid. NPS decreased basal and ethanol-induced increases in duodenal motility as well as bethanechol stimulated colonic motility in a dose-dependent manner. Furthermore, NPS was shown to inhibit basal duodenal bicarbonate secretion, stimulate mucosal fluid absorption and increase mucosal paracellular permeability. In response to luminal exposure of acid, NPS increased bicarbonate secretion and mucosal paracellular permeability. All effects induced by the administration of NPS were dependent on nitrergic pathways. In rats, administration of NPS increased the tissue protein levels of the inflammatory biomarkers IL-1β and CXCL1. Immunohistochemistry showed that NPS was localized at myenteric nerve cell bodies and fibers, while NPSR1 and nNOS were only confined to the myenteric nerve cell bodies. Perfusing the duodenal segment with the SCFAs acetate or propionate reduced the duodenal mucosal paracellular permeability, decreased transepithelial net fluid secretion and increased bicarbonate secretion. An i.v. infusion of SCFAs reduces mucosal paracellular permeability without any effects on mucosal net fluid flux. However, it significantly decreased bicarbonate secretion. Luminal SCFAs changed the duodenal motility pattern from fasting to feeding motility while i.v. SCFAs was without effect on motility. The systemic administration of glucagon-like peptide-2 (GLP-2) induced increases in mucosal bicarbonate secretion and fluid absorption. An i.v. GLP-2 infusion during a luminal perfusion of SCFAs significantly reduced the duodenal motility. In conclusion, the results in the present thesis show that melatonin, NPS and SCFAs influence the neurohumoral regulation of intestinal mucosal barrier function and motility. Aberrant signaling in response to melatonin, NPS and to luminal fatty acids might be involved in the symptom or the onset of disease related to intestinal dysfunction in humans. / <p>Research funders and strategic development areas:</p><p>- Bengt Ihre Foundation (grant SLS-177521)</p><p>- Socialstyrelsen(grant SLS-176671)</p><p>- Erik, Karin, and Gösta Selanders Foundation</p><p>- Emil and Ragna Börjesson Foundation</p><p>- Uppsala University </p><p>- Ministry of Education of Malaysia</p><p>- Universiti Malaysia Sabah, Malaysia</p>

<p>The stomach is frequently exposed to hazardous agents and to resist this harsh environment, several protective mechanisms exist. Of special interest is the gastric pathogen <i>Helicobacter pylori </i>which causes gastritis, ulcers and cancer but the mechanism leading to these diseases are still unclear. However it is very likely that <i>H. pylori </i>negatively influence the protection mechanisms that exist in the stomach. </p><p>The aims of the present investigation were first to develop an in vivo mouse model in which different protection mechanisms could be studied, and second to investigate the influence of <i>H. pylori</i> on these mechanisms. </p><p>An in vivo preparation of the gastric mucosa in mice was developed. This preparation allows studies of different gastric mucosal variables and can also be applied for studies in other gastro-intestinal organs. </p><p>Mice chronically infected with <i>H. pylori</i>, were shown to have a reduced ability of the mucosa to maintain a neutral pH at the epithelial cell surface. This could be due to the thinner inner, firmly adherent mucus gel layer, and/or to defective bicarbonate transport across the epithelium. The Cl^-/HCO₃^- exchanger SLC26A9 was inhibited by NH₄⁺, which also is produced by <i>H. pylori</i>. The mRNA levels of SLC26A9 were upregulated in infected mice, suggesting a way to overcome the inhibition of the transporter. Furthermore, the hyperemic response to acid pH 2 and 1.5 was abolished in these mice. The mechanisms by which the bacteria could alter the blood flow response might involve inhibition of the epithelial iNOS.</p><p>Water extracts of <i>H. pylori </i>(HPE) reduces the blood flow acutely through an iNOS and nerve-mediated pathway, possibly through the endogenous iNOS inhibitor ADMA. Furthermore, HPE alters the blood flow response to acid as the hyperemic response to acid pH 0.8 is accentuated in mice treated with HPE. </p>

The stomach is frequently exposed to hazardous agents and to resist this harsh environment, several protective mechanisms exist. Of special interest is the gastric pathogen Helicobacter pylori which causes gastritis, ulcers and cancer but the mechanism leading to these diseases are still unclear. However it is very likely that H. pylori negatively influence the protection mechanisms that exist in the stomach. The aims of the present investigation were first to develop an in vivo mouse model in which different protection mechanisms could be studied, and second to investigate the influence of H. pylori on these mechanisms. An in vivo preparation of the gastric mucosa in mice was developed. This preparation allows studies of different gastric mucosal variables and can also be applied for studies in other gastro-intestinal organs. Mice chronically infected with H. pylori, were shown to have a reduced ability of the mucosa to maintain a neutral pH at the epithelial cell surface. This could be due to the thinner inner, firmly adherent mucus gel layer, and/or to defective bicarbonate transport across the epithelium. The Cl-/HCO3- exchanger SLC26A9 was inhibited by NH4+, which also is produced by H. pylori. The mRNA levels of SLC26A9 were upregulated in infected mice, suggesting a way to overcome the inhibition of the transporter. Furthermore, the hyperemic response to acid pH 2 and 1.5 was abolished in these mice. The mechanisms by which the bacteria could alter the blood flow response might involve inhibition of the epithelial iNOS. Water extracts of H. pylori (HPE) reduces the blood flow acutely through an iNOS and nerve-mediated pathway, possibly through the endogenous iNOS inhibitor ADMA. Furthermore, HPE alters the blood flow response to acid as the hyperemic response to acid pH 0.8 is accentuated in mice treated with HPE.