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M2035 Submucosal Histamine Release Involves Emodin-Induced Cl- Secretion in the Distal Colon of Rat
person) (P <0.001). [Conclusions] 1) Data showed a good comparability of these modalities in recording peristalsis, with a few of missing of secondary peristalsis with manometry. The new evaluation with unsedated transnasal endoscopy may enable a simpler and more sensitive recording for esophageal peristalsis. 2) The decreased frequency of acid-induced peristalsis may lead the esophageal acid environment as more acidic and be involved in the pathogenesis of BE. M2035 Submucosal Histamine Release Involves Emodin-Induced Cl- Secretion in the Distal Colon of Rat Jingdong Xu, Wen Wang, Jin Song, Yue Xu, Jin-xia Zhu Background and aim: Emodin, an anthraquinone derivative, has been reported to induce histamine release from isolated rat mast cells, and increase the level of intestinal mucosa histamine in the rats with intestinal obstruction. Histamine has also been found to activate enteric nerve cells via H2 receptors. We currently have demonstrated the stimulatory effect of emodin on colonic anion secretion. This effect was significantly abolished by tetrodotoxin through inhibition of submucosal plexus activity. In this study we investigated whether the submucosal histamine release contributes to the emodin-induced colonic anion secretion in rats and whether H2 receptor mediated this effect. Methods: Histochemistry and immunofluorescence were used to investigate the distribution of mast cells in the mucosa and submucosa of distal colon. Radioimmunoassay (RIA) was used to measure the amount of histamine release. Scanning ion-selective electrode technique (SIET) and short circuit current (ISC) recording were utilized to examine the epithelial Cl—flux and ion transport. Results: Histochemistry and Immunofluorescence results demonstrated that mast cells were located in the lamina propria and submucosa of distal colon of rats, and the immunoreactive tryptase was more concentrated in the cytoplasm of mast cells in emodin treated group. Treatment of mucosa/submucosa preparations with emodin at concentration of 10μmol/L and 100μmol/ L increased histamine release by 60.0% and 71.42%, respectively. Basolateral application of emodin (100μmol/L) induced increases of ISC from 21.2 ± 3.9 μA.cm-2 to 79.68±9.31μA.cm-2 (p<0.01). Furthermore this increase was blocked by apical addition of glibenclymide or basolateral application of bumetanide, but not affected by apical addition of amiloride. Compared with the results measured by ISC recording technique, similar tendency was observed by using SIET, which was also blocked by glibenclymide and bumetanide, but not by amiloride. These results indicated that emodin-induced increase in ISC was mediated by Cl- secretion. Pretreatment of mucosa/submucosa preparations with TTX inhibited emodin-induced ISC response by 81.6% (p<0.001). Basolateral addition of M receptor blocker, atropine (10μM) attenuated emodin-induced ISC response by 36.8%. However cimetidine (100μM) and ranitidine (10μM), H2 receptor antagonists, induced a reduction of 68.8% (p<0.001) and 72.5% (p<0.001), respectively. The blocking effect of cimetidine on emodin-induced ISC response was in dose-dependent manner (not shown). Conclusions: Emodin induces colonic Cl- secretion and submucosa histamine release, and histamine mediates Cl- secretion by activating H2 receptor.
M2033 Acetylcholine (ACh)-Induced Enhanced Actin Polymerization is Associated With Small Heat Shock Protien Relocation Robert R. Gilmont, Sita Somara, Saranyaraajan Varadarajan, Khalil Bitar Background: Polymerized actin is required for contraction as treatment with actin depolymerizing agents abolishes cellular response to contractile agonists. There are three forms of actin in colonic smooth muscle cells (α, β and γ). α and γ forms comprise the thin filament component of the contractile apparatus while the cytoskeleton is composed of β and γ forms. For cellular contraction to occur the contractile apparatus must be anchored to the cytoskeleton. The integrity of cellular actin networks may be compromised with aging or during inflammation, leading to colonic dysfunction. Objectives: To determine the effect of Ach stimulation on colonic circular smooth muscle cell (CSMC) actin polymerization. To determine the effect of Ach-stimulation on small heat shock protein (HSP20 and HSP27) associations with F-actin. Methods: Confluent cultures of colonic circular smooth muscle cells (CSMC) were serum starved before being treated with 10-7 M Ach for various times. Cells were solubilized in the presence of 0.1 % N P40, 0.1 % Triton X-100 and 0.1 % Tween 20 and separated into particulate (F-actin) and cytosol (G-actin) fractions by ultracentrifugation at 37oC. Under these conditions cellular membranes are solubilized and do not pellet. The relative abundance of F and G-actin fractions was determined by immunoblotting using antibodies directed against total actin (all isoforms) or antibodies specific for α, β or γ actin isoforms. Additionally, blots were probed for HSP20, HSP27, tropomyosin and caldesmon. Results: Under control conditions, HSP27, tropomyosin and caldesmon preferentially associated with F-actin while a minority of total HSP20 (28 %) associated with F-actin. Ach stimulation induced an increase in total actin polymerization of 6.7%, an increase in HSP27 association with F-actin of 6% and a decrease in association of HSP20 with F-actin of 18.8 % (~ 3 fold) by 30 sec. By 15 sec Ach stimulation induced a modest 3% increase in α-actin and 3.5 % increase in γ-actin polymerization, while the increase in the β isoform of F-actin was slightly over 10%. Summary: Ach stimulation of CSMC induces rapid (within 15 sec) actin polymerization, causing an increase in cellular F-actin primarily by increasing the cytoskeleton F-actin. A rapid redistribution of HSP20/HSP27 with Factin is coupled to increased actin polymerization. Conclusion: These data demonstrate a redistribution of HSP20/HSP27 temporally coupled to actin polymerization during smooth muscle contraction and implies a role for small HSPs in regulating actin dynamics. Supported by NIH/NIDDK RO1DK 057020
M2036 Modulation of NHE8 by Somatostatin in Human Intestinal Epithelial Cells Chunhui Wang, Hua Xu, Huacong Chen, Fayez K. Ghishan Introduction: Somatostatin (SST) is an important neuropeptide of the human gastrointestinal tract. SST has been known to stimulate sodium absorption in the gut for decades. Its longer acting analog, octreotide (OCT), has been widely used as an antidiarrheal agent. Na+/H+ exchanger (NHE) is a large transporter family with nine members that play important roles in intracellular pH regulation and sodium absorption in many cell types. Specifically, NHE2, NHE3 and NHE8 are well known transporters involved in sodium absorption in the intestinal tract. Although SST and its analog have been shown to be able to enhance sodium absorption, whether SST alters NHEs activity and expression in intestinal epithelial cells is unknown. The primary aim of the study was to investigate the role of SST on NHE8 expression in human intestinal epithelial cells. Methods: Human intestinal epithelial cells (Caco2 cells) were treated with 100 nM SST for various duration before isolating protein and RNA. Membrane protein was extracted either by density centrifuge or by biotinylation method. Total RNA was isolated using Trizol. Western blotting was used to analysis the expression of NHE-8 protein in Caco-2 cells, and Real-time PCR was used to measure NHE8 mRNA expression level. Results: Western blots of total membrane protein preps showed that NHE8 protein expression was increased about 57% and 88% after SST treatment for 1 hour and 14 hours, respectively. Cell surface protein biotinylation result also showed that SST treatment increased surface NHE8 protein expression without changing the surface expression of EGFR. Real-time PCR data demonstrated that NHE8 mRNA abundance was also increased by SST treatment. Conclusion: SST specifically stimulates NHE8 expression in human intestinal epithelial cells, which supports the role of SST in enhancing the process of sodium absorption. This is the only physiological factor identified so far that enhances NHE8 expression since the discovery of NHE8.
M2034 Evaluation of Esophageal Peristalsis With a New Method Using Unsedated Transnasal Endoscopy Go Kobayashi, Mitsuru Kaise, Hiroshi Arakawa, Hisao Tajiri [Background and aim] The increasing prevalence of acid-related esophageal disorders in Asian countries enhances the necessity of esophageal function tests. However, these tests are not commonly performed because of their complicated features. Our aim was to develop a simpler and more efficient esophageal function test that can be applicable in a standard clinical setting. [Methods] We designed an examination that simultaneously evaluated abnormalities of esophageal motility and morphology. Using unsedated transnasal endoscopy, morphological abnormalities were evaluated first. Esophageal secondary peristalsis was evaluated next during acid (0.1N HCl) or saline infusion via endoscopic channel (10 ml/min for 10 min for each). Secondary peristalsis designated E-2ndP was defined as a complete contraction that swept out solution retained in the esophagus. Complete contraction without sweeping, incomplete contraction with and without sweeping were designated as E-SynchrW, E-small2ndP and E-smallSynchrW, respectively. We also confirmed the relationship between endoscopy- and manometry-defined peristalsis by simultaneous recording with endoscopy and conventional manometry. Wave amplitudes more than 25 mmHg with a propagate velocity of not more than 6 cm/s and more than 6 cm/s were defined secondary peristalsis (M-2ndP) and synchronous pressure wave (M-SynchrW). [Results] 1) In total, 51 individuals were enrolled in the study; 25 healthy volunteers (HV, 32±8 years old) and 26 patients with Barrett's esophagus (BE, 40±15 years old, short segment=17 and long segment =9). 2) The simultaneous recording, performed in 5 volunteers, showed the relationships between those two modalities. All 73 of M-2ndP were recorded as E-2ndP, but 2 and 1 of 76 E2ndPs were recorded as M-SynchrWs and not recorded with mamometry. Two, 4, 5 and 19 of 30 M-SynchrW were recorded as E-2ndP, E-SynchrW, E-small2ndP and E-smallSynchrW, meaning that 23% of M-SynchrWs swept out solution retained in the esophagus. 3) In 51 individuals, infusion-induced esophageal contractions were evaluated with unsedated endoscopy. E-2ndP were induced less frequently in BE (5.3±1.2/person) than HV (8.2±2.5/
M2037 The Role of Submucosal 5-HT3 Receptor and Somatostatin Neuron in the Regulation of Rat Colonic Secretion Yun Li, Xiaofeng Li, Xinliang Mi, Yue Zhang, Jinxia Zhu Background and Aims: 5-hydroxytryptamine (5-HT) is a key regulator in gastrointestinal (GI) tract. Our previous study has demonstrated that submucous 5-HT3 receptor and somatostatin play an inhibitory role in 5-HT-induced colonic ion transport. In this study we investigated the inhibitory pathway between activation of submucous 5-HT3 receptor and neuronal somatostatin release, and the effect of acute stress on this pathway. Methods: Radioimmunoassay (RIA) was used to determine 5-HT-induced submucous somatostatin release. Short circuit current (ISC) recording were utilized to examine the ion transport. Rats were subjected to water-immersion restraint stress (WIR) for 2h to make acute stress
S-463
AGA Abstracts
AGA Abstracts
from rabbit sigmoid colon were assayed for expression and phosphorylation of contractile proteins by immunoblotting. Basal force generation was measured in 3D longitudinal tissue bioengineered from LSMC, and from 3D bioengineered circular rings. Results: At rest, compared to CSMC, LSMC exhibited (A) Expression of contractile proteins: (i) 3 fold increase in HSP27 expression; (ii) 2 fold increase in Tropomyosin (TM) expression; (iii) 1.7 fold increase in Caldesmon (CaD) expression; (iv) 1.4 fold increase in HSP20 expression; (B) Phosphorylation of contractile proteins: (i) 200% increase in HSP27 phosphorylation; (ii) 50% increase in CaD phosphorylation; (iii) 65% increase in PKCα phosphorylation. (C) Force generation: The 3D bioengineered longitudinal tissue (i) did not display phasic characteristics of basal force generated by 3D bioengineered circular rings; (ii) displayed higher magnitude of absolute basal force when compared to 3D bioengineered circular rings. Summary: LSMC expresses significantly higher levels of contractile regulatory proteins TM, CaD, HSP27 and HSP20 with significantly enhanced basal phosphorylation levels of CaD, HSP27, PKCα. Further, basal tone of 3D bioengineered longitudinal tissue displayed features of tonic muscle. Conclusion: The fundamental differences in basal expression and phosphorylation levels of contractile proteins could contribute to difference in the basal tone of longitudinal and circular muscle. Significance: These studies provide basis for the myogenic differences intrinsic to the longitudinal smooth muscle layer of the colon and contributes to better understanding the pathophysiology of dysfunctional colonic motility. Supported by NIH/NIDDK RO1DK 057020
M2033 Acetylcholine (ACh)-Induced Enhanced Actin Polymerization is Associated With Small Heat Shock Protien Relocation Robert R. Gilmont, Sita Somara, Saranyaraajan Varadarajan, Khalil Bitar Background: Polymerized actin is required for contraction as treatment with actin depolymerizing agents abolishes cellular response to contractile agonists. There are three forms of actin in colonic smooth muscle cells (α, β and γ). α and γ forms comprise the thin filament component of the contractile apparatus while the cytoskeleton is composed of β and γ forms. For cellular contraction to occur the contractile apparatus must be anchored to the cytoskeleton. The integrity of cellular actin networks may be compromised with aging or during inflammation, leading to colonic dysfunction. Objectives: To determine the effect of Ach stimulation on colonic circular smooth muscle cell (CSMC) actin polymerization. To determine the effect of Ach-stimulation on small heat shock protein (HSP20 and HSP27) associations with F-actin. Methods: Confluent cultures of colonic circular smooth muscle cells (CSMC) were serum starved before being treated with 10-7 M Ach for various times. Cells were solubilized in the presence of 0.1 % N P40, 0.1 % Triton X-100 and 0.1 % Tween 20 and separated into particulate (F-actin) and cytosol (G-actin) fractions by ultracentrifugation at 37oC. Under these conditions cellular membranes are solubilized and do not pellet. The relative abundance of F and G-actin fractions was determined by immunoblotting using antibodies directed against total actin (all isoforms) or antibodies specific for α, β or γ actin isoforms. Additionally, blots were probed for HSP20, HSP27, tropomyosin and caldesmon. Results: Under control conditions, HSP27, tropomyosin and caldesmon preferentially associated with F-actin while a minority of total HSP20 (28 %) associated with F-actin. Ach stimulation induced an increase in total actin polymerization of 6.7%, an increase in HSP27 association with F-actin of 6% and a decrease in association of HSP20 with F-actin of 18.8 % (~ 3 fold) by 30 sec. By 15 sec Ach stimulation induced a modest 3% increase in α-actin and 3.5 % increase in γ-actin polymerization, while the increase in the β isoform of F-actin was slightly over 10%. Summary: Ach stimulation of CSMC induces rapid (within 15 sec) actin polymerization, causing an increase in cellular F-actin primarily by increasing the cytoskeleton F-actin. A rapid redistribution of HSP20/HSP27 with Factin is coupled to increased actin polymerization. Conclusion: These data demonstrate a redistribution of HSP20/HSP27 temporally coupled to actin polymerization during smooth muscle contraction and implies a role for small HSPs in regulating actin dynamics. Supported by NIH/NIDDK RO1DK 057020
M2036 Modulation of NHE8 by Somatostatin in Human Intestinal Epithelial Cells Chunhui Wang, Hua Xu, Huacong Chen, Fayez K. Ghishan Introduction: Somatostatin (SST) is an important neuropeptide of the human gastrointestinal tract. SST has been known to stimulate sodium absorption in the gut for decades. Its longer acting analog, octreotide (OCT), has been widely used as an antidiarrheal agent. Na+/H+ exchanger (NHE) is a large transporter family with nine members that play important roles in intracellular pH regulation and sodium absorption in many cell types. Specifically, NHE2, NHE3 and NHE8 are well known transporters involved in sodium absorption in the intestinal tract. Although SST and its analog have been shown to be able to enhance sodium absorption, whether SST alters NHEs activity and expression in intestinal epithelial cells is unknown. The primary aim of the study was to investigate the role of SST on NHE8 expression in human intestinal epithelial cells. Methods: Human intestinal epithelial cells (Caco2 cells) were treated with 100 nM SST for various duration before isolating protein and RNA. Membrane protein was extracted either by density centrifuge or by biotinylation method. Total RNA was isolated using Trizol. Western blotting was used to analysis the expression of NHE-8 protein in Caco-2 cells, and Real-time PCR was used to measure NHE8 mRNA expression level. Results: Western blots of total membrane protein preps showed that NHE8 protein expression was increased about 57% and 88% after SST treatment for 1 hour and 14 hours, respectively. Cell surface protein biotinylation result also showed that SST treatment increased surface NHE8 protein expression without changing the surface expression of EGFR. Real-time PCR data demonstrated that NHE8 mRNA abundance was also increased by SST treatment. Conclusion: SST specifically stimulates NHE8 expression in human intestinal epithelial cells, which supports the role of SST in enhancing the process of sodium absorption. This is the only physiological factor identified so far that enhances NHE8 expression since the discovery of NHE8.
M2034 Evaluation of Esophageal Peristalsis With a New Method Using Unsedated Transnasal Endoscopy Go Kobayashi, Mitsuru Kaise, Hiroshi Arakawa, Hisao Tajiri [Background and aim] The increasing prevalence of acid-related esophageal disorders in Asian countries enhances the necessity of esophageal function tests. However, these tests are not commonly performed because of their complicated features. Our aim was to develop a simpler and more efficient esophageal function test that can be applicable in a standard clinical setting. [Methods] We designed an examination that simultaneously evaluated abnormalities of esophageal motility and morphology. Using unsedated transnasal endoscopy, morphological abnormalities were evaluated first. Esophageal secondary peristalsis was evaluated next during acid (0.1N HCl) or saline infusion via endoscopic channel (10 ml/min for 10 min for each). Secondary peristalsis designated E-2ndP was defined as a complete contraction that swept out solution retained in the esophagus. Complete contraction without sweeping, incomplete contraction with and without sweeping were designated as E-SynchrW, E-small2ndP and E-smallSynchrW, respectively. We also confirmed the relationship between endoscopy- and manometry-defined peristalsis by simultaneous recording with endoscopy and conventional manometry. Wave amplitudes more than 25 mmHg with a propagate velocity of not more than 6 cm/s and more than 6 cm/s were defined secondary peristalsis (M-2ndP) and synchronous pressure wave (M-SynchrW). [Results] 1) In total, 51 individuals were enrolled in the study; 25 healthy volunteers (HV, 32±8 years old) and 26 patients with Barrett's esophagus (BE, 40±15 years old, short segment=17 and long segment =9). 2) The simultaneous recording, performed in 5 volunteers, showed the relationships between those two modalities. All 73 of M-2ndP were recorded as E-2ndP, but 2 and 1 of 76 E2ndPs were recorded as M-SynchrWs and not recorded with mamometry. Two, 4, 5 and 19 of 30 M-SynchrW were recorded as E-2ndP, E-SynchrW, E-small2ndP and E-smallSynchrW, meaning that 23% of M-SynchrWs swept out solution retained in the esophagus. 3) In 51 individuals, infusion-induced esophageal contractions were evaluated with unsedated endoscopy. E-2ndP were induced less frequently in BE (5.3±1.2/person) than HV (8.2±2.5/
M2037 The Role of Submucosal 5-HT3 Receptor and Somatostatin Neuron in the Regulation of Rat Colonic Secretion Yun Li, Xiaofeng Li, Xinliang Mi, Yue Zhang, Jinxia Zhu Background and Aims: 5-hydroxytryptamine (5-HT) is a key regulator in gastrointestinal (GI) tract. Our previous study has demonstrated that submucous 5-HT3 receptor and somatostatin play an inhibitory role in 5-HT-induced colonic ion transport. In this study we investigated the inhibitory pathway between activation of submucous 5-HT3 receptor and neuronal somatostatin release, and the effect of acute stress on this pathway. Methods: Radioimmunoassay (RIA) was used to determine 5-HT-induced submucous somatostatin release. Short circuit current (ISC) recording were utilized to examine the ion transport. Rats were subjected to water-immersion restraint stress (WIR) for 2h to make acute stress
S-463
AGA Abstracts
AGA Abstracts
from rabbit sigmoid colon were assayed for expression and phosphorylation of contractile proteins by immunoblotting. Basal force generation was measured in 3D longitudinal tissue bioengineered from LSMC, and from 3D bioengineered circular rings. Results: At rest, compared to CSMC, LSMC exhibited (A) Expression of contractile proteins: (i) 3 fold increase in HSP27 expression; (ii) 2 fold increase in Tropomyosin (TM) expression; (iii) 1.7 fold increase in Caldesmon (CaD) expression; (iv) 1.4 fold increase in HSP20 expression; (B) Phosphorylation of contractile proteins: (i) 200% increase in HSP27 phosphorylation; (ii) 50% increase in CaD phosphorylation; (iii) 65% increase in PKCα phosphorylation. (C) Force generation: The 3D bioengineered longitudinal tissue (i) did not display phasic characteristics of basal force generated by 3D bioengineered circular rings; (ii) displayed higher magnitude of absolute basal force when compared to 3D bioengineered circular rings. Summary: LSMC expresses significantly higher levels of contractile regulatory proteins TM, CaD, HSP27 and HSP20 with significantly enhanced basal phosphorylation levels of CaD, HSP27, PKCα. Further, basal tone of 3D bioengineered longitudinal tissue displayed features of tonic muscle. Conclusion: The fundamental differences in basal expression and phosphorylation levels of contractile proteins could contribute to difference in the basal tone of longitudinal and circular muscle. Significance: These studies provide basis for the myogenic differences intrinsic to the longitudinal smooth muscle layer of the colon and contributes to better understanding the pathophysiology of dysfunctional colonic motility. Supported by NIH/NIDDK RO1DK 057020