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Alpha 2 adrenergic receptors are involved in central apolipoprotein A-IV-induced acid inhibition in rats
April 1995
• INCREASED SMALL BOWEL GROWTH IN IGF-1 TRANSGENIC MICE I£. Ohneda, M.H.Ulshen, C.R.Fuller, A.J.D'Ercole, and P.K.Lund. Dept. of Physiology, Pediatrics and Center for gastrointestinal Biology and Disease, Univ. of North Carolina at Chapel Hill, NC Introductiort : Previous studies demonstrate that a line of transgenic mice with methallothionein promoter driven 0VIT-1)overexpression of human IGF-1Ea precursor show increased duodenal weight (Behringer et al Endocrinol 1990, 126 : 299) but small bowel growth has not been examined in detail. In the present study, duodenal, jejunal and ileal wet weight (mg/cm) were compared in a second, recently developed MT-1hlGF-1Ea transgenic (TG) mouse line and wild type littermates (WT). Mucosal DNA content, protein content and sucrase activities were analyzed in jejunum. Northern and in situ hybridizations were performed to examine levels and cellular sites of expression of the hIGF-IEa transgene and the mRNA encoding IGF binding protein 3 (IGFBP3)which may modulate IGF action. All results are expressed as ratios of values from TG to those from WT (TG/WT, m e ~ E ) . R e s u l t s : In mice fed ad lib (n=5), wet weights (mg/cm) of duodenum and jejunum were greater in TG than in WT (p<0.05). TG/WT of duodenal wet weight (2.00"&-0.02) was greater than the same values in jejunum (1.44--+0.08,p<0.05) and ileum (1.57_+0.3) and of body weight (1.34_+0.07, p<0.02). In jejunum, protein content and DNA content were greater in TG than in WT (p<0.05). TG/WT of protein content (2.04-+0.16) was greater than that of body weight (p<0.005). Sucrase activity did not differ between TG and WT. In pair-fed mice (n=4), wet weights (mg/cm) of duodenum and jejunum were also greater in TG than in WT (p<0.05) with TG/WTs of 1.51_+0.02 and 1.34_+0.11, respectively. Studies in ad lib fed mice showed that transgene expression was similar in duodenum, jejunum and ileum. In situ hybridization revealed that the hIGF-IEa transgene derived mRNA is expressed throughout the bowel wall,but expression is especially high in villus epithelial cells. IGFBP3 mRNA, expressed in lamina propria of smaU bowel, was increased in TG compared with WT. Conclusions : Excess IGF-I promotes increased small bowel mass with duodenum being particularly responsive. Excess IGF-I increases mucosai DNA and protein indicating effects on cell number and protein synthesis. Excess IGF-I induces increased intestinal IGFBP3mRNA, consistent with previous reports of increased serum IGFBP3. Increased bowel growth in hIGF-IEa TG, despite increased bowel IGFBP3 mRNA expression, indicates that IGFBP3 does not inhibit the enterotrophic actions of excess IGF-I.
TIME RELATED BIPHASIC ACTIVITY OF CARBAMYLCHOLINE CHLORIDE ON CANINE ANTRAL GASTRIN GENE EXPRESSION. K.Okazaki. A. Miyata, S. Sano, and Y. Yamamoto The First Department of Internal Medicine, Kochi Medical School, Kochi, 783, Japan. The effects of the muscarinic receptor agonist, carbamylcholinechloride, on gastrin release and gastrin mRNA levels in the dog antral mucosa were determined. Steady-state gastrin mRNA levels in controls were constant during the entire period (l-12h) of observation. At all concentrations (10"7-10 -4 M) examined during a 2 h-incubation, carbachol caused a si.tgnificant and dose dependent decrease in gastrin mRNA levels: at 1 0 - / , 1 0 -6, 10-5 and 10-'tM, gastrin mRNA levels were 94+12 (P:NS), 71+8 (P<0.001), 37+6 (P<0.001), and 33+5% of control (P<0.00 I), respectively. During the 2 h-incubation, carbachol increased in gastrin release (from 612+52 to 922+71 pg/mg tissue protein) from canine antral tissue and carbachol-stimulated gastrin release was linked to a decrease in intmceilulargastrin content (from 1602+96 to 1280+122 pg/mg tissue protein). Gastrin mRNA levels gradually increased: after 6 and 9-h of culture, they were 96+12 and 126+23 % of control, respectively. Atropine sulfate (I 0-6M) did not alter the basal gastrin release or gastrin gene expression but completely inhibited carbachol-stimulated changes. During a 6-h incubation period, the concentration of gastrin in the medium progressively increased (612-+52 to 14230-+52 pg/mg tissue protein), as did the tissue gastrin content (1602-+120 to 2860-+128 pg/mg tissue protein). Cycloheximide remarkably decreased the tissue gastrin concentration (90%<) but increased gastrin mRNA levels, whereas it had no effects on gastrin release. In conclusion, these studies indicated that carbachol has a time-related biphasic activity upon canine antral gastrin biosynthesis.
Hormones and Receptors
A995
NO NECESSITY FOR CHOLECYSTOKININ (CCK)-A-RECEPTOR FOR NORMAL PANCREATIC GROWTH AFFER WEANING: A STUDY IN A NEW RAT MODEL WITHOUT GENE EXPRESSION OF CCK-A-RECEPTOR. M. Ohta, S. Kanai, M. Masuda, K M. Matsumoto, A. Funakoshi. Department o--'f hysiology, Tokyo-173, National Kyushu Cancer Center, Fukuoka-815 This work extends a recent observation that Otsuka L o n g - E v a n s T o k u s h i m a Fatty (OLETF) rats s h o w no expression of the CCK-A-receptor gene in the pancreas. We examined whether the CCK-A receptor is mandatory for the natural g r o w t h of the pancreas, using OLETF rats, and c o m p a r e d with control (Long-Evans-Tokushima = LETO) rats. M e t h o d s : Male OLETF and LETO rats were used at 5-8 weeks and 24-25 weeks of age. The changes in pancreatic wet weight, protein, DNA, insulin, chymotrypsin and amylase contents were examined. For the functional examination, rats were prepared with cannulas draining bile and pancreatic juice separately, and with a duodenal cannula and an extrajugular vein cannula. The pancreatic responses to graded doses of CCK-8 (30, 100, 300 pmol/kg, bolus injection) and bile-pancreatic juice diversion (BPJD) were examined. Results: The pancreatic wet weight increased significantly with age in both OLETF and LETO rats, but was significantly less in OLETF rats than in LETO rats. The changes in concentrations (per g wet tissue) of protein, amylase, chymotrypsin and insulin were comparable i n OLETF and LETO rats and, except for the chymotrypsin and DNA concentrations, they all increased significantly increased with age. The chymotrypsin concentration decreased and the DNA concentration did not change with age. However total contents of these parameters in the whole pancreas significantly increased with age, except for the chymotrypsin content, which did not change with age. Basal pancreatic exocrine secretion of protein increased significantly with age in both strains. The protein Secretions in response t o CCK and/or BPJD were significantly higher at 24-25 weeks than 5-8 weeks in LETO rats, whereas no stimulation was observed in OLETF rats. These results suggest that the absence of the CCK-A-receptor did not affect normal pancreatic growth from 5 to 25 weeks of age (after weaning).
• ALPHA 2 ADRENERGIC RECEPTORS ARE INVOLVED IN CENTRAL APOLIPOPROTEIN A-IV-INDUCED ACID INHIBITION IN RATS T. Okumura*#, K. Fukagawa**, P. Tso**, I.L. Taylor***, T.N. Pappas*, A. Uehara#, Y. Kohgo# *Dept of Surgery, Duke University Medical Center, Durham NC, **Dept of Physiology, Louisiana State University Medical Center, Shreveport LA, ***Dept of Medicine, Medical University of South Carolina, Charleston SC, Dept of Internal Medicine (liI), Asahikawa Medical College, Asahikawa, Japan Intracistemal injection of apolipoprotein A-IV (apo A-IV) inhibits gastric acid secretion in pyloms-ligated conscious rats (T. Okumura et al., Gastroenterology in press). In the present study, we examined the mechanism by which centrally administered apo A-IV suppressed gastric acid secretion using Spmgue-Dawley rats with pylorus ligation. First, effects of intmeistemal apo A-IV (4ug) on bethanechol-, thyrotropinreleasing hormone (TRH) analogue- or pentagastrin-stimulated gastric acid output were examined. Second, effect of vagotomy on the acid inhibition by central apo A-IV was examined. Finally, role of the adrenergic system was investigated using adrenergic receptor antagonism, tntracistemally administered apo A-IV (4 Hg)significantly inhibited all the stimulated gastric acid output by bethanechol (1 mg/kg, se), TRH analogue (5 ng, ic) or pentagastrin (75 ,ug/kg, ip) in a similar fashion. Inhibition of pentagastrin-stimulated gastric acid by apo A-IV still occurred even in vagotomized rats. Yohimbine (4 mg/kg, sc, Figure), but not propranolol (4 mg/kg, sc) abolished acid inhibition by apo A-IV. These results suggest that apo A-IV acts in the brain to inhibit gastric acid secretion through alpha 2 adrenergic receptors. Gastric acid output ~ E q / 2 h) [] saline
300 200 tO0
vehicle
Yohimbine
• INCREASED SMALL BOWEL GROWTH IN IGF-1 TRANSGENIC MICE I£. Ohneda, M.H.Ulshen, C.R.Fuller, A.J.D'Ercole, and P.K.Lund. Dept. of Physiology, Pediatrics and Center for gastrointestinal Biology and Disease, Univ. of North Carolina at Chapel Hill, NC Introductiort : Previous studies demonstrate that a line of transgenic mice with methallothionein promoter driven 0VIT-1)overexpression of human IGF-1Ea precursor show increased duodenal weight (Behringer et al Endocrinol 1990, 126 : 299) but small bowel growth has not been examined in detail. In the present study, duodenal, jejunal and ileal wet weight (mg/cm) were compared in a second, recently developed MT-1hlGF-1Ea transgenic (TG) mouse line and wild type littermates (WT). Mucosal DNA content, protein content and sucrase activities were analyzed in jejunum. Northern and in situ hybridizations were performed to examine levels and cellular sites of expression of the hIGF-IEa transgene and the mRNA encoding IGF binding protein 3 (IGFBP3)which may modulate IGF action. All results are expressed as ratios of values from TG to those from WT (TG/WT, m e ~ E ) . R e s u l t s : In mice fed ad lib (n=5), wet weights (mg/cm) of duodenum and jejunum were greater in TG than in WT (p<0.05). TG/WT of duodenal wet weight (2.00"&-0.02) was greater than the same values in jejunum (1.44--+0.08,p<0.05) and ileum (1.57_+0.3) and of body weight (1.34_+0.07, p<0.02). In jejunum, protein content and DNA content were greater in TG than in WT (p<0.05). TG/WT of protein content (2.04-+0.16) was greater than that of body weight (p<0.005). Sucrase activity did not differ between TG and WT. In pair-fed mice (n=4), wet weights (mg/cm) of duodenum and jejunum were also greater in TG than in WT (p<0.05) with TG/WTs of 1.51_+0.02 and 1.34_+0.11, respectively. Studies in ad lib fed mice showed that transgene expression was similar in duodenum, jejunum and ileum. In situ hybridization revealed that the hIGF-IEa transgene derived mRNA is expressed throughout the bowel wall,but expression is especially high in villus epithelial cells. IGFBP3 mRNA, expressed in lamina propria of smaU bowel, was increased in TG compared with WT. Conclusions : Excess IGF-I promotes increased small bowel mass with duodenum being particularly responsive. Excess IGF-I increases mucosai DNA and protein indicating effects on cell number and protein synthesis. Excess IGF-I induces increased intestinal IGFBP3mRNA, consistent with previous reports of increased serum IGFBP3. Increased bowel growth in hIGF-IEa TG, despite increased bowel IGFBP3 mRNA expression, indicates that IGFBP3 does not inhibit the enterotrophic actions of excess IGF-I.
TIME RELATED BIPHASIC ACTIVITY OF CARBAMYLCHOLINE CHLORIDE ON CANINE ANTRAL GASTRIN GENE EXPRESSION. K.Okazaki. A. Miyata, S. Sano, and Y. Yamamoto The First Department of Internal Medicine, Kochi Medical School, Kochi, 783, Japan. The effects of the muscarinic receptor agonist, carbamylcholinechloride, on gastrin release and gastrin mRNA levels in the dog antral mucosa were determined. Steady-state gastrin mRNA levels in controls were constant during the entire period (l-12h) of observation. At all concentrations (10"7-10 -4 M) examined during a 2 h-incubation, carbachol caused a si.tgnificant and dose dependent decrease in gastrin mRNA levels: at 1 0 - / , 1 0 -6, 10-5 and 10-'tM, gastrin mRNA levels were 94+12 (P:NS), 71+8 (P<0.001), 37+6 (P<0.001), and 33+5% of control (P<0.00 I), respectively. During the 2 h-incubation, carbachol increased in gastrin release (from 612+52 to 922+71 pg/mg tissue protein) from canine antral tissue and carbachol-stimulated gastrin release was linked to a decrease in intmceilulargastrin content (from 1602+96 to 1280+122 pg/mg tissue protein). Gastrin mRNA levels gradually increased: after 6 and 9-h of culture, they were 96+12 and 126+23 % of control, respectively. Atropine sulfate (I 0-6M) did not alter the basal gastrin release or gastrin gene expression but completely inhibited carbachol-stimulated changes. During a 6-h incubation period, the concentration of gastrin in the medium progressively increased (612-+52 to 14230-+52 pg/mg tissue protein), as did the tissue gastrin content (1602-+120 to 2860-+128 pg/mg tissue protein). Cycloheximide remarkably decreased the tissue gastrin concentration (90%<) but increased gastrin mRNA levels, whereas it had no effects on gastrin release. In conclusion, these studies indicated that carbachol has a time-related biphasic activity upon canine antral gastrin biosynthesis.
Hormones and Receptors
A995
NO NECESSITY FOR CHOLECYSTOKININ (CCK)-A-RECEPTOR FOR NORMAL PANCREATIC GROWTH AFFER WEANING: A STUDY IN A NEW RAT MODEL WITHOUT GENE EXPRESSION OF CCK-A-RECEPTOR. M. Ohta, S. Kanai, M. Masuda, K M. Matsumoto, A. Funakoshi. Department o--'f hysiology, Tokyo-173, National Kyushu Cancer Center, Fukuoka-815 This work extends a recent observation that Otsuka L o n g - E v a n s T o k u s h i m a Fatty (OLETF) rats s h o w no expression of the CCK-A-receptor gene in the pancreas. We examined whether the CCK-A receptor is mandatory for the natural g r o w t h of the pancreas, using OLETF rats, and c o m p a r e d with control (Long-Evans-Tokushima = LETO) rats. M e t h o d s : Male OLETF and LETO rats were used at 5-8 weeks and 24-25 weeks of age. The changes in pancreatic wet weight, protein, DNA, insulin, chymotrypsin and amylase contents were examined. For the functional examination, rats were prepared with cannulas draining bile and pancreatic juice separately, and with a duodenal cannula and an extrajugular vein cannula. The pancreatic responses to graded doses of CCK-8 (30, 100, 300 pmol/kg, bolus injection) and bile-pancreatic juice diversion (BPJD) were examined. Results: The pancreatic wet weight increased significantly with age in both OLETF and LETO rats, but was significantly less in OLETF rats than in LETO rats. The changes in concentrations (per g wet tissue) of protein, amylase, chymotrypsin and insulin were comparable i n OLETF and LETO rats and, except for the chymotrypsin and DNA concentrations, they all increased significantly increased with age. The chymotrypsin concentration decreased and the DNA concentration did not change with age. However total contents of these parameters in the whole pancreas significantly increased with age, except for the chymotrypsin content, which did not change with age. Basal pancreatic exocrine secretion of protein increased significantly with age in both strains. The protein Secretions in response t o CCK and/or BPJD were significantly higher at 24-25 weeks than 5-8 weeks in LETO rats, whereas no stimulation was observed in OLETF rats. These results suggest that the absence of the CCK-A-receptor did not affect normal pancreatic growth from 5 to 25 weeks of age (after weaning).
• ALPHA 2 ADRENERGIC RECEPTORS ARE INVOLVED IN CENTRAL APOLIPOPROTEIN A-IV-INDUCED ACID INHIBITION IN RATS T. Okumura*#, K. Fukagawa**, P. Tso**, I.L. Taylor***, T.N. Pappas*, A. Uehara#, Y. Kohgo# *Dept of Surgery, Duke University Medical Center, Durham NC, **Dept of Physiology, Louisiana State University Medical Center, Shreveport LA, ***Dept of Medicine, Medical University of South Carolina, Charleston SC, Dept of Internal Medicine (liI), Asahikawa Medical College, Asahikawa, Japan Intracistemal injection of apolipoprotein A-IV (apo A-IV) inhibits gastric acid secretion in pyloms-ligated conscious rats (T. Okumura et al., Gastroenterology in press). In the present study, we examined the mechanism by which centrally administered apo A-IV suppressed gastric acid secretion using Spmgue-Dawley rats with pylorus ligation. First, effects of intmeistemal apo A-IV (4ug) on bethanechol-, thyrotropinreleasing hormone (TRH) analogue- or pentagastrin-stimulated gastric acid output were examined. Second, effect of vagotomy on the acid inhibition by central apo A-IV was examined. Finally, role of the adrenergic system was investigated using adrenergic receptor antagonism, tntracistemally administered apo A-IV (4 Hg)significantly inhibited all the stimulated gastric acid output by bethanechol (1 mg/kg, se), TRH analogue (5 ng, ic) or pentagastrin (75 ,ug/kg, ip) in a similar fashion. Inhibition of pentagastrin-stimulated gastric acid by apo A-IV still occurred even in vagotomized rats. Yohimbine (4 mg/kg, sc, Figure), but not propranolol (4 mg/kg, sc) abolished acid inhibition by apo A-IV. These results suggest that apo A-IV acts in the brain to inhibit gastric acid secretion through alpha 2 adrenergic receptors. Gastric acid output ~ E q / 2 h) [] saline
300 200 tO0
vehicle
Yohimbine