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Functional characterization of tachykinin receptors mediating ion transport in porcine jejunum
A700
AGA ABSTRACTS
GASTROENTEROLOGY, Vol. 108, No. 4
• FUNCTIONAL CHARACTERISATION OF TACHYKININ RECEPTORS MEDIATING ION TRANSPORT IN PORCINE JEJUNUM. J.E. Thorbell, M.B. Hansen, E. Skadhauge. Department of Anatomy and Physiology, The Royal Veterinary and Agricultural University, Copenhagen, Denmark. Substance P (SP) and neurokinin A (NKA) are the major tachykinln neurotransmitters in the porcine small intestine. The development of highaffinity and selective nonpeptide antagonists for tachykinin receptors has provided possibilities for a detailed understanding of the physiological role of taehykinins. The purpose of the present study was to functionally characterise tachykinin receptors involved in ion transport in the porcine jejunum. Methods: Stripped porcine jejunal tissue preparations with intact mucosa and submucosa were mounted in Ussing chambers containing oxygenated Ringer-solution. The tissue was short-circuited and corresponding values of electrical parameters (resistance and open circuit potential difference) were measured and calculated by computer. All compounds were added to the serosal side, antagonists 15 minutes prior to subsequent tachykinln addition (unless otherwise stated). Results: SP produced, within 5 minutes, a transient increase in short-circuit current (SCC). The response was concentration dependent over the range of 10 nM to 10 ~tM, the curve having a double sigmoidal form. The nonpeptide NK-1 antagonist, CP 99,994 (1 ~tM), caused a dextral shift of the SP concentration response curve and totally inhibited the first sigmoidal response (UP to 0,5 ~M). NKA produced, within 5 minutes, a transient increase in SCC, which was concentration dependent over the range of 50 riM to 1 ~tM. The NKA concentration response curve was not significantly changed (!0>0.05) by the nonpeptide NK-2 antagonist, SR 48,968 (0.1 laM). To further evaluate the NKA-response, NKA (1 ~tM) was added when tissues were pre-treated with either SK 48,968 (0.1 ~tM, incubation time 15 minutes or 30 minutes) or the peptide NK-2 antagonist GR 94,800 (1 /aM) or CP 99,994 (1 laM). Only CP 99,994 significantly reduced (p<0.05) the NKA-response. Conclusion: The present results indicate, that tachykinin secretory responses in the porcine jejunum are mediated by at least NK-1 receptors. NK-2 receptors do not seem to be functionally present.
EFFECTS OF WHEAT BRAN AND PECTIN ON THE PRODUCTION OF FECAL SHORT-CHAIN FATTY ACIDS (SCFAs). W Treem, N Ahsan, G Kastoff, M Shoup, T Lerer, C Justinieh, J Hyams. Division of Pediatric GI and Nutrition, Hartford Hospital, University of Connecticut School of Medicine, Farmington, CT. Salts of the SCFA butyrate have been used in enemas to treat patients with refractory ulcerative proetitis and as antiproliferative and differentiating agents in animal models of colon cancer. METHODS: To determine the effects of chronic fiber supplementation on human colonic fermentation of carbohydrates and production of SCFAs and butyrate, we studied fecal homogenates from 16 normal controls, 8 of whom received 5 g of wheat bran/day and the other half 5 g of citrus pectin/day in addition to their normal diets. Stool samples were collected in the morning at baseline (wk 0) and after 4 weeks (wk 4l of supplementation. SCFAs were measured by gas chromatography. Fecal homogenates (1.25 g stool/25 ml) were incubated with no additional carbohydrate, 100 mg of laetulose, and 100 mg potato starch under anaerobic conditions. Changes in total SCFA (TOT) and butyrate (BUT) concentrations (/tmols) were measured at 6,16, and 24 hr, and net production rates of total SCFAs (PR, umol/hr) calculated for the first 6 hr. RESULTS: The table shows changes in TOT and BUT concentrations, BUT as a % of TOT (%BUT), and PR with no additional carbohydrate PR(NC) after 4 weeks of wheat bran or pectin. Values (median range), are compared using non-parametric measures. Wheat bran
I TOT BUT
Wk 0 366(227-384)
I 5(4-e)
%BUT
PR(NC) I
1.4(1.1-1.7)
Wk 4 485(325-662)*
Pectin
Wk 0
Wk 4
302(287-450)
354(261-81o)
24(8-39)*
8(5-20)
9(7-45)*
4.8(1.8-8.3)*
2.6(1.6-6.5)
2.8(1.7-7,0)f
121(62-15351" 74(15-201) 244(20-379)* 70(14-1131 * =p<0.01 t =p
• THE EFFECT OF SUPPLEMENTAL PECTIN ON FECAL SHORT-CHAIN FATTY ACID (SCFA) PRODUCTION IN PATIENTS WITH ULCERATIVE COLITIS (UC) AND IRRITABLE BOWEL SYNDROME (IBS).W Tr0em,N Ahsan,M Shoup,G Kastoff, T Lerer,C Justinich,J Hyams. Division of Pediatric GI and Nutrition, Hartford Hospital, University of Connecticut School of Medicine, Farmington, CT. Abnormalities in the production and metabolism of SCFAs have been implicated in the pathogenesis of diarrhea in patients with UC and IBS.METHODS: To determine whether these patients differ in the response of their colonic flora to supplemental fiber,we studied changes in fecal SCFAs with the addition of 5 g citrus pectin (PEC) to the diet of controls(n = 8),lBS(n = 5),and UC patients (n =4) over 4 wk.Patients consumed their normal diets and were taking no supplemental fiber, anticholinergie agents, antibiotics, or sulfasalazlne for at least one month prior to the study.UC patients had inactive disease.SCFAs were measured in fresh morning stool by gas chromatography at entry(wk 0),and after 4 wk (wk 4) of PEC.Fecal homogenates (1.25 g stool/25 ml) were incubated anaerobically with no additional carbohydrate (NC) and with 100 mg PEC for 6 hr to determine net production rates (PR) of total SCFAs.Data are shown for total SCFAs (TOT, #regis), butyrate (BUT,/~mols),the percentage of the TOT of BUT (%BUT),and the PR ~umols/hr) with NC and PEC.Data (median and range) are compared using the Kruakal-Wallis 1-way ANOVA test.RESULTS: At wk 0, TOT are higher in controls than either IBS or UC patients (p< O.01), however butyrate concentrations are greater in both IBS and UC patients than controls (p<0.05), and the %BUT is markedly increased in 16S and UC patients over controls (p<0,01).PR(NC) is not significantly different but PR(PEC) is greater in controls than either IBS or UC patients.The table shows data for wk 4. Control
IBS
UC
p value
TOT
J 354(261-810)
212(139-252)
J 203(116-a61)
<0.02
BUT
I 917-4S)
30(10-551
J 43129-621
<0.02
%BUT
J 2.8(1.7-7.o)
21(e-22)
J 17(15-42)
<0.01
PRINC)
J 244120-a791
831ss-a46)
J 29(e-e2)
<0.06
PR(PEC) 237(as-s75) 927(382-1373) 94(s2-187) <0.02 PR PEC at wk 4 increased over baseline (wk 0) to control level in patients with IBS but was unchanged in patients with UC.CONCLUSIONS: Baseline fecal SCFA profiles are similar in IBS and UC patients with low total SCFAs, increased botyrate and low in-vitro production rates compared to controls.Pectin supplementation results in enhancement of SCFA production in IBS but not in UC patients. Failure to respond to pectin supplementation may signify an abnormality of colonic bacterial fermentation in UC.
Q INHIBITION OF PROGRESSING ESOPHAGEAL PERISTALSIS BY PHARYNGEAL WATER STIMULATION IN HUMANS. A. Trifan, R. Shaker, J. Ren, C. Hofmann, R.C. Amdoffer. MCW Dysphagia Institute, Departments of Medicine and Radiology. Medical College of Wisconsin, Milwaukee, WI Earlier studies have shown that pharyngeal water stimulation inhibits the lower esophageal sphincter (LES) resting pressure. However, it is not known whether this is an isolated effect or involves the entire esophagus. Our aim was to detemxine the effect of pharyngeal water stimulation on the progressing esophageal peristalsis. We studied 11 normal volunteers age 22+4 (5F, 6M) in supine position. Presence of normal peristalsis was verified before the study, as well as before and after each water injection. Esophageal peristalsis was recorded at 3,6,9,12,15 and 18 cm above the LES. LES and LIES were simultaneously monitored by sleeve sensors. Pharyngeal stimulation was done by injecting a predetermined threshold volume into the pharynx 2 cm above the UES directed posteriorly. The injections were timed to coincide with the arrival of the peristaltic wave induced by dry swallows at respective recording sites. The dry swallows used for inhibition were done at least 30 see. after a previous swallow and followed by another swallow 30 sec. later for comparison. Each test was repeated x3 for each recording site. In 5 subjects the LES pressure response to pharyngeal water injection was also determined. Studies were repeated after pharyngeal topical anesthesia in the same session. Results: Injection of the threshold volume (0.5_+0.1SE) stopped the progression of peristalsis at both striated as well as smooth muscle part of the esophagus (p<0.01 chi squared). Topical pharyngeal anesthesia blocked this inhibitory effect (Table) (*p<0.01 ANOVA). % Inhibition of Peristalsis at Various Sites Above LES JF-sophagealSites ] lgcm i 15cm i 12cm i 9cm ] 6cm [ Before Anesthesia 78 92 183. 94, 100 [ After Anesthesia 0* 8* 0* Injection of similar volumes at resting state induced isolated LES relaxation, whereas larger volumes induced an irrepressible swallow. Conclusions: 1) Pharyngeal water stimulation at a threshold volume a) inhibits the progression of the peristalsis in striated as well as the smooth muscle portion of the esophagus and b) LES pressure. 2) These findings suggest that pharyngeal water stimulation induces inhibition of cholinergic excitatory and stimulation of nonadrenergic, nonchoinergic inhibitory neural pathways to the esophagus. 3) These findings may explain the mechanism for some of the failed esophageal peristalsis.
AGA ABSTRACTS
GASTROENTEROLOGY, Vol. 108, No. 4
• FUNCTIONAL CHARACTERISATION OF TACHYKININ RECEPTORS MEDIATING ION TRANSPORT IN PORCINE JEJUNUM. J.E. Thorbell, M.B. Hansen, E. Skadhauge. Department of Anatomy and Physiology, The Royal Veterinary and Agricultural University, Copenhagen, Denmark. Substance P (SP) and neurokinin A (NKA) are the major tachykinln neurotransmitters in the porcine small intestine. The development of highaffinity and selective nonpeptide antagonists for tachykinin receptors has provided possibilities for a detailed understanding of the physiological role of taehykinins. The purpose of the present study was to functionally characterise tachykinin receptors involved in ion transport in the porcine jejunum. Methods: Stripped porcine jejunal tissue preparations with intact mucosa and submucosa were mounted in Ussing chambers containing oxygenated Ringer-solution. The tissue was short-circuited and corresponding values of electrical parameters (resistance and open circuit potential difference) were measured and calculated by computer. All compounds were added to the serosal side, antagonists 15 minutes prior to subsequent tachykinln addition (unless otherwise stated). Results: SP produced, within 5 minutes, a transient increase in short-circuit current (SCC). The response was concentration dependent over the range of 10 nM to 10 ~tM, the curve having a double sigmoidal form. The nonpeptide NK-1 antagonist, CP 99,994 (1 ~tM), caused a dextral shift of the SP concentration response curve and totally inhibited the first sigmoidal response (UP to 0,5 ~M). NKA produced, within 5 minutes, a transient increase in SCC, which was concentration dependent over the range of 50 riM to 1 ~tM. The NKA concentration response curve was not significantly changed (!0>0.05) by the nonpeptide NK-2 antagonist, SR 48,968 (0.1 laM). To further evaluate the NKA-response, NKA (1 ~tM) was added when tissues were pre-treated with either SK 48,968 (0.1 ~tM, incubation time 15 minutes or 30 minutes) or the peptide NK-2 antagonist GR 94,800 (1 /aM) or CP 99,994 (1 laM). Only CP 99,994 significantly reduced (p<0.05) the NKA-response. Conclusion: The present results indicate, that tachykinin secretory responses in the porcine jejunum are mediated by at least NK-1 receptors. NK-2 receptors do not seem to be functionally present.
EFFECTS OF WHEAT BRAN AND PECTIN ON THE PRODUCTION OF FECAL SHORT-CHAIN FATTY ACIDS (SCFAs). W Treem, N Ahsan, G Kastoff, M Shoup, T Lerer, C Justinieh, J Hyams. Division of Pediatric GI and Nutrition, Hartford Hospital, University of Connecticut School of Medicine, Farmington, CT. Salts of the SCFA butyrate have been used in enemas to treat patients with refractory ulcerative proetitis and as antiproliferative and differentiating agents in animal models of colon cancer. METHODS: To determine the effects of chronic fiber supplementation on human colonic fermentation of carbohydrates and production of SCFAs and butyrate, we studied fecal homogenates from 16 normal controls, 8 of whom received 5 g of wheat bran/day and the other half 5 g of citrus pectin/day in addition to their normal diets. Stool samples were collected in the morning at baseline (wk 0) and after 4 weeks (wk 4l of supplementation. SCFAs were measured by gas chromatography. Fecal homogenates (1.25 g stool/25 ml) were incubated with no additional carbohydrate, 100 mg of laetulose, and 100 mg potato starch under anaerobic conditions. Changes in total SCFA (TOT) and butyrate (BUT) concentrations (/tmols) were measured at 6,16, and 24 hr, and net production rates of total SCFAs (PR, umol/hr) calculated for the first 6 hr. RESULTS: The table shows changes in TOT and BUT concentrations, BUT as a % of TOT (%BUT), and PR with no additional carbohydrate PR(NC) after 4 weeks of wheat bran or pectin. Values (median range), are compared using non-parametric measures. Wheat bran
I TOT BUT
Wk 0 366(227-384)
I 5(4-e)
%BUT
PR(NC) I
1.4(1.1-1.7)
Wk 4 485(325-662)*
Pectin
Wk 0
Wk 4
302(287-450)
354(261-81o)
24(8-39)*
8(5-20)
9(7-45)*
4.8(1.8-8.3)*
2.6(1.6-6.5)
2.8(1.7-7,0)f
121(62-15351" 74(15-201) 244(20-379)* 70(14-1131 * =p<0.01 t =p
• THE EFFECT OF SUPPLEMENTAL PECTIN ON FECAL SHORT-CHAIN FATTY ACID (SCFA) PRODUCTION IN PATIENTS WITH ULCERATIVE COLITIS (UC) AND IRRITABLE BOWEL SYNDROME (IBS).W Tr0em,N Ahsan,M Shoup,G Kastoff, T Lerer,C Justinich,J Hyams. Division of Pediatric GI and Nutrition, Hartford Hospital, University of Connecticut School of Medicine, Farmington, CT. Abnormalities in the production and metabolism of SCFAs have been implicated in the pathogenesis of diarrhea in patients with UC and IBS.METHODS: To determine whether these patients differ in the response of their colonic flora to supplemental fiber,we studied changes in fecal SCFAs with the addition of 5 g citrus pectin (PEC) to the diet of controls(n = 8),lBS(n = 5),and UC patients (n =4) over 4 wk.Patients consumed their normal diets and were taking no supplemental fiber, anticholinergie agents, antibiotics, or sulfasalazlne for at least one month prior to the study.UC patients had inactive disease.SCFAs were measured in fresh morning stool by gas chromatography at entry(wk 0),and after 4 wk (wk 4) of PEC.Fecal homogenates (1.25 g stool/25 ml) were incubated anaerobically with no additional carbohydrate (NC) and with 100 mg PEC for 6 hr to determine net production rates (PR) of total SCFAs.Data are shown for total SCFAs (TOT, #regis), butyrate (BUT,/~mols),the percentage of the TOT of BUT (%BUT),and the PR ~umols/hr) with NC and PEC.Data (median and range) are compared using the Kruakal-Wallis 1-way ANOVA test.RESULTS: At wk 0, TOT are higher in controls than either IBS or UC patients (p< O.01), however butyrate concentrations are greater in both IBS and UC patients than controls (p<0.05), and the %BUT is markedly increased in 16S and UC patients over controls (p<0,01).PR(NC) is not significantly different but PR(PEC) is greater in controls than either IBS or UC patients.The table shows data for wk 4. Control
IBS
UC
p value
TOT
J 354(261-810)
212(139-252)
J 203(116-a61)
<0.02
BUT
I 917-4S)
30(10-551
J 43129-621
<0.02
%BUT
J 2.8(1.7-7.o)
21(e-22)
J 17(15-42)
<0.01
PRINC)
J 244120-a791
831ss-a46)
J 29(e-e2)
<0.06
PR(PEC) 237(as-s75) 927(382-1373) 94(s2-187) <0.02 PR PEC at wk 4 increased over baseline (wk 0) to control level in patients with IBS but was unchanged in patients with UC.CONCLUSIONS: Baseline fecal SCFA profiles are similar in IBS and UC patients with low total SCFAs, increased botyrate and low in-vitro production rates compared to controls.Pectin supplementation results in enhancement of SCFA production in IBS but not in UC patients. Failure to respond to pectin supplementation may signify an abnormality of colonic bacterial fermentation in UC.
Q INHIBITION OF PROGRESSING ESOPHAGEAL PERISTALSIS BY PHARYNGEAL WATER STIMULATION IN HUMANS. A. Trifan, R. Shaker, J. Ren, C. Hofmann, R.C. Amdoffer. MCW Dysphagia Institute, Departments of Medicine and Radiology. Medical College of Wisconsin, Milwaukee, WI Earlier studies have shown that pharyngeal water stimulation inhibits the lower esophageal sphincter (LES) resting pressure. However, it is not known whether this is an isolated effect or involves the entire esophagus. Our aim was to detemxine the effect of pharyngeal water stimulation on the progressing esophageal peristalsis. We studied 11 normal volunteers age 22+4 (5F, 6M) in supine position. Presence of normal peristalsis was verified before the study, as well as before and after each water injection. Esophageal peristalsis was recorded at 3,6,9,12,15 and 18 cm above the LES. LES and LIES were simultaneously monitored by sleeve sensors. Pharyngeal stimulation was done by injecting a predetermined threshold volume into the pharynx 2 cm above the UES directed posteriorly. The injections were timed to coincide with the arrival of the peristaltic wave induced by dry swallows at respective recording sites. The dry swallows used for inhibition were done at least 30 see. after a previous swallow and followed by another swallow 30 sec. later for comparison. Each test was repeated x3 for each recording site. In 5 subjects the LES pressure response to pharyngeal water injection was also determined. Studies were repeated after pharyngeal topical anesthesia in the same session. Results: Injection of the threshold volume (0.5_+0.1SE) stopped the progression of peristalsis at both striated as well as smooth muscle part of the esophagus (p<0.01 chi squared). Topical pharyngeal anesthesia blocked this inhibitory effect (Table) (*p<0.01 ANOVA). % Inhibition of Peristalsis at Various Sites Above LES JF-sophagealSites ] lgcm i 15cm i 12cm i 9cm ] 6cm [ Before Anesthesia 78 92 183. 94, 100 [ After Anesthesia 0* 8* 0* Injection of similar volumes at resting state induced isolated LES relaxation, whereas larger volumes induced an irrepressible swallow. Conclusions: 1) Pharyngeal water stimulation at a threshold volume a) inhibits the progression of the peristalsis in striated as well as the smooth muscle portion of the esophagus and b) LES pressure. 2) These findings suggest that pharyngeal water stimulation induces inhibition of cholinergic excitatory and stimulation of nonadrenergic, nonchoinergic inhibitory neural pathways to the esophagus. 3) These findings may explain the mechanism for some of the failed esophageal peristalsis.