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Effects of Orexins, Guanylins and Feeding on Duodenal Bicarbonate Secretion and Enterocyte Intracellular Signaling
Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Neuroscience.
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The duodenal epithelium secretes bicarbonate ions and this is regarded as the primary defence mechanism against the acid discharged from the stomach. For an efficient protection, the duodenum must also function as a sensory organ identifying luminal factors. Enteroendocrine cells are well-established intestinal “taste” cells that express signaling peptides such as orexins and guanylins. Luminal factors affect the release of these peptides, which may modulate the activity of nearby epithelial and neural cells.

The present thesis considers the effects of orexins and guanylins on duodenal bicarbonate secretion. The duodenal secretory response to the peptides was examined in anaesthetised rats in situ and the effects of orexin-A on intracellular calcium signaling by human as well as rat duodenal enterocytes were studied in vitro.

Orexin-A, guanylin and uroguanylin were all stimulants of bicarbonate secretion. The stimulatory effect of orexin-A was inhibited by the OX1-receptor selective antagonist SB-334867. The muscarinic antagonist atropine on the other hand, did not affect the orexin-A-induced secretion, excluding involvement of muscarinic receptors. Orexin-A induced calcium signaling in isolated duodenocytes suggesting a direct effect at these cells. Interestingly, orexin-induced secretion and calcium signaling as well as mucosal orexin-receptor mRNA and OX1-receptor protein levels were all substantially downregulated in overnight fasted rats compared with animals with continuous access to food. Further, secretion induced by Orexin-A was shown to be dependent on an extended period of glucose priming.

The uroguanylin-induced bicarbonate secretion was reduced by atropine suggesting involvement of muscarinic receptors. The melatonin receptor antagonist luzindole attenuated the secretory response to intra-arterially administered guanylins but had no effect on secretion when the guanylins were given luminally.

In conclusion, the results suggest that orexin-A as well as guanylins may participate in the regulation of duodenal bicarbonate secretion. Further, the duodenal orexin system is dependent on the feeding status of the animals.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2008. , p. 70
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 337
Keywords [en]
Physiology, alkaline secretion, carbohydrates, central nervous system, cholinergic stimulation, duodenum, enteric nervous system, enterochromaffin cell, fasting, feeding, glucose, guanylyl cyclase C, humans, hypocretin, intra-arterial, in situ, intracerebroventricular, luminal acid, luzindole, orexin-B, SB-334867
Keywords [sv]
Fysiologi
Identifiers
URN: urn:nbn:se:uu:diva-8664ISBN: 978-91-554-7173-6 (print)OAI: oai:DiVA.org:uu-8664DiVA, id: diva2:171902
Public defence
2008-05-15, B21, Uppsala Biomedicinska Centrum (BMC), Norra vägen, Uppsala, 13:15
Opponent
Supervisors
Available from: 2008-04-23 Created: 2008-04-23Bibliographically approved
List of papers
1. Food-induced expression of orexin receptors in rat duodenal mucosa regulates the bicarbonate secretory response to orexin-A
Open this publication in new window or tab >>Food-induced expression of orexin receptors in rat duodenal mucosa regulates the bicarbonate secretory response to orexin-A
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2007 (English)In: American Journal of Physiology - Gastrointestinal and Liver Physiology, ISSN 0193-1857, E-ISSN 1522-1547, Vol. 293, no 2, p. G501-G509Article in journal (Refereed) Published
Abstract [en]

Presence of appetite-regulating peptides orexin-A and orexin-B in mucosal endocrine cells suggests a role in physiological control of the intestine. Our aim was to characterize orexin-induced stimulation of duodenal bicarbonate secretion and modulation of secretory responses and mucosal orexin receptors by overnight food deprivation. Lewis x Dark Agouti rats were anesthetized and proximal duodenum cannulated in situ. Mucosal bicarbonate secretion (pH stat) and mean arterial blood pressure were continuously recorded. Orexin-A was administered intra-arterially close to the duodenum, intraluminally, or into the brain ventricles. Total RNA was extracted from mucosal specimens, reverse transcribed to cDNA and expression of orexin receptors 1 and 2 (OX1 and OX2) measured by quantitative real-time PCR. OX1 protein was measured by Western blot. Intra-arterial orexin-A (60–600 nmol·h–1·kg–1) increased (P < 0.01) the duodenal secretion in fed but not in fasted animals. The OX1 receptor antagonist SB-334867, which was also found to have a partial agonist action, abolished the orexin-induced secretory response but did not affect secretion induced by the muscarinic agonist bethanechol. Atropine, in contrast, inhibited bethanechol but not orexin-induced secretion. Orexin-A infused into the brain ventricles (2–20 nmol·kg–1·h–1) or added to luminal perfusate (1.0–100 nM) did not affect secretion, indicating that orexin-A acts peripherally and at basolateral receptors. Overnight fasting decreased mucosal OX1 and OX2 mRNA expression (P < 0.01) as well as OX1 protein expression (P < 0.05). We conclude that stimulation of secretion by orexin-A may involve both receptor types and is independent of cholinergic pathways. Intestinal OX receptors and secretory responses are markedly related to food intake.

Keywords
bicarbonate secretion, enteroendocrine cells, fed and fasting state, perfused duodenum in situ, TRH
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-97103 (URN)10.1152/ajpgi.00514.2006 (DOI)000248487800012 ()
Available from: 2008-04-23 Created: 2008-04-23 Last updated: 2017-12-14Bibliographically approved
2. Short food deprivation inhibits orexin receptor 1 expression and orexin-A induced intracellular calcium signaling in acutely isolated duodenal enterocytes
Open this publication in new window or tab >>Short food deprivation inhibits orexin receptor 1 expression and orexin-A induced intracellular calcium signaling in acutely isolated duodenal enterocytes
2009 (English)In: American Journal of Physiology - Gastrointestinal and Liver Physiology, ISSN 0193-1857, E-ISSN 1522-1547, Vol. 296, no 3, p. G651-G658Article in journal (Refereed) Published
Abstract [en]

Bengtsson MW, Makela K, Herzig KH, Flemstrom G. Short food deprivation   inhibits orexin receptor 1 expression and orexin-A induced   intracellular calcium signaling in acutely isolated duodenal   enterocytes. Am J Physiol Gastrointest Liver Physiol 296: G651-G658,   2009. First published December 31, 2008;   doi:10.1152/ajpgi.90387.2008.-Close intra-arterial infusion of the   appetite regulating peptide orexin-A stimulates bicarbonate secretion   from the duodenal mucosa. The aim of the present study was to elucidate   the ability of orexin-A to induce intracellular calcium signaling in   acutely isolated duodenal enterocytes. Freshly isolated clusters of   enterocytes, obtained from rat duodenal mucosa or human duodenal   biopsies, were loaded with fura 2-AM and mounted in a perfusion   chamber. Cryptlike enterocytes were selected (caged), and changes in   intracellular calcium concentration ([Ca2+](i)) were evaluated by   fluorescence imaging. Total RNA was extracted from pellets of   enterocytes and reverse transcribed to cDNA, and expression of orexin   receptors 1 and 2 (OX1R and OX2R) was measured by quantitative   real-time PCR. Orexin-A at all concentrations tested (1-100 nM)   increased [Ca2+](i) in enterocytes isolated from continuously fed rats,   and the OX1R-antagonist SB-334867 (10 nM) attenuated the response. The   primary [Ca2+](i) response was a slow increase to a sustained plateau   persisting after orexin-A removal, and a similar response was observed   in enterocytes from human biopsies. In contrast to orexin-A, the OX2R   agonist (Ala(11), D-Leu(15))orexin-B (1-10 nM) did not induce calcium   signaling. There were no significant [Ca2+](i) responses in enterocytes   from animals food deprived overnight, and overnight fasting decreased   (P < 0.01) enterocyte OX1R as well as OX2R mRNA. Induction of   intracellular calcium signaling in isolated duodenal enterocytes is   thus mediated primarily by OX1R receptors. Short (overnight) food   deprivation markedly depresses receptor expression and inhibits   orexin-A induced increases in [Ca2+](i). Studies of enterocyte   signaling and intestinal secretion requires particular evaluation   regarding feeding status.

Keywords
(Ala(11), D-Leu(15))-orexin-B, bicarbonate secretion, fasting, feeding, SB-334867
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-97104 (URN)10.1152/ajpgi.90387.2008 (DOI)000263900800022 ()19118115 (PubMedID)
Available from: 2009-04-02 Created: 2008-05-15 Last updated: 2017-12-14Bibliographically approved
3. Presence of glucose/carbohydrate but not other nutrients in the duodenal lumen influences duodenal secretory response to orexin-A
Open this publication in new window or tab >>Presence of glucose/carbohydrate but not other nutrients in the duodenal lumen influences duodenal secretory response to orexin-A
Manuscript (Other academic)
Identifiers
urn:nbn:se:uu:diva-97105 (URN)
Available from: 2008-04-23 Created: 2008-04-23 Last updated: 2010-01-13Bibliographically approved
4. Duodenal bicarbonate secretion in rats: Stimulation by intra-arterial and luminal guanylin and uroguanylin
Open this publication in new window or tab >>Duodenal bicarbonate secretion in rats: Stimulation by intra-arterial and luminal guanylin and uroguanylin
2007 (English)In: Acta Physiologica, ISSN 1748-1708, E-ISSN 1748-1716, Vol. 191, no 4, p. 309-317Article in journal (Refereed) Published
Abstract [en]

Aim: Uroguanylin and guanylin are endogenous ligands for guanylate cyclase C, an upstream regulator of the cystic fibrosis transmembrane resistance (CFTR) anion channel, and both peptides increase intestinal anion export in vitro. We have compared the effects of close intra-arterial and luminal administration of uroguanylin and guanylin on duodenal bicarbonate secretion in vivo and studied the interactions with melatonin and cholinergic stimulation.

Methods: Lewis × Dark Agouti rats were anaesthetized and a segment of the proximal duodenum with intact blood supply was cannulated in situ. Mucosal bicarbonate secretion (pH stat) was continuously recorded and peptides were infused intra-arterially or added to the luminal perfusate.

Results: Intra-arterial (50–1000 pmol kg−1 h−1) as well as luminal administration (50–500 nmol L−1) of guanylin or uroguanylin caused dose-dependent increases in the duodenal secretion. Luminal administration induced more rapidly appearing rises in secretion and the two peptides induced secretory responses of similar shape and magnitude. The melatonin MT2-selective antagonist luzindole (600 nmol kg−1) significantly depressed the response to intra-arterial guanylins but did not affect secretion induced by luminal guanylins. Similarly, the muscarinic antagonist atropine (0.75 μmol kg−1 followed by 0.15 μmol kg−1 h−1) abolished the response to intra-arterial uroguanylin but caused only slight suppression of the response to luminal uroguanylin.

Conclusions: Intra-arterial as well as luminal uroguanylin and guanylin are potent stimuli of duodenal mucosal bicarbonate secretion in vivo. The response to luminal guanylins reflects an action at apical receptors. Stimulation by parenteral guanylins, in contrast, is under cholinergic influence and interacts with melatonin produced by mucosal enteroendocrine cells.

Keywords
bicarbonate secretion, cholinergic stimulation, duodenum in situ, enteroendocrine cells, guanylyl cyclase C, luzindole, melatonin
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-97106 (URN)10.1111/j.1748-1716.2007.01759.x (DOI)000250795300006 ()17995576 (PubMedID)
Available from: 2008-04-23 Created: 2008-04-23 Last updated: 2017-12-14Bibliographically approved

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