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Expression of reg gene and its product are enhanced in gastric endocrine cells aroud water immersion-induced gastric lesions in rat
Abstracts from the 1 l th International Symposium on Regulatory Peptides
7
Is cyclic AMP involved in the contractile response of rainbow trout gastrointestinal smooth muscle to bombesin and trout gastrin releasing peptide? U. Aronsson, J. Jensen and S. Holmgren, Dept of Zoophysiology, University of GOteborg, Sweden. Bombesin and gastrin releasing peptide (GRP) contract smooth muscle of the gastrointestinal tract in both mammals and fish. In mammals the signal transduction from receptor to contraction involves release of calcium from extracellular and/or intracellular stores and a reduction of the cyclic AMP levels. By using two agents known to increase the concentration of cyclic AMP in mammals, the phosphodiesterase inhibitor 3-isobuthyl-l-methylxanthine (IBMX) and the adenylyl cyclase stimulator forskolin, we wanted to investigate whether cyclic AMP plays a role in the bombesin/trout GRP-stimulated contraction of smooth muscle from the rainbow trout, O,corhynchus mykiss,. Trout GRP was added in single doses (0.1 p.M) to isolated circular muscle strip preparations from the cardiac stomach and bombesin (0.1 pM) was added to longitudinal strip preparations from the proximal intestine. IBMX (1 mM) blocked >95% of the contractile response to both trout GRP and bombesin. Forskolin (10BM) completely blocked the effect of trout GRP on the stomach but did not significantly affect the bombesin-induced contraction of the intestine. Measurement of the levels of cyclic AMP in gastrointestinal smooth muscle tissue showed that IBMX increased the levels of cyclic AMP six fold in the intestine and five fold in the stomach. Forskolin raised the levels of cyclic AMP 2-3 times. Bombesin and trout GRP did not significantly alter the basal levels of cyclic AMP. When the peptides were added together with either forskolin or IBMX, the elevated levels were not changed significantly. These results show that IBMX and forskolin increase the cellular content of cyclic AMP and suggest that cyclic AMP is not involved in the bombesin/trout GRP-stimulated contraction of the gastrointestinal tract in rainbow trout. An explanation of the inhibitory effect of IBMX on the response to bombesin and trout GRP could be an increase in cyclic GMP levels since, in mammals, IBMX has been shown to inhibit cyclic GMP phosphodiesterases as well as cyclic AMP phosphodiesterases.
]rgPRZSSIOM O F R Z G GEh'E A N D CELLS
AROUD
WATER
ITS
PRODUCT
IMMERSION-INDUCED
ARZ
ENHANCED
GASTRIC
LESIONS
IN GASTRIC IN
RAT
ENDOCRINE M. Asahara,
K. Fukui Y. Kinoshita, K. Kishi, Y. Matsushima, N.Fujita il, T. Kaw~nami, T. Chiba. Division of Gerontology, Department of Internal Medicine, Kohe University School of Medicine, Kobe 650, i l D e p a r ~ t of Internal Medicine, Rokko Hospital Kobe 657, Japan. Reg (regenerating gene) was first isolated by screening cDNA library derived from the regenerating pancreatic islets. Subsequently, it was found that reg gene is expressed in the regenerating islets but not in the normal islets. Further studies demonstrated that reg protein has a trophic effect on pancreatic islets and can ameliorate diabetes in partially depancreatized rats. Although these studies suggest that reg gene is involved in regeneration of pancreatic beta cells, little is known about the role of reg gene in the stomach. The aim of this study was, therefore, to investigate the reg gene expression and its product in the damaged gastric mucosa and to clarify which type of cells in the stomach express it. Method: Water irmaersion-induced gastric lesions were prepared in male Sprague-Dawly rats (250-300g) . Northern blot analysis was performed to investigate changes of the levels of reg gene mRNA. Furthermore, in situ hybridization (ISH) and inrm~nocytochemical studies were carried out in order to clarify localization of reg mRNA and Reg p r o t e i n - ~ o r e a c t i v i t y . Result: Reg gene mRNA was increased 5 and i0 fold at 6 and 24 hr after the start of water-immersion, respectively, in Northern blot analysis as quantified by BAS 2000. In ISH study, small amount of reg mRNA was expressed in the basal part of the fundic glands of normal stomach. Interestingly, reg gene expression was markedly increased in gastric ECL cells around the gastric erosions in water-irm~rsed rat stc~achs. In agreement with the levels of reg mRNA, Reg protein-~oreactivity was enhanced around the erosions and found to be present in chromogranin A-positive endocrine cells. Conclusion: Expression of reg mRNA and its product are enhanced in gastric ent erochrcr0a f f in- 1 ike cells around water inrnersion-ir~duced gastric lesions.
7
Is cyclic AMP involved in the contractile response of rainbow trout gastrointestinal smooth muscle to bombesin and trout gastrin releasing peptide? U. Aronsson, J. Jensen and S. Holmgren, Dept of Zoophysiology, University of GOteborg, Sweden. Bombesin and gastrin releasing peptide (GRP) contract smooth muscle of the gastrointestinal tract in both mammals and fish. In mammals the signal transduction from receptor to contraction involves release of calcium from extracellular and/or intracellular stores and a reduction of the cyclic AMP levels. By using two agents known to increase the concentration of cyclic AMP in mammals, the phosphodiesterase inhibitor 3-isobuthyl-l-methylxanthine (IBMX) and the adenylyl cyclase stimulator forskolin, we wanted to investigate whether cyclic AMP plays a role in the bombesin/trout GRP-stimulated contraction of smooth muscle from the rainbow trout, O,corhynchus mykiss,. Trout GRP was added in single doses (0.1 p.M) to isolated circular muscle strip preparations from the cardiac stomach and bombesin (0.1 pM) was added to longitudinal strip preparations from the proximal intestine. IBMX (1 mM) blocked >95% of the contractile response to both trout GRP and bombesin. Forskolin (10BM) completely blocked the effect of trout GRP on the stomach but did not significantly affect the bombesin-induced contraction of the intestine. Measurement of the levels of cyclic AMP in gastrointestinal smooth muscle tissue showed that IBMX increased the levels of cyclic AMP six fold in the intestine and five fold in the stomach. Forskolin raised the levels of cyclic AMP 2-3 times. Bombesin and trout GRP did not significantly alter the basal levels of cyclic AMP. When the peptides were added together with either forskolin or IBMX, the elevated levels were not changed significantly. These results show that IBMX and forskolin increase the cellular content of cyclic AMP and suggest that cyclic AMP is not involved in the bombesin/trout GRP-stimulated contraction of the gastrointestinal tract in rainbow trout. An explanation of the inhibitory effect of IBMX on the response to bombesin and trout GRP could be an increase in cyclic GMP levels since, in mammals, IBMX has been shown to inhibit cyclic GMP phosphodiesterases as well as cyclic AMP phosphodiesterases.
]rgPRZSSIOM O F R Z G GEh'E A N D CELLS
AROUD
WATER
ITS
PRODUCT
IMMERSION-INDUCED
ARZ
ENHANCED
GASTRIC
LESIONS
IN GASTRIC IN
RAT
ENDOCRINE M. Asahara,
K. Fukui Y. Kinoshita, K. Kishi, Y. Matsushima, N.Fujita il, T. Kaw~nami, T. Chiba. Division of Gerontology, Department of Internal Medicine, Kohe University School of Medicine, Kobe 650, i l D e p a r ~ t of Internal Medicine, Rokko Hospital Kobe 657, Japan. Reg (regenerating gene) was first isolated by screening cDNA library derived from the regenerating pancreatic islets. Subsequently, it was found that reg gene is expressed in the regenerating islets but not in the normal islets. Further studies demonstrated that reg protein has a trophic effect on pancreatic islets and can ameliorate diabetes in partially depancreatized rats. Although these studies suggest that reg gene is involved in regeneration of pancreatic beta cells, little is known about the role of reg gene in the stomach. The aim of this study was, therefore, to investigate the reg gene expression and its product in the damaged gastric mucosa and to clarify which type of cells in the stomach express it. Method: Water irmaersion-induced gastric lesions were prepared in male Sprague-Dawly rats (250-300g) . Northern blot analysis was performed to investigate changes of the levels of reg gene mRNA. Furthermore, in situ hybridization (ISH) and inrm~nocytochemical studies were carried out in order to clarify localization of reg mRNA and Reg p r o t e i n - ~ o r e a c t i v i t y . Result: Reg gene mRNA was increased 5 and i0 fold at 6 and 24 hr after the start of water-immersion, respectively, in Northern blot analysis as quantified by BAS 2000. In ISH study, small amount of reg mRNA was expressed in the basal part of the fundic glands of normal stomach. Interestingly, reg gene expression was markedly increased in gastric ECL cells around the gastric erosions in water-irm~rsed rat stc~achs. In agreement with the levels of reg mRNA, Reg protein-~oreactivity was enhanced around the erosions and found to be present in chromogranin A-positive endocrine cells. Conclusion: Expression of reg mRNA and its product are enhanced in gastric ent erochrcr0a f f in- 1 ike cells around water inrnersion-ir~duced gastric lesions.