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Sa2050 Corticotropin-Releasing Factor (CRF) Peptides Modulates Rat Colonic Neuronal Tau Phosphorylation: Differential Role of CRF Receptors
AGA Abstracts
Sa2050
Sa2052
Corticotropin-Releasing Factor (CRF) Peptides Modulates Rat Colonic Neuronal Tau Phosphorylation: Differential Role of CRF Receptors Hung Pham, Shuping S. Wu, Perozi Eslami, Marni E. Harris-White, Mulugeta Million
Role of Stretch-Activated Ion Channels in Esophageal Inhibitory Motor Neurons Hui Dong, Yanfen Jiang, Ravinder K. Mittal
BACKGROUND: Corticotropin releasing factor receptor-1 (CRF1)-overexpression enhances experimental stress or CRF-induced tau (TAU) phosphorylation in the mouse brain (PNAS, 2012: 109(16):6277-82). CRF is a key mediator of the gut response to stress. Whether CRF affect enteric TAU and whether enteric neuronal TAU modulation impacts gastrointestinal tract functions are not known. AIM: 1) Determine 1) the effect of selective activation of CRF receptors on rat colon primary myneteric neuron Tau 2) the colonic motor response of Tau transgenic mice to acute stress. METHODS: Rat distal colonic primary myenteric neurons were incubated with non-selective CRF1 and 2 receptor agonists (CRF, urocortin 1 (Ucn1)) and selective CRF2 receptor agonist (Ucn2) for 30 min (10 or 100 nM) and pTAU probed by western blot. Rat colon primary myenteric neurons were processed for immunostaining of Tau. Human TauP301L overexpressing and wild-type (WT) mice were exposed to 1-hour novel environment stress and cumulative Fecal Pellet Output (FPO) at 5, 15, 30, 45 and 60 min was monitored. The overexpression of human Tau in the TauOE mice was confirmed by western blotting. RESULTS: Rat colon primary neurons have constitutively active pTau that is suppressed by selective CRF2 receptor activation but not by the non-selective CRF receptor ligand, CRF. Rat colon primary myenteric neurons have abundant Tau. Compared to WT, Tau overexpressing mice have 2-4X increased total Tau mRNA expression, increased defecation (60.7±6.5% increase in 60 min cumulative FPO) response to mild novel environment stress and display altered ileal and colonic CRF2 receptor expression. CONCLUSION: The differential modulation of colonic primary myenteric TAU phophorylation by CRF peptides and the altered colonic motor response to stress in the mice that express phospho TAU in the intestine and the modulation of colonic CRF2 receptor mRNA profile in these mice, suggest a possible interplay between myenteric TAU and CRFsignaling in the colonic response to stress. Supported by R01DK078676 (MM); Veterans Administration Merit Review (MEH-W)
Background & Aims: Inhibitory motor neurons located in the esophageal wall play an important role in regulating function of the lower esophageal sphincter (LES). Our earlier whole animal studies revealed that mechanical stretch of the esophagus in the axial direction induces neurally mediated LES relaxation, but the mechanisms underlying mechanosensitive process is currently unknown. The aims of our study were to investigate, 2) if esophageal inhibitory motor neurons are mechanosensitive and 2) if stretch-activated ion channels (SACs) play a role in this mechanosensitivy. Methods: Esophageal myenteric neurons were freshly isolated from the esophagus of postnatal rats (5 days) and primary cultured for two weeks, immmunocytochemical characteristics of these neurons was conducted using NOS staining. Neurons in the culture were stretched by hypotonic solution (170 mOsm) to induce cell swelling and stretch. Three parameters were monitored with fluorescence dyes, fura 2AM and DAF-FM in single neurons using a digital cell imaging system: 1) cytoplasmic volume of neurons, 2) cytoplasmic free Ca2+ concentrations ([Ca2+]cyt) in the neurons, a cell signaling essential for neurotransmitter release, and 3) intracellular nitric oxide (NO), an important neurotransmitter in the inhibitory motor neurons. Results: Iimmmunocytochemical analysis revealed that >95% cells were positive for neuronal marker PGP 9.5. Sixtysix percent of the PGP 9.5 positive neurons co-stained with nNOS. Hypotonic solution induced changes in the cytoplasmic volume in all tested cells, which is independent of the presence or absence of extracellular Ca2+ Hypotonic solution induced [Ca2+]cyt signaling in ~ 65% of the neurons, in the presence but not the absence of extracellular Ca2+. Hypotonic stretch-induced [Ca2+]cyt signaling was not affected by extracellular Mg2+ and nifedipine, but was attenuated by potent blockers of SACs, Gd3+ and GsMTx4. Finally, hypotonic swelling induced NO production in about 57% of the neurons detected with DAF during cell stretch. Removal of extracellular Ca2+ markedly reduced hypotonic stretch-induced NO production in these single neurons. Conclusion: Our data suggest that esophageal inhibitory motor neurons are mechanosensitive, and that SACs on the neurons may play an important role in this mechanosensitivy through a novel Ca2+-NO signaling pathway.
Sa2051 A Phase II, Randomized, Double-Blind, Placebo-Controlled, Multiple-Dose, Parallel-Group Study to Evaluate the Efficacy, Safety, and Pharmacodynamics of RM-131 in Patients With Chronic Constipation Andres Acosta, Gururaj Kolar, Johanna Iturrino, Lawrence A. Szarka, Amy Boldingh, Duane D. Burton, Michael Ryks, Deborah L. Rhoten, Alan R. Zinsmeister, Michael Camilleri Background: Some patients do not achieve satisfactory relief of chronic constipation (CC). RM-131 is a pentapeptide, selective ghrelin receptor-1a agonist that increased gastric emptying (GE) in patients with diabetes mellitus. Aim: To evaluate effects of RM-131 on GE, small bowel transit (SBT) and colonic transit (CT) in patients with CC. Methods: As part of a trial of 48 patients assessing symptoms (NCT01781104), we conducted a randomized (1:1), double-blind, placebo (pbo)-controlled, parallel-group pharmacodynamics (PD) substudy in a single-center, evaluating effects of RM-131, 100μg s.c. once daily for 14 days, on GE, SBT [colonic filling at 6h (CF6)] and CT in patients with CC. Subjects (18-65y; BMI 19-40 kg/m2) were stratified by baseline GC24 value (≤1.6 vs. >1.6,<2.4). During 14-day baseline, placebo-treatment period, and the double-blind treatment periods, daily diaries of bowel habits, abdominal symptoms, global measures and baseline CT were collected. Main inclusion criteria were baseline GC24<2.4 and average spontaneous BMs ≤4/week. GI and CT of solids were measured during the last 48h of treatment using a standard, validated scintigraphic method (Deiteren et al NGM 22:415-423, 2010). The 24h image was obtained before the medication on treatment day 13. Safety was evaluated including adverse events, 12-lead electrocardiogram, and clinical laboratory assessments. The primary PD efficacy analysis (for CT and GE parameters) was by analysis of covariance (ANCOVA) models using baseline measurements (GC24 for CT data; BMI for CF6 and AC T½; and age for GE) as covariates. The PD substudy required 12 patients per treatment arm. Results: Baseline demographics and GC24 were similar in both treatment groups: the participants were all female, mean age was 40.5±10.3y; mean BMI was 24.8±2.9kg/m2. RM-131 was associated with a significant (table) acceleration of GE (GE T½ p=0.027), colonic filling at 6h [CF6% (surrogate for SBT), p=0.051], and CT at 32 and 48h (p=0.040 and p=0.017, respectively). There were no significant increases in CT at 24h (p=0.44) or ascending colon emptying (AC T½ p=0.43). There were no drop outs or withdrawals. Clinical endpoints will be analyzed upon completion of the 48th participant in the overall study. Conclusion: RM131, 100μg s.c. daily, significantly accelerates CT in patients with CC, in addition to accelerating GE and demonstrating stimulation of both upper and lower GI motility. The magnitude of effect on CT GC48 is associated with a 1 point difference in stool consistency on the Bristol Stool Form scale (Deiteren et al NGM 2010), suggesting that the PD effect is clinically relevant. Funding: Rhythm Pharmaceuticals
Figure. Mechanosensitivity of esophageal enteric neurons and the involvement of stretchactivated ion channels. Summary data showing the time courses of neuronal responses to Hypot in the presence of nifedipine (10 μM) or GsMTx4 (10 μM). After F340/380 baselines were stable in PSS, the first switch of extracellular solutions to Hypot as a control. Before the second or the third switches of extracellular solutions to Hypot, the neurons were pretreated with nifedipine or GsMTx4 for 5 min. Values are means ± SEM. n=55 cells from 6 rats. Sa2053 Understanding the Effects of Propofol on Serotonergic Signalling and Colonic Motility Lucy B. Diss, Kim R. Pearce, Bhavik A. Patel Introduction: Propofol (2,6 diisopropylphenol) is the most widely used intravenous anaesthetic agent for induction and maintenance of anaesthesia and sedation. Propofol is known to have an antagonistic effect on 5-hydroxytryptamine receptor (5-HT3R) but its actions on motility are not fully understood. We have investigated the effect of propofol on mucosal serotonergic signalling, colonic migrating motor complex (CMMC) and faecal pellet colonic motility. Methods: Colonic tissue samples were obtained from 10-12 weeks male C57BL/6 mice. For all studies tissues were treated with or without 1 μM propofol for 30 minutes. High-performance liquid chromatography with electrochemical detection (HPLC-ED) was utilised to detect levels of mucosal serotonin (5-HT), precursors and metabolites from proximal and distal colon mucosa and longitudinal muscle myenteric plexus (LMMP). CMMCs were monitored using isometric force transducers along the length of the colon. An artificial epoxy-coated fecal pellet was utilised to monitor colonic motility. The influence of 5-HT3R antagonist, ondansetron and nitric oxide synthase blocker, L-NNA were assessed on tissue exposed to propofol. Results: Following treatment in propofol, no significant differences were observed in the signalling process in the proximal colon mucosa and LMMP. However in the distal colon, there was a significant increase in mucosal 5-HT levels (p<0.05, n=6) and the ratio of 5-HT:5-HTP (p<0.05, n=6) in the presence of propofol. During CMMC recordings, the amplitude of contractions in the presence of 1 μM propofol was unchanged
Data show least squares mean (LSM) ± SEM: *p≤0.05; CI=confidence interval
AGA Abstracts
S-364
Sa2050
Sa2052
Corticotropin-Releasing Factor (CRF) Peptides Modulates Rat Colonic Neuronal Tau Phosphorylation: Differential Role of CRF Receptors Hung Pham, Shuping S. Wu, Perozi Eslami, Marni E. Harris-White, Mulugeta Million
Role of Stretch-Activated Ion Channels in Esophageal Inhibitory Motor Neurons Hui Dong, Yanfen Jiang, Ravinder K. Mittal
BACKGROUND: Corticotropin releasing factor receptor-1 (CRF1)-overexpression enhances experimental stress or CRF-induced tau (TAU) phosphorylation in the mouse brain (PNAS, 2012: 109(16):6277-82). CRF is a key mediator of the gut response to stress. Whether CRF affect enteric TAU and whether enteric neuronal TAU modulation impacts gastrointestinal tract functions are not known. AIM: 1) Determine 1) the effect of selective activation of CRF receptors on rat colon primary myneteric neuron Tau 2) the colonic motor response of Tau transgenic mice to acute stress. METHODS: Rat distal colonic primary myenteric neurons were incubated with non-selective CRF1 and 2 receptor agonists (CRF, urocortin 1 (Ucn1)) and selective CRF2 receptor agonist (Ucn2) for 30 min (10 or 100 nM) and pTAU probed by western blot. Rat colon primary myenteric neurons were processed for immunostaining of Tau. Human TauP301L overexpressing and wild-type (WT) mice were exposed to 1-hour novel environment stress and cumulative Fecal Pellet Output (FPO) at 5, 15, 30, 45 and 60 min was monitored. The overexpression of human Tau in the TauOE mice was confirmed by western blotting. RESULTS: Rat colon primary neurons have constitutively active pTau that is suppressed by selective CRF2 receptor activation but not by the non-selective CRF receptor ligand, CRF. Rat colon primary myenteric neurons have abundant Tau. Compared to WT, Tau overexpressing mice have 2-4X increased total Tau mRNA expression, increased defecation (60.7±6.5% increase in 60 min cumulative FPO) response to mild novel environment stress and display altered ileal and colonic CRF2 receptor expression. CONCLUSION: The differential modulation of colonic primary myenteric TAU phophorylation by CRF peptides and the altered colonic motor response to stress in the mice that express phospho TAU in the intestine and the modulation of colonic CRF2 receptor mRNA profile in these mice, suggest a possible interplay between myenteric TAU and CRFsignaling in the colonic response to stress. Supported by R01DK078676 (MM); Veterans Administration Merit Review (MEH-W)
Background & Aims: Inhibitory motor neurons located in the esophageal wall play an important role in regulating function of the lower esophageal sphincter (LES). Our earlier whole animal studies revealed that mechanical stretch of the esophagus in the axial direction induces neurally mediated LES relaxation, but the mechanisms underlying mechanosensitive process is currently unknown. The aims of our study were to investigate, 2) if esophageal inhibitory motor neurons are mechanosensitive and 2) if stretch-activated ion channels (SACs) play a role in this mechanosensitivy. Methods: Esophageal myenteric neurons were freshly isolated from the esophagus of postnatal rats (5 days) and primary cultured for two weeks, immmunocytochemical characteristics of these neurons was conducted using NOS staining. Neurons in the culture were stretched by hypotonic solution (170 mOsm) to induce cell swelling and stretch. Three parameters were monitored with fluorescence dyes, fura 2AM and DAF-FM in single neurons using a digital cell imaging system: 1) cytoplasmic volume of neurons, 2) cytoplasmic free Ca2+ concentrations ([Ca2+]cyt) in the neurons, a cell signaling essential for neurotransmitter release, and 3) intracellular nitric oxide (NO), an important neurotransmitter in the inhibitory motor neurons. Results: Iimmmunocytochemical analysis revealed that >95% cells were positive for neuronal marker PGP 9.5. Sixtysix percent of the PGP 9.5 positive neurons co-stained with nNOS. Hypotonic solution induced changes in the cytoplasmic volume in all tested cells, which is independent of the presence or absence of extracellular Ca2+ Hypotonic solution induced [Ca2+]cyt signaling in ~ 65% of the neurons, in the presence but not the absence of extracellular Ca2+. Hypotonic stretch-induced [Ca2+]cyt signaling was not affected by extracellular Mg2+ and nifedipine, but was attenuated by potent blockers of SACs, Gd3+ and GsMTx4. Finally, hypotonic swelling induced NO production in about 57% of the neurons detected with DAF during cell stretch. Removal of extracellular Ca2+ markedly reduced hypotonic stretch-induced NO production in these single neurons. Conclusion: Our data suggest that esophageal inhibitory motor neurons are mechanosensitive, and that SACs on the neurons may play an important role in this mechanosensitivy through a novel Ca2+-NO signaling pathway.
Sa2051 A Phase II, Randomized, Double-Blind, Placebo-Controlled, Multiple-Dose, Parallel-Group Study to Evaluate the Efficacy, Safety, and Pharmacodynamics of RM-131 in Patients With Chronic Constipation Andres Acosta, Gururaj Kolar, Johanna Iturrino, Lawrence A. Szarka, Amy Boldingh, Duane D. Burton, Michael Ryks, Deborah L. Rhoten, Alan R. Zinsmeister, Michael Camilleri Background: Some patients do not achieve satisfactory relief of chronic constipation (CC). RM-131 is a pentapeptide, selective ghrelin receptor-1a agonist that increased gastric emptying (GE) in patients with diabetes mellitus. Aim: To evaluate effects of RM-131 on GE, small bowel transit (SBT) and colonic transit (CT) in patients with CC. Methods: As part of a trial of 48 patients assessing symptoms (NCT01781104), we conducted a randomized (1:1), double-blind, placebo (pbo)-controlled, parallel-group pharmacodynamics (PD) substudy in a single-center, evaluating effects of RM-131, 100μg s.c. once daily for 14 days, on GE, SBT [colonic filling at 6h (CF6)] and CT in patients with CC. Subjects (18-65y; BMI 19-40 kg/m2) were stratified by baseline GC24 value (≤1.6 vs. >1.6,<2.4). During 14-day baseline, placebo-treatment period, and the double-blind treatment periods, daily diaries of bowel habits, abdominal symptoms, global measures and baseline CT were collected. Main inclusion criteria were baseline GC24<2.4 and average spontaneous BMs ≤4/week. GI and CT of solids were measured during the last 48h of treatment using a standard, validated scintigraphic method (Deiteren et al NGM 22:415-423, 2010). The 24h image was obtained before the medication on treatment day 13. Safety was evaluated including adverse events, 12-lead electrocardiogram, and clinical laboratory assessments. The primary PD efficacy analysis (for CT and GE parameters) was by analysis of covariance (ANCOVA) models using baseline measurements (GC24 for CT data; BMI for CF6 and AC T½; and age for GE) as covariates. The PD substudy required 12 patients per treatment arm. Results: Baseline demographics and GC24 were similar in both treatment groups: the participants were all female, mean age was 40.5±10.3y; mean BMI was 24.8±2.9kg/m2. RM-131 was associated with a significant (table) acceleration of GE (GE T½ p=0.027), colonic filling at 6h [CF6% (surrogate for SBT), p=0.051], and CT at 32 and 48h (p=0.040 and p=0.017, respectively). There were no significant increases in CT at 24h (p=0.44) or ascending colon emptying (AC T½ p=0.43). There were no drop outs or withdrawals. Clinical endpoints will be analyzed upon completion of the 48th participant in the overall study. Conclusion: RM131, 100μg s.c. daily, significantly accelerates CT in patients with CC, in addition to accelerating GE and demonstrating stimulation of both upper and lower GI motility. The magnitude of effect on CT GC48 is associated with a 1 point difference in stool consistency on the Bristol Stool Form scale (Deiteren et al NGM 2010), suggesting that the PD effect is clinically relevant. Funding: Rhythm Pharmaceuticals
Figure. Mechanosensitivity of esophageal enteric neurons and the involvement of stretchactivated ion channels. Summary data showing the time courses of neuronal responses to Hypot in the presence of nifedipine (10 μM) or GsMTx4 (10 μM). After F340/380 baselines were stable in PSS, the first switch of extracellular solutions to Hypot as a control. Before the second or the third switches of extracellular solutions to Hypot, the neurons were pretreated with nifedipine or GsMTx4 for 5 min. Values are means ± SEM. n=55 cells from 6 rats. Sa2053 Understanding the Effects of Propofol on Serotonergic Signalling and Colonic Motility Lucy B. Diss, Kim R. Pearce, Bhavik A. Patel Introduction: Propofol (2,6 diisopropylphenol) is the most widely used intravenous anaesthetic agent for induction and maintenance of anaesthesia and sedation. Propofol is known to have an antagonistic effect on 5-hydroxytryptamine receptor (5-HT3R) but its actions on motility are not fully understood. We have investigated the effect of propofol on mucosal serotonergic signalling, colonic migrating motor complex (CMMC) and faecal pellet colonic motility. Methods: Colonic tissue samples were obtained from 10-12 weeks male C57BL/6 mice. For all studies tissues were treated with or without 1 μM propofol for 30 minutes. High-performance liquid chromatography with electrochemical detection (HPLC-ED) was utilised to detect levels of mucosal serotonin (5-HT), precursors and metabolites from proximal and distal colon mucosa and longitudinal muscle myenteric plexus (LMMP). CMMCs were monitored using isometric force transducers along the length of the colon. An artificial epoxy-coated fecal pellet was utilised to monitor colonic motility. The influence of 5-HT3R antagonist, ondansetron and nitric oxide synthase blocker, L-NNA were assessed on tissue exposed to propofol. Results: Following treatment in propofol, no significant differences were observed in the signalling process in the proximal colon mucosa and LMMP. However in the distal colon, there was a significant increase in mucosal 5-HT levels (p<0.05, n=6) and the ratio of 5-HT:5-HTP (p<0.05, n=6) in the presence of propofol. During CMMC recordings, the amplitude of contractions in the presence of 1 μM propofol was unchanged
Data show least squares mean (LSM) ± SEM: *p≤0.05; CI=confidence interval
AGA Abstracts
S-364