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Characteristics of rat colonic muscularis mucosae responses to inflammatory mediators in vitro
A668
AGA ABSTRACTS
GASTROENTEROLOGY,Vol. 108, No. 4
• CHARACTERISTICS OF RAT COLONIC MUSCULARIS MUCOSAE RESPONSES TO INFLAMMATORY MEDIATORS IN VITRO. W H P e r c v & LM Anderson, DePar[ment of Physiology & Pharmacology, Sch6ol of Medicine, University of South Da~ta, Vermillion, SD 57069. Rat colon is frequently used in studies of the pathophysiologic consequences of inflammation of the intestine. It is known that the motor effects of inflammatory mediators on the colonic muscularispropria of the rat are atypical (Percy et al., J. Gastro. Motil. 3: 229-23-6, -1991). The aim of this study was to assess their effects on the corresponding muscularis mucosae (MM). METHODS: 4-5cm segments of distaF, mid and proximal colon were removed from euthanized 350g SpragueDawley rats. Strips of MM 3era x 4mm were prepared for isometric recording usingthe technique described by Percy & Christensen (Am. J. Physiol. 250: G98-G102,1986) and mounted in 2ml tissue baths under a tension equivalent to 0.5g (4.gmN). All responses were expressed as a percentage of each tissue's maximum response to acetylcholine (Ach). RESULTS: Resting tone of MM from the proximal, mid and distal colon did not differ significantly (3.58_+0.27, 3.28_+0.26 and 2.71+0.18mN (n ~ 9:p > 0.05). All preparations gave atrol~ine 00"6M) sensitive, concentration-dependent contractions to Ach (10"J-10"~M)~ On colonic 3 MM bradykinin (BK)(2.3x10"13-2.3x10~6M) his~mi~e (10- -10" M), prostaglandi~s (PG=~)c,~2and F2a (2.8x10 "l~' 2.8x10 M) and substance P (3.3x10 -3.3x10 M)were potent agonists, whoseconcentration-response curves lay to the leff of that for acetylcholine, with maxima of 250-300% of .the largest response to Ach (n_>5 e a c h ) . In the6~n.xi_~ddcolon the concentration-response curve for BK (2.3x10~-2.3x10 M) lay to the left of that for Ach, with its largest responses being 355.4_+71.5% of the Ach maximum (n_~4); the concentration-response curve for substance P (3.3x10" -3.3x10 M) also lay to the left of that for Ach and it caused ~ontr~actions 152.6+36.2% of the Ach maximum (n>__5). Histamine (10--10-~M'I ~vas 10 times less > potent than Ach (n_5); PGE2 and PGF2a (2.8x1(i- 1 -2.8x10- o M) were without a significant effect (~o=4). Distal colonic MM was refractory to PGE2 and PGF2a (2.8x10- -2.~xl0_~M) (n>_5) and was only w e ~ y stimulated by histamine (10" -10- M)(26.5+3.4% maximal:n=6L Substance P (3.3x10I°-3.3x]0:6M) was equipote0t with A_~h (n=5~. The concentration-response curve for BK (2.3x10-~-2.3x10 M) lay to left of that for ACh, but responses to this agent did not exceed the acetylcholine maximum (n=4). CONCLUSIONS: 1. Unlike other rat intestinal muscles proximal colonic MM is contracted by histamine. 2. Rat mid and distal colonic MM are refractory to inflammatory mediators that have significant motor effects on intestinal muscles from humans and other species. 3. The anomalous responses of rat colonic MM to inflammatory mediators are unrelated to resting tone and illustrate its unique pharmacologic profile in different colonic regions.
IN VITRO STUDIES ON THE RELATIONSHIP BETWEEN MUSCULARIS MUCOSAE CONTRACTION AND CHANGES IN TRANSEPITHELIALPOTENTIAL DIFFERENCE IN RABBIT DISTAL COLON. ~ Department of Physiology & Pharmacology, School of Medicine, University of South Dakota, Vermillion, SD 57069. Previous studies have suggested that there is a relationship between intestinal muscle contraction and changes in transepithelial potential difference (PD). Due to the proximity of the museularis mueosae (MM) to the epithelium the aim of this study was to assess the possible contribution of this muscle to such phenomena. METHODS: A 5era segment of distal colon was removed from euthanized, 3-5kg New Zealand white rabbits. The longitudinal and circular muscle layers were removed, leaving a cylinder composed of submucosa, MM, lamina propria and epitlielium. The preparation was placed in an organ bath and luminally perrused with Krebs r solution at 12ml/min. One agar salt bridge electrode was placed in the bathing medium and another was inserted into a luminal outflow "T" piece onto which was tied the distal end of the preparation. PD was measured between these electrodes. The proximal end of the preparation was connected to an isometric transducer which measured MM contractions. ~ Both MM tone and PD exhibited oscillations under basal conditions. These had amplitudes of 0.5-1.0mN and 015-1.0mV and frequencies of 5.52 + 1.68 and 6.10 + 0.59 cycles Per minute respectively (n---5 each). The frequencies were not significantly different. However, when the contraeble frequency from each preparation was plotted against the corresponding frequency of its PD oscillation the resulting line was described by the equationy = 0.32x + 4.31 (r=0.91:n=5) and had a sloPe significantly differ9ent l.~om 1. Both MM and epithelium responded to acetylcholine (10- -10" M) with concentration-dependent increases in tension and in PD (n=5). When expressed as a percentage of their respective maximal responses to aeetylcholinethe concentration-response curves for electrical and mechanical activity were supe~mposable. The EC~o values for both contractilg(4.82 + 1.63xlO-IM) and epithelial ~esponses (1.51 + 1.01xl0"°M) were not significantly different when compared by a MannWhitney U test (p=0.66:n=5L Responses of both the MM and epithelium to histamine (10:9-1~-3M) were indistinguishable from those to equimolar concentrations of acetylcholine. Responses of the MM to both acetylchotine and histamine were reversed within 5 minules by changing the bathing medium. In contrast, at concentrations > 10~M the responses of the epithelium to these agents persisted for up to 20 minutes after washing. CONCLUSIONS: 1. Spontaneous oscillations in PD are independent of MM activity. 2. Phasic responses of the epithelium to acetylcholine and histamine are linearly related to the amplitude of MM contractions. 3. Sustained, agonist-induced elevations in PD do not require concurrent MM contraction.
• ACETORPHAN INHIBITS CHOLERA TOXIN-INDUCED WATER AND ELECTROLYTE SECRETION 1N THE HUMAN JEJUNUM.. W. Petritsch. T.~. Hinterleitner, *H. Berard, *J-M.Lecor~te, G.J. KreJs. Dept. of Int. Medicine, Karl Franzens University, Graz, Austria, *BioproJet. 75003 Paris, France
DIARRHEA IN ALCOHOLICS: PREVALENCE, PATHOGENETIC FACTORS AND CLINICAL COURSE. A.Pfeiffer, T.Sehmidt, O. Kuntzen, H.Kaess. Department of Gastroenterology, Hospital MfincbenBogeahausen, Munich, Germany.
A c e t o r p h a n is a n orally a d m i n i s t e r e d i n h i b i t o r of enkephalinase. T h e physiological role of this endopeptidase lies in t h e d e g r a d a t i o n of e n d o g e n o u s e n k e p h a l i n s released by s u b m u c o s a l a n d myenteric n e u r o n s . Therefore, a n inhibitor could a u g m e n t t h e effect of t h e s e n e u r o t r a n s m l t t e r s on local delta opiate receptors w h i c h were located by autoradiography in t h e villous core a n d crypt area of the small intestine. Previous studies in m a n have s h o w n a significant antidiarrheal activity in a c u t e a n d castor oil-induced diarrhea. In addition, a c e t o r p h a n significantly reduced cholera toxin (CT)-induced secretion in dogs. We investigated whether this agent can also inhibit C T ; i n d u c e d s e c r e t i o n in t h e h u m a n J e j u n u m . A s e g m e n t a l ' p e r f u s l o n t e c h n i q u e with a proximal occfuding balloon w a s u s e d in 6 h e a l t h y subjects. A 30-cm test s e g m e n t was perfused u n d e r steady state conditions u s i n g a plasma-like electrolyte solution. Polyethylene glycol {PEG 4000) served a s a n o n a b s o r h a b l e volume marker. After the tube h a d reached its correct position 300 m g of acetorphan was given orally. The perfusion w a s started after 60 m i n allowing for sufficient drug absorption. S e r u m levels of t h e active d r u g metaboltte R,Sthiorphan showed a significant i n c r e a s e above baseline. After a control period of one h o u r 6.25 ~tg CT w a s a d m i n i s t e r e d intraJeJunally a s a bolus. The J e j u n u m was t h e n perfused for four hours. Intestinal water a n d electrolyte transport was a s s e s s e d in the fourth h o u r in experiments with a n d without acetorphan. The table gives m e a n n e t m o v e m e n t s (ml or m m o l / 3 0 cm.h); (-) indicates absorption, (+) denotes secretion; *p<0.05
Background: Diarrhea is a common complaint in chronic alcoholics (Am J Med 1959;27:575-85). The aim was to obtain data on the prevalence, possible pathoganefic factors and the clinical course of this diarrhea. Methods: 35 alcoholic patients without liver cirrhosis or history of pancreatitis who had been admitted for somatic alcohol withdrawal (clomethiazol, propranolol) were interviewed about the presence of diarrhea (_>3 stools daily) during the week before, during day 2-4, and during day 8-10 after alcohol withdrawal. The duration and amount of alcohol consumption was compared between patients with and without diarrhea. During day 1-7, a D-xylose absorption test, an H2-hreath-test, duodenal mucosai biopsies (for disaccharidase activities and histology) and a pancreolauryl test were performed in each patient. Results (median, range, Wilcoxon test): Before alcohol withdrawal, 19 patients (54.3 %) had a diarrhea with 4 (3-15) stools daily. On day 2-4 and day 8-10, the daily stool frequency diminished significantly (p<0.001) to 1 (0.3-7), and 1 (0.3-4), respectively, and only 2 patients (5.7 %) continued to have diarrhea. Patients with diarrhea and patients without diarrhea were neither different in the duration of alcohol abuse (10 [1-50] vs 9 [4-28] years), nor in the amount of alcohol consumed daily during the 6 months (140 [0-400] vs 125 [0-380] g) and 4 weeks (140 [80-425] vs 150 [100700] g) before admission. Investigation of the absorptive function of the upper gastrointestinal tract revealed normal results except an isolated trahahse deficiency in 2 patients, and an abnormal H2-breath-test in 1 patient, respectively. Conclusion: Diarrhea is a common finding in chronic alcoholics. It resolves spontanvouslywithin a few days after alcohol withdrawal. The duration and the amount of alcohol consumption are excluded as pathogenetic factors. Clinical routine tests of the absorptive function of the upper gastrointestinal tract are nondiagnostic in the workup ofdiarrbea in chronic alcoholics.
..........................................................................
CTalone (n=4) CT +Acetorphan (n=6}
H20 +131 - 26*
Na + +44 0*
K+ +0.9 - 0.1"
CI+55 +14
HCO 3+ 3.5 - 1.4
.........................................................................
Our s t u d i e s indicate t h a t a c e t o r p h a n abolishes CT-induced secretion in t h e h u m a n J e j u n u m . These experiments w a r r a n t further s t u d i e s of a c e t o r p h a n in various forms of secretory diarrhea.
AGA ABSTRACTS
GASTROENTEROLOGY,Vol. 108, No. 4
• CHARACTERISTICS OF RAT COLONIC MUSCULARIS MUCOSAE RESPONSES TO INFLAMMATORY MEDIATORS IN VITRO. W H P e r c v & LM Anderson, DePar[ment of Physiology & Pharmacology, Sch6ol of Medicine, University of South Da~ta, Vermillion, SD 57069. Rat colon is frequently used in studies of the pathophysiologic consequences of inflammation of the intestine. It is known that the motor effects of inflammatory mediators on the colonic muscularispropria of the rat are atypical (Percy et al., J. Gastro. Motil. 3: 229-23-6, -1991). The aim of this study was to assess their effects on the corresponding muscularis mucosae (MM). METHODS: 4-5cm segments of distaF, mid and proximal colon were removed from euthanized 350g SpragueDawley rats. Strips of MM 3era x 4mm were prepared for isometric recording usingthe technique described by Percy & Christensen (Am. J. Physiol. 250: G98-G102,1986) and mounted in 2ml tissue baths under a tension equivalent to 0.5g (4.gmN). All responses were expressed as a percentage of each tissue's maximum response to acetylcholine (Ach). RESULTS: Resting tone of MM from the proximal, mid and distal colon did not differ significantly (3.58_+0.27, 3.28_+0.26 and 2.71+0.18mN (n ~ 9:p > 0.05). All preparations gave atrol~ine 00"6M) sensitive, concentration-dependent contractions to Ach (10"J-10"~M)~ On colonic 3 MM bradykinin (BK)(2.3x10"13-2.3x10~6M) his~mi~e (10- -10" M), prostaglandi~s (PG=~)c,~2and F2a (2.8x10 "l~' 2.8x10 M) and substance P (3.3x10 -3.3x10 M)were potent agonists, whoseconcentration-response curves lay to the leff of that for acetylcholine, with maxima of 250-300% of .the largest response to Ach (n_>5 e a c h ) . In the6~n.xi_~ddcolon the concentration-response curve for BK (2.3x10~-2.3x10 M) lay to the left of that for Ach, with its largest responses being 355.4_+71.5% of the Ach maximum (n_~4); the concentration-response curve for substance P (3.3x10" -3.3x10 M) also lay to the left of that for Ach and it caused ~ontr~actions 152.6+36.2% of the Ach maximum (n>__5). Histamine (10--10-~M'I ~vas 10 times less > potent than Ach (n_5); PGE2 and PGF2a (2.8x1(i- 1 -2.8x10- o M) were without a significant effect (~o=4). Distal colonic MM was refractory to PGE2 and PGF2a (2.8x10- -2.~xl0_~M) (n>_5) and was only w e ~ y stimulated by histamine (10" -10- M)(26.5+3.4% maximal:n=6L Substance P (3.3x10I°-3.3x]0:6M) was equipote0t with A_~h (n=5~. The concentration-response curve for BK (2.3x10-~-2.3x10 M) lay to left of that for ACh, but responses to this agent did not exceed the acetylcholine maximum (n=4). CONCLUSIONS: 1. Unlike other rat intestinal muscles proximal colonic MM is contracted by histamine. 2. Rat mid and distal colonic MM are refractory to inflammatory mediators that have significant motor effects on intestinal muscles from humans and other species. 3. The anomalous responses of rat colonic MM to inflammatory mediators are unrelated to resting tone and illustrate its unique pharmacologic profile in different colonic regions.
IN VITRO STUDIES ON THE RELATIONSHIP BETWEEN MUSCULARIS MUCOSAE CONTRACTION AND CHANGES IN TRANSEPITHELIALPOTENTIAL DIFFERENCE IN RABBIT DISTAL COLON. ~ Department of Physiology & Pharmacology, School of Medicine, University of South Dakota, Vermillion, SD 57069. Previous studies have suggested that there is a relationship between intestinal muscle contraction and changes in transepithelial potential difference (PD). Due to the proximity of the museularis mueosae (MM) to the epithelium the aim of this study was to assess the possible contribution of this muscle to such phenomena. METHODS: A 5era segment of distal colon was removed from euthanized, 3-5kg New Zealand white rabbits. The longitudinal and circular muscle layers were removed, leaving a cylinder composed of submucosa, MM, lamina propria and epitlielium. The preparation was placed in an organ bath and luminally perrused with Krebs r solution at 12ml/min. One agar salt bridge electrode was placed in the bathing medium and another was inserted into a luminal outflow "T" piece onto which was tied the distal end of the preparation. PD was measured between these electrodes. The proximal end of the preparation was connected to an isometric transducer which measured MM contractions. ~ Both MM tone and PD exhibited oscillations under basal conditions. These had amplitudes of 0.5-1.0mN and 015-1.0mV and frequencies of 5.52 + 1.68 and 6.10 + 0.59 cycles Per minute respectively (n---5 each). The frequencies were not significantly different. However, when the contraeble frequency from each preparation was plotted against the corresponding frequency of its PD oscillation the resulting line was described by the equationy = 0.32x + 4.31 (r=0.91:n=5) and had a sloPe significantly differ9ent l.~om 1. Both MM and epithelium responded to acetylcholine (10- -10" M) with concentration-dependent increases in tension and in PD (n=5). When expressed as a percentage of their respective maximal responses to aeetylcholinethe concentration-response curves for electrical and mechanical activity were supe~mposable. The EC~o values for both contractilg(4.82 + 1.63xlO-IM) and epithelial ~esponses (1.51 + 1.01xl0"°M) were not significantly different when compared by a MannWhitney U test (p=0.66:n=5L Responses of both the MM and epithelium to histamine (10:9-1~-3M) were indistinguishable from those to equimolar concentrations of acetylcholine. Responses of the MM to both acetylchotine and histamine were reversed within 5 minules by changing the bathing medium. In contrast, at concentrations > 10~M the responses of the epithelium to these agents persisted for up to 20 minutes after washing. CONCLUSIONS: 1. Spontaneous oscillations in PD are independent of MM activity. 2. Phasic responses of the epithelium to acetylcholine and histamine are linearly related to the amplitude of MM contractions. 3. Sustained, agonist-induced elevations in PD do not require concurrent MM contraction.
• ACETORPHAN INHIBITS CHOLERA TOXIN-INDUCED WATER AND ELECTROLYTE SECRETION 1N THE HUMAN JEJUNUM.. W. Petritsch. T.~. Hinterleitner, *H. Berard, *J-M.Lecor~te, G.J. KreJs. Dept. of Int. Medicine, Karl Franzens University, Graz, Austria, *BioproJet. 75003 Paris, France
DIARRHEA IN ALCOHOLICS: PREVALENCE, PATHOGENETIC FACTORS AND CLINICAL COURSE. A.Pfeiffer, T.Sehmidt, O. Kuntzen, H.Kaess. Department of Gastroenterology, Hospital MfincbenBogeahausen, Munich, Germany.
A c e t o r p h a n is a n orally a d m i n i s t e r e d i n h i b i t o r of enkephalinase. T h e physiological role of this endopeptidase lies in t h e d e g r a d a t i o n of e n d o g e n o u s e n k e p h a l i n s released by s u b m u c o s a l a n d myenteric n e u r o n s . Therefore, a n inhibitor could a u g m e n t t h e effect of t h e s e n e u r o t r a n s m l t t e r s on local delta opiate receptors w h i c h were located by autoradiography in t h e villous core a n d crypt area of the small intestine. Previous studies in m a n have s h o w n a significant antidiarrheal activity in a c u t e a n d castor oil-induced diarrhea. In addition, a c e t o r p h a n significantly reduced cholera toxin (CT)-induced secretion in dogs. We investigated whether this agent can also inhibit C T ; i n d u c e d s e c r e t i o n in t h e h u m a n J e j u n u m . A s e g m e n t a l ' p e r f u s l o n t e c h n i q u e with a proximal occfuding balloon w a s u s e d in 6 h e a l t h y subjects. A 30-cm test s e g m e n t was perfused u n d e r steady state conditions u s i n g a plasma-like electrolyte solution. Polyethylene glycol {PEG 4000) served a s a n o n a b s o r h a b l e volume marker. After the tube h a d reached its correct position 300 m g of acetorphan was given orally. The perfusion w a s started after 60 m i n allowing for sufficient drug absorption. S e r u m levels of t h e active d r u g metaboltte R,Sthiorphan showed a significant i n c r e a s e above baseline. After a control period of one h o u r 6.25 ~tg CT w a s a d m i n i s t e r e d intraJeJunally a s a bolus. The J e j u n u m was t h e n perfused for four hours. Intestinal water a n d electrolyte transport was a s s e s s e d in the fourth h o u r in experiments with a n d without acetorphan. The table gives m e a n n e t m o v e m e n t s (ml or m m o l / 3 0 cm.h); (-) indicates absorption, (+) denotes secretion; *p<0.05
Background: Diarrhea is a common complaint in chronic alcoholics (Am J Med 1959;27:575-85). The aim was to obtain data on the prevalence, possible pathoganefic factors and the clinical course of this diarrhea. Methods: 35 alcoholic patients without liver cirrhosis or history of pancreatitis who had been admitted for somatic alcohol withdrawal (clomethiazol, propranolol) were interviewed about the presence of diarrhea (_>3 stools daily) during the week before, during day 2-4, and during day 8-10 after alcohol withdrawal. The duration and amount of alcohol consumption was compared between patients with and without diarrhea. During day 1-7, a D-xylose absorption test, an H2-hreath-test, duodenal mucosai biopsies (for disaccharidase activities and histology) and a pancreolauryl test were performed in each patient. Results (median, range, Wilcoxon test): Before alcohol withdrawal, 19 patients (54.3 %) had a diarrhea with 4 (3-15) stools daily. On day 2-4 and day 8-10, the daily stool frequency diminished significantly (p<0.001) to 1 (0.3-7), and 1 (0.3-4), respectively, and only 2 patients (5.7 %) continued to have diarrhea. Patients with diarrhea and patients without diarrhea were neither different in the duration of alcohol abuse (10 [1-50] vs 9 [4-28] years), nor in the amount of alcohol consumed daily during the 6 months (140 [0-400] vs 125 [0-380] g) and 4 weeks (140 [80-425] vs 150 [100700] g) before admission. Investigation of the absorptive function of the upper gastrointestinal tract revealed normal results except an isolated trahahse deficiency in 2 patients, and an abnormal H2-breath-test in 1 patient, respectively. Conclusion: Diarrhea is a common finding in chronic alcoholics. It resolves spontanvouslywithin a few days after alcohol withdrawal. The duration and the amount of alcohol consumption are excluded as pathogenetic factors. Clinical routine tests of the absorptive function of the upper gastrointestinal tract are nondiagnostic in the workup ofdiarrbea in chronic alcoholics.
..........................................................................
CTalone (n=4) CT +Acetorphan (n=6}
H20 +131 - 26*
Na + +44 0*
K+ +0.9 - 0.1"
CI+55 +14
HCO 3+ 3.5 - 1.4
.........................................................................
Our s t u d i e s indicate t h a t a c e t o r p h a n abolishes CT-induced secretion in t h e h u m a n J e j u n u m . These experiments w a r r a n t further s t u d i e s of a c e t o r p h a n in various forms of secretory diarrhea.