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Lithocholyltaurine (LCT), a conjugated 3α-mono (hydroxy) bile acid, binds to M3 subtype muscarinic cholinergic receptors
A1126 AGAABSTRACTS
GASTROENTEROLOGY Vol. 114, No. 4
• G4607 INCREASED ACTIVITY OF HYPOTHALAMIC 5-HT IN ANOREXIA INDUCED BY EXPERIMENTAL COLITIS. lAB Ballin~er. iT Elhaj, Ij Turvill, 2G Williams, lMJG Farthing. Digestive Diseases Research Centre, St Bartholomew's & The Royal London School of Medicine & Dentistry, London, UK l, Dept. of Medicine, The University of Liverpool, UK2. Inflammatory bowel disease and other chronic inflammatory conditions are often associated with anorexia and marked weight loss. In children and adolescents this contributes to linear growth retardation and delayed puberty. In health, serotonin (5-HT) is a potent anorexic agent with effects centered on the medial hypothalamus, a major appetite regulating centre. We have explored the hypothesis that anorexia associated with intestinal inflammation is related to increased 5-HT activity. Methods. Colitis was induced in Wistar rats by intrarectai administration of trinitrobenzene (TNBS) in ethanol. Two control groups were used: healthy free-feeding and a pair-fed (PF) group whose food intake was matched to the colitic group. 5-HT concentrations were measured by HPLC in microdissected hypothaiamic nuclei 1 day after induction of colitis when anorexia was maximal. Inflammation was assessed by measurement of intestinal myeloperoxidase (MPO). Results: In TNBScolitis there was increased MPO concentrations (colitic, 0.64 -+0.06; controls, 0.35 -+0.06; PF, 0.19 -+0.04 U/g), hypophagia (colitic, 1.8 -+0.4; controls 25 -+ 1 g/24-h) and weight loss. By definition food intake in PF and colitic groups were equal. Within the paraventricular (PVN), dorsomedial (DMH) and ventromedial hypothalamus (VMH), 5-HT concentrations in the PF (food restricted), but not the colitic group, were 2 -5-fold higher than controls. pmol/mg protein
PVN
DMH
VMH
Fed controls 5 -+ 1.7 3.4 -+0.9 20.9 + 5 Colitic 2.4 -+ 0.73 4.5 +- 1.3 11.3 -+ 1.6 Pair-fed 21.4 -+ 6.5t 20.0 -+ 6.7t 43 -+ 16* * P = 0.01 vs controls and colitis, * P < 0.05 vs controls and colitis
Taken together with the known anorexic effects of 5-HT, results in the PF group suggest that in response to food restriction there is inhibition of neuronal 5-HT release resulting in higher tissue concentrations in the medial hypothalamus. In the colitic group results suggest continuing 5-HT release which would inhibit the hunger and hyperphagia normally induced by weight loss. • G4608 MECHANISMS OF LINEAR GROWTH FAILURE ASSOCIATED WITH INTESTINAL INFLAMMATION. AB Ballinaer. O Azooz, MJG Farthing. Digestive Diseases Research Centre, St Bartholomew's & The Royal London School of Medicine & Dentistry, London, UK.
Suppression of linear growth is a major complication of inflammatory bowel disease and is thought to be due primarily to reduced food intake induced by inflammatory cytokines. However, cytokines may also directly inhibit growth; transgenic mice which overexpress IL-6 are growth retarded with inhibition of growth hormone (GH) stimulated hepatic insulin-like growth factor (IGF-1) secretion. It has been difficult to disentangle these aetiological factors when studying mechanisms of growth retardation. We have explored the hypothesis that inflammation and reduced feeding have independent but cumulative effects on growth. Methods: Colitis was induced in 10 prepubertal Wistar rats by intrarectal administration of trinitrobenzene (TNBS) in ethanol. Two age and weight matched control groups were used: healthy free-feeding and a pair-fed (PF) group whose food intake was matched to the colitic group. Food intake and body weight was measured daily and nose-base of tail length assessed at induction and at sacrifice. Animals were sacrificed 5 days after induction and blood collected for hormone assay. Inflammation was assessed by measurement of intestinal myeloperoxidase (MPO) concentrations. Results. TNBS induced marked distal colitis with a 3-fold increase in IL-6 concentrations, increased MPO concentrations (colitis 165 -+52; controls 33 -+ 6; PF 49 + 10 mU/g), hypophagia and reduced weight gain (colitis 6.7 -+0.7g, controls 37.5 -+ 5.6 g/5 days). By definition food intake in the PF group was the same as the colitic group. PF (food restricted) animals were growth retarded with reduced plasma IGF-1 but increased GH concentrations compared to fed controls. Growth was further reduced in the colitic group with reduced GH and IGF-1 concentrations compared with PF. Controls Colitic Pair-fed
Growth (cm/5 days) GH (ng/ml) ! IGF-1 (ng/mi) 1.7 -+ 0.18 90 -+36 753 -+93 0.5 -+0.06* 153 -+ 27* 267 -+ 17t 1 -+0.18 270 + 54 433 -+64 * P < 0.05 vs PF; * P < 0.02 vs PF; * P < 0.005 vs PF
Conclusions. Experimental colitis impairs growth independent of nutritional factors. Reduced GH and IGF-1 concentrations suggest that inflammatory mediators act at both central and peripheral sites. G4609 OXYTOCIN RECEPTORS IN THE BRAIN ARE INVOLVED IN ENDOTOXIN-INDUCED INHIBITION OF GASTRIC ACID SECRETION. M.D. Barrachina, S. Calatayud, B. Beltrfin, A. Canet, J.V. Esplugues. Dept. of Pharmacology, University of Valencia, Valencia, Spain. ' Background: Endotoxin stimulates the release of endogenous mediators some of which modulate gastric function acting either in the periphery or in
the central nervous system. Aim: We have analyzed the effects of oxytocin, vasopressin and CRF on distension-stimulated gastric acid secretion and the endogenous role of these mediators in the acute inhibitory effects of low doses of endotoxin on gastric function. Methods: Sprague-Dawley (220-280g) rats were anaesthetized with urethane (1.5 g kg -1, i.p.), the stomachs continuously perfused with saline (0.9 ml min-l) and acid output measured. Oxytocin (0.2, 2 or 4 nmol rat-1, i.e.), vasopressin (0.1, 2 or 5 nmol rat-1, i.e.), CRF (0.5, 1 or 2 nmol rat -1, i.e.) or endotoxin (E coli LPS, 5 lag kg "l, i.v.) were administered once H÷ output remained constant for 60 min and 15 min before distension of the stomach with an intragastric pressure of 20 cm H20. In some experiments, 15 min before endotoxin animals received, a single injection of the antagonists of oxytocin [Compound VI(d(CH2)5.)OVT, llag rat-l, i.e. or i.v.], CRF [a-Helical CRF (9-41), 20 lag rat-l, i.e.[ or vasopressin [[3-mercapto13[3-cyclopentamethylenepropionyl, O-Et-Tyr2, Val4, Arg8)-vasopressin, 20 lag rat -l, i.e.). Results: Net gastric acid secretion (A) induced by distension (44. 1-+ 10.6 AlaEq H÷ 100g-1 90min-1, n=10) was reduced in a dosedependent manner by oxytocin (25.7 - 26.6%, n=4; 55.1 -+ 14.1%*, n=17 and 81.2-+3.5%*, n=5) or CRF (65.3-25.8%*, n=3, 77.1 +-13.0%*, n=6 and 85.9-+7.6%*, n=12) (results expressed as percentage of reduction over vehicle). Similar doses of vasopressin, a closely-related peptide to oxytocin did not significantly modify acid output (-19.9 _+16.3%, n=3, 37.9 _+32.3%, n=3 and 33.6_+20.1%, n=6). Endotoxin-induced inhibition (11.8-+ 1.9", AlaEq H÷ 100g-1 90min -1, n=10) of distension-stimulated gastric acid secretion (27.3-+7.3 AlaEq H÷ 100g-1 90min-1 n=9) was prevented by i.e. administration of oxytocin antagonist (29.1 -+9.4 & AlaEq H÷ 100g-1 90min -1, n=5). However, when administered i.v., the antagonist failed to modify the antisecretory effect of endotoxin. Pretreatment with either CRF (13.2 + 5.9 AlaEq H÷ 100g-1 90min -l, n=7) or vasopressin (5.2 -+ 3.2 AlaEq H÷ 100g-1 90min -1, n=5) antagonists did not significantly reverse the inhibitory effects of endotoxin (11.8-+ 1.9" AlaEq H÷ 100g-1 90min-1, n=10). Conclusion: Oxytocin or CRF, but no vasopressin act in the brain to inhibit distensionstimulated gastric acid secretion. However, only activation of central oxytocin receptors is involved in endotoxin-induced inhibition of acid output. *p<0.05 vs the respective vehicle-treated group &p<0.05 vs endotoxin-treated group • G4610
LITHOCHOLYLTAURINE (LCT), A CONJUGATED 3cc-MONO (HYDROXY) BILE ACID, BINDS TO M 3 SUBTYPE MUSCARINIC CHOLINERGIC RECEPTORS. A. Bartoszko-Malik P. Zimniak, and J-P. Raufman. Department of Internal Medicine, Division of Gastroenterology, University of Arkansas for Medical Sciences, Little Rock, AR. In the course of examining the actions of bile acids on dispersed chief cells from guinea pig stomach, we observed that micromolar concentrations of LCT stimulate a 3-fold increase in pepsinogen secretion. Because (a) the secretory actions of LCT are inhibited by atropine; (b) increasing concentrations of LCT cause a progressive reduction in carbachol-induced pepsinogen secretion; and (c) M3 subtype muscarinic cholinergic receptors are the only cholinergic receptors expressed on chief ceils, we hypothesized that LCT binds to M3 receptors. To test this hypothesis, we examined the effects of LCT and other bile acids on binding of a cholinergic radioligand, 3H-N-methylscopolamine (3H-NMS), to a highly homogeneous population of gastric chief cells (prepared from guinea pig stomach by collagenase digestion of gastric mucosa and separation of dispersed cells on a Percoll density gradient) and to Chinese hamster ovary (CHO) ceils expressing the 5 known muscarinic receptor subtypes (designated M l - Ms) (kindly provided by Dr. T. Bonner, NIMH). To control for different levels of M 3 receptor expression, gastric chief cells (61 fmol M3 receptor/cell) and CHO cells (690 fmol M 3 receptor/cell) were tested at a density of 107 and 106 cells/ml, respectively. Unlabeled NMS (1/aM) was used to determine non-specific binding. 3H-NMS binding to both cell types was time- and temperature-dependent, saturable and reversible. Increasing concentrations of LCT (10-8 to 10.3 M) resulted in progressive inhibition of 3H-NMS binding to chief and CHO cells expressing M3 receptors. TLC did not alter 3H-NMS binding to CHO cells expressing MI, M4, and M5 receptors, and only the highest concentration of LCT tested (1 mM) inhibited binding to CHO cells expressing M 2 receptors (40% inhibition of maximal 3H-NMS binding). Taurine, cholyltaurine, and lithocholylglycine did not alter 3H-NMS binding to chief or CHO ceils. Inhibition of 3H-NMS binding to M3-CHO and chief cells was first detected with 1 and 100/aM LCT, respectively. Half-maximal inhibition of 3H-NMS binding to M3-CHO cells was achieved with approx. 10-fold lower concentrations of LCT when compared to those in chief cells. In both cell types, maximal inhibition of 3H-NMS binding by LCT was approx. 50% of that observed with maximal concentrations of carbachol, atropine or unlabeled NMS. In control experiments, performed to exclude non-specific effects on radioligand binding, LCT did not alter the binding of 12sI-CCK-8 to chief cells. These data indicate that, at 'physiological' concentrations, LCT binds specifically to M3 subtype muscarinic cholinergic receptors. Binding of LCT to M 3 receptors on chief cells stimulates pepsinogen secretion. It is noteworthy that, other than acetylcholine, LCT appears to be the only known endogenous ligand specific for muscarinic receptors. Moreover, LCT-induced cholinergic stimulation of gastric secretory cells may contribute to mucosal injury in bile gastritis.
GASTROENTEROLOGY Vol. 114, No. 4
• G4607 INCREASED ACTIVITY OF HYPOTHALAMIC 5-HT IN ANOREXIA INDUCED BY EXPERIMENTAL COLITIS. lAB Ballin~er. iT Elhaj, Ij Turvill, 2G Williams, lMJG Farthing. Digestive Diseases Research Centre, St Bartholomew's & The Royal London School of Medicine & Dentistry, London, UK l, Dept. of Medicine, The University of Liverpool, UK2. Inflammatory bowel disease and other chronic inflammatory conditions are often associated with anorexia and marked weight loss. In children and adolescents this contributes to linear growth retardation and delayed puberty. In health, serotonin (5-HT) is a potent anorexic agent with effects centered on the medial hypothalamus, a major appetite regulating centre. We have explored the hypothesis that anorexia associated with intestinal inflammation is related to increased 5-HT activity. Methods. Colitis was induced in Wistar rats by intrarectai administration of trinitrobenzene (TNBS) in ethanol. Two control groups were used: healthy free-feeding and a pair-fed (PF) group whose food intake was matched to the colitic group. 5-HT concentrations were measured by HPLC in microdissected hypothaiamic nuclei 1 day after induction of colitis when anorexia was maximal. Inflammation was assessed by measurement of intestinal myeloperoxidase (MPO). Results: In TNBScolitis there was increased MPO concentrations (colitic, 0.64 -+0.06; controls, 0.35 -+0.06; PF, 0.19 -+0.04 U/g), hypophagia (colitic, 1.8 -+0.4; controls 25 -+ 1 g/24-h) and weight loss. By definition food intake in PF and colitic groups were equal. Within the paraventricular (PVN), dorsomedial (DMH) and ventromedial hypothalamus (VMH), 5-HT concentrations in the PF (food restricted), but not the colitic group, were 2 -5-fold higher than controls. pmol/mg protein
PVN
DMH
VMH
Fed controls 5 -+ 1.7 3.4 -+0.9 20.9 + 5 Colitic 2.4 -+ 0.73 4.5 +- 1.3 11.3 -+ 1.6 Pair-fed 21.4 -+ 6.5t 20.0 -+ 6.7t 43 -+ 16* * P = 0.01 vs controls and colitis, * P < 0.05 vs controls and colitis
Taken together with the known anorexic effects of 5-HT, results in the PF group suggest that in response to food restriction there is inhibition of neuronal 5-HT release resulting in higher tissue concentrations in the medial hypothalamus. In the colitic group results suggest continuing 5-HT release which would inhibit the hunger and hyperphagia normally induced by weight loss. • G4608 MECHANISMS OF LINEAR GROWTH FAILURE ASSOCIATED WITH INTESTINAL INFLAMMATION. AB Ballinaer. O Azooz, MJG Farthing. Digestive Diseases Research Centre, St Bartholomew's & The Royal London School of Medicine & Dentistry, London, UK.
Suppression of linear growth is a major complication of inflammatory bowel disease and is thought to be due primarily to reduced food intake induced by inflammatory cytokines. However, cytokines may also directly inhibit growth; transgenic mice which overexpress IL-6 are growth retarded with inhibition of growth hormone (GH) stimulated hepatic insulin-like growth factor (IGF-1) secretion. It has been difficult to disentangle these aetiological factors when studying mechanisms of growth retardation. We have explored the hypothesis that inflammation and reduced feeding have independent but cumulative effects on growth. Methods: Colitis was induced in 10 prepubertal Wistar rats by intrarectal administration of trinitrobenzene (TNBS) in ethanol. Two age and weight matched control groups were used: healthy free-feeding and a pair-fed (PF) group whose food intake was matched to the colitic group. Food intake and body weight was measured daily and nose-base of tail length assessed at induction and at sacrifice. Animals were sacrificed 5 days after induction and blood collected for hormone assay. Inflammation was assessed by measurement of intestinal myeloperoxidase (MPO) concentrations. Results. TNBS induced marked distal colitis with a 3-fold increase in IL-6 concentrations, increased MPO concentrations (colitis 165 -+52; controls 33 -+ 6; PF 49 + 10 mU/g), hypophagia and reduced weight gain (colitis 6.7 -+0.7g, controls 37.5 -+ 5.6 g/5 days). By definition food intake in the PF group was the same as the colitic group. PF (food restricted) animals were growth retarded with reduced plasma IGF-1 but increased GH concentrations compared to fed controls. Growth was further reduced in the colitic group with reduced GH and IGF-1 concentrations compared with PF. Controls Colitic Pair-fed
Growth (cm/5 days) GH (ng/ml) ! IGF-1 (ng/mi) 1.7 -+ 0.18 90 -+36 753 -+93 0.5 -+0.06* 153 -+ 27* 267 -+ 17t 1 -+0.18 270 + 54 433 -+64 * P < 0.05 vs PF; * P < 0.02 vs PF; * P < 0.005 vs PF
Conclusions. Experimental colitis impairs growth independent of nutritional factors. Reduced GH and IGF-1 concentrations suggest that inflammatory mediators act at both central and peripheral sites. G4609 OXYTOCIN RECEPTORS IN THE BRAIN ARE INVOLVED IN ENDOTOXIN-INDUCED INHIBITION OF GASTRIC ACID SECRETION. M.D. Barrachina, S. Calatayud, B. Beltrfin, A. Canet, J.V. Esplugues. Dept. of Pharmacology, University of Valencia, Valencia, Spain. ' Background: Endotoxin stimulates the release of endogenous mediators some of which modulate gastric function acting either in the periphery or in
the central nervous system. Aim: We have analyzed the effects of oxytocin, vasopressin and CRF on distension-stimulated gastric acid secretion and the endogenous role of these mediators in the acute inhibitory effects of low doses of endotoxin on gastric function. Methods: Sprague-Dawley (220-280g) rats were anaesthetized with urethane (1.5 g kg -1, i.p.), the stomachs continuously perfused with saline (0.9 ml min-l) and acid output measured. Oxytocin (0.2, 2 or 4 nmol rat-1, i.e.), vasopressin (0.1, 2 or 5 nmol rat-1, i.e.), CRF (0.5, 1 or 2 nmol rat -1, i.e.) or endotoxin (E coli LPS, 5 lag kg "l, i.v.) were administered once H÷ output remained constant for 60 min and 15 min before distension of the stomach with an intragastric pressure of 20 cm H20. In some experiments, 15 min before endotoxin animals received, a single injection of the antagonists of oxytocin [Compound VI(d(CH2)5.)OVT, llag rat-l, i.e. or i.v.], CRF [a-Helical CRF (9-41), 20 lag rat-l, i.e.[ or vasopressin [[3-mercapto13[3-cyclopentamethylenepropionyl, O-Et-Tyr2, Val4, Arg8)-vasopressin, 20 lag rat -l, i.e.). Results: Net gastric acid secretion (A) induced by distension (44. 1-+ 10.6 AlaEq H÷ 100g-1 90min-1, n=10) was reduced in a dosedependent manner by oxytocin (25.7 - 26.6%, n=4; 55.1 -+ 14.1%*, n=17 and 81.2-+3.5%*, n=5) or CRF (65.3-25.8%*, n=3, 77.1 +-13.0%*, n=6 and 85.9-+7.6%*, n=12) (results expressed as percentage of reduction over vehicle). Similar doses of vasopressin, a closely-related peptide to oxytocin did not significantly modify acid output (-19.9 _+16.3%, n=3, 37.9 _+32.3%, n=3 and 33.6_+20.1%, n=6). Endotoxin-induced inhibition (11.8-+ 1.9", AlaEq H÷ 100g-1 90min -1, n=10) of distension-stimulated gastric acid secretion (27.3-+7.3 AlaEq H÷ 100g-1 90min-1 n=9) was prevented by i.e. administration of oxytocin antagonist (29.1 -+9.4 & AlaEq H÷ 100g-1 90min -1, n=5). However, when administered i.v., the antagonist failed to modify the antisecretory effect of endotoxin. Pretreatment with either CRF (13.2 + 5.9 AlaEq H÷ 100g-1 90min -l, n=7) or vasopressin (5.2 -+ 3.2 AlaEq H÷ 100g-1 90min -1, n=5) antagonists did not significantly reverse the inhibitory effects of endotoxin (11.8-+ 1.9" AlaEq H÷ 100g-1 90min-1, n=10). Conclusion: Oxytocin or CRF, but no vasopressin act in the brain to inhibit distensionstimulated gastric acid secretion. However, only activation of central oxytocin receptors is involved in endotoxin-induced inhibition of acid output. *p<0.05 vs the respective vehicle-treated group &p<0.05 vs endotoxin-treated group • G4610
LITHOCHOLYLTAURINE (LCT), A CONJUGATED 3cc-MONO (HYDROXY) BILE ACID, BINDS TO M 3 SUBTYPE MUSCARINIC CHOLINERGIC RECEPTORS. A. Bartoszko-Malik P. Zimniak, and J-P. Raufman. Department of Internal Medicine, Division of Gastroenterology, University of Arkansas for Medical Sciences, Little Rock, AR. In the course of examining the actions of bile acids on dispersed chief cells from guinea pig stomach, we observed that micromolar concentrations of LCT stimulate a 3-fold increase in pepsinogen secretion. Because (a) the secretory actions of LCT are inhibited by atropine; (b) increasing concentrations of LCT cause a progressive reduction in carbachol-induced pepsinogen secretion; and (c) M3 subtype muscarinic cholinergic receptors are the only cholinergic receptors expressed on chief ceils, we hypothesized that LCT binds to M3 receptors. To test this hypothesis, we examined the effects of LCT and other bile acids on binding of a cholinergic radioligand, 3H-N-methylscopolamine (3H-NMS), to a highly homogeneous population of gastric chief cells (prepared from guinea pig stomach by collagenase digestion of gastric mucosa and separation of dispersed cells on a Percoll density gradient) and to Chinese hamster ovary (CHO) ceils expressing the 5 known muscarinic receptor subtypes (designated M l - Ms) (kindly provided by Dr. T. Bonner, NIMH). To control for different levels of M 3 receptor expression, gastric chief cells (61 fmol M3 receptor/cell) and CHO cells (690 fmol M 3 receptor/cell) were tested at a density of 107 and 106 cells/ml, respectively. Unlabeled NMS (1/aM) was used to determine non-specific binding. 3H-NMS binding to both cell types was time- and temperature-dependent, saturable and reversible. Increasing concentrations of LCT (10-8 to 10.3 M) resulted in progressive inhibition of 3H-NMS binding to chief and CHO cells expressing M3 receptors. TLC did not alter 3H-NMS binding to CHO cells expressing MI, M4, and M5 receptors, and only the highest concentration of LCT tested (1 mM) inhibited binding to CHO cells expressing M 2 receptors (40% inhibition of maximal 3H-NMS binding). Taurine, cholyltaurine, and lithocholylglycine did not alter 3H-NMS binding to chief or CHO ceils. Inhibition of 3H-NMS binding to M3-CHO and chief cells was first detected with 1 and 100/aM LCT, respectively. Half-maximal inhibition of 3H-NMS binding to M3-CHO cells was achieved with approx. 10-fold lower concentrations of LCT when compared to those in chief cells. In both cell types, maximal inhibition of 3H-NMS binding by LCT was approx. 50% of that observed with maximal concentrations of carbachol, atropine or unlabeled NMS. In control experiments, performed to exclude non-specific effects on radioligand binding, LCT did not alter the binding of 12sI-CCK-8 to chief cells. These data indicate that, at 'physiological' concentrations, LCT binds specifically to M3 subtype muscarinic cholinergic receptors. Binding of LCT to M 3 receptors on chief cells stimulates pepsinogen secretion. It is noteworthy that, other than acetylcholine, LCT appears to be the only known endogenous ligand specific for muscarinic receptors. Moreover, LCT-induced cholinergic stimulation of gastric secretory cells may contribute to mucosal injury in bile gastritis.