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Cellular mechanisms of the no-induced inhibition in rat ileum: Involvement of intracellular Ca2+ stores
April 1995
Motility and Nerve-Gut Interactions
A601
• ENHANCED SENSITIVITY O F THE WHOLE GUT IN PATIENTS WITH IRRITABLE BOWEL SYNDROME (IBS), 1C Y Francis. ~L A Houghton, IP J Whorwell, 2j Morris, 3j Bradbury. Departments of 1Medicine and 2Statistics, University Hospital of South Manchester, and 3Glaxo Research and Development, U.K. Previous studies have shown that various regions of the gut, in partienlar the colon and rectum, are abnormally sensitive to balloon distension in patients with IBS. However, these studies have only examined up to two anatomical regions in the same patient at any one time and thus it is not known whether all or just specific areas of the gut are abnormally sensitive. Sensory and motility responses to balloon distension of the oesophagus [01, duodenum/D], jejunum [J], ileum //], colon [CI and rectum/'R1 were therefore evaluated in random order in 20 patients with diarrhoea predominant IBS (aged 22-57, 11 females) and compared with 20 matched healthy volunteers (aged 20-57, 10 females). All patients had loose stools (> 3 bowel movements per day) and fulfilled the Rome criteria for IBS. In addition, patient anxiety scores were evaluated using the Hospital Anxiety and Depression questionnaire (abnormal > 10). Results In accordance with previous studies, IBS patients had significantly lower sensory thresholds in the R [vol to discomfort (ml): IBS 109(60,225) geometric mean (range) v controls 175(80,325); p=0.001] and C [100(50,180) v166(60,500); p=0.005] compared with controls. Furthermore, IBS patients exhibited significantly lower thresholds for discomfort, provoked by distension of the O [10(6,20) v 20(8,110); p=0.002], D [45(15,170) v 82(40,150); p=0.002], J [38(20,140) v 73(40,170); p<0.001], and 1 [38(15,120) v 55(30,150); p=0.03]. However, these sensory changes were not associated with any change in compliance [ml/cmI-I~O at mean volume to discomfort: O, 0.45(0.26,0.96) v 0.49(0.22,2.38); D, 3.4(1.47,9.63) v 3.21(1.59,9.28); J, 2.44(1.28,6.13) v 2.11(1.07,4.76); 1, 1.76(0.99,14.5) v 1.4(0.77,3.15); C, 3.4(1.08,6.53) v 3.44(1.65,6.2); R, 5.69(2.82,12.5) v 5.68(2.33,10.3)]. In addition, although patients had higher anxiety scores [9(2,19), median (range)] than controls [4.5(2,7); p<0.001], there was no correlation between their anxiety score and sensory threshold in the D (r= -0.21; p=0.39), J (r= -0.18; p=0.46), I (r-0.23; p=0.45), C (r= -0.28; p=0.38) and R (r= -0.41; p=0.08), with the exception of the O (r= -0.54; p=0.027). Conclusion This is the first study to confirm the suspicion that IBS represents a generalized disorder of visceral sensation. Furthermore, although these results do not exclude a sensory defect at a local level within the gut, they lend some support to the concept of there being an abnormality of the visceral afferent nervous system or its central modulation.
• CELLULAR MECHANISMS OF THE NO-INDUCED INHIBITION IN RAT ILEUM: INVOLVEMENT OF INTRACELLULAR CA2+ STORES. H. Franck, M. Storr, A. Puschmann, H.-D. Allescher. Department of Internal Medicine lI, Tech. University of Munich, Germany
• EFFECTS OF PATHOPHYSIOLOGIC LEVELS OF GLUCAGON ON GLUCOSE DELIVERY, ABSORPTION AND HOMEOSTASIS IN NONDIABETIC HUMANS. J.W. Frank, G.M. Thomforde, S.F. Dineen, R.A. Rizza, M. CamiUeri. Mayo Clinic, Rochester, MN
• EFFECTS OF H Y P E R G L Y C E M I A ON SMALL INTESTINAL MOTILITY AND TRANSIT IN HEALTHY HUMANS. R Fraser, A Rosso, J Dent, M Horowitz. Depts Medicine & Gastrointestinal Medicine, Royal Adelaide Hospital, Adelaide, Australia
Pharmacologic levels of glucagon impair gastric emptying. Failure of postprandial giucagon suppression is an important determinant of postprandial hyperglycemia in diabetes mellitns (DM) by hepatic glucose release (HGR). However, it is unclear whether such pathophysiologic levels compensate for enhanced HGR by reducing glucose delivery from the stomach or its absorption. Our aim was to characterize the effect of physiologic and supraphysiologic levels of glucagon on gastric emptying, carbohydrate absorption, glucose homeostasis, and plasma levels of selected gut neuropeptides in healthy subjects. Methods: We assessed the effects of physiologic and supraphysiologic (n=6 each) levels of glucagon and of saline (placebo) in 12 healthy subjects. We measured gastric emptying of solids and liquids seintigraphically, as well as appearance of meal glucose and HGR using a well-validated isotopic dilution technique. Results: The groups were comparable for age, body mass index, and fasting blood glucose. Low and high dose (0.65 and 3 ng/kg/min) glucagon infusions resulted in mean peak glucagon concentrations of 70 and 350 ng/ml respectively, levels observed postprandially in DM and severe stress respectively. Glucagon did not alter gastric emptying of liquids or solids, rate of appearance of glucose from the meal in plasma, or disappearance of glucose from plasma. However, D-xylose was more slowly absorbed (P=0.009) in the group receiving the higher dose of glucagon. The high, but not low dose of glucagon infusion increased HGR during the first 2 hours after meal ingestion. Plasma insulin, CCK, GIP, neurotensin and PYY did not differ between the 3 groups (i.e., saline, high dose and low dose glucagon). Conclusion: Neither physiologic nor supraphysiologie levels of glecagon alter gastric emptying; higher levels transiently increase HGR postprandially and reduce absorption of D-xylose (which occurs passively) but do not alter the rate of appearance of meal glucose in peripheral plasma. These data are consistent with known inhibition of gut mucosal blood flow by glucagon and/or other effects on the unstirred layer and permeability. The motor effects of physiologic levels of glucagon have minimal impact on glucose homeostasis in nondiabetic humans.
The aim of the present study was to investigate the cellular mechanisms of the inhibition induced by the NO-donor SIN-1 and two membrane-permeable proteinkinase G activators in rat ileum smooth muscle in vitro. Isolated muscle strips were stimulated in an organ bath with carbachol (1045 M) or with the Ca2+ channel activator Bay-K-8644 (10-7 M). The NO-donor S1N-I (ED50: 4.5 x 10-4 M, n=12), 8-Br-cGMP 0ED50:7.4 x 10"4" M, n=12) and 8-pCPTcGMP (ED50:8.1 + 3.0 x 10-5 M, n=8) caused a dose-dependent TTXinsensitive inhibition of the ileal muscle, which was net influenced by pyroganol (10 -4 M). The inhibitor of cGMP-dependent proteinkinase I~, Rp-8-pCPTcGMPS (10-4 M), showed no influence on the SIN-I- and 8-pCPT-cGMPinduced inhibition. The K + channel blockers apamin (10-7 M), charybdotoxin (10 -7 M) glibenclamide (10-5 M) and TEA (10-1 M) had no influence on the iruhibitory effects. Blockade of the sarcoplasmic Ca2+ATPase with cyclopiazonic acid (CPA)(10 -5 M) induced a contraction which could be inhibited completely by 8-pCPT-cGMP and by SIN-1. However, SIN-I or 8pCPT-cGMP showed no inhibition when CPA and carbachol where applied simultaneously. After incubation in Ca2÷ free medium containing CPA ~10'5 M) and subsequent stimulation with carbachol in order to empty the Ca2+ stores, the inhibitory effect of SIN-1 was still completely abolished and the effect of 8-Br-cGMP was significantly reduced, when CPA and CCH were added simultanously. Calphostin C (10-6 M! which is selective for blocking proteinkinase C-dependent mechanisms in the applied dosage, caused a recovery to 41 + 13 % (n=9) of the inhibitory action of SIN-I in presence of both CPA and CCH (compared to controls: 18 -r_ 14 %, n = l l ) . In contrast, Calphostin C I10-6 M) did not change the inhibitory action of SIN-1 on CCHprestimulation alone From these results it can be concluded: a~ The inhibitory effect of SIN-1 seems not to be mediated by proteinkinase G 1~, suggesting a possible role of proteinkinase G In. b) Blockade of the Ca2+ ATPase alone does not affect the inhibitory effect of SIN-I. 8-Br-cGMP and 8-pCPT-cGMP. However_ the inhibitory action of the tested mediators of NO-signaltransduction is dependent on functional intraceilular Ca2+ stores. The recovery of NOinduced Inhibition with Calphostin C demonstrates participation of proteinkinase C-signaltransduction in the described effect. DFG-AI-245/8-1 and DAAD 322.
Recent studies indicate that the blood glucose concentration has a major impact on gastrointestinal motility in both normal subjects and patients with diabetes mellitus. Hyperglycemia slows small intestinal transit, but the motor mechanisms responsible for this effect are unknown. 6 healthy volunteers (aged 22-41yr) underwent paired studies during euglycemia (47 mmol/1) and hyperglycemia (12-16 mmol/1) in randomised order. Pressures in the distal antrum (1 site), duodenum (3 sites) and jejunum (3 sites) were measured with a multi-lumen manometric assembly. Sideholes were spaced at 10 cm intervals and catheter position was monitored using measurements of antroduodenal transmueosal potential difference. Hyperglycemia was induced by rapid infusion of 150 mi 25% dextrose and maintained with an iv dextrose infusion. In the study performed during euglycemia a similar volume of iv normal saline was given. On each study day 10 rain after commencement of iv dextrose or saline a liquid meal comprising 100 ml Ensure and 15g lactulose was instilled into the duodenum over 20 rain. The number of duodenal and jejunal pressure waves was counted. Phase 3 activity was defined as bursts of pressure waves (>10/min) with a duration of more than 3 minutes which began in the duodenum and propagated distally. Small intestinal U'ansit was quantified by breath hydrogen measurements. Data are shown as mean values + SEM. Small intestinal transit was slower during hyperglycemia than euglycemia (120 + 9 rain v 60 + 8 min, p<0.05). During euglycemia, instillation of the meal induced a fed pattern of motor activity within 15 rain and the first episode of intestinal phase 3 activity occurred 116+ 26 rain following the start of the iv infusion. During hyperglycemia phase 3 activity occurred much earlier 33+14 min (p<0.05). However when phase 3 activity was excluded, in the first 60 min, there were less pressure waves in both the duodenum (33 :t: 10 v 182 + 48, p<0.05) and jejunum (114 + 14 v 210 + 21, p<0.05) during hyperglycemia compared to euglycemia. We conclude that in healthy subjects acute hyperglycemia stimulates premature phase 3 activity, but is associated with an overall reduction in postprandial duodenal and jejunal motility. The effects of hyperglycemia are likely to contribute to disordered small intestinal motility in patients with diabetes mellitus.
Motility and Nerve-Gut Interactions
A601
• ENHANCED SENSITIVITY O F THE WHOLE GUT IN PATIENTS WITH IRRITABLE BOWEL SYNDROME (IBS), 1C Y Francis. ~L A Houghton, IP J Whorwell, 2j Morris, 3j Bradbury. Departments of 1Medicine and 2Statistics, University Hospital of South Manchester, and 3Glaxo Research and Development, U.K. Previous studies have shown that various regions of the gut, in partienlar the colon and rectum, are abnormally sensitive to balloon distension in patients with IBS. However, these studies have only examined up to two anatomical regions in the same patient at any one time and thus it is not known whether all or just specific areas of the gut are abnormally sensitive. Sensory and motility responses to balloon distension of the oesophagus [01, duodenum/D], jejunum [J], ileum //], colon [CI and rectum/'R1 were therefore evaluated in random order in 20 patients with diarrhoea predominant IBS (aged 22-57, 11 females) and compared with 20 matched healthy volunteers (aged 20-57, 10 females). All patients had loose stools (> 3 bowel movements per day) and fulfilled the Rome criteria for IBS. In addition, patient anxiety scores were evaluated using the Hospital Anxiety and Depression questionnaire (abnormal > 10). Results In accordance with previous studies, IBS patients had significantly lower sensory thresholds in the R [vol to discomfort (ml): IBS 109(60,225) geometric mean (range) v controls 175(80,325); p=0.001] and C [100(50,180) v166(60,500); p=0.005] compared with controls. Furthermore, IBS patients exhibited significantly lower thresholds for discomfort, provoked by distension of the O [10(6,20) v 20(8,110); p=0.002], D [45(15,170) v 82(40,150); p=0.002], J [38(20,140) v 73(40,170); p<0.001], and 1 [38(15,120) v 55(30,150); p=0.03]. However, these sensory changes were not associated with any change in compliance [ml/cmI-I~O at mean volume to discomfort: O, 0.45(0.26,0.96) v 0.49(0.22,2.38); D, 3.4(1.47,9.63) v 3.21(1.59,9.28); J, 2.44(1.28,6.13) v 2.11(1.07,4.76); 1, 1.76(0.99,14.5) v 1.4(0.77,3.15); C, 3.4(1.08,6.53) v 3.44(1.65,6.2); R, 5.69(2.82,12.5) v 5.68(2.33,10.3)]. In addition, although patients had higher anxiety scores [9(2,19), median (range)] than controls [4.5(2,7); p<0.001], there was no correlation between their anxiety score and sensory threshold in the D (r= -0.21; p=0.39), J (r= -0.18; p=0.46), I (r-0.23; p=0.45), C (r= -0.28; p=0.38) and R (r= -0.41; p=0.08), with the exception of the O (r= -0.54; p=0.027). Conclusion This is the first study to confirm the suspicion that IBS represents a generalized disorder of visceral sensation. Furthermore, although these results do not exclude a sensory defect at a local level within the gut, they lend some support to the concept of there being an abnormality of the visceral afferent nervous system or its central modulation.
• CELLULAR MECHANISMS OF THE NO-INDUCED INHIBITION IN RAT ILEUM: INVOLVEMENT OF INTRACELLULAR CA2+ STORES. H. Franck, M. Storr, A. Puschmann, H.-D. Allescher. Department of Internal Medicine lI, Tech. University of Munich, Germany
• EFFECTS OF PATHOPHYSIOLOGIC LEVELS OF GLUCAGON ON GLUCOSE DELIVERY, ABSORPTION AND HOMEOSTASIS IN NONDIABETIC HUMANS. J.W. Frank, G.M. Thomforde, S.F. Dineen, R.A. Rizza, M. CamiUeri. Mayo Clinic, Rochester, MN
• EFFECTS OF H Y P E R G L Y C E M I A ON SMALL INTESTINAL MOTILITY AND TRANSIT IN HEALTHY HUMANS. R Fraser, A Rosso, J Dent, M Horowitz. Depts Medicine & Gastrointestinal Medicine, Royal Adelaide Hospital, Adelaide, Australia
Pharmacologic levels of glucagon impair gastric emptying. Failure of postprandial giucagon suppression is an important determinant of postprandial hyperglycemia in diabetes mellitns (DM) by hepatic glucose release (HGR). However, it is unclear whether such pathophysiologic levels compensate for enhanced HGR by reducing glucose delivery from the stomach or its absorption. Our aim was to characterize the effect of physiologic and supraphysiologic levels of glucagon on gastric emptying, carbohydrate absorption, glucose homeostasis, and plasma levels of selected gut neuropeptides in healthy subjects. Methods: We assessed the effects of physiologic and supraphysiologic (n=6 each) levels of glucagon and of saline (placebo) in 12 healthy subjects. We measured gastric emptying of solids and liquids seintigraphically, as well as appearance of meal glucose and HGR using a well-validated isotopic dilution technique. Results: The groups were comparable for age, body mass index, and fasting blood glucose. Low and high dose (0.65 and 3 ng/kg/min) glucagon infusions resulted in mean peak glucagon concentrations of 70 and 350 ng/ml respectively, levels observed postprandially in DM and severe stress respectively. Glucagon did not alter gastric emptying of liquids or solids, rate of appearance of glucose from the meal in plasma, or disappearance of glucose from plasma. However, D-xylose was more slowly absorbed (P=0.009) in the group receiving the higher dose of glucagon. The high, but not low dose of glucagon infusion increased HGR during the first 2 hours after meal ingestion. Plasma insulin, CCK, GIP, neurotensin and PYY did not differ between the 3 groups (i.e., saline, high dose and low dose glucagon). Conclusion: Neither physiologic nor supraphysiologie levels of glecagon alter gastric emptying; higher levels transiently increase HGR postprandially and reduce absorption of D-xylose (which occurs passively) but do not alter the rate of appearance of meal glucose in peripheral plasma. These data are consistent with known inhibition of gut mucosal blood flow by glucagon and/or other effects on the unstirred layer and permeability. The motor effects of physiologic levels of glucagon have minimal impact on glucose homeostasis in nondiabetic humans.
The aim of the present study was to investigate the cellular mechanisms of the inhibition induced by the NO-donor SIN-1 and two membrane-permeable proteinkinase G activators in rat ileum smooth muscle in vitro. Isolated muscle strips were stimulated in an organ bath with carbachol (1045 M) or with the Ca2+ channel activator Bay-K-8644 (10-7 M). The NO-donor S1N-I (ED50: 4.5 x 10-4 M, n=12), 8-Br-cGMP 0ED50:7.4 x 10"4" M, n=12) and 8-pCPTcGMP (ED50:8.1 + 3.0 x 10-5 M, n=8) caused a dose-dependent TTXinsensitive inhibition of the ileal muscle, which was net influenced by pyroganol (10 -4 M). The inhibitor of cGMP-dependent proteinkinase I~, Rp-8-pCPTcGMPS (10-4 M), showed no influence on the SIN-I- and 8-pCPT-cGMPinduced inhibition. The K + channel blockers apamin (10-7 M), charybdotoxin (10 -7 M) glibenclamide (10-5 M) and TEA (10-1 M) had no influence on the iruhibitory effects. Blockade of the sarcoplasmic Ca2+ATPase with cyclopiazonic acid (CPA)(10 -5 M) induced a contraction which could be inhibited completely by 8-pCPT-cGMP and by SIN-1. However, SIN-I or 8pCPT-cGMP showed no inhibition when CPA and carbachol where applied simultaneously. After incubation in Ca2÷ free medium containing CPA ~10'5 M) and subsequent stimulation with carbachol in order to empty the Ca2+ stores, the inhibitory effect of SIN-1 was still completely abolished and the effect of 8-Br-cGMP was significantly reduced, when CPA and CCH were added simultanously. Calphostin C (10-6 M! which is selective for blocking proteinkinase C-dependent mechanisms in the applied dosage, caused a recovery to 41 + 13 % (n=9) of the inhibitory action of SIN-I in presence of both CPA and CCH (compared to controls: 18 -r_ 14 %, n = l l ) . In contrast, Calphostin C I10-6 M) did not change the inhibitory action of SIN-1 on CCHprestimulation alone From these results it can be concluded: a~ The inhibitory effect of SIN-1 seems not to be mediated by proteinkinase G 1~, suggesting a possible role of proteinkinase G In. b) Blockade of the Ca2+ ATPase alone does not affect the inhibitory effect of SIN-I. 8-Br-cGMP and 8-pCPT-cGMP. However_ the inhibitory action of the tested mediators of NO-signaltransduction is dependent on functional intraceilular Ca2+ stores. The recovery of NOinduced Inhibition with Calphostin C demonstrates participation of proteinkinase C-signaltransduction in the described effect. DFG-AI-245/8-1 and DAAD 322.
Recent studies indicate that the blood glucose concentration has a major impact on gastrointestinal motility in both normal subjects and patients with diabetes mellitus. Hyperglycemia slows small intestinal transit, but the motor mechanisms responsible for this effect are unknown. 6 healthy volunteers (aged 22-41yr) underwent paired studies during euglycemia (47 mmol/1) and hyperglycemia (12-16 mmol/1) in randomised order. Pressures in the distal antrum (1 site), duodenum (3 sites) and jejunum (3 sites) were measured with a multi-lumen manometric assembly. Sideholes were spaced at 10 cm intervals and catheter position was monitored using measurements of antroduodenal transmueosal potential difference. Hyperglycemia was induced by rapid infusion of 150 mi 25% dextrose and maintained with an iv dextrose infusion. In the study performed during euglycemia a similar volume of iv normal saline was given. On each study day 10 rain after commencement of iv dextrose or saline a liquid meal comprising 100 ml Ensure and 15g lactulose was instilled into the duodenum over 20 rain. The number of duodenal and jejunal pressure waves was counted. Phase 3 activity was defined as bursts of pressure waves (>10/min) with a duration of more than 3 minutes which began in the duodenum and propagated distally. Small intestinal U'ansit was quantified by breath hydrogen measurements. Data are shown as mean values + SEM. Small intestinal transit was slower during hyperglycemia than euglycemia (120 + 9 rain v 60 + 8 min, p<0.05). During euglycemia, instillation of the meal induced a fed pattern of motor activity within 15 rain and the first episode of intestinal phase 3 activity occurred 116+ 26 rain following the start of the iv infusion. During hyperglycemia phase 3 activity occurred much earlier 33+14 min (p<0.05). However when phase 3 activity was excluded, in the first 60 min, there were less pressure waves in both the duodenum (33 :t: 10 v 182 + 48, p<0.05) and jejunum (114 + 14 v 210 + 21, p<0.05) during hyperglycemia compared to euglycemia. We conclude that in healthy subjects acute hyperglycemia stimulates premature phase 3 activity, but is associated with an overall reduction in postprandial duodenal and jejunal motility. The effects of hyperglycemia are likely to contribute to disordered small intestinal motility in patients with diabetes mellitus.