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T1793 Both Campylobacter Jejuni and CDT-C. Jejuni Acutely Reduce the Number of Deep Muscular Plexus Interstitial Cells of Cajal
where slow waves did not propagate; the first in the oral medial fundus and the second distal in the antrum, adjacent to the pylorus. Finally, recordings from the entire ventral surface revealed the presence of 3 to 5 simultaneously aborally propagating slow waves (see diagram). Conclusions: High resolution mapping of the origin and propagation of the slow wave in the canine stomach revealed areas of high amplitude and rapid velocity, areas with fractionated low amplitude and low velocity and areas with no propagation. These components together constitute the elements of the gastric conduction system.
T1793 Both Campylobacter Jejuni and CDT- C. Jejuni Acutely Reduce the Number of Deep Muscular Plexus Interstitial Cells of Cajal Venkata B. Pokkunuri, Walter Morales, Amy L. Zhu, Kimberly Low, Laura J. Hwang, Jeffrey L. Conklin, Christopher Chang, Mark Pimentel Four slow waves propagating simultaneously in the canine stomach.
Campylobacter jejuni is a leading cause of post-infectious IBS. Cytolethal distending toxin (CDT) is produced by C. jejuni and other pathogenic bacteria, and leads to In Vitro cell cycle arrest and eventual death. In a recent rat model of post-infectious IBS, C. jejuni but not CDT- C. jejuni produces chronic altered stool form and a reduction in the deep muscular plexus interstitial cells of Cajal (DMP-ICC) 3 months after clearance of pathogen. In this study, we aim to evaluate the acute effect of C. jejuni and a CDT- C. jejuni on DMP-ICC. Methods: 100 adult male Sprague-Dawley rats were divided into two groups 1)rats gavaged with 108 cfu wild-type C. jejuni 18-176 (C+) and 2)rats gavaged with 108 cfu C. jejuni cdtB mutant (C+/CDT-). Ten rats from each group were euthanized on days 2, 4, 8, 16, and 32 post-inoculation. A third group was gavaged with PBS alone (Controls). Predefined segments of duodenum, jejunum and ileum were resected. Tissue was fixed in formalin and then 80% ethanol, mounted and immunstained using anti-CD117. Slides were coded, randomized and blinded readers counted the number of intestinal villi and DMP-ICC cell bodies. Results: All rats were colonized with Campy. For both groups there was a rapid reduction in the number of DMP-ICC. This was most pronounced on day 2 for C+/CDT- and on day 4 for C+ compared to control (see figure). The overall recovery from the effect was more rapid in C+/CDT-. By day 8 the reduction persisted in C+ and was still significantly less than control (P<0.001) and C+/CDT- (P<0.05) . Full recovery of all groups was seen by day 16. Conclusions: C. jejuni produces an acute reduction in DMP-ICC cells. Recovery of ICC is seen by day 16.
T1795 Involvement of BKCa Channel in the Inhibitory Effect of Magnolol On Rat Colonic Spontaneous Contractility Ao Pan, Siliang Chen, Zihuan Yang, Haijie Yu, Wenliang Zhou The objective of the present study was to investigate the mechanism underlying the inhibitory effect of Magnolol on isolated colonic spontaneous contraction, and find its specific potential for therapy of intestinal motility disorders. Magnolol (5 μM - 1 mM) induced a concentrationdependent inhibition on rat colonic longitude smooth muscle spontaneous contraction. In primary cultured ICCs, Magnolol (50 μM) abolished the intracellular calcium oscillation. Magnolol-induced significant membrane potential hyperpolarization attenuated by specific large-conductance calcium-activated K+ (BKCa) channel inhibitor iberiotoxin. Under whole cell patch-clamp condition, ICCs increased a potassium outward current. Iberiotoxin (200 nM) did reversal effect on the inhibition of mechanical activity of rat colon by Magnolol, which consisted with the other experiments above. In addition, Magnolol could also suppress colonic contraction enhanced by Neostigmine. In conclusion, the inhibitory effect of Magnolol on rat colonic spontaneous contractility was due to abolishing the intracellular calcium oscillation via opening BKCa channels on ICC. The potent effect of Magnolol on ICC seems to make it a potential anti-spasmodic applying in multiple organs. T1796 Stretch Activated Inhibitory and Excitatory Responses of the Lower Esophageal Sphincter: Lack of Evidence for the Role of Interstitial Cells of Cajal in the Neuromuscular Transmission Yanfen Jiang, Valmik Bhargava, Ravinder K. Mittal Background/Aim: Axial and circumferential stretch of the esophagus induce relaxation and contraction of the lower esophageal sphincter (LES) respectively; both of which are mediated at the level of myenteric plexus. The goal of our study was to determine if interstitial cells of Cajal (ICC) play an intermediary role in the neuromuscular transmission of these responses. Methods: In-Vivo and in-vitro experiments were conducted in the ICC deficient (W/Wv), which lack ICC-IM, and its background mice (WT). For In-Vivo studies, bilateral vagotomy was performed and through a midline laprotomy sutures were placed just above the LES and withdrawn through the mouth to exert axial stretch on the LES. Sutures were also used to exert stretch in the circumferential direction. The LES pressure was monitored with a 2F solid-state transducer placed through the gastrostomy and anchored in the LES. Muscle strips from the LES were studied in-vitro (muscle bath) and assessed histologically. Results: Axial and circumference stretch induced LES relaxation and contraction respectively both in WT and W/Wv mice. At the maximal stretch, the amplitude of LES relaxation and contraction were slightly but significantly smaller in W/Wv as compared to WT. Nitric oxide synthase blocker(L-NAME) blocked the stretch activated relaxation. SNP (NO donor), which acts directly on the muscle induced smaller LES relaxation in W/Wv as compared to WT (Figure A and B). Similarly, carbachol, which acts directly on the muscle, induced smaller contraction in W/Wv mice, both In-Vivo and in-vitro. Histology revealed that the LES muscle is significantly thicker (44%) in W/Wv mice as compared to WT. Conclusion: Stretch activated LES responses are predominantly present in W/Wv mice. Smaller quantitative responses by SNP and carbachol in the ICC deficient mice suggest that the muscle rather than neuromuscular transmission is defective in the W/Wv mice.
T1794 Slow Wave Origin and Pattern of Propagation in the Canine Stomach In Vivo Wim Lammers, Luc Ver Donck, Betty Stephen, Dirk Smets, Jan A. Schuurkes Aim: Slow waves are known to originate orally in the stomach and to propagate towards the antrum. However, the exact location of the pacemaker and the precise pattern of propagation have not yet been studied. We analyzed simultaneous recordings from 240 extracellular electrodes distributed over the stomach. Methods: Using an assembly of 240 extracellular electrodes, simultaneous recordings of spontaneous electrical activity were made on the fundus, corpus and antrum in 18 open-abdomen anesthetized dogs. The signals were analyzed off-line, pathways of slow wave propagation were re-constructed and local slow wave velocities and amplitudes were measured. Results: The gastric pacemaker is located in the upper part of the fundus, close to the oesophageal junction, along the greater curvature. Extracellularly recorded slow waves in the pacemaker area exhibited large amplitudes (1.8±1.0 mV) and rapid velocities (1.5±0.9 cm/s) whereas propagation in the remainder of the fundus and in the corpus was slow (0.5±0.2 cm/s; p<0.05) with low amplitude and fractionated waveforms (0.8±0.5 mV; p<0.05). In the antrum, slow wave propagation was fast (1.5±0.6 cm/s) with large amplitude deflections (2.0±1.3 mV). Two areas were identified
A-581
AGA Abstracts
AGA Abstracts
: The gastric antral circular muscle strips were separated from the longitudinal muscle layer of male guinea-pigs. The force of contraction was recorded isometrically. Muscle preparations were attached to a holder under a resting force of 5 mN and equilibrated for 60-90 min. During this period, 70 mM K+ was repeatedly applied until the sustained force became reproducible. [Ca2+]i was measured by using the fluorescent Ca2+ indicator fura-2. Muscle strips were exposed to the acethoxymethyl ester of fura-2 (fura-2/AM, 10 mM) in the presence of 0.02 % cremophor EL for 5 - 6 h at room temperature, and was transferred to the muscle bath integrated in the fluorimeter. The muscle strips was illuminated alternately (48Hz) with 340 nm and 380 nm light. The light emitted from the muscle strip was collected by a photomultiplier through a 500 nm filter. Results : Spontaneous slow waves and associated Ca2+ transients and contractions were uniform in amplitude (2.3±0.4/min, n=4). Cyclopiazonic acid (CPA, 10 μM), known as selective Ca2+ store inhibitor, reduced spontaneous frequency (1.4±0.5/min, n=5) and the first application of caffeine consistently abolished slow wave activity. Activity could be restored by the addition of Ca2+ (5 mM). Acetylcholine (Ach, 1 μM) caused an significant increase in the amplitude of Ca2+ transients and phasic contractions, as well as an increase in the frequency that consistently produced mechanically productive slow waves (up to 6/min). Spontaneous activity restored by ACh revealed a pattern in which every other slow waves was mechanically non-productive at frequencies as low as 2.5/min. Mechanically non-productive slow waves were reduced in amplitude and duration. Furthermore, these slow waves were initiated prior to the full recovery of [Ca2+]i to basal levels following the previous event. Conclusions : These results suggest that CPAsensitive intracellular Ca2+ stores participate in the recovery of [Ca2+]i following a slow wave.
T1793 Both Campylobacter Jejuni and CDT- C. Jejuni Acutely Reduce the Number of Deep Muscular Plexus Interstitial Cells of Cajal Venkata B. Pokkunuri, Walter Morales, Amy L. Zhu, Kimberly Low, Laura J. Hwang, Jeffrey L. Conklin, Christopher Chang, Mark Pimentel Four slow waves propagating simultaneously in the canine stomach.
Campylobacter jejuni is a leading cause of post-infectious IBS. Cytolethal distending toxin (CDT) is produced by C. jejuni and other pathogenic bacteria, and leads to In Vitro cell cycle arrest and eventual death. In a recent rat model of post-infectious IBS, C. jejuni but not CDT- C. jejuni produces chronic altered stool form and a reduction in the deep muscular plexus interstitial cells of Cajal (DMP-ICC) 3 months after clearance of pathogen. In this study, we aim to evaluate the acute effect of C. jejuni and a CDT- C. jejuni on DMP-ICC. Methods: 100 adult male Sprague-Dawley rats were divided into two groups 1)rats gavaged with 108 cfu wild-type C. jejuni 18-176 (C+) and 2)rats gavaged with 108 cfu C. jejuni cdtB mutant (C+/CDT-). Ten rats from each group were euthanized on days 2, 4, 8, 16, and 32 post-inoculation. A third group was gavaged with PBS alone (Controls). Predefined segments of duodenum, jejunum and ileum were resected. Tissue was fixed in formalin and then 80% ethanol, mounted and immunstained using anti-CD117. Slides were coded, randomized and blinded readers counted the number of intestinal villi and DMP-ICC cell bodies. Results: All rats were colonized with Campy. For both groups there was a rapid reduction in the number of DMP-ICC. This was most pronounced on day 2 for C+/CDT- and on day 4 for C+ compared to control (see figure). The overall recovery from the effect was more rapid in C+/CDT-. By day 8 the reduction persisted in C+ and was still significantly less than control (P<0.001) and C+/CDT- (P<0.05) . Full recovery of all groups was seen by day 16. Conclusions: C. jejuni produces an acute reduction in DMP-ICC cells. Recovery of ICC is seen by day 16.
T1795 Involvement of BKCa Channel in the Inhibitory Effect of Magnolol On Rat Colonic Spontaneous Contractility Ao Pan, Siliang Chen, Zihuan Yang, Haijie Yu, Wenliang Zhou The objective of the present study was to investigate the mechanism underlying the inhibitory effect of Magnolol on isolated colonic spontaneous contraction, and find its specific potential for therapy of intestinal motility disorders. Magnolol (5 μM - 1 mM) induced a concentrationdependent inhibition on rat colonic longitude smooth muscle spontaneous contraction. In primary cultured ICCs, Magnolol (50 μM) abolished the intracellular calcium oscillation. Magnolol-induced significant membrane potential hyperpolarization attenuated by specific large-conductance calcium-activated K+ (BKCa) channel inhibitor iberiotoxin. Under whole cell patch-clamp condition, ICCs increased a potassium outward current. Iberiotoxin (200 nM) did reversal effect on the inhibition of mechanical activity of rat colon by Magnolol, which consisted with the other experiments above. In addition, Magnolol could also suppress colonic contraction enhanced by Neostigmine. In conclusion, the inhibitory effect of Magnolol on rat colonic spontaneous contractility was due to abolishing the intracellular calcium oscillation via opening BKCa channels on ICC. The potent effect of Magnolol on ICC seems to make it a potential anti-spasmodic applying in multiple organs. T1796 Stretch Activated Inhibitory and Excitatory Responses of the Lower Esophageal Sphincter: Lack of Evidence for the Role of Interstitial Cells of Cajal in the Neuromuscular Transmission Yanfen Jiang, Valmik Bhargava, Ravinder K. Mittal Background/Aim: Axial and circumferential stretch of the esophagus induce relaxation and contraction of the lower esophageal sphincter (LES) respectively; both of which are mediated at the level of myenteric plexus. The goal of our study was to determine if interstitial cells of Cajal (ICC) play an intermediary role in the neuromuscular transmission of these responses. Methods: In-Vivo and in-vitro experiments were conducted in the ICC deficient (W/Wv), which lack ICC-IM, and its background mice (WT). For In-Vivo studies, bilateral vagotomy was performed and through a midline laprotomy sutures were placed just above the LES and withdrawn through the mouth to exert axial stretch on the LES. Sutures were also used to exert stretch in the circumferential direction. The LES pressure was monitored with a 2F solid-state transducer placed through the gastrostomy and anchored in the LES. Muscle strips from the LES were studied in-vitro (muscle bath) and assessed histologically. Results: Axial and circumference stretch induced LES relaxation and contraction respectively both in WT and W/Wv mice. At the maximal stretch, the amplitude of LES relaxation and contraction were slightly but significantly smaller in W/Wv as compared to WT. Nitric oxide synthase blocker(L-NAME) blocked the stretch activated relaxation. SNP (NO donor), which acts directly on the muscle induced smaller LES relaxation in W/Wv as compared to WT (Figure A and B). Similarly, carbachol, which acts directly on the muscle, induced smaller contraction in W/Wv mice, both In-Vivo and in-vitro. Histology revealed that the LES muscle is significantly thicker (44%) in W/Wv mice as compared to WT. Conclusion: Stretch activated LES responses are predominantly present in W/Wv mice. Smaller quantitative responses by SNP and carbachol in the ICC deficient mice suggest that the muscle rather than neuromuscular transmission is defective in the W/Wv mice.
T1794 Slow Wave Origin and Pattern of Propagation in the Canine Stomach In Vivo Wim Lammers, Luc Ver Donck, Betty Stephen, Dirk Smets, Jan A. Schuurkes Aim: Slow waves are known to originate orally in the stomach and to propagate towards the antrum. However, the exact location of the pacemaker and the precise pattern of propagation have not yet been studied. We analyzed simultaneous recordings from 240 extracellular electrodes distributed over the stomach. Methods: Using an assembly of 240 extracellular electrodes, simultaneous recordings of spontaneous electrical activity were made on the fundus, corpus and antrum in 18 open-abdomen anesthetized dogs. The signals were analyzed off-line, pathways of slow wave propagation were re-constructed and local slow wave velocities and amplitudes were measured. Results: The gastric pacemaker is located in the upper part of the fundus, close to the oesophageal junction, along the greater curvature. Extracellularly recorded slow waves in the pacemaker area exhibited large amplitudes (1.8±1.0 mV) and rapid velocities (1.5±0.9 cm/s) whereas propagation in the remainder of the fundus and in the corpus was slow (0.5±0.2 cm/s; p<0.05) with low amplitude and fractionated waveforms (0.8±0.5 mV; p<0.05). In the antrum, slow wave propagation was fast (1.5±0.6 cm/s) with large amplitude deflections (2.0±1.3 mV). Two areas were identified
A-581
AGA Abstracts
AGA Abstracts
: The gastric antral circular muscle strips were separated from the longitudinal muscle layer of male guinea-pigs. The force of contraction was recorded isometrically. Muscle preparations were attached to a holder under a resting force of 5 mN and equilibrated for 60-90 min. During this period, 70 mM K+ was repeatedly applied until the sustained force became reproducible. [Ca2+]i was measured by using the fluorescent Ca2+ indicator fura-2. Muscle strips were exposed to the acethoxymethyl ester of fura-2 (fura-2/AM, 10 mM) in the presence of 0.02 % cremophor EL for 5 - 6 h at room temperature, and was transferred to the muscle bath integrated in the fluorimeter. The muscle strips was illuminated alternately (48Hz) with 340 nm and 380 nm light. The light emitted from the muscle strip was collected by a photomultiplier through a 500 nm filter. Results : Spontaneous slow waves and associated Ca2+ transients and contractions were uniform in amplitude (2.3±0.4/min, n=4). Cyclopiazonic acid (CPA, 10 μM), known as selective Ca2+ store inhibitor, reduced spontaneous frequency (1.4±0.5/min, n=5) and the first application of caffeine consistently abolished slow wave activity. Activity could be restored by the addition of Ca2+ (5 mM). Acetylcholine (Ach, 1 μM) caused an significant increase in the amplitude of Ca2+ transients and phasic contractions, as well as an increase in the frequency that consistently produced mechanically productive slow waves (up to 6/min). Spontaneous activity restored by ACh revealed a pattern in which every other slow waves was mechanically non-productive at frequencies as low as 2.5/min. Mechanically non-productive slow waves were reduced in amplitude and duration. Furthermore, these slow waves were initiated prior to the full recovery of [Ca2+]i to basal levels following the previous event. Conclusions : These results suggest that CPAsensitive intracellular Ca2+ stores participate in the recovery of [Ca2+]i following a slow wave.