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The inlfuence of loperamide on relaxation of the internal anal sphincter in the opossum
1064
ABSTRACTS
OF PAPERS
GASTROENTEROLOGY
THE INLFUENCEOF LOPERAMIDEON RELAKATION OF THE INTERNAL ANAL SPHINCTER IN THE OPOSSUM. S. Rattan and P.J. Culver, Jr. Harvard Digestive Diseases Center, Harvard-Thorndike Laboratory, Charles A. Dana Research Institute, GI Division, Beth Israel Hospital, Boston, MA 02215. Studies were performed on oc-chloralose anesthetized opossums subsequently treated with pancuronium. Loperamide was administered through a femoral artery catheter advanced to the level of the aortic bifurcation. Internal anal sphincter pressures (IASP) were monitored using a water perfused catheter assembly with an inflatable balloon at the proximal end, 6 cm from the recording port. Varying volumes (2 to 15 cc) of rectal balloon distention (RD) were used. The ventral root of the third sacral nerve was stimulated (SNS) with 5 mA, 0.5 ms pulses, for 2 sec. at 0.5 to 10 Hz. RD and SNS produced stimulus intensity-dependent relaxation of IASP. Loperamide (2.4 umolslkg) significantly (*) antagonized this relaxation (Table). RD
(cc) 2 5 10 15
Vol
X Fall in IASP Cont Loperamide l+-2* 18+6 44+6 3oi3* 68+5 48?6* 7723 61+10*
SNS (Hz) 0.5 2 5 10
X Fall Cont 1928 31t5 57t3 69A2
in IASP Loperamide o+o* 6t2* 1925* 2023*
Values represent mean t SE of 25 observations in five animals. The inhibitory action of loperamide was blocked by naloxone (4 mg/kg). The calcium channel blocker, verapamil (128 “g/kg), failed to modify the IASP relaxation in response to RD and SNS. These results suggest that loperamide may act on an opioid receptor which supresses the reflex relaxation of the sphincter by inhibition of inhibitory sacral efferents to the IAS.
CALCITONIN GENE-RELATED PEPTIDE (CGRP) LOCALIZATION IN THE ESOPHAGUSAND ITS EFFECT ON THE LOWER ESOPHAGEALSPHINCTER. S. Rattan, P. Gonella and R.K. Goyal. Harvard Digestive Diseases Center, Harvard-Thorndike Laboratory, Charles A. Dana Research Institute, GI Divison, Beth Israel Hospital, Boston, MA 02215. Manometric and immunocytochemical studies were done in opossums. Lower esophageal sphincter pressures were monitored in anesthetized animals with a specially designed continuously perfused catheter assembly which was placed in the lower esophageal sphincter. A branch of the left gastric artery to the sphincter was cannulated for intra -arterial administrations. CGRP caused a dose-dependent fall in the sphincter pressure with a calculated ED50 of 80 fmoles/kg. CGRP administered intravenously also caused a dose-dependent fall in the sphincter pressure with a ED of 50 nmoleslkg. The inhibitory effect of CGRP was no 5Oantagonized by atropine 30 “g/kg plus hexamethonium 20 mg/kg, propranolol 1 rag/kg, haloperidol 3 rag/kg, bulbocaponine 5 mg/kg, 5 methoxy N,N,dimethyltryptamine 200 “g/kg, pyrlamine 5 rug/kg plus metiamide 2 mg/kg. indomethacin 10 rag/kg or naloxone 4 mg/kg. These studies suggest that the inhibitory effect of CGRP was neither mediated by cholinergic, adrenergic, serotonergic, or histaminergic receptors, nor via prostaglandins or opioid receptors. Tetrodotoxin in doses which blocked the effect of vagal stimulation or esophageal distention on the lower esophageal sphincter only partially antagonized the sphincter relaxation caused by CGRP. Immunohistochemically CGRP was localized in the myenteric neurons and in many varicosities around the cells in the myenteric ganglia. CGRP has also been localized in the afferent fibers and dorsal motor nucleus of the vagus. In conclusion, it is possible that CGRP may act at several different sites in the neural pathways to the lower esophageal sphincter including synaptic transmission between pre- and postganglionic inhibitory neurons as well as at the inhibitory neuromuscular transmission.
Vol. 91, No.
4
FREE INTRACELLULARCALCIUM ([Ca2] ) IN UNSTIMULATEDAND STIMULATEDLOWER ESOPHAGEALSPHINETER (TONIC) AND ESOPHAGEALBODY (PHASIC) SMOOTHMUSCLES. S. Rattan, and R.K Goyal. Harvard Digestive Diseases Center, Harvard-Thorndike Laboratory, Charles A. Dana Research Institute, GI Division Beth Israel Hospital and Harvard Medica$+School, Boston, MA 02215. Ii is thought to play an important role in tonic ICa and phasic contractions of smooth muscles. It has been suggqsted that myosin light chain phosphorylation and [Ca Ii increases during shortening but may fall to near resting levg)s during contraction maintenance. Information on [Ca Ii levels in unstimulated tonic and phasic muscles is not available. We used aequorin b&luminiscence to estimate the free intracellular [Ca ] . Thin muscle strips taken from manometrically ide&ified lower esophageal sphincter (tonic) and esophageal body (phasic) 5 cm above. The strips were loaded with aequorin by transiently increasing the permeability of the smooth muscle cells. The aequorin biluminiscence and the tension signals were recorded simultaneously by using a light-tight apparatus. In the unstimulated the calcula$sd free intracellular in the sphincfer (assumiqg [Mg Ii-1.2) ranged from,4.8 to 7.1~12~ M (mean- 7x10 M) and from 2.7 to 6.3x10 (mean- 5x10 M) in the esophageal body (p 2f.05). Bethanecpl (10%) causgd an increase in the [Ca Ii fr9m 5.3x10 M-&o 9.5 x10 M in the body, and from 5.0x19+ M to 1.9x10 M in the sphincter. The rise in [Ca ]i in the esophageal body was transient and it correlated with phasi5+contraction of the esophageal muscle. The rise in [Ca ] in the sphincter was more prolonged and correlated wi i h prolonged contractile responses of the sphincter. These studies suggest that: 1) the tonically contracted sphincter muscle has higher free intracellular calcium than esophageal body muscle; and 2) bethanechol induced contraction in the esophageal body @haqp) muscle is associated with transient rise in [Ca ] and in the sphincter (tonic) muscle it is associated wf th more protracted rise in intracellular calcium.
ASSESSMENT OF THE COUPLING OF COLONIC SLOW WAVES BY FFT ANALYSIS TECHNIQUES. S.N. Reddy, Dept. of Neurosciences, flctiaster University, Hamilton, ON. Canada LEN 325. Assessment of coupling between two adjacent gastrointestinal (GI) slow waves or electric control activities (ECAS) is important in understanding the GI physiology and motility in health and disease. In the proximal gut, coupling characteristics can be evaluated by visual or period analysis techniques. In the colon, where poor coupling may often lead to random ECA, advanced spectral (FFT) analysis techniques are needed. This study explores unsmoothed cross spectrum (CS), smoothed CS. and coherence function (which is a measure, between sero and unity, of linearity between two signals) to study coupling of distal colonic ECAs recorded from two sites, 3cm apart, by implanted serosal electrodes in three chronic dogs. The results were corroborated on simulated signals. Unsmoothed CS results often provided spurious peaks. Plotting smoothed CS with its highest peak as unity, resulted in a false impression of common frequencies occurring between the two ECAs. This ambiguity was minimized when CS was evaluated in conjunction with the corresponding power spectra. However, because of leakage problems associated with digital processing, there were often CS peaks with no corresponding peaks in the power spectra. Coherence could not be evaluated from unsmoothed spectra but only from smoothed spectra. Uhen peaks in smoothed power spectra and CS corresponded with each other, coherence values were very high; otherwise they were small when there was no correspondence. The common frequency peaks occurred at either or both 7 and 15 c/m (Hz x 60). In conclusion, in the colon: 1) unsmoothed cross spectral analysis is an inappropriate method to evaluate common oscillations between two adjacent ECAs; 2) evaluation of CS must be in conjunction with corresponding power spectra or preferably the coherence function; and, 3) the coherence function provides a good measure of coupling between adjacent oecillators in the colon and may, therefore, serve as a powerful tool for the evaluation of the organization of ECA in the colon. Supported by MRC of Canada.
ABSTRACTS
OF PAPERS
GASTROENTEROLOGY
THE INLFUENCEOF LOPERAMIDEON RELAKATION OF THE INTERNAL ANAL SPHINCTER IN THE OPOSSUM. S. Rattan and P.J. Culver, Jr. Harvard Digestive Diseases Center, Harvard-Thorndike Laboratory, Charles A. Dana Research Institute, GI Division, Beth Israel Hospital, Boston, MA 02215. Studies were performed on oc-chloralose anesthetized opossums subsequently treated with pancuronium. Loperamide was administered through a femoral artery catheter advanced to the level of the aortic bifurcation. Internal anal sphincter pressures (IASP) were monitored using a water perfused catheter assembly with an inflatable balloon at the proximal end, 6 cm from the recording port. Varying volumes (2 to 15 cc) of rectal balloon distention (RD) were used. The ventral root of the third sacral nerve was stimulated (SNS) with 5 mA, 0.5 ms pulses, for 2 sec. at 0.5 to 10 Hz. RD and SNS produced stimulus intensity-dependent relaxation of IASP. Loperamide (2.4 umolslkg) significantly (*) antagonized this relaxation (Table). RD
(cc) 2 5 10 15
Vol
X Fall in IASP Cont Loperamide l+-2* 18+6 44+6 3oi3* 68+5 48?6* 7723 61+10*
SNS (Hz) 0.5 2 5 10
X Fall Cont 1928 31t5 57t3 69A2
in IASP Loperamide o+o* 6t2* 1925* 2023*
Values represent mean t SE of 25 observations in five animals. The inhibitory action of loperamide was blocked by naloxone (4 mg/kg). The calcium channel blocker, verapamil (128 “g/kg), failed to modify the IASP relaxation in response to RD and SNS. These results suggest that loperamide may act on an opioid receptor which supresses the reflex relaxation of the sphincter by inhibition of inhibitory sacral efferents to the IAS.
CALCITONIN GENE-RELATED PEPTIDE (CGRP) LOCALIZATION IN THE ESOPHAGUSAND ITS EFFECT ON THE LOWER ESOPHAGEALSPHINCTER. S. Rattan, P. Gonella and R.K. Goyal. Harvard Digestive Diseases Center, Harvard-Thorndike Laboratory, Charles A. Dana Research Institute, GI Divison, Beth Israel Hospital, Boston, MA 02215. Manometric and immunocytochemical studies were done in opossums. Lower esophageal sphincter pressures were monitored in anesthetized animals with a specially designed continuously perfused catheter assembly which was placed in the lower esophageal sphincter. A branch of the left gastric artery to the sphincter was cannulated for intra -arterial administrations. CGRP caused a dose-dependent fall in the sphincter pressure with a calculated ED50 of 80 fmoles/kg. CGRP administered intravenously also caused a dose-dependent fall in the sphincter pressure with a ED of 50 nmoleslkg. The inhibitory effect of CGRP was no 5Oantagonized by atropine 30 “g/kg plus hexamethonium 20 mg/kg, propranolol 1 rag/kg, haloperidol 3 rag/kg, bulbocaponine 5 mg/kg, 5 methoxy N,N,dimethyltryptamine 200 “g/kg, pyrlamine 5 rug/kg plus metiamide 2 mg/kg. indomethacin 10 rag/kg or naloxone 4 mg/kg. These studies suggest that the inhibitory effect of CGRP was neither mediated by cholinergic, adrenergic, serotonergic, or histaminergic receptors, nor via prostaglandins or opioid receptors. Tetrodotoxin in doses which blocked the effect of vagal stimulation or esophageal distention on the lower esophageal sphincter only partially antagonized the sphincter relaxation caused by CGRP. Immunohistochemically CGRP was localized in the myenteric neurons and in many varicosities around the cells in the myenteric ganglia. CGRP has also been localized in the afferent fibers and dorsal motor nucleus of the vagus. In conclusion, it is possible that CGRP may act at several different sites in the neural pathways to the lower esophageal sphincter including synaptic transmission between pre- and postganglionic inhibitory neurons as well as at the inhibitory neuromuscular transmission.
Vol. 91, No.
4
FREE INTRACELLULARCALCIUM ([Ca2] ) IN UNSTIMULATEDAND STIMULATEDLOWER ESOPHAGEALSPHINETER (TONIC) AND ESOPHAGEALBODY (PHASIC) SMOOTHMUSCLES. S. Rattan, and R.K Goyal. Harvard Digestive Diseases Center, Harvard-Thorndike Laboratory, Charles A. Dana Research Institute, GI Division Beth Israel Hospital and Harvard Medica$+School, Boston, MA 02215. Ii is thought to play an important role in tonic ICa and phasic contractions of smooth muscles. It has been suggqsted that myosin light chain phosphorylation and [Ca Ii increases during shortening but may fall to near resting levg)s during contraction maintenance. Information on [Ca Ii levels in unstimulated tonic and phasic muscles is not available. We used aequorin b&luminiscence to estimate the free intracellular [Ca ] . Thin muscle strips taken from manometrically ide&ified lower esophageal sphincter (tonic) and esophageal body (phasic) 5 cm above. The strips were loaded with aequorin by transiently increasing the permeability of the smooth muscle cells. The aequorin biluminiscence and the tension signals were recorded simultaneously by using a light-tight apparatus. In the unstimulated the calcula$sd free intracellular in the sphincfer (assumiqg [Mg Ii-1.2) ranged from,4.8 to 7.1~12~ M (mean- 7x10 M) and from 2.7 to 6.3x10 (mean- 5x10 M) in the esophageal body (p 2f.05). Bethanecpl (10%) causgd an increase in the [Ca Ii fr9m 5.3x10 M-&o 9.5 x10 M in the body, and from 5.0x19+ M to 1.9x10 M in the sphincter. The rise in [Ca ]i in the esophageal body was transient and it correlated with phasi5+contraction of the esophageal muscle. The rise in [Ca ] in the sphincter was more prolonged and correlated wi i h prolonged contractile responses of the sphincter. These studies suggest that: 1) the tonically contracted sphincter muscle has higher free intracellular calcium than esophageal body muscle; and 2) bethanechol induced contraction in the esophageal body @haqp) muscle is associated with transient rise in [Ca ] and in the sphincter (tonic) muscle it is associated wf th more protracted rise in intracellular calcium.
ASSESSMENT OF THE COUPLING OF COLONIC SLOW WAVES BY FFT ANALYSIS TECHNIQUES. S.N. Reddy, Dept. of Neurosciences, flctiaster University, Hamilton, ON. Canada LEN 325. Assessment of coupling between two adjacent gastrointestinal (GI) slow waves or electric control activities (ECAS) is important in understanding the GI physiology and motility in health and disease. In the proximal gut, coupling characteristics can be evaluated by visual or period analysis techniques. In the colon, where poor coupling may often lead to random ECA, advanced spectral (FFT) analysis techniques are needed. This study explores unsmoothed cross spectrum (CS), smoothed CS. and coherence function (which is a measure, between sero and unity, of linearity between two signals) to study coupling of distal colonic ECAs recorded from two sites, 3cm apart, by implanted serosal electrodes in three chronic dogs. The results were corroborated on simulated signals. Unsmoothed CS results often provided spurious peaks. Plotting smoothed CS with its highest peak as unity, resulted in a false impression of common frequencies occurring between the two ECAs. This ambiguity was minimized when CS was evaluated in conjunction with the corresponding power spectra. However, because of leakage problems associated with digital processing, there were often CS peaks with no corresponding peaks in the power spectra. Coherence could not be evaluated from unsmoothed spectra but only from smoothed spectra. Uhen peaks in smoothed power spectra and CS corresponded with each other, coherence values were very high; otherwise they were small when there was no correspondence. The common frequency peaks occurred at either or both 7 and 15 c/m (Hz x 60). In conclusion, in the colon: 1) unsmoothed cross spectral analysis is an inappropriate method to evaluate common oscillations between two adjacent ECAs; 2) evaluation of CS must be in conjunction with corresponding power spectra or preferably the coherence function; and, 3) the coherence function provides a good measure of coupling between adjacent oecillators in the colon and may, therefore, serve as a powerful tool for the evaluation of the organization of ECA in the colon. Supported by MRC of Canada.