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μ-Opioid Receptors Co-Expressed in Cholinergic Neurons of Mouse Ileum Myenteric Plexus Develop Tolerance to Chronic Morphine Exposure
AGA Abstracts
included in this analysis. Studies must have mentioned regarding mortality and morbidity associated with POEM. Data collection & extraction: Articles were searched in Medline, Pubmed, Ovid journals, CINAH, International pharmaceutical abstracts, old Medline, Medline nonindexed citations, and Cochrane Central Register of Controlled Trials & Database of Systematic Reviews. Two reviewers independently searched and extracted data. Any differences were resolved by mutual agreement. Statistical Method: Pooled proportions were calculated using both Mantel-Haenszel method (fixed effects model) and DerSimonian Laird method (random effects model). The heterogeneity among studies was tested using I2 statistic. Results: Initial search identified 168 reference articles, of which 31 articles were selected and reviewed. Data was extracted from 9 studies (N=587) which met the inclusion criteria. Median age of the patients was 15 years, with 52% males. Median follow up period was 12 months. The p for chi-squared heterogeneity for all the pooled accuracy estimates was > 0.10. In the pooled patient population that underwent POEM, adverse events occurred in 18.29% (95% CI = 14.63 to 22.25). I2 heterogeneity calculated for the POEM related adverse events was 88.4%. In the pooled patient population that underwent POEM, postop GERD occurred in 21.68% (95% CI = 11.34 to 34.26). I2 heterogeneity calculated for the POEM related post-op GERD was 84.4%. Effect size of length of hospital stay in pooled patients that underwent POEM was 4.32 (95% CI = 3.91 to 4.72), I2 heterogeneity 94.7%. There were no POEM related deaths. Conclusions: In pediatric patients with achalasia, POEM was associated with minor adverse events. Length of hospital stay after in POEM patients seemed to be slightly more in pediatric patients compared to adult patients (around 2.5 days based on the current estimates).
RESULTS: RNA-seq of FACS TPH1-CFP EC cells from duodenum showed a robust expression of several potassium channel subunits (e.g. Kcnh2/6 [HERG1/2], Kcnc1 [KV3.1], Kcnq1 [delayed rectifier] and Kcnj2 [KATP]), but a single highly expressed voltage-gated sodium α-subunit, Scn3a, accounted for 97% of voltage-gated sodium channel expression (n=2 animals). IHC showed that in epithelium Scn3a-encoded NaV1.3 was specifically localized in TPH1-CFP (EC) cells in small bowel (89.4±2.0%, n=3 animals) and colon (88.4±4.4%, n=3 animals). Single cell PCR of primary cultures (24-72 hours) showed robust Scn3a expression in small bowel and colon EC cells. Voltage-clamp electrophysiology of primary EC cells from both small bowel and colon showed robust voltage-dependent sodium current (small bowel: 78% of cells, -86±16 pA/pF, n=28 and colon: 65% of cells, -66±12 pA/pF, n=14) carried mostly by NaV1.3, based on Na+ substitution and pharmacologic blockade with ICA-121431. Current clamp recordings showed that EC cells had discrete resting and plateau potentials of -70 mV and -45 mV, respectively, and EC cells discharged both elicited and spontaneous action potentials that were NaV1.3-dependent. The likelihood of an action potential showed a Gaussian distribution around a peak of -30 mV. Both spontaneous and elicited action potentials showed bursting at a frequency that matched NaV1.3 recovery from activation. CONCLUSION: The EC cells of mouse small and large bowel are electrically excitable and display complex spontaneous bursting activity. Scn3a-encoded NaV1.3 is necessary for EC cell electrical excitability.
Mo1539 µ-OPIOID RECEPTORS CO-EXPRESSED IN CHOLINERGIC NEURONS OF MOUSE ILEUM MYENTERIC PLEXUS DEVELOP TOLERANCE TO CHRONIC MORPHINE EXPOSURE Karan Muchhala, Aravind Gade, William L. Dewey, Hamid I. Akbarali
Mo1537 IMPACT OF ANTIBIOTIC-INDUCED MICROBIOTA DEPLETION ON SMALL BOWEL EXCITATORY AND INHIBITORY NEUROMUSCULAR PATHWAYS IN ADOLESCENT MICE Valentina Caputi, Ilaria Marsilio, Silvia Cerantola, Viviana Filpa, Isabella Lante, Patrizia Debetto, Genny Orso, Francesca Crema, Rocchina Colucci, Cristina Giaroni, Maria Cecilia Giron
Opioids, including morphine decrease gastrointestinal transit time. This is due to activation of µ -opioid receptors (MOR) expressed on enteric neurons, which leads to inhibition of enteric neuronal excitability. The AIMS of this study were to 1) determine the localization of MOR on enteric neuronal subpopulations, and 2) to examine the effect of acute and chronic morphine on neuronal excitability. METHODS: MOR expression was determined in whole-mount longitudinal muscle myenteric plexus and in isolated primary cultures of myenteric neurons from the ileum. Transgenic mice expressing tdTomato in choline acetyl transferase (ChAT)-expressing neurons were developed by crossing Gt(ROSA)26SORtm9(CAGtdTomato)Hze/ J with ChATtm2(cre)Lowl/J mice. VIP (vasoactive intestinal peptide) expression was similarly developed using VIPtm1(cre)Zhj/J mice. MOR and NOS expression were identified using specific antibodies in whole mount and in single neurons. Acute (3 µM) morphine effects on neuronal excitability were tested in distinct neuronal subtypes using whole-cell patch clamp electrophysiology in the current clamp mode. Ileum neurons were also cultured in 10 µM morphine for 16-48 hours to investigate the effect of prolonged morphine exposure on neuronal excitability. RESULTS: MORs co-expressed with ChAT (48/125; 38.4%) to a greater extent than VIP (9/92; 9.7%) or NOS1 (10/87; 11.4%) in myenteric ganglia of the ileum. The pattern of co-expression was similar in isolated neurons in primary culture with MOR co-expressing with ChAT neurons. In current-clamp studies, VIP neurons were significantly more excitable than ChAT neurons. The rheobase was 13.5 ± 3.5 pA in VIP positive neurons and 51.2 ± 9.4 pA in ChAT neurons. The input resistance in VIP neurons (1466 ± 177 MΩ) was significantly greater than ChAT neurons (905 ± 104 MΩ). Acute morphine perfusion (3 µM) shifted the threshold potential for the generation of the action potential from -20.3 ± 3.7 mV to -13.9 ± 5.3 mV. The rheobase also increased from 27.0 ± 5.0 pA to 44.0 ± 9.0 pA, while the height of the action potential spike at rheobase reduced from 89.6 ± 6.8 mV to 75.1 ± 7.2mV (n= 7). The increase in threshold potential and rheobase, and decrease in action potential spike height are indicative of reduced excitability. These changes were not accompanied by shifts in resting membrane potential or input resistance. When neurons were exposed to morphine for 16-48 hrs, an acute challenge of morphine did not induce shifts in threshold potential, rheobase or action potential spike height, indicating the development of tolerance in single neurons. CONCLUSION: These findings demonstrate that 1) MORs are primarily expressed in cholinergic neurons of ileum myenteric ganglia, and 2) acute morphine significantly reduces excitability of these neurons, but tolerance develops after prolonged in-vitro morphine exposure.
Background Gut microbiota appears as a key regulator in the development of bowel neuromotor activity by promoting several crucial functions. Early life perturbations of microbiota may impact gut maturation leading to functional disorders later in life. Aim The present study examines the role of gut microbiota depletion on small bowel excitatory and inhibitory neuromuscular pathways during mouse adolescence. Methods Depletion of gut microbiota was performed on male C57Bl/6 mice (4±1 weeks old) by orally administering an antibiotic cocktail (ABX) consisting of vancomycin (50 mg/kg), neomycin (100 mg/kg), metronidazol (100 mg/kg), ampicillin (100 mg/kg) every 12 hours for 14 days. Gastrointestinal (GI) transit was assessed in ABX and control (CNTR) mice 30 minutes after administration of a nonabsorbable fluorescent-labeled dextran probe by oral gavage. Isolated ileum segments were isometrically mounted along the longitudinal axis in organ baths. Changes in muscle tension were recorded following electric field stimulation (EFS, 0-40 or 10 Hz) in either the presence or absence of 1 µM atropine + 1 µM guanethidine (NANC conditions), 100 µM Nω-nitroL-arginine methyl ester (L-NAME, a pan-NOS inhibitor) and 10 µM L732138 (NK1 receptor antagonist). In ileal longitudinal muscle myenteric plexus preparations (LMMP), the distribution of glial (glial fibrillary acidic protein,GFAP and S100β) and neuronal (HuC/D) markers, nNOS and substance P (SP) was determined by confocal immunofluorescence. Acetylcholinesterase (AChE) biochemical assay and GluN1 protein levels analysis were performed in LMMP. Results In ABX mice, electrically-stimulated cholinergic contractions were significantly reduced (-60±7% at 10 Hz), whereas the excitatory NK1R-mediated tachykininergic response, measured in NANC conditions in presence of L-NAME, increased. Moreover, changes in NANC relaxant response were found and were partially abolished by L-NAME. GI transit significantly decreased in ABX mice (-52±3%). In the enteric nervous system (ENS) of ABX mice, S100β immunoreactivity significantly increased (+40±5%) and was associated with altered density of GFAP+ glial processes. The ENS neurochemical coding was significantly influenced by ABX treatment, as evidenced by the reduction of HuC/D+(29±2%) and nNOS+ (-35±4%) myenteric neurons and of AChE+ fibers (-34±6%) in LMMP. An increased number of SP+ neuronal cells (+31±5%) was associated with higher GluN1 protein level expression (-33±5%) in the myenteric plexus of ABX mice. Conclusion Our findings agree with the current cutting edge hypothesis of the existence of a symbiotic relationship between the enteric microbiota and the ENS. In this view, the commensal microbiota is proposed to play an outstanding role in the fine tuning of ENS maturation processes, consequently contributing to preserve gut homeostasis and promoting healthy adulthood.
Mo1540 TOLL-LIKE RECEPTOR 4 MODULATES NEUROMUSCULAR FUNCTION THROUGH NITRERGIC AND PURINERGIC PATHWAYS IN MOUSE SMALL INTESTINE Ilaria Marsilio, Valentina Caputi, Silvia Cerantola, Cristina Ravazzolo, Annalisa Brigato, Isabella Lante, Eleonora Napoli, Cecilia Giulivi, Genny Orso, Maria Cecilia Giron
Mo1538
Background Toll-like receptors (TLRs) play a pivotal role in the homoeostasis of the microbiota-host crosstalk. TLR4-mediated modulation of both motility and enteric neuronal survival has been reported for colon but limited information is available on the TLR4mediated control of small bowel motility by enteric nervous system (ENS). Aim This study aimed to assess the role of TLR4 signaling in controlling ENS homoeostasis and gut motor function. Methods Male TLR4 knockout (TLR4-/-, 9±1 weeks old) and sex- and age-matched wild-type (WT) C57BL/6J mice were used for the experiments. In distal ileum segments, mounted along the longitudinal axis in organ baths, the relaxation was evaluated as changes in muscle tension recorded by isometric transducers, following electric field stimulation (EFS, 10 Hz) in non-adrenergic non-cholinergic (NANC) conditions (1 µM atropine and 1 µM guanethidine) with and without i) 10 µM 1400W (inhibitor of inducible NOS, iNOS), ii) 0.1 mM Nω-nitro-L-arginine methyl ester (L-NAME, a pan-NOS inhibitor); iii) 0.1 mM theophylline (P1 receptor antagonist); iv) 0.1 mM suramin (P2 receptor antagonist). To evaluate the involvement of P2Y1 receptor in ileal relaxation, segments from WT and TLR4-/- mice were exposed to ADP (1 µM to 1 mM) added cumulatively to organ baths. Immunoreactivity of both glial fibrillary acidic protein (GFAP) and S100β was determined by confocal microscopy in ileal frozen sections from TLR4-/- and WT mice. The integrity of ENS neurochemical code was assessed by HuC/D, neuronal nitric oxide synthase (nNOS), iNOS, vasoactive intestinal peptide (VIP) and P2X7 receptor (P2X7R) immunohistochemistry in ileal longitudinal muscle-myenteric plexus (LMMP) whole mount preparations. Results
SCN3A-ENCODED VOLTAGE-GATED SODIUM CHANNEL NAV1.3 BESTOWS MOUSE ENTEROCHROMAFFIN CELLS WITH PATTERNS OF BURSTING ELECTRICAL ACTIVITY Peter R. Strege, Kaitlyn R. Knutson, Samuel Eggers, Fan Wang, Hui Joyce Li, Andrew B. Leiter, Gianrico Farrugia, Arthur Beyder INTRODUCTION: Endocrine and neuroendocrine cells, such as the beta-cell in the pancreas and chromaffin cell in the adrenal gland, are electrically excitable. Patterns of electrical excitability encode complex exocytosis phenotypes. In the gastrointestinal (GI) epithelium, the enterochromaffin (EC) cell is an enteroendocrine cell that uses tryptophan-hydroxylase 1 (TPH-1) to produce serotonin (5-hydroxytryptamine, 5-HT). The EC cell responds to chemical and mechanical stimuli by complex patterns of 5-HT release, which has important paracrine and endocrine functions. Therefore, the EC cell's electrophysiology may be important. However, it is unknown whether the EC cells are electrically excitable. AIM: To determine if the EC cells are electrically excitable. METHODS: Murine TPH1-CFP small bowel was dissociated and fluorescence activated cell sorted (FACS) for TPH1-CFP cells, and ion channel expression was analyzed by RNA-seq. TPH1-CFP small bowel and colon were also fixed in paraformaldehyde and examined by immunohistochemistry (IHC). TPH1CFP small bowel and colon were dissociated and kept in primary cultures up to 72 hours. CFP+ and CFP- cultured cells were analyzed by single cell RT-qPCR and electrophysiology.
AGA Abstracts
S-710
included in this analysis. Studies must have mentioned regarding mortality and morbidity associated with POEM. Data collection & extraction: Articles were searched in Medline, Pubmed, Ovid journals, CINAH, International pharmaceutical abstracts, old Medline, Medline nonindexed citations, and Cochrane Central Register of Controlled Trials & Database of Systematic Reviews. Two reviewers independently searched and extracted data. Any differences were resolved by mutual agreement. Statistical Method: Pooled proportions were calculated using both Mantel-Haenszel method (fixed effects model) and DerSimonian Laird method (random effects model). The heterogeneity among studies was tested using I2 statistic. Results: Initial search identified 168 reference articles, of which 31 articles were selected and reviewed. Data was extracted from 9 studies (N=587) which met the inclusion criteria. Median age of the patients was 15 years, with 52% males. Median follow up period was 12 months. The p for chi-squared heterogeneity for all the pooled accuracy estimates was > 0.10. In the pooled patient population that underwent POEM, adverse events occurred in 18.29% (95% CI = 14.63 to 22.25). I2 heterogeneity calculated for the POEM related adverse events was 88.4%. In the pooled patient population that underwent POEM, postop GERD occurred in 21.68% (95% CI = 11.34 to 34.26). I2 heterogeneity calculated for the POEM related post-op GERD was 84.4%. Effect size of length of hospital stay in pooled patients that underwent POEM was 4.32 (95% CI = 3.91 to 4.72), I2 heterogeneity 94.7%. There were no POEM related deaths. Conclusions: In pediatric patients with achalasia, POEM was associated with minor adverse events. Length of hospital stay after in POEM patients seemed to be slightly more in pediatric patients compared to adult patients (around 2.5 days based on the current estimates).
RESULTS: RNA-seq of FACS TPH1-CFP EC cells from duodenum showed a robust expression of several potassium channel subunits (e.g. Kcnh2/6 [HERG1/2], Kcnc1 [KV3.1], Kcnq1 [delayed rectifier] and Kcnj2 [KATP]), but a single highly expressed voltage-gated sodium α-subunit, Scn3a, accounted for 97% of voltage-gated sodium channel expression (n=2 animals). IHC showed that in epithelium Scn3a-encoded NaV1.3 was specifically localized in TPH1-CFP (EC) cells in small bowel (89.4±2.0%, n=3 animals) and colon (88.4±4.4%, n=3 animals). Single cell PCR of primary cultures (24-72 hours) showed robust Scn3a expression in small bowel and colon EC cells. Voltage-clamp electrophysiology of primary EC cells from both small bowel and colon showed robust voltage-dependent sodium current (small bowel: 78% of cells, -86±16 pA/pF, n=28 and colon: 65% of cells, -66±12 pA/pF, n=14) carried mostly by NaV1.3, based on Na+ substitution and pharmacologic blockade with ICA-121431. Current clamp recordings showed that EC cells had discrete resting and plateau potentials of -70 mV and -45 mV, respectively, and EC cells discharged both elicited and spontaneous action potentials that were NaV1.3-dependent. The likelihood of an action potential showed a Gaussian distribution around a peak of -30 mV. Both spontaneous and elicited action potentials showed bursting at a frequency that matched NaV1.3 recovery from activation. CONCLUSION: The EC cells of mouse small and large bowel are electrically excitable and display complex spontaneous bursting activity. Scn3a-encoded NaV1.3 is necessary for EC cell electrical excitability.
Mo1539 µ-OPIOID RECEPTORS CO-EXPRESSED IN CHOLINERGIC NEURONS OF MOUSE ILEUM MYENTERIC PLEXUS DEVELOP TOLERANCE TO CHRONIC MORPHINE EXPOSURE Karan Muchhala, Aravind Gade, William L. Dewey, Hamid I. Akbarali
Mo1537 IMPACT OF ANTIBIOTIC-INDUCED MICROBIOTA DEPLETION ON SMALL BOWEL EXCITATORY AND INHIBITORY NEUROMUSCULAR PATHWAYS IN ADOLESCENT MICE Valentina Caputi, Ilaria Marsilio, Silvia Cerantola, Viviana Filpa, Isabella Lante, Patrizia Debetto, Genny Orso, Francesca Crema, Rocchina Colucci, Cristina Giaroni, Maria Cecilia Giron
Opioids, including morphine decrease gastrointestinal transit time. This is due to activation of µ -opioid receptors (MOR) expressed on enteric neurons, which leads to inhibition of enteric neuronal excitability. The AIMS of this study were to 1) determine the localization of MOR on enteric neuronal subpopulations, and 2) to examine the effect of acute and chronic morphine on neuronal excitability. METHODS: MOR expression was determined in whole-mount longitudinal muscle myenteric plexus and in isolated primary cultures of myenteric neurons from the ileum. Transgenic mice expressing tdTomato in choline acetyl transferase (ChAT)-expressing neurons were developed by crossing Gt(ROSA)26SORtm9(CAGtdTomato)Hze/ J with ChATtm2(cre)Lowl/J mice. VIP (vasoactive intestinal peptide) expression was similarly developed using VIPtm1(cre)Zhj/J mice. MOR and NOS expression were identified using specific antibodies in whole mount and in single neurons. Acute (3 µM) morphine effects on neuronal excitability were tested in distinct neuronal subtypes using whole-cell patch clamp electrophysiology in the current clamp mode. Ileum neurons were also cultured in 10 µM morphine for 16-48 hours to investigate the effect of prolonged morphine exposure on neuronal excitability. RESULTS: MORs co-expressed with ChAT (48/125; 38.4%) to a greater extent than VIP (9/92; 9.7%) or NOS1 (10/87; 11.4%) in myenteric ganglia of the ileum. The pattern of co-expression was similar in isolated neurons in primary culture with MOR co-expressing with ChAT neurons. In current-clamp studies, VIP neurons were significantly more excitable than ChAT neurons. The rheobase was 13.5 ± 3.5 pA in VIP positive neurons and 51.2 ± 9.4 pA in ChAT neurons. The input resistance in VIP neurons (1466 ± 177 MΩ) was significantly greater than ChAT neurons (905 ± 104 MΩ). Acute morphine perfusion (3 µM) shifted the threshold potential for the generation of the action potential from -20.3 ± 3.7 mV to -13.9 ± 5.3 mV. The rheobase also increased from 27.0 ± 5.0 pA to 44.0 ± 9.0 pA, while the height of the action potential spike at rheobase reduced from 89.6 ± 6.8 mV to 75.1 ± 7.2mV (n= 7). The increase in threshold potential and rheobase, and decrease in action potential spike height are indicative of reduced excitability. These changes were not accompanied by shifts in resting membrane potential or input resistance. When neurons were exposed to morphine for 16-48 hrs, an acute challenge of morphine did not induce shifts in threshold potential, rheobase or action potential spike height, indicating the development of tolerance in single neurons. CONCLUSION: These findings demonstrate that 1) MORs are primarily expressed in cholinergic neurons of ileum myenteric ganglia, and 2) acute morphine significantly reduces excitability of these neurons, but tolerance develops after prolonged in-vitro morphine exposure.
Background Gut microbiota appears as a key regulator in the development of bowel neuromotor activity by promoting several crucial functions. Early life perturbations of microbiota may impact gut maturation leading to functional disorders later in life. Aim The present study examines the role of gut microbiota depletion on small bowel excitatory and inhibitory neuromuscular pathways during mouse adolescence. Methods Depletion of gut microbiota was performed on male C57Bl/6 mice (4±1 weeks old) by orally administering an antibiotic cocktail (ABX) consisting of vancomycin (50 mg/kg), neomycin (100 mg/kg), metronidazol (100 mg/kg), ampicillin (100 mg/kg) every 12 hours for 14 days. Gastrointestinal (GI) transit was assessed in ABX and control (CNTR) mice 30 minutes after administration of a nonabsorbable fluorescent-labeled dextran probe by oral gavage. Isolated ileum segments were isometrically mounted along the longitudinal axis in organ baths. Changes in muscle tension were recorded following electric field stimulation (EFS, 0-40 or 10 Hz) in either the presence or absence of 1 µM atropine + 1 µM guanethidine (NANC conditions), 100 µM Nω-nitroL-arginine methyl ester (L-NAME, a pan-NOS inhibitor) and 10 µM L732138 (NK1 receptor antagonist). In ileal longitudinal muscle myenteric plexus preparations (LMMP), the distribution of glial (glial fibrillary acidic protein,GFAP and S100β) and neuronal (HuC/D) markers, nNOS and substance P (SP) was determined by confocal immunofluorescence. Acetylcholinesterase (AChE) biochemical assay and GluN1 protein levels analysis were performed in LMMP. Results In ABX mice, electrically-stimulated cholinergic contractions were significantly reduced (-60±7% at 10 Hz), whereas the excitatory NK1R-mediated tachykininergic response, measured in NANC conditions in presence of L-NAME, increased. Moreover, changes in NANC relaxant response were found and were partially abolished by L-NAME. GI transit significantly decreased in ABX mice (-52±3%). In the enteric nervous system (ENS) of ABX mice, S100β immunoreactivity significantly increased (+40±5%) and was associated with altered density of GFAP+ glial processes. The ENS neurochemical coding was significantly influenced by ABX treatment, as evidenced by the reduction of HuC/D+(29±2%) and nNOS+ (-35±4%) myenteric neurons and of AChE+ fibers (-34±6%) in LMMP. An increased number of SP+ neuronal cells (+31±5%) was associated with higher GluN1 protein level expression (-33±5%) in the myenteric plexus of ABX mice. Conclusion Our findings agree with the current cutting edge hypothesis of the existence of a symbiotic relationship between the enteric microbiota and the ENS. In this view, the commensal microbiota is proposed to play an outstanding role in the fine tuning of ENS maturation processes, consequently contributing to preserve gut homeostasis and promoting healthy adulthood.
Mo1540 TOLL-LIKE RECEPTOR 4 MODULATES NEUROMUSCULAR FUNCTION THROUGH NITRERGIC AND PURINERGIC PATHWAYS IN MOUSE SMALL INTESTINE Ilaria Marsilio, Valentina Caputi, Silvia Cerantola, Cristina Ravazzolo, Annalisa Brigato, Isabella Lante, Eleonora Napoli, Cecilia Giulivi, Genny Orso, Maria Cecilia Giron
Mo1538
Background Toll-like receptors (TLRs) play a pivotal role in the homoeostasis of the microbiota-host crosstalk. TLR4-mediated modulation of both motility and enteric neuronal survival has been reported for colon but limited information is available on the TLR4mediated control of small bowel motility by enteric nervous system (ENS). Aim This study aimed to assess the role of TLR4 signaling in controlling ENS homoeostasis and gut motor function. Methods Male TLR4 knockout (TLR4-/-, 9±1 weeks old) and sex- and age-matched wild-type (WT) C57BL/6J mice were used for the experiments. In distal ileum segments, mounted along the longitudinal axis in organ baths, the relaxation was evaluated as changes in muscle tension recorded by isometric transducers, following electric field stimulation (EFS, 10 Hz) in non-adrenergic non-cholinergic (NANC) conditions (1 µM atropine and 1 µM guanethidine) with and without i) 10 µM 1400W (inhibitor of inducible NOS, iNOS), ii) 0.1 mM Nω-nitro-L-arginine methyl ester (L-NAME, a pan-NOS inhibitor); iii) 0.1 mM theophylline (P1 receptor antagonist); iv) 0.1 mM suramin (P2 receptor antagonist). To evaluate the involvement of P2Y1 receptor in ileal relaxation, segments from WT and TLR4-/- mice were exposed to ADP (1 µM to 1 mM) added cumulatively to organ baths. Immunoreactivity of both glial fibrillary acidic protein (GFAP) and S100β was determined by confocal microscopy in ileal frozen sections from TLR4-/- and WT mice. The integrity of ENS neurochemical code was assessed by HuC/D, neuronal nitric oxide synthase (nNOS), iNOS, vasoactive intestinal peptide (VIP) and P2X7 receptor (P2X7R) immunohistochemistry in ileal longitudinal muscle-myenteric plexus (LMMP) whole mount preparations. Results
SCN3A-ENCODED VOLTAGE-GATED SODIUM CHANNEL NAV1.3 BESTOWS MOUSE ENTEROCHROMAFFIN CELLS WITH PATTERNS OF BURSTING ELECTRICAL ACTIVITY Peter R. Strege, Kaitlyn R. Knutson, Samuel Eggers, Fan Wang, Hui Joyce Li, Andrew B. Leiter, Gianrico Farrugia, Arthur Beyder INTRODUCTION: Endocrine and neuroendocrine cells, such as the beta-cell in the pancreas and chromaffin cell in the adrenal gland, are electrically excitable. Patterns of electrical excitability encode complex exocytosis phenotypes. In the gastrointestinal (GI) epithelium, the enterochromaffin (EC) cell is an enteroendocrine cell that uses tryptophan-hydroxylase 1 (TPH-1) to produce serotonin (5-hydroxytryptamine, 5-HT). The EC cell responds to chemical and mechanical stimuli by complex patterns of 5-HT release, which has important paracrine and endocrine functions. Therefore, the EC cell's electrophysiology may be important. However, it is unknown whether the EC cells are electrically excitable. AIM: To determine if the EC cells are electrically excitable. METHODS: Murine TPH1-CFP small bowel was dissociated and fluorescence activated cell sorted (FACS) for TPH1-CFP cells, and ion channel expression was analyzed by RNA-seq. TPH1-CFP small bowel and colon were also fixed in paraformaldehyde and examined by immunohistochemistry (IHC). TPH1CFP small bowel and colon were dissociated and kept in primary cultures up to 72 hours. CFP+ and CFP- cultured cells were analyzed by single cell RT-qPCR and electrophysiology.
AGA Abstracts
S-710