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Localization of Ca2+ channel subtypes in the enteric nervous system and pancreas
April 1998
Motility and Nerve-Gut Interactions A793
postprandial hour and the percentage of gastric retention at 2 hours (r=0.647, P=0.009) but not for the first postprandial hour. Conclusions: 1) The postprandial EGG power change during the second postprandial hour is highly correlated with the percentage of gastric retention at 2 hours. 2) Recording postprandial EGG after a solid meal for 2 hours maximizes the correlation between EGG parameters and gastric emptying and adopting this methodology may begin to make recording techniques more uniform and hence facilitate the clinical application of the EGG. • G3262 INSULIN PER SE MODIFIES GASTROINTESTINAL MOTOR REFLEXES. Th. Lin~,enfelser. R. Kadow, W. M. Sun, T. Hinrichs, J. Dent, V. Gross. Department of Gastroenterology, Klinikum Amberg, Germany, and Gastrointestinal Medicine, Royal Adelaide Hospital, South Australia. In healthy subjects physiological hyperglycaemia (~10 raM) modifies gastrointestinal motor function. Subsequent hyperinsulinaemia due to hyperglycaemia may mediate this phenomenon. We investigated the effect of physiological hyperinsulinaemia on antropyloroduodenal motility in 10 healthy volunteers. Blood samples were taken during normoinsulinaemia (50 pM) and mild hyperinsulinaemia (300 pM) in randomized order and cross-over fashion, i.e. hyperinsulinaemia-normoinsulinaemia (H/N) vs. normoinsulinaemia-hyperinsulinaemia (N/H). Antropyloroduodenal motor responses were evaluated after repetitive proximal duodenal distension with a non-compliant ultrathin bag during phase I activity. Intrabag volumes increased by 4 mL at each distension step (from 12 to 48 mL) of 2.5 rain duration (10 min intervals). A manometric sleeve-sidehole assembly recorded number and amplitude of antral, pyloric and duodenal contractions, tonic and phasic activity of isolated pyloric pressure waves (IPPW), and intraduodenal pressure. Proximal duodenal distension induced IPPW and duodenal pressure waves, and raised basal pyloric and intraduodenal pressure. In both studies, hyperinsulinaemia increased these motor responses at each distension step (e.g. at 48 mL): number of IPPW (1.9-+0.6/2.0+0.8, N/H vs. 2.5 -+0.8/2.0 -+ 0.6, H/N, p<0.05), amplitude of IPPW (32.5 _+0.8/77.0 _+6.9, N/H vs. 58.5_+1.1/38.8_+2.5, H/N, p<0.05), basal pyloric pressure (7.3 _+1.1/9.0 _+1.3, N/H vs. 8.2 _+1.7/7.1 + 1.2, H/N, p<0.05), and intraduodenal pressure (8.3-+ 1.1/12.6_+ 1.5, N/H vs. 12.9 _+ 1.2/11.2_+ 1.3, H/N, p<0.05). Insulin per se has a major impact on gastrointestinal motor reflexes after duodenal distension in healthy subjects. G3263 ANAL ENDOSONOGRAPHY IN THE EVALUATION OF FECAL INCONTINENCE. S.D. Lippe, J. Tripodi, D. Rex, R. Burakoff, F. Gress Division of Gastroenterology, Winthrop-University Hospital, SUNY Stony Brook School of Medicine and Indiana University Medical Center. Introduction: Fecal Incontinence (FI) can be related to anatomic problems,
such as trauma or childbirth, neumlogic disorders like diabetes, or can be idiopathic. Aim: The purpose of this study was to investigate the role of anal endosonography (AE) in evaluating patients with FI. Method: A total of 21 patients presenting for evaluation of FI underwent rectal manometric studies. Parameters included resting pressures (RP) and squeeze pressures (SP) using the stationary pull-through technique, as well as sensation. These patients then underwent AE with the examiner blinded to the manometric results. AE was performed using a 7 MHz rotating transducer (Bruel and Kjaer, Denmark) to evaluate the integrity of the internal anal sphincter (IAS) and external anal sphincter (EAS). Results: In 11 patients with low IAS resting pressure, AE identified 8 [specificity 73%]. In those 10 patients with a normal IAS resting pressure, AE did not discern any defects. In the 13 patients with an abnormal EAS squeeze pressure, AE found defects in 10 patients [specificity 77%]. In the eight patients with a normal squeeze pressure, AE found no defects in seven cases. pressure (mmHg) RP < 40 RP ->40
#pts IAS defect present absent 8 3 2 8
pressure nl > 2x RP abnormal normal
# pts EAS defect present absent 10 3 1 7
Conclusions: AE is a safe and useful modality in the evaluation of patients
with the complaint of FI. AE can provide the specific diagnosis in cases where an anatomic problem exists (ie. sphincter defect from trauma). In patients with FI and sphincter defects, AE was helpful since it identified those individuals who were more likely to respond to biofeedback techniques. If AE fails to elicit an abnormality and manometry is abnormal, one must consider other causes such as neurological disease or idiopathic disorders. This would suggest that other studies such as EMG be undertaken. The data from AE and manometric studies are complementary in the evaluation of FI. Further studies using controls will be undertaken to evaluate sensitivity of AE in detecting rectal motility disorders.
• G3264 NITRIC OXIDE INHIBITS ACETYLCHOLINE RELEASE AT T H E NEUROMUSCULAR JUNCTIONS OF MOUSE CRURAL DIAPHRAGM. J. Liu, E. H. Middlekauff, H. C. Lee, Y. I. Kim, R. K. Mittai. University of Virginia School of Medicine, Charlottesville, Virginia; C. Singaram, University of Wisconsin, Madison, Wisconsin. Background: Mechanism of selective inhibition of the crurai diaphragm
(CD) during transient lower esophageal sphincter relaxation (TLESR) is not known. Such inhibition of the CD induced by esophageal distension is thought to occur at the level of the brain stem. However, we recently observed a peripheral mechanism of CD inhibition in cats. Aims: To determine if nitric oxide, a neurotransmitter responsible for relaxation of the smooth muscle, can induce relaxation of the skeletal msuc!e of the crural diaphragm. We studied the effects of nitric oxide on the parameters of neuromuscular transmission in the mouse CD in vitro. We also examined if NADPH diaphorase was present in the mouse CD using immunohistochemistry. Methods: Neuromuscular junctions (NMJs) in phrenic nerve-crural diaphragm muscle preparations were studied in 33 female Swiss-Webster mice. Pre- and postjunctional effects of NO were assessed by the standard intracelhiler microelectrode recording technique. Spontaneouse miniature end-plate potentials (MEPPs), nerveevoked end-plate potentials (EPPs) and EPP quantal content (# of ACh quata released per nerve impulse) were measured from 10-15 NMJs of each mouse in the control solution and in the presence of a nitric oxide donor [Spermine NONOate (SNt)]. Immunohistochemistry was performed using a standard technique. Data are shown as mean + SEM. P < 0.05 was considered significant. Results: NADPH diaphorase activity was seen in the neuromuscular junction of the CD. SNt decreased the EPP amplitude and quantal content in response to phrenic nerve stimulus at all the doses tested. This effect ensued without corresponding alteration in MEPP amplitude, indicating that NO acts presynapticaily. % Reduction of EPP quantal content in response to nitric oxide donor SNt Concentration I 100~tM [ 3 0 0 ~ I , ~ I~0~ I %quantalcontentreduction 30.5±14.3" 28.7±7.6* 42.7±5.7* 54.5±7.3* * p < 0.05 by Student's t-test compared to the control. [
Conclusions: Nitric oxide system is present in the skeletal muscle of the crural diaphragm. Exogenous nitric oxide inhibits neuromuscular transmission presynaptically by suppressing the evoked release of ACh at the NMJs of crural diaphragm. This observation indicates that nitric oxide may play a role in the function of crural diaphragm muscle at the level of neuromuscular junction. This study was supported by an NIH grant.
• G3265 LOCALIZATION OF Ca 2÷ CHANNEL SUBTYPES IN THE ENTERIC NERVOUS SYSTEM AND PANCREAS. M.-T. Liu A. Kirchgessner, Dept. of Anatomy and Cell Biology, Columbia University, New York, NY. Voltage-gated Ca2+ channels control many aspects of cellular function in the gut and pancreas, including the regulation of neurotransmitter release; however, the distribution of these channels in either organ has not previously been determined. Anti-peptide antibodies specific for the class A (P/Q-type), class B (N-type), and class C (L-type) Ca2+ channel ctI subunits (Alomone Labs, Israel) were utilized to examine the distribution of these channels in the gut and pancreas of the rat and guinea pig. Ca2+ channels were localized by immunofluorescence in conjunction with confocal microscopy. cqB immunoreactivity was abundant in all regions of the bowel and pancreas. Immunoreactivity was punctate and appeared to be predominantly associated with nerve terminals. ¢qB-immunoreactive nerve processes were found in ganglia, islets, muscle, the lamina propria, among the acini, and surrounding blood vessels. In the guinea pig, punctate clusters of crib immunoreactivity appeared to be closely associated with ChAT-immunoreactive varicosities. In the rat, am immunoreactivity was detected in neurites that displayed vesicular acetylcholine transporter (VAChT) and calretinin immunoreactivities. A subset of calbindin/ChAT-immunoreactive neurons of the guinea pig myenteric plexus, and SP/ShAT- and VIP-immunoreactive submucosal neurons, displayed cytoplasmic ctm immunoreactivity, ct m immunoreactivity was also found in a subset of islet cells. In the gut and pancreas, large numbers of nerve fibers (in ganglia, muscle, and mucosa) and neuronal cell bodies were also cqc-immunoreactive. Immunoreactivity was largely confined to nerve cell membrane. Most of the Cqc-immunoreactive cell bodies were also VAChT-positive. In addition, Cqc-immunoreactivity was observed i n a subset of 5-HT-immunoreactive enterochromaffin cells. Punctate cqA immunoreactivity was also observed in the bowel; however, it was restricted to a small number of cell bodies and fibers. Our results demonstrate that N-, L-, and P/Q-type Ca2+ channels are differentially localized on neural structures in the gut and pancreas and are consistent with the idea that each channel Subtype serves a distinct role in neuronal signal transduction. Supported by NIH grants NS27645 and NS15982.
Motility and Nerve-Gut Interactions A793
postprandial hour and the percentage of gastric retention at 2 hours (r=0.647, P=0.009) but not for the first postprandial hour. Conclusions: 1) The postprandial EGG power change during the second postprandial hour is highly correlated with the percentage of gastric retention at 2 hours. 2) Recording postprandial EGG after a solid meal for 2 hours maximizes the correlation between EGG parameters and gastric emptying and adopting this methodology may begin to make recording techniques more uniform and hence facilitate the clinical application of the EGG. • G3262 INSULIN PER SE MODIFIES GASTROINTESTINAL MOTOR REFLEXES. Th. Lin~,enfelser. R. Kadow, W. M. Sun, T. Hinrichs, J. Dent, V. Gross. Department of Gastroenterology, Klinikum Amberg, Germany, and Gastrointestinal Medicine, Royal Adelaide Hospital, South Australia. In healthy subjects physiological hyperglycaemia (~10 raM) modifies gastrointestinal motor function. Subsequent hyperinsulinaemia due to hyperglycaemia may mediate this phenomenon. We investigated the effect of physiological hyperinsulinaemia on antropyloroduodenal motility in 10 healthy volunteers. Blood samples were taken during normoinsulinaemia (50 pM) and mild hyperinsulinaemia (300 pM) in randomized order and cross-over fashion, i.e. hyperinsulinaemia-normoinsulinaemia (H/N) vs. normoinsulinaemia-hyperinsulinaemia (N/H). Antropyloroduodenal motor responses were evaluated after repetitive proximal duodenal distension with a non-compliant ultrathin bag during phase I activity. Intrabag volumes increased by 4 mL at each distension step (from 12 to 48 mL) of 2.5 rain duration (10 min intervals). A manometric sleeve-sidehole assembly recorded number and amplitude of antral, pyloric and duodenal contractions, tonic and phasic activity of isolated pyloric pressure waves (IPPW), and intraduodenal pressure. Proximal duodenal distension induced IPPW and duodenal pressure waves, and raised basal pyloric and intraduodenal pressure. In both studies, hyperinsulinaemia increased these motor responses at each distension step (e.g. at 48 mL): number of IPPW (1.9-+0.6/2.0+0.8, N/H vs. 2.5 -+0.8/2.0 -+ 0.6, H/N, p<0.05), amplitude of IPPW (32.5 _+0.8/77.0 _+6.9, N/H vs. 58.5_+1.1/38.8_+2.5, H/N, p<0.05), basal pyloric pressure (7.3 _+1.1/9.0 _+1.3, N/H vs. 8.2 _+1.7/7.1 + 1.2, H/N, p<0.05), and intraduodenal pressure (8.3-+ 1.1/12.6_+ 1.5, N/H vs. 12.9 _+ 1.2/11.2_+ 1.3, H/N, p<0.05). Insulin per se has a major impact on gastrointestinal motor reflexes after duodenal distension in healthy subjects. G3263 ANAL ENDOSONOGRAPHY IN THE EVALUATION OF FECAL INCONTINENCE. S.D. Lippe, J. Tripodi, D. Rex, R. Burakoff, F. Gress Division of Gastroenterology, Winthrop-University Hospital, SUNY Stony Brook School of Medicine and Indiana University Medical Center. Introduction: Fecal Incontinence (FI) can be related to anatomic problems,
such as trauma or childbirth, neumlogic disorders like diabetes, or can be idiopathic. Aim: The purpose of this study was to investigate the role of anal endosonography (AE) in evaluating patients with FI. Method: A total of 21 patients presenting for evaluation of FI underwent rectal manometric studies. Parameters included resting pressures (RP) and squeeze pressures (SP) using the stationary pull-through technique, as well as sensation. These patients then underwent AE with the examiner blinded to the manometric results. AE was performed using a 7 MHz rotating transducer (Bruel and Kjaer, Denmark) to evaluate the integrity of the internal anal sphincter (IAS) and external anal sphincter (EAS). Results: In 11 patients with low IAS resting pressure, AE identified 8 [specificity 73%]. In those 10 patients with a normal IAS resting pressure, AE did not discern any defects. In the 13 patients with an abnormal EAS squeeze pressure, AE found defects in 10 patients [specificity 77%]. In the eight patients with a normal squeeze pressure, AE found no defects in seven cases. pressure (mmHg) RP < 40 RP ->40
#pts IAS defect present absent 8 3 2 8
pressure nl > 2x RP abnormal normal
# pts EAS defect present absent 10 3 1 7
Conclusions: AE is a safe and useful modality in the evaluation of patients
with the complaint of FI. AE can provide the specific diagnosis in cases where an anatomic problem exists (ie. sphincter defect from trauma). In patients with FI and sphincter defects, AE was helpful since it identified those individuals who were more likely to respond to biofeedback techniques. If AE fails to elicit an abnormality and manometry is abnormal, one must consider other causes such as neurological disease or idiopathic disorders. This would suggest that other studies such as EMG be undertaken. The data from AE and manometric studies are complementary in the evaluation of FI. Further studies using controls will be undertaken to evaluate sensitivity of AE in detecting rectal motility disorders.
• G3264 NITRIC OXIDE INHIBITS ACETYLCHOLINE RELEASE AT T H E NEUROMUSCULAR JUNCTIONS OF MOUSE CRURAL DIAPHRAGM. J. Liu, E. H. Middlekauff, H. C. Lee, Y. I. Kim, R. K. Mittai. University of Virginia School of Medicine, Charlottesville, Virginia; C. Singaram, University of Wisconsin, Madison, Wisconsin. Background: Mechanism of selective inhibition of the crurai diaphragm
(CD) during transient lower esophageal sphincter relaxation (TLESR) is not known. Such inhibition of the CD induced by esophageal distension is thought to occur at the level of the brain stem. However, we recently observed a peripheral mechanism of CD inhibition in cats. Aims: To determine if nitric oxide, a neurotransmitter responsible for relaxation of the smooth muscle, can induce relaxation of the skeletal msuc!e of the crural diaphragm. We studied the effects of nitric oxide on the parameters of neuromuscular transmission in the mouse CD in vitro. We also examined if NADPH diaphorase was present in the mouse CD using immunohistochemistry. Methods: Neuromuscular junctions (NMJs) in phrenic nerve-crural diaphragm muscle preparations were studied in 33 female Swiss-Webster mice. Pre- and postjunctional effects of NO were assessed by the standard intracelhiler microelectrode recording technique. Spontaneouse miniature end-plate potentials (MEPPs), nerveevoked end-plate potentials (EPPs) and EPP quantal content (# of ACh quata released per nerve impulse) were measured from 10-15 NMJs of each mouse in the control solution and in the presence of a nitric oxide donor [Spermine NONOate (SNt)]. Immunohistochemistry was performed using a standard technique. Data are shown as mean + SEM. P < 0.05 was considered significant. Results: NADPH diaphorase activity was seen in the neuromuscular junction of the CD. SNt decreased the EPP amplitude and quantal content in response to phrenic nerve stimulus at all the doses tested. This effect ensued without corresponding alteration in MEPP amplitude, indicating that NO acts presynapticaily. % Reduction of EPP quantal content in response to nitric oxide donor SNt Concentration I 100~tM [ 3 0 0 ~ I , ~ I~0~ I %quantalcontentreduction 30.5±14.3" 28.7±7.6* 42.7±5.7* 54.5±7.3* * p < 0.05 by Student's t-test compared to the control. [
Conclusions: Nitric oxide system is present in the skeletal muscle of the crural diaphragm. Exogenous nitric oxide inhibits neuromuscular transmission presynaptically by suppressing the evoked release of ACh at the NMJs of crural diaphragm. This observation indicates that nitric oxide may play a role in the function of crural diaphragm muscle at the level of neuromuscular junction. This study was supported by an NIH grant.
• G3265 LOCALIZATION OF Ca 2÷ CHANNEL SUBTYPES IN THE ENTERIC NERVOUS SYSTEM AND PANCREAS. M.-T. Liu A. Kirchgessner, Dept. of Anatomy and Cell Biology, Columbia University, New York, NY. Voltage-gated Ca2+ channels control many aspects of cellular function in the gut and pancreas, including the regulation of neurotransmitter release; however, the distribution of these channels in either organ has not previously been determined. Anti-peptide antibodies specific for the class A (P/Q-type), class B (N-type), and class C (L-type) Ca2+ channel ctI subunits (Alomone Labs, Israel) were utilized to examine the distribution of these channels in the gut and pancreas of the rat and guinea pig. Ca2+ channels were localized by immunofluorescence in conjunction with confocal microscopy. cqB immunoreactivity was abundant in all regions of the bowel and pancreas. Immunoreactivity was punctate and appeared to be predominantly associated with nerve terminals. ¢qB-immunoreactive nerve processes were found in ganglia, islets, muscle, the lamina propria, among the acini, and surrounding blood vessels. In the guinea pig, punctate clusters of crib immunoreactivity appeared to be closely associated with ChAT-immunoreactive varicosities. In the rat, am immunoreactivity was detected in neurites that displayed vesicular acetylcholine transporter (VAChT) and calretinin immunoreactivities. A subset of calbindin/ChAT-immunoreactive neurons of the guinea pig myenteric plexus, and SP/ShAT- and VIP-immunoreactive submucosal neurons, displayed cytoplasmic ctm immunoreactivity, ct m immunoreactivity was also found in a subset of islet cells. In the gut and pancreas, large numbers of nerve fibers (in ganglia, muscle, and mucosa) and neuronal cell bodies were also cqc-immunoreactive. Immunoreactivity was largely confined to nerve cell membrane. Most of the Cqc-immunoreactive cell bodies were also VAChT-positive. In addition, Cqc-immunoreactivity was observed i n a subset of 5-HT-immunoreactive enterochromaffin cells. Punctate cqA immunoreactivity was also observed in the bowel; however, it was restricted to a small number of cell bodies and fibers. Our results demonstrate that N-, L-, and P/Q-type Ca2+ channels are differentially localized on neural structures in the gut and pancreas and are consistent with the idea that each channel Subtype serves a distinct role in neuronal signal transduction. Supported by NIH grants NS27645 and NS15982.