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Intracisternal TRH analog-induced gastric hyperemia in anesthetized rats: role of 5-HT and histamine receptors
GASTROENTEROLOGY Vol. 118, No.4
ABO AGA ABSTRACTS
591
593
INTRACEREBROVENTRICULAR APPLICATION OF LEPTIN PROTECTS GASTRIC MUCOSA VIA ENHANCEMENT OF NI· TRIC OXIDE (NO) AND SENSORY AFFERENTS. Tomasz Brzozowski, Slawomir Kwiecien, Peter Ch Konturek, Wladyslaw Bielenski, Stanislaw 1. Konturek, Eckhart G. Hahn, Dept Physiol , Univ Med Sch, Cracow, Poland; Dept Med I , Univ Med, Erlangen, Germany. Leptin, an adipoctye product that controls food intake via hypothalamus, has been recently detected in the stomach and found to be released by cholecystokinin (CCK) and to protect gastric mucosa but it is unknown whether centrally applied leptin influences gastric secretion and mucosal integrity. We compared the effects of leptin and CCK -8 applied intracerebroventricularly (i.c.v.) on gastric acid secretion and mucosal lesions induced by topical application of 75% ethanol. Several series of Wistar rats(A-E)were used to determine the gastroprotective activity of exogenous leptin and CCK-8 applied i.c.v. against ethanol injury in animals with or without: A)blockade of CCK A receptors with loxiglumide (30 mglkg i.p.), B) cutting of vagal nerves (vagotomy), C) inactivation of sensory nerves by capsaicin (125 mglkg s.c.), D) inhibition of CGRP with CGRP S_37 (100 /Lglkg i.p.) and Ejsuppression of NO-synthase with L-NAME (5 mglkg i.v.), Rats were killed I h after ethanol administration and the area of gastric lesions was measured by planimetry and the gastric blood flow (GBF) was determined by Hz-gas clearance technique. Blood was withdrawn for the measurement of plasma leptin and CCK by radioimmunoassay (RIA)and gastric biopsy samples were collected for the determination of cNOS and iNOS mRNA by RT-PCR. Leptin and CCK (0.01-5 /Lglkg i.c.v.) dose-dependently attenuated gastric lesions induced by 75% ethanol; the dose reducing these lesions by 50% (ED 50) was 0.8 /Lglkg and 1.2 /Lglkg, respectively, and this was accompanied by a significant rise in plasma levels of leptin and CCK and an increase in GBF. Loxiglumide abolished the protective and hyperemic effects of central CCK but not those of central leptin. The protective and hyperemic effects of central leptin were completely abolished by vagotomy and significantly attenuated by the capsaicin-denervation, the blockade of CGRP with CGRP S_37 or the pretreatment with L-NAME. Strong signal for iNOS mRNA was recorded in gastric mucosa of leptin and CCK-treated animals, whereas cNOS mRNA was unaffected. We conclude that: I) central leptin exerts a potent gastroprotective action on the stomach depending upon vagal activity and sensory nerves and involving hyperemia probably mediated by NO and 2)leptin mimics the protective effect of CCK and may be implicated in the gastroprotective and hyperemic actions of this peptide.
DISCRETE LOCALIZATION WITHIN THE RAT NODOSE GANGLION OF VAGAL AFFERENT SENSORY NEURONS INNERVATING THE GASTROINTESTINAL TRACT. Jinfeng Guo, Olufemi Abiodun, Kirsteen N. Browning, Henry Ford Health Sci Ctr, Detroit, MI. The nodose ganglia contain the nerve cell bodies of vagal afferent neurons that receive sensory innervation from the abdominal and thoracic viscera. Nodose ganglion neurons are non-uniform with respect to several different properties such as neurochemical phenotype and electrophysiological properties. The aim of this study was to investigate whether neurons that innervate the gastrointestinal tract exhibit a viscerotopic organization within the nodose ganglion. The anterogradely transported fluorescent tracer Dil was applied to either the gastric fundus (n =6), corpus (n = 10) or antrum/pylorus (n=7), the duodenum (n=6) or cecum (n=9) in 12 day old rat pups of either sex. After 10-12 days, the left and right nodose ganglia were removed and fixed separately prior to freezing and cutting in 50/Lm sections using a cryostat. Slices were then examined to reconstruct the location of the anterogradely-Iabeled neurons within the ganglion. While neurons that innervate the gastric regions were located almost exclusively in the caudal poles of the nodose ganglia, neurons innervating the intestinal regions were located more in the mid-pole of the ganglion, with only a few caudal cells found on the convex side of the ganglion. These findings provide preliminary evidence for a discrete viscerotopic organization of vagal sensory afferent neurons within the nodose ganglion. Together with the evidence of non-uniform immunohistochemical and electrophysiological properties, such a somatotropic classification may indicate that the vagal sensory neurons are organized into discrete subpopulations based upon their location within the nodose ganglion, their electrophysiological and neurochemical properties according to the region of the gastrointestinal tract they innervate
592 REGIONAL BRAIN SEROTONIN SYNTHESIS RATES IN MALE AND FEMALE IRRITABLE BOWEL SYNDROME (IBS) PATIENTS. Mirko Diksic, A. Nakai, Y. Kumakura, Michel Boivin, Kathryn E. Kersey, McGill Univ, Montreal, ON, Canada; Ctr Hosptialier de l'Universite de Montreal, Montreal, ON, Canada; Glaxo Wellcome, Mississauga, ON, Canada. Introduction: Serotonergic pathways are involved in visceral sensation and gastrointestinal motility, which playa role in the pathogenesis of IBS. Differences have been observed in regional brain activation during visceral perception between normal and IBS subjects (Gastroenterology 1997; 112:64-72) and between males and females (J Nuc Med 1997; 38:273P). No study to date has directly examined serotonin (5-HT) metabolism in the brain in IBS patients. Aim: To determine the rates of brain 5-HT synthesis in male and female IBS patients. Methods: 5-HT synthesis was measured with positron emission tomography (PET) using a-[IIC] methyl-L-tryptophan (aMTrp) as the tracer in 12 (6F, 6M) non-constipated IBS patients. Medications used to treat IBS, such as anticholinergics, as well as other gastrointestinal medications, antidepressants, anxiolytics, narcotics, other 5-HT 3 antagonists, and tryptophan supplements were prohibited for 7 days prior to the PET scan. The PET images were converted to 5-HT synthesis rates by calculating the brain uptake constant for data collected between 20 to 60min post-tracer injection. All subjects also had a magnetic resonance image (MRI) performed which was used to transform subjects' brains to Talairach's space, where PET and MRI images were co-localized. A comparison was done with II normal controls (5F, 6M). Comparisons between groups were done using statistical parametric mapping (SPM) with proportional scaling. Differences were transformed to t-maps which were converted to z-scores. Clusters having z-scores for height of ::0:2.58 (p<0.005) and size ::0: 100 voxels were considered significantly different. A Bonferroni multiple comparison correction was done to avoid significant Type I errors. Results: No significant differences in 5-HT synthesis rates were observed between male IBS patients and male controls. Female IBS patients had several areas in which the 5-HT synthesis rate was higher than, and none lower than, female controls. Significant differences were found in Brodmann areas 21, 10,32, as well as midbrain, radiatio optica, tapetum and fasciculus longitudinalis inferior. Conclusion: An imbalance in serotonergic pathways may exist between female IBS and female control subjects. Study S3BI0901 was supported by Glaxo Wellcome.
594 INTRACISTERNAL TRH ANALOG-INDUCED GASTRIC HYPEREMIA IN ANESTHETIZED RATS: ROLE OF 5-HT AND HISTAMINE RECEPTORS. Keishi Kawakubo, Yasutada Akiba, Paul H. Guth, Jonathan D. Kaunitz, Yvette Tache, Kyushu Univ, Fukuoka, Japan; CURElDDRC, VAMC, Dept of Med , UCLA, Los Angeles, CA. BACKGROUND: The TRH analog, RX 77368 (RX) injected intracisternally (Ie) at cytoprotective doses induced a vagal atropine sensitive gastric mucosal hyperemia mediated by CGRP contained in capsaicin-sensitive afferent fibers . The hyperemic response is also accompanied by the degranulation of mucosal and submucosal mast cells (MCs), and prevented by ketotifen, doxantrazole and cromolyn, MC stabilizers. MCs-derived substances activate capsaicin-sensitive afferent fibers. AIM: To investigate the roles of 5-hydroxytryptamine (5-HT) and histamine receptor subtypes in RX-induced gastric hyperemia, using selective receptor subtype antagonists. METHODS: Gastric mucosal blood flow (GMBF; measured by laser-Doppler flowmetry), mean arterial blood pressure and gastric acid secretion (GAS) were assessed simultaneously for 30 min before and 60 min after IC injection of RX (2.5 ng) or saline in urethane-anesthetized rats using an ex vivo chamber technique. Ketanserin (Ket, 5-HT 2 receptor antagonist (RA); 3 mg/kg, sc), ondansetron (Ond, 5HT 3 RA; I mg/kg, bolus iv), and RS-039604-190 (RS, 5HT 4 RA; 300 p..glkg, bolus iv), pyrilamine (Pyr, HI RA; 2 mglkg, iv), and famotidine (Fam, Hz RA; 2 mg/kg, iv) were administered 20 min before IC RX. RESULTS: The increase of GMBF and the decreased of GMVR after IC RX was prevented by Ond, RS or Fam, but not by Ket or Pyr. Conclusions: These results show that the hyperemic response induced by a cytoprotective dose of IC RX is mediated via 5-HT 3 , 5-HT 4 and Hz receptors. MCs have an important role in gastric mucosal protection through the releases of 5-HT and histamine. Pretreatment + RX (IC, 2.5 ng) Time (after RX, min)
!:J.GMBF
!:J.GMVR
GAS (flEq
20
20
Eq 160 min
ABO AGA ABSTRACTS
591
593
INTRACEREBROVENTRICULAR APPLICATION OF LEPTIN PROTECTS GASTRIC MUCOSA VIA ENHANCEMENT OF NI· TRIC OXIDE (NO) AND SENSORY AFFERENTS. Tomasz Brzozowski, Slawomir Kwiecien, Peter Ch Konturek, Wladyslaw Bielenski, Stanislaw 1. Konturek, Eckhart G. Hahn, Dept Physiol , Univ Med Sch, Cracow, Poland; Dept Med I , Univ Med, Erlangen, Germany. Leptin, an adipoctye product that controls food intake via hypothalamus, has been recently detected in the stomach and found to be released by cholecystokinin (CCK) and to protect gastric mucosa but it is unknown whether centrally applied leptin influences gastric secretion and mucosal integrity. We compared the effects of leptin and CCK -8 applied intracerebroventricularly (i.c.v.) on gastric acid secretion and mucosal lesions induced by topical application of 75% ethanol. Several series of Wistar rats(A-E)were used to determine the gastroprotective activity of exogenous leptin and CCK-8 applied i.c.v. against ethanol injury in animals with or without: A)blockade of CCK A receptors with loxiglumide (30 mglkg i.p.), B) cutting of vagal nerves (vagotomy), C) inactivation of sensory nerves by capsaicin (125 mglkg s.c.), D) inhibition of CGRP with CGRP S_37 (100 /Lglkg i.p.) and Ejsuppression of NO-synthase with L-NAME (5 mglkg i.v.), Rats were killed I h after ethanol administration and the area of gastric lesions was measured by planimetry and the gastric blood flow (GBF) was determined by Hz-gas clearance technique. Blood was withdrawn for the measurement of plasma leptin and CCK by radioimmunoassay (RIA)and gastric biopsy samples were collected for the determination of cNOS and iNOS mRNA by RT-PCR. Leptin and CCK (0.01-5 /Lglkg i.c.v.) dose-dependently attenuated gastric lesions induced by 75% ethanol; the dose reducing these lesions by 50% (ED 50) was 0.8 /Lglkg and 1.2 /Lglkg, respectively, and this was accompanied by a significant rise in plasma levels of leptin and CCK and an increase in GBF. Loxiglumide abolished the protective and hyperemic effects of central CCK but not those of central leptin. The protective and hyperemic effects of central leptin were completely abolished by vagotomy and significantly attenuated by the capsaicin-denervation, the blockade of CGRP with CGRP S_37 or the pretreatment with L-NAME. Strong signal for iNOS mRNA was recorded in gastric mucosa of leptin and CCK-treated animals, whereas cNOS mRNA was unaffected. We conclude that: I) central leptin exerts a potent gastroprotective action on the stomach depending upon vagal activity and sensory nerves and involving hyperemia probably mediated by NO and 2)leptin mimics the protective effect of CCK and may be implicated in the gastroprotective and hyperemic actions of this peptide.
DISCRETE LOCALIZATION WITHIN THE RAT NODOSE GANGLION OF VAGAL AFFERENT SENSORY NEURONS INNERVATING THE GASTROINTESTINAL TRACT. Jinfeng Guo, Olufemi Abiodun, Kirsteen N. Browning, Henry Ford Health Sci Ctr, Detroit, MI. The nodose ganglia contain the nerve cell bodies of vagal afferent neurons that receive sensory innervation from the abdominal and thoracic viscera. Nodose ganglion neurons are non-uniform with respect to several different properties such as neurochemical phenotype and electrophysiological properties. The aim of this study was to investigate whether neurons that innervate the gastrointestinal tract exhibit a viscerotopic organization within the nodose ganglion. The anterogradely transported fluorescent tracer Dil was applied to either the gastric fundus (n =6), corpus (n = 10) or antrum/pylorus (n=7), the duodenum (n=6) or cecum (n=9) in 12 day old rat pups of either sex. After 10-12 days, the left and right nodose ganglia were removed and fixed separately prior to freezing and cutting in 50/Lm sections using a cryostat. Slices were then examined to reconstruct the location of the anterogradely-Iabeled neurons within the ganglion. While neurons that innervate the gastric regions were located almost exclusively in the caudal poles of the nodose ganglia, neurons innervating the intestinal regions were located more in the mid-pole of the ganglion, with only a few caudal cells found on the convex side of the ganglion. These findings provide preliminary evidence for a discrete viscerotopic organization of vagal sensory afferent neurons within the nodose ganglion. Together with the evidence of non-uniform immunohistochemical and electrophysiological properties, such a somatotropic classification may indicate that the vagal sensory neurons are organized into discrete subpopulations based upon their location within the nodose ganglion, their electrophysiological and neurochemical properties according to the region of the gastrointestinal tract they innervate
592 REGIONAL BRAIN SEROTONIN SYNTHESIS RATES IN MALE AND FEMALE IRRITABLE BOWEL SYNDROME (IBS) PATIENTS. Mirko Diksic, A. Nakai, Y. Kumakura, Michel Boivin, Kathryn E. Kersey, McGill Univ, Montreal, ON, Canada; Ctr Hosptialier de l'Universite de Montreal, Montreal, ON, Canada; Glaxo Wellcome, Mississauga, ON, Canada. Introduction: Serotonergic pathways are involved in visceral sensation and gastrointestinal motility, which playa role in the pathogenesis of IBS. Differences have been observed in regional brain activation during visceral perception between normal and IBS subjects (Gastroenterology 1997; 112:64-72) and between males and females (J Nuc Med 1997; 38:273P). No study to date has directly examined serotonin (5-HT) metabolism in the brain in IBS patients. Aim: To determine the rates of brain 5-HT synthesis in male and female IBS patients. Methods: 5-HT synthesis was measured with positron emission tomography (PET) using a-[IIC] methyl-L-tryptophan (aMTrp) as the tracer in 12 (6F, 6M) non-constipated IBS patients. Medications used to treat IBS, such as anticholinergics, as well as other gastrointestinal medications, antidepressants, anxiolytics, narcotics, other 5-HT 3 antagonists, and tryptophan supplements were prohibited for 7 days prior to the PET scan. The PET images were converted to 5-HT synthesis rates by calculating the brain uptake constant for data collected between 20 to 60min post-tracer injection. All subjects also had a magnetic resonance image (MRI) performed which was used to transform subjects' brains to Talairach's space, where PET and MRI images were co-localized. A comparison was done with II normal controls (5F, 6M). Comparisons between groups were done using statistical parametric mapping (SPM) with proportional scaling. Differences were transformed to t-maps which were converted to z-scores. Clusters having z-scores for height of ::0:2.58 (p<0.005) and size ::0: 100 voxels were considered significantly different. A Bonferroni multiple comparison correction was done to avoid significant Type I errors. Results: No significant differences in 5-HT synthesis rates were observed between male IBS patients and male controls. Female IBS patients had several areas in which the 5-HT synthesis rate was higher than, and none lower than, female controls. Significant differences were found in Brodmann areas 21, 10,32, as well as midbrain, radiatio optica, tapetum and fasciculus longitudinalis inferior. Conclusion: An imbalance in serotonergic pathways may exist between female IBS and female control subjects. Study S3BI0901 was supported by Glaxo Wellcome.
594 INTRACISTERNAL TRH ANALOG-INDUCED GASTRIC HYPEREMIA IN ANESTHETIZED RATS: ROLE OF 5-HT AND HISTAMINE RECEPTORS. Keishi Kawakubo, Yasutada Akiba, Paul H. Guth, Jonathan D. Kaunitz, Yvette Tache, Kyushu Univ, Fukuoka, Japan; CURElDDRC, VAMC, Dept of Med , UCLA, Los Angeles, CA. BACKGROUND: The TRH analog, RX 77368 (RX) injected intracisternally (Ie) at cytoprotective doses induced a vagal atropine sensitive gastric mucosal hyperemia mediated by CGRP contained in capsaicin-sensitive afferent fibers . The hyperemic response is also accompanied by the degranulation of mucosal and submucosal mast cells (MCs), and prevented by ketotifen, doxantrazole and cromolyn, MC stabilizers. MCs-derived substances activate capsaicin-sensitive afferent fibers. AIM: To investigate the roles of 5-hydroxytryptamine (5-HT) and histamine receptor subtypes in RX-induced gastric hyperemia, using selective receptor subtype antagonists. METHODS: Gastric mucosal blood flow (GMBF; measured by laser-Doppler flowmetry), mean arterial blood pressure and gastric acid secretion (GAS) were assessed simultaneously for 30 min before and 60 min after IC injection of RX (2.5 ng) or saline in urethane-anesthetized rats using an ex vivo chamber technique. Ketanserin (Ket, 5-HT 2 receptor antagonist (RA); 3 mg/kg, sc), ondansetron (Ond, 5HT 3 RA; I mg/kg, bolus iv), and RS-039604-190 (RS, 5HT 4 RA; 300 p..glkg, bolus iv), pyrilamine (Pyr, HI RA; 2 mglkg, iv), and famotidine (Fam, Hz RA; 2 mg/kg, iv) were administered 20 min before IC RX. RESULTS: The increase of GMBF and the decreased of GMVR after IC RX was prevented by Ond, RS or Fam, but not by Ket or Pyr. Conclusions: These results show that the hyperemic response induced by a cytoprotective dose of IC RX is mediated via 5-HT 3 , 5-HT 4 and Hz receptors. MCs have an important role in gastric mucosal protection through the releases of 5-HT and histamine. Pretreatment + RX (IC, 2.5 ng) Time (after RX, min)
!:J.GMBF
!:J.GMVR
GAS (flEq
20
20
Eq 160 min