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Functional evidence that the medullary parapyramidal regions participate in the medullary regulation of gastric function
April 1998
Hormones, Transmitters, G r o w t h Factors, a n d t h e i r Receptors A l 1 9 3
• G4875
Results:
SOMATOSTATIN RECEPTOR TYPE 2 MEDIATED SUPPRESSION OF HELICOBACTER PYLORI PROLIFERATION IN VITRO. K. Yamashita, H. Kaneko, T. Konagaya, S. Yamamoto, *K. Kusugami & T. Mitsuma. Fourth Dept. of Intern. Med. Aichi Med. University, Aichi & *First Dept. of Intem. Med. Nagoya University, School of Med., Nagoya, Japan.
Backeround & Aims: As is shown in somatostatin action to cell proliferation, somatostatin shows antiproliferative effect on Helicobacter pylori (H. pylori) in vitro from our preliminary data. At least five different types of somatostatin receptor (SSTR-1 to SSTR-5) have been cloned from various tissues. We investigated which type(s) of receptor may be involved in somatostatin action against H. pylori proliferation in vitro. Methods: H. pylori (ATCC43504, 103 CFU/mL) was cultured in 5 mL Brucella broth. Bacterial number was calculated 48 h after incubation by counting the colonies on the blood agar. Chemicals were dissolved in absolute methanol and added into the broth at a final methanol concentration of 1%. Somatostatin analogs (MK-678: high affinity for type 2, BIM-23052: for type 3 & 5, BIM-23268: for type 5 with some affinity for type 1 & 3, and BIM-23295: for type 2, 3 & 4) are gifts from Dr. D. H. Coy (Tulane Univ. Med. Cntr. New Orleans, LO). Exp.1) The effect of somatostatin and somatostatin analogs (10 -n mol/L) on H. pylori proliferation was investigated. Exp. 2) The influence of purified IgG against SSTR-1 peptide [41-52] (SSTR-1-Ab: 1.0 og/mL), IgG against SSTR-2 peptide [31-41] (SSTR-2-Ab: 1.0 pg/mL) produced and characterized in our laboratory (Endocrine Regul. 29:189,1995 & 30:67,1996) or IgG from nonimmune rabbit serum (Control-IgG: 1.0 lag/mL) on somatostatin (10 -n mol/L)-induced antiproliferative effect on H. pylori was tested. Results: 1) Somatostatin at 10-H mol/L significantly inhibited proliferation of ATCC43504 by 92%. MK-678 and BIM-23295 also dramatically suppressed proliferation by 89% and 79%, respectively (P<0.01). BIM-23268 less potent, but significantly (P<0.05) showed an antiproliferative effect. In contrast, BIM-23052 had no effect. 2) Neither SSTR-1-Ab nor SSTR-2-Ab itself modified H. pylori proliferation. In Control-lgG treated group, somatostatin inhibited H. pylori proliferation by 88%. This antiproliferative effect of somatostatin was partially blocked by SSTR-2-Ab co-incubation by 52% (P<0.05). SSTR-1-Ab had no effect. (Each bar represents the mean _+SEM of 5 samples. **P<0.01 vs respective vehicle, #P<0.05 vs Control-IgG+Somatostatin) 40-
s0-
=-so~m.~,~,
I
I
~20100
CordrolqgQ SSTR-1-Ab SSTR-2-Ab
Conclusions: These data indicate that somatostatin-induced suppression in H. pylori proliferation mainly mediates SSTR-2 in vitro.
• G4876 FUNCTIONAL EVIDENCE THAT THE MEDULLARY PARAPYRAMIDAL REGIONS PARTICIPATE IN THE MEDULLARY REGULATION OF GASTRIC FUNCTION. H. Yang, P.Q. Yuan and Y.Tach6. CURE: Digestive Diseases Research Center, VA Medical Center, Dept. of Medicine, UCLA, Los Angeles, CA 90073, USA.
Background: Neurons synthesizing thyrotropin-releasing hormone, substance P and serotonin in the caudal medullary raphe nuclei project to the dorsal vagal complex and play a important role in the central vagal regulation of gastric function (Tachs et al, Peptides, 16:431, 1995). Neurons in the parapyramidal regions (paPy) have similar peptides and projections as in the midline raphe nuclei (Sasek et al, Neuroscience, 35:105, 1990. Lynn et al, J Comp Neurol, 311:271, 1991), however the functional relevance of these neurons on gastric regulation is unknown. Aim: To provide functional evidence that the paPy is another important area in the caudal medulla in the central regulation of gastric function. Methods: Chemical activation of paPy cell bodies was achieved by microinjection of kainic acid (in 50 nl 0.1 M phosphate buffer) in 24 h fasted, urethane-anesthetized SD male rats (260-300 g). The coordinates used for paPy microinjection was (in mm): ventral from the surface of the brain, 3.7; anterior from the obex, 1.5; lateral from the midline, 1.2. The gastric acid secretion was measured by flushing technique and monitored before and after the microinjection. Gastric erosions Was induced by intragastric administration of 45% ethanol (5 ml/kg) 20 rain after the microinjection and measured by HIN Image 1 h after the intragastric ethanol. Cold restraint stress (4°C, 2 h) induced neuronal activation was assessed by immunohistochemical detection of Fos positive neurons in the paPy. Bilateral cervical vagotomy was performed 1 h before microinjection.
A. Effect of rnicroinjection of kainic acid into the paPy on 1) gastric acid secretion 2) ethanol induced gastric erosions Kainic acid dose Net acid output Kaiuic acid dose Erosion area (ng) n (/Jmol/90min) (ng) n (%) 0 5 2±2 0.0 y 30±5 10 5 29 ± 14 0.5 4 15 ± 5* 15 5 46 ± 10" 1.0 4 13 ± 2* 20 7 90±20* 2.0 6 10±3" 20 7 -3 ± 3# 5.0 5 15 ± 4* 15 or 20 13 9±3@ 0.5-5.0 5 31±0@ * P < 0.05 vs dose 0. # Vagotomized rats. @ microinjection outside of the paPy B. Effect of cold restraint stress on Fos expression in the paPy Group n Number of Fos positive neurons/section Room temperature 4 0.33 ± 0.27 Col 4 14.81 ± 2.00* * P < 0.01 vs room temperature.
Conclusion: Chemical stimulation of the paPy cell bodies induced a doserelated, site specific and vagally mediated gastric acid secretion and gastric cytoprotection. Cold exposure linked with vagal stimulation of gastric function activates paPy neurons. These findings indicate that the paPy, like the midline raphe nuclei, are important caudal medullary regions participating in the central regulation of gastric function. This research is supported by NIH grants DK-301 l0 and DK-50255. G4877 GASTRIC INHIBITORY POLYPElrrIDE (GIP) RECEPTORS ARE PRESENT AND FUNCTIONAL IN FAT CELLS, R.G.C. Yip and M.M. Wolfe. Section of Gastroenterology, Boston University School of Medicine and Boston Medical Center, Boston, MA. GIP is a 42-amino acid peptide that was originally named on the basis of its ability to inhibit gastric acid secretion. Following nutrient ingestion, GIP released into the circulation augments the release of insulin in the presence of postprandial increases in serum glucose. Recently, it has become apparent that GIP is also directly involved in different steps of glucose and fat metabolism. GIP has been shown not only to stimulate the release of lipoprotein lipase from fat, but also to increase the rate of fat incorporation into adipose tissue. In addition, GIP increases the clearance rate of chylomicrons in the circulation and inhibits the action of glucagon. Despite evidence for GIP effects on fat tissue, GIP receptors have not previously been identified in fat cells or tissues. The present study was undertaken to identify GIP receptors in isolated adipocytes and in established fat cell lines and to determine whether these receptors might be functional. RNAse protection assays demonstrated the presence of GIP receptor transcripts in rat adipocytes. A polyclonal GIP receptor antibody directed at the variable N-terminus of the receptor was used to probe Western blots of fat cells derived from the rat epididymal fat pads and differentiated 3T3-L1 adipocytes. These studies detected the presence of GIP receptors in both rat fat and differentiated 3T3-L1 cells. Moreover, 125I-GIP binding assays revealed both specific and displaceable GIP binding sites on rat fat and differentiated 3T3-LI cells. When undifferentiated 3T3-L1 cells, which appear to express relatively few GIP receptors, were incubated in the presence of GIP, no effect on intracellular cAMP accumulation was detected. In contract, the inclusion of 10 nM GIP in the incubation medium increased cAMP accumulation in rat fat cells and differentiated 3T3-L1 cells. This increase in cAMP accumulation was abolished by the specific GIP receptor antagonist GIP(7-30)NH 2. The results of these studies indicate that GIP receptors are present on rat fat cells and appear to be up-regulated when 3T3-L1 cells undergo differentiation to become fat cells. Furthermore, as an increase in intracellular cAMP accumulation was detected upon ligand binding, these receptors appear to be both functional and physiologicallyrelevant. This research was funded by grant DK53158 from the National Institutes of Health.
Hormones, Transmitters, G r o w t h Factors, a n d t h e i r Receptors A l 1 9 3
• G4875
Results:
SOMATOSTATIN RECEPTOR TYPE 2 MEDIATED SUPPRESSION OF HELICOBACTER PYLORI PROLIFERATION IN VITRO. K. Yamashita, H. Kaneko, T. Konagaya, S. Yamamoto, *K. Kusugami & T. Mitsuma. Fourth Dept. of Intern. Med. Aichi Med. University, Aichi & *First Dept. of Intem. Med. Nagoya University, School of Med., Nagoya, Japan.
Backeround & Aims: As is shown in somatostatin action to cell proliferation, somatostatin shows antiproliferative effect on Helicobacter pylori (H. pylori) in vitro from our preliminary data. At least five different types of somatostatin receptor (SSTR-1 to SSTR-5) have been cloned from various tissues. We investigated which type(s) of receptor may be involved in somatostatin action against H. pylori proliferation in vitro. Methods: H. pylori (ATCC43504, 103 CFU/mL) was cultured in 5 mL Brucella broth. Bacterial number was calculated 48 h after incubation by counting the colonies on the blood agar. Chemicals were dissolved in absolute methanol and added into the broth at a final methanol concentration of 1%. Somatostatin analogs (MK-678: high affinity for type 2, BIM-23052: for type 3 & 5, BIM-23268: for type 5 with some affinity for type 1 & 3, and BIM-23295: for type 2, 3 & 4) are gifts from Dr. D. H. Coy (Tulane Univ. Med. Cntr. New Orleans, LO). Exp.1) The effect of somatostatin and somatostatin analogs (10 -n mol/L) on H. pylori proliferation was investigated. Exp. 2) The influence of purified IgG against SSTR-1 peptide [41-52] (SSTR-1-Ab: 1.0 og/mL), IgG against SSTR-2 peptide [31-41] (SSTR-2-Ab: 1.0 pg/mL) produced and characterized in our laboratory (Endocrine Regul. 29:189,1995 & 30:67,1996) or IgG from nonimmune rabbit serum (Control-IgG: 1.0 lag/mL) on somatostatin (10 -n mol/L)-induced antiproliferative effect on H. pylori was tested. Results: 1) Somatostatin at 10-H mol/L significantly inhibited proliferation of ATCC43504 by 92%. MK-678 and BIM-23295 also dramatically suppressed proliferation by 89% and 79%, respectively (P<0.01). BIM-23268 less potent, but significantly (P<0.05) showed an antiproliferative effect. In contrast, BIM-23052 had no effect. 2) Neither SSTR-1-Ab nor SSTR-2-Ab itself modified H. pylori proliferation. In Control-lgG treated group, somatostatin inhibited H. pylori proliferation by 88%. This antiproliferative effect of somatostatin was partially blocked by SSTR-2-Ab co-incubation by 52% (P<0.05). SSTR-1-Ab had no effect. (Each bar represents the mean _+SEM of 5 samples. **P<0.01 vs respective vehicle, #P<0.05 vs Control-IgG+Somatostatin) 40-
s0-
=-so~m.~,~,
I
I
~20100
CordrolqgQ SSTR-1-Ab SSTR-2-Ab
Conclusions: These data indicate that somatostatin-induced suppression in H. pylori proliferation mainly mediates SSTR-2 in vitro.
• G4876 FUNCTIONAL EVIDENCE THAT THE MEDULLARY PARAPYRAMIDAL REGIONS PARTICIPATE IN THE MEDULLARY REGULATION OF GASTRIC FUNCTION. H. Yang, P.Q. Yuan and Y.Tach6. CURE: Digestive Diseases Research Center, VA Medical Center, Dept. of Medicine, UCLA, Los Angeles, CA 90073, USA.
Background: Neurons synthesizing thyrotropin-releasing hormone, substance P and serotonin in the caudal medullary raphe nuclei project to the dorsal vagal complex and play a important role in the central vagal regulation of gastric function (Tachs et al, Peptides, 16:431, 1995). Neurons in the parapyramidal regions (paPy) have similar peptides and projections as in the midline raphe nuclei (Sasek et al, Neuroscience, 35:105, 1990. Lynn et al, J Comp Neurol, 311:271, 1991), however the functional relevance of these neurons on gastric regulation is unknown. Aim: To provide functional evidence that the paPy is another important area in the caudal medulla in the central regulation of gastric function. Methods: Chemical activation of paPy cell bodies was achieved by microinjection of kainic acid (in 50 nl 0.1 M phosphate buffer) in 24 h fasted, urethane-anesthetized SD male rats (260-300 g). The coordinates used for paPy microinjection was (in mm): ventral from the surface of the brain, 3.7; anterior from the obex, 1.5; lateral from the midline, 1.2. The gastric acid secretion was measured by flushing technique and monitored before and after the microinjection. Gastric erosions Was induced by intragastric administration of 45% ethanol (5 ml/kg) 20 rain after the microinjection and measured by HIN Image 1 h after the intragastric ethanol. Cold restraint stress (4°C, 2 h) induced neuronal activation was assessed by immunohistochemical detection of Fos positive neurons in the paPy. Bilateral cervical vagotomy was performed 1 h before microinjection.
A. Effect of rnicroinjection of kainic acid into the paPy on 1) gastric acid secretion 2) ethanol induced gastric erosions Kainic acid dose Net acid output Kaiuic acid dose Erosion area (ng) n (/Jmol/90min) (ng) n (%) 0 5 2±2 0.0 y 30±5 10 5 29 ± 14 0.5 4 15 ± 5* 15 5 46 ± 10" 1.0 4 13 ± 2* 20 7 90±20* 2.0 6 10±3" 20 7 -3 ± 3# 5.0 5 15 ± 4* 15 or 20 13 9±3@ 0.5-5.0 5 31±0@ * P < 0.05 vs dose 0. # Vagotomized rats. @ microinjection outside of the paPy B. Effect of cold restraint stress on Fos expression in the paPy Group n Number of Fos positive neurons/section Room temperature 4 0.33 ± 0.27 Col 4 14.81 ± 2.00* * P < 0.01 vs room temperature.
Conclusion: Chemical stimulation of the paPy cell bodies induced a doserelated, site specific and vagally mediated gastric acid secretion and gastric cytoprotection. Cold exposure linked with vagal stimulation of gastric function activates paPy neurons. These findings indicate that the paPy, like the midline raphe nuclei, are important caudal medullary regions participating in the central regulation of gastric function. This research is supported by NIH grants DK-301 l0 and DK-50255. G4877 GASTRIC INHIBITORY POLYPElrrIDE (GIP) RECEPTORS ARE PRESENT AND FUNCTIONAL IN FAT CELLS, R.G.C. Yip and M.M. Wolfe. Section of Gastroenterology, Boston University School of Medicine and Boston Medical Center, Boston, MA. GIP is a 42-amino acid peptide that was originally named on the basis of its ability to inhibit gastric acid secretion. Following nutrient ingestion, GIP released into the circulation augments the release of insulin in the presence of postprandial increases in serum glucose. Recently, it has become apparent that GIP is also directly involved in different steps of glucose and fat metabolism. GIP has been shown not only to stimulate the release of lipoprotein lipase from fat, but also to increase the rate of fat incorporation into adipose tissue. In addition, GIP increases the clearance rate of chylomicrons in the circulation and inhibits the action of glucagon. Despite evidence for GIP effects on fat tissue, GIP receptors have not previously been identified in fat cells or tissues. The present study was undertaken to identify GIP receptors in isolated adipocytes and in established fat cell lines and to determine whether these receptors might be functional. RNAse protection assays demonstrated the presence of GIP receptor transcripts in rat adipocytes. A polyclonal GIP receptor antibody directed at the variable N-terminus of the receptor was used to probe Western blots of fat cells derived from the rat epididymal fat pads and differentiated 3T3-L1 adipocytes. These studies detected the presence of GIP receptors in both rat fat and differentiated 3T3-L1 cells. Moreover, 125I-GIP binding assays revealed both specific and displaceable GIP binding sites on rat fat and differentiated 3T3-LI cells. When undifferentiated 3T3-L1 cells, which appear to express relatively few GIP receptors, were incubated in the presence of GIP, no effect on intracellular cAMP accumulation was detected. In contract, the inclusion of 10 nM GIP in the incubation medium increased cAMP accumulation in rat fat cells and differentiated 3T3-L1 cells. This increase in cAMP accumulation was abolished by the specific GIP receptor antagonist GIP(7-30)NH 2. The results of these studies indicate that GIP receptors are present on rat fat cells and appear to be up-regulated when 3T3-L1 cells undergo differentiation to become fat cells. Furthermore, as an increase in intracellular cAMP accumulation was detected upon ligand binding, these receptors appear to be both functional and physiologicallyrelevant. This research was funded by grant DK53158 from the National Institutes of Health.