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Activation of delayed rectifier K+ channels by vasoactive intestinal peptide
A690
AGA ABSTRACTS
GASTROENTEROLOGY, VOl. 108, NO. 4
• DOPAMIN CONTAINING CELL AND DOPAMINE R E C E P T O R E X P R E S S I O N IN THE STOMACH. K. Shichijo, *Y. S.-Yamashita, T. Nakayama, M. Ito, I. Sekine and *K. Taniyama Department of Pathology, Atomic Disease Institute and *Department of Pharmacology II, Nagasaki University School of Medicine, Nagasaki 852, Japan. Dopamine plays some role in the gastrointestinal tract possibly via dopamine receptor. Particularly in the stomach, dopamine modulates gastric motility and gastric acid secretion, and dopamine receptor antagonists prevent ulcer relapse. We have demonstrated dopamine is present in neuron of the myenteric plexus and the dopaminergic neuron regulates aeetylcholine release from the cholinergic neurons in the stomach ( Gastroenterology 106: A566, 1994 ). The present study was attempted to examine the extra neural localization of dopamine and dopamine receptor expression in the mucosal layer of the stomach. M e t h o d s a n d Results: When the dopamine content was determined by HPLC in the mucosal layer and the mucosa-free layer (muscle layers including myenteric plexus) of rat stomach, dopamine content was higher in the mucosa-free layer (29:3 ng/g wet wt) than in the mucosal layer (12.5 ng/g wet wt). To denervate the fibers supplying the stomach bilateral abdominal vagotomy and celiac ganglionectomy were performed surgically. 7 days after the vagotomy and celiac ganglionectomy induced no change in the dopamine content of the mucosal layer in the stomach. Immunohistochemical experiments using specific antiserum against dopamine were studied in the preparation from the stomach. A few endocrine cells and nerve fibers in the lamina propria of the stomach contained clearly immunoreactivity for dopamine. The 35S-labeled dopamine D1 and D2 olygoprobes were used for in situ hybridization. There were numerous cells in the lamina pr0pria of the stomach containing m R N A for dopamine Dland D2 receptors. The probes directed to dopamine D1 and D2 receptor mRNAs gave the strong signals. Conclusion: These results revealed that endocrine dopamine containing-cells are present in the epithelium, and dopaminergic nerve fibers and cells containing dopamine D1 and D2 receptor mRNAs are present in the lamina propria of stomach.
• ACTIVATION O F DELAYED R E c I ' I F t t ~ IC CHANNELS BY VASOACTIVE INTF_,STINAL PEP'FIDE. C.W.R. Shuttleworth, S.D. Koh, O. Bayginov, and K.M. Sanders. Department of Physiology, University of Nevada, Reno, NV 89557.
• RECYCLING
• CANINE MULTI-VISCERAL AUTOTRANSPLANTATION OF THE ENTIRE UPPER GUT: EFFECTS ON FASTING AND FED MOTILITY PATI'ERNS. M.R. Siadati. M.M. Mulr, M.IC Foley. J.L. Steers. M.G. Sarr. Dept. of Surgery, Mayo Clinic, Rochester, MN BACKGROUND: Multi-organ upper gut transplantation is becoming clinically feasible; however, the effects of multi-visceral transplantations on gastrointestinal motility is unknown. AIM: To determine the neural and hormonal mechanisms controlling motility patterns after autotrausplantation of the upper gut. METHODS: Five dogs underwent a model of orthotopic autotransplantation of the stomach, entire small intestine, proximal colon. liver, and pancreas by transecting all connections (distal esophagus, midcolon, all nerves, lymphatics, etc.) to this multi-visceral complex except the celiac and superior mesenteric arteries and the supra- and infrahepatic cava; these vessels were stripped meticulously of advenfitia under optical magnification. After a 1-2 wk recovery, myoelectric and manometric recordings of stomach and myoelectric recordings of small bowel were obtained from conscious animals while fasting, after meals of 50 and 500 g liver, and after motilin injections (0.I /~g/kg). RESULTS: During fasting, a characteristic cyclic migrating motor complex (MMC) was observed with parameters shown in the table; a gastric MMC was present in only 3 of 5 dogs. Regular cycling of the MMC during fasting, however, was intermittently disrupted (despite fasting) and replaced by a non-cyclic pattern of intermittent spikes/contractions i n 2 of 5 dogs about 43% of the time. A small meal (50 g liver) did not abolish the MMC as occurs in normal dogs; in contrast, 500 g liver abolished the MMC regularly in all regions. Exogenous motilin (0.1 tzg/kg IV) induced a premature MMC beginning in gastroduodenum and migrating along small intestine. SUMMARY: A cyclic small bowel MMC persists after complete upper gut extrinsic denervation, but gastric MMCs are partially disrupted. Large but not small caloric meals disrupt the MMC. CONCLUSIONS: These data suggest that extrinsic innervation to the upper gut modulates but is not requisite for interdigestive and postprandial motiii~ of the stomach. Parameter* " Stomach Duodenum Jejunum Ileum Phase I 17~-3 17±3 13±2 15±2 Phase II 49±4 51±2 59±6 56±3 Phase III 14±1 7±1 5±1 6±1 Phase IV 25±4 27±1 24±2 24±3 Period of MMC 104±5 102±4 102±4 102±4 *,Duration (rain), ~±SE, n =5 dogs. Support: NIH DK 39337
OF
L-CI'I~ULLINE
MAINTAINS
NITRIC-OXIDE
DEPENDENT ENTERIC NEUROTRANSMISSION.
C.W.R. Shuttleworth. AJ. Burns, S.M. Ward, W.E. O'Brien" and K.M. Sanders. Department of Physiology, University of Nevada, Reno, NV 89557 and "The Institute for Molecular Genetics, Baylor College of Medicine, Houston, TX 77030. Synthesis of nitric oxide from arginme produces stoichiometrie amounts of citrulline. We studied whether NO-releasing enteric neurons can recycle citmlline back to arginine to sustain uitrergic neurotransmission. Serial activity of two enzymes, argininosuccinate synthetase (ASS) and argininosuccmate lyase (ASL),- can convert citmlline to arginine. Immunohistochemic~l studies were performed to determine the distribution of ASS and ASL in cryostat sections of canine colon, ASS- and ASL-like unmunoreactivities (ASS-LI and ASL-LI) were found in discrete populations of nerve cell bodies within myenteric and submucosal ganglia. ASS-LI and ASL-LI were also found in varicose nerve fibers within the circular and longitudinal muscle layers. ASS-LI and ASL-LI co-localized with neuronal NADPH diaphorase staining, demonstrating nitric oxide synthase (NOS) containing neurons also contain the apparatus to recycle citmlline. Citmlline recycling should provide a source of arginine for NO synthesis. To test this hypothesis, the effects of inhibitory nerve stimulation were evaluated on contractions, inhibitory junction potentials (IJPs) and electrical slow waves. Citmfiine or arginme alone (0.1-2mM) had no effect on electrical or mechanical activity, or on control nitrergic neurotransmissinn. When inhibitory transmission was inhibited by arginine analogues (e.g. Lnitroarginine methyl ester (L-NAME) or L-nitroarginine (L-NNA); 100pM), citrulline (0.1-2mM), but not other neutral amino acids, reversed the effects of L-NAME and L-NNA, similar to the effects of arginine. For example, LNAME reduced nerve-stimulated inhibition of contractile activity from 80.3~:4.0% to 7.8~:12% (0.5ms pulses of electrical field stimulation delivered at 5Hz, n=5). In the continued presence of L-NAME, addition of citrulline (2mM) restored nerve-stimulated inhibition to 77.0~3.2% (n=5), Citrulline did not affect the sensitivity of muscle strips to sodium nitropmsside (ECs0 [.26 x 10"TMand 1.35x10"TM in absence and presence of citmlline, respectively). Taken together, these data demonstrate the presence and activity of the apparatus needed to recycle intranenronal ciWalline to arginine. This pathway may be a primary means of maintaining sufficient intranenrunal arginine levels for NO synthesis. (Supported by PO1 DK 41315)
Vasoactive intestinal peptide (VIP) inhibits contractile activity of canine proximal colon circular muscle and may co-mediate inhibitory neurotransmission in this tissue. To characterize the mechanism of VIP responses, electrical slow waves were recorded from the submucosal region of the circular muscle layer. VIP (]0"7M) decreased the frequency of spontaneous slow waves, but had no effect on slow wave amplitude or duration (3.8±0.7s and 3.6±0.4s duration in control and VIP, respectively n=5). In the presence of excitatory agonists such as Bay K 8644 (10"tM) or histamine (10"~Vl) which enhance the amplitude and duration of slow waves, VIP (10"~M) decreased the duration of slow waves (15.3±4.3s and 5.2±0.4s in control and VIP, respectively n=4) and inhibited contractions. These effects could be due to the enhancement o f K + conductance by VIP. Tetraethylammonium chloride (TEA, 10"2M) increased the force of contractions, and did not block the effect of VIP (90.5 ± 3.2% inhibition with 10"1M VIP, n=4). 4-aminopyridine (4-AP; 10"2M) also increased contractile force and' significantly reduced the inhibitory effects of ~ (27.1:t:4.0% inhibition with 10"TM VIP, n=6). These data suggest that VIP might activate a 4-AP-sansitive K ÷ conductance. Patch clamp experiments on isolated circular muscle cells were performed to test this hypothesis. Voltage-dependent, 4-AP sensitive, TEA insensitive K + channels were recorded in "on-cell" patches. The conductance of these channels was 8.4 pS at room temperature in a symmetrical K ÷ gradient. VIP (10"tM) reversibly increased the mean open probability of these channels from 0.22--1:0.026 to 0.44±0.043 (n=5). Two additional 4-AP-insensitive K ÷ channels (80 pS and <4 pS) that were often observed in these patches were not influenced by VIP. In summary, the effects of VIP on electrical slow waves may b e due to the activation of a group of 4-AP sensitive, "delayed rectifier" K ÷ channels that participate in the repolarization of electrical slow waves. (Supported by DK 41315).
AGA ABSTRACTS
GASTROENTEROLOGY, VOl. 108, NO. 4
• DOPAMIN CONTAINING CELL AND DOPAMINE R E C E P T O R E X P R E S S I O N IN THE STOMACH. K. Shichijo, *Y. S.-Yamashita, T. Nakayama, M. Ito, I. Sekine and *K. Taniyama Department of Pathology, Atomic Disease Institute and *Department of Pharmacology II, Nagasaki University School of Medicine, Nagasaki 852, Japan. Dopamine plays some role in the gastrointestinal tract possibly via dopamine receptor. Particularly in the stomach, dopamine modulates gastric motility and gastric acid secretion, and dopamine receptor antagonists prevent ulcer relapse. We have demonstrated dopamine is present in neuron of the myenteric plexus and the dopaminergic neuron regulates aeetylcholine release from the cholinergic neurons in the stomach ( Gastroenterology 106: A566, 1994 ). The present study was attempted to examine the extra neural localization of dopamine and dopamine receptor expression in the mucosal layer of the stomach. M e t h o d s a n d Results: When the dopamine content was determined by HPLC in the mucosal layer and the mucosa-free layer (muscle layers including myenteric plexus) of rat stomach, dopamine content was higher in the mucosa-free layer (29:3 ng/g wet wt) than in the mucosal layer (12.5 ng/g wet wt). To denervate the fibers supplying the stomach bilateral abdominal vagotomy and celiac ganglionectomy were performed surgically. 7 days after the vagotomy and celiac ganglionectomy induced no change in the dopamine content of the mucosal layer in the stomach. Immunohistochemical experiments using specific antiserum against dopamine were studied in the preparation from the stomach. A few endocrine cells and nerve fibers in the lamina propria of the stomach contained clearly immunoreactivity for dopamine. The 35S-labeled dopamine D1 and D2 olygoprobes were used for in situ hybridization. There were numerous cells in the lamina pr0pria of the stomach containing m R N A for dopamine Dland D2 receptors. The probes directed to dopamine D1 and D2 receptor mRNAs gave the strong signals. Conclusion: These results revealed that endocrine dopamine containing-cells are present in the epithelium, and dopaminergic nerve fibers and cells containing dopamine D1 and D2 receptor mRNAs are present in the lamina propria of stomach.
• ACTIVATION O F DELAYED R E c I ' I F t t ~ IC CHANNELS BY VASOACTIVE INTF_,STINAL PEP'FIDE. C.W.R. Shuttleworth, S.D. Koh, O. Bayginov, and K.M. Sanders. Department of Physiology, University of Nevada, Reno, NV 89557.
• RECYCLING
• CANINE MULTI-VISCERAL AUTOTRANSPLANTATION OF THE ENTIRE UPPER GUT: EFFECTS ON FASTING AND FED MOTILITY PATI'ERNS. M.R. Siadati. M.M. Mulr, M.IC Foley. J.L. Steers. M.G. Sarr. Dept. of Surgery, Mayo Clinic, Rochester, MN BACKGROUND: Multi-organ upper gut transplantation is becoming clinically feasible; however, the effects of multi-visceral transplantations on gastrointestinal motility is unknown. AIM: To determine the neural and hormonal mechanisms controlling motility patterns after autotrausplantation of the upper gut. METHODS: Five dogs underwent a model of orthotopic autotransplantation of the stomach, entire small intestine, proximal colon. liver, and pancreas by transecting all connections (distal esophagus, midcolon, all nerves, lymphatics, etc.) to this multi-visceral complex except the celiac and superior mesenteric arteries and the supra- and infrahepatic cava; these vessels were stripped meticulously of advenfitia under optical magnification. After a 1-2 wk recovery, myoelectric and manometric recordings of stomach and myoelectric recordings of small bowel were obtained from conscious animals while fasting, after meals of 50 and 500 g liver, and after motilin injections (0.I /~g/kg). RESULTS: During fasting, a characteristic cyclic migrating motor complex (MMC) was observed with parameters shown in the table; a gastric MMC was present in only 3 of 5 dogs. Regular cycling of the MMC during fasting, however, was intermittently disrupted (despite fasting) and replaced by a non-cyclic pattern of intermittent spikes/contractions i n 2 of 5 dogs about 43% of the time. A small meal (50 g liver) did not abolish the MMC as occurs in normal dogs; in contrast, 500 g liver abolished the MMC regularly in all regions. Exogenous motilin (0.1 tzg/kg IV) induced a premature MMC beginning in gastroduodenum and migrating along small intestine. SUMMARY: A cyclic small bowel MMC persists after complete upper gut extrinsic denervation, but gastric MMCs are partially disrupted. Large but not small caloric meals disrupt the MMC. CONCLUSIONS: These data suggest that extrinsic innervation to the upper gut modulates but is not requisite for interdigestive and postprandial motiii~ of the stomach. Parameter* " Stomach Duodenum Jejunum Ileum Phase I 17~-3 17±3 13±2 15±2 Phase II 49±4 51±2 59±6 56±3 Phase III 14±1 7±1 5±1 6±1 Phase IV 25±4 27±1 24±2 24±3 Period of MMC 104±5 102±4 102±4 102±4 *,Duration (rain), ~±SE, n =5 dogs. Support: NIH DK 39337
OF
L-CI'I~ULLINE
MAINTAINS
NITRIC-OXIDE
DEPENDENT ENTERIC NEUROTRANSMISSION.
C.W.R. Shuttleworth. AJ. Burns, S.M. Ward, W.E. O'Brien" and K.M. Sanders. Department of Physiology, University of Nevada, Reno, NV 89557 and "The Institute for Molecular Genetics, Baylor College of Medicine, Houston, TX 77030. Synthesis of nitric oxide from arginme produces stoichiometrie amounts of citrulline. We studied whether NO-releasing enteric neurons can recycle citmlline back to arginine to sustain uitrergic neurotransmission. Serial activity of two enzymes, argininosuccinate synthetase (ASS) and argininosuccmate lyase (ASL),- can convert citmlline to arginine. Immunohistochemic~l studies were performed to determine the distribution of ASS and ASL in cryostat sections of canine colon, ASS- and ASL-like unmunoreactivities (ASS-LI and ASL-LI) were found in discrete populations of nerve cell bodies within myenteric and submucosal ganglia. ASS-LI and ASL-LI were also found in varicose nerve fibers within the circular and longitudinal muscle layers. ASS-LI and ASL-LI co-localized with neuronal NADPH diaphorase staining, demonstrating nitric oxide synthase (NOS) containing neurons also contain the apparatus to recycle citmlline. Citmlline recycling should provide a source of arginine for NO synthesis. To test this hypothesis, the effects of inhibitory nerve stimulation were evaluated on contractions, inhibitory junction potentials (IJPs) and electrical slow waves. Citmfiine or arginme alone (0.1-2mM) had no effect on electrical or mechanical activity, or on control nitrergic neurotransmissinn. When inhibitory transmission was inhibited by arginine analogues (e.g. Lnitroarginine methyl ester (L-NAME) or L-nitroarginine (L-NNA); 100pM), citrulline (0.1-2mM), but not other neutral amino acids, reversed the effects of L-NAME and L-NNA, similar to the effects of arginine. For example, LNAME reduced nerve-stimulated inhibition of contractile activity from 80.3~:4.0% to 7.8~:12% (0.5ms pulses of electrical field stimulation delivered at 5Hz, n=5). In the continued presence of L-NAME, addition of citrulline (2mM) restored nerve-stimulated inhibition to 77.0~3.2% (n=5), Citrulline did not affect the sensitivity of muscle strips to sodium nitropmsside (ECs0 [.26 x 10"TMand 1.35x10"TM in absence and presence of citmlline, respectively). Taken together, these data demonstrate the presence and activity of the apparatus needed to recycle intranenronal ciWalline to arginine. This pathway may be a primary means of maintaining sufficient intranenrunal arginine levels for NO synthesis. (Supported by PO1 DK 41315)
Vasoactive intestinal peptide (VIP) inhibits contractile activity of canine proximal colon circular muscle and may co-mediate inhibitory neurotransmission in this tissue. To characterize the mechanism of VIP responses, electrical slow waves were recorded from the submucosal region of the circular muscle layer. VIP (]0"7M) decreased the frequency of spontaneous slow waves, but had no effect on slow wave amplitude or duration (3.8±0.7s and 3.6±0.4s duration in control and VIP, respectively n=5). In the presence of excitatory agonists such as Bay K 8644 (10"tM) or histamine (10"~Vl) which enhance the amplitude and duration of slow waves, VIP (10"~M) decreased the duration of slow waves (15.3±4.3s and 5.2±0.4s in control and VIP, respectively n=4) and inhibited contractions. These effects could be due to the enhancement o f K + conductance by VIP. Tetraethylammonium chloride (TEA, 10"2M) increased the force of contractions, and did not block the effect of VIP (90.5 ± 3.2% inhibition with 10"1M VIP, n=4). 4-aminopyridine (4-AP; 10"2M) also increased contractile force and' significantly reduced the inhibitory effects of ~ (27.1:t:4.0% inhibition with 10"TM VIP, n=6). These data suggest that VIP might activate a 4-AP-sansitive K ÷ conductance. Patch clamp experiments on isolated circular muscle cells were performed to test this hypothesis. Voltage-dependent, 4-AP sensitive, TEA insensitive K + channels were recorded in "on-cell" patches. The conductance of these channels was 8.4 pS at room temperature in a symmetrical K ÷ gradient. VIP (10"tM) reversibly increased the mean open probability of these channels from 0.22--1:0.026 to 0.44±0.043 (n=5). Two additional 4-AP-insensitive K ÷ channels (80 pS and <4 pS) that were often observed in these patches were not influenced by VIP. In summary, the effects of VIP on electrical slow waves may b e due to the activation of a group of 4-AP sensitive, "delayed rectifier" K ÷ channels that participate in the repolarization of electrical slow waves. (Supported by DK 41315).