- Email: [email protected]
Hypoxia down-regulates Na-K-2Cl cotransporter (NKCC1) gene expression and function
AG08AGA ABSTRACTS
GASTROENTEROLOGY Vol. 118, No.4
3132
3134
LEVAMISOLE INHmITS INTESTINAL EPITHELIAL CHLORIDE SECRETION: MULTIPLE ACTIONS AT APICAL AND BASOLATERAL MEMBRANE SITES. Edward C. Mun, Celina M. Hanson, Isabel Calvo, Jaekyung C. Song, Amanda Hillman, Jeffrey B. Matthews, Beth Israel Deaconess Med Ctr & Harvard Med Sch, Boston, MA.
SMALL INTESTINAL GLUCOSE ABSORPTION IS INCREASED IN TYPE 1 DIABETES MELLITUS. Christopher Keith Rayner, Matthijs P. Schwartz, P. Sytze van Dam, Willem Renooij, Martin de Smet, Michael Horowitz, Andre 1. Smout, Melvin Samsom, Royal Adelaide Hosp, Adelaide, Australia; Univ Hosp Utrecht, Utrecht, Netherlands. Intestinal glucose absorption is increased in animal models of diabetes mellitus, but little data is available in humans. Disordered small intestinal motility in diabetes potentially affects glucose absorption. Our aim was to evaluate duodenal motility and glucose absorption in patients with type I diabetes mellitus (DM). 9 healthy volunteers (median 26 yrs, 22-54) and 6 patients with type I DM (37 yrs, 30-46), 2 of whom had evidence of cardiac autonomic neuropathy, were studied. A manometric catheter with 12 duodenal sideholes was positioned across the pylorus. During euglycemia, a nutrient liquid was infused intraduodenally for 30 min (Nutridrink, 3 kcallmin), followed by 4 g/25 mL 3-0-methylglucose (30MG) over 2 minutes. Blood was sampled every 5 min for the first hour, and then every 30 min; 30MG concentrations were measured by gas-liquid chromatography. Automated analysis of duodenal manometry was performed. Comparisons were made using Student's t test. Both the absorption rate coefficient (ka) (P<0.05) and the peak concentration of 30MG (P=0.05) were greater in DM patients than controls, with no difference in the timing of the peak. The area under the 30MG curve in the first 30 minutes tended to be greater (P=0.09) in the patients. There was no overall difference in the frequency of duodenal pressure waves and propagated sequences between patients and controls, although there was a non-significant trend for more duodenal waves (785::':127 v 536::':59, P=0.08) and antegrade duodenal sequences (167::':34 v 100::':12, P=0.06) in DM patients in the 30 minute period after 3-0MG infusion. We conclude that absorption of glucose in the small intestine is increased in patients with type I DM when compared with healthy controls, which may potentially relate to increased duodenal motility.
Levamisole (LEV) weakly activates CFfR, the apical Cl channel of cAMP-mediated Cl secretion, via effects on an alkaline phosphatase. Despite activating CFrR, we found that LEV inhibits cAMP-sensitive basolateral K+ channels. We now examine LEV action on the major alternative (Ca2+) pathway of CI- secretion and on Ca2+-regulated K+ channels. Methods: In T84 intestinal epithelial cells grown on permeable supports, CI- secretion was measured as short-circuit current (Ise> by voltage clamp. Basolateral K+ and apical Cl currents (IK and Id were measured after nystatin permeabilization of the contralateral membrane. Data are mean ::': SEM, *=p<.05 by ANOV A. Results: LEV inhibited the I se response to the muscarinic M 3 receptor (M3R) Ca 2+ -agonist carbachol with IC50~300MM (Ise=94::':6 vs. 47::':6MAJcm2 for control vs. 300MM LEV, n = 120*). LEV right-shifted the dose-response to carbachol, consistent with competitive blockade of M 3R (total n>IOO*). In contrast, LEV enhanced the I se to two Ca2+-agonists acting distal to M 3R (Isc = 156::':13 and 187::': 23 percent control for A23187 and thapsigargin respectively, n= 13*). In apically permeabilized cells, LEV abolished the I K elicited by carbachol (*) but not by A23187 or by thapsigargin (n=4). In basolaterally permeabilized cells, LEV did not augment Iel in the presence of A23187 or thapsigargin (n=4). A brominated D-isomer derivative of LEV (D-bromotetramisole, inactive against alkaline phosphatase) blocked I se with greater potency than either the L-isomer or LEV (n=72*). Conclusions: LEV inhibits cholinergic secretion via competitive M 3R blockade. LEV does not inhibit Ca 2+ -regulated K+ channels when activated distal to M 3R activation, but instead enhances net Cl secretion due to LEV action at apical CFfR. Our data illustrate that CI- secretion involves integrated events at both apical and basolateral membranes, and that single agents may exert multiple and even opposing actions on these sites. Because LEV and its D-brominated derivatives inhibit both cAMP and cholinergic Cl secretory responses (albeit by different mechanisms), these agents hold promise as novel antisecretory therapy.
3133 HYPOXIA DOWN·REGULATES NA-K·2CL COTRANSPORTER (NKCC1) GENE EXPRESSION AND FUNCTION. Edward C. Mun, G.D. Vivek Sagar, Celina M. Hanson, Jaekyung C. Song, Jeffrey B. Matthews, Beth Israel Deaconess Med Ctr & Harvard Med Sch, Boston, MA. A variety of intestinal disorders are associated with tissue hypoxia, a stimulus that is increasingly recognized to alter patterns of gene expression and protein function that favor cell adaptation and survival. Taylor et al (J Pharmacol Exp Ther, 1998) showed that hypoxia inhibits cAMP-mediated cr secretion in cultured intestinal epithelial cells, an effect that at least in part involves down-regulation of apical CFfR CI- channels. We have shown that NKCC I, the transport site responsible for basolateral chloride entry, is a major determinant of the rate of transepithelial Cl secretion, and that NKCCI gene expression may be regulated independently of apical CFrR. Aim: To examine whether hypoxia-mediated inhibition of Cl secretion is regulated by changes in NKCCI function and gene expression. Methods: T84 intestinal epithelial cells grown on flasks or permeable supports were subjected to low (1%) O2 in hypoxic chambers. NKCCI activity was measured by bumetanide-sensitive 86Rb+ trans-flux across monolayers that were apically permeabilized with nystatin (allowing NKCCI function to be assessed independent of changes in driving force and other transport sites). NKCCI mRNA expression was assessed by Northern assay, and NKCCI protein levels were assayed by irnmunoprecipitationlWestern blotting. Surface expression of NKCCI was examined by selective basolateral membrane biotinylation. Relative amounts of mRNA and protein bands were quantified by densitometry. Results: Hypoxia inhibited cAMP-elicited cr secretion in a time-dependent manner (~40% reduction at 12 hrs, p<.05, n=3), consistent with earlier reports. Following 24 hr hypoxia, both basal and stimulated 86Rb+ transflux were decreased (27.0% inhibition for basal activity, p<.Ol, and 37.5% reduction for stimulated NKCCI activity, p<.05, n=3 each). Hypoxia decreased NKCCI mRNA in a time-dependent fashion (-75% decrease at 24 hrs). Total cellular NKCCI protein level was also decerased by hypoxia (57% inhibition at 24 hrs). Additionally, the relative amount of NKCCI expressed in the basolateral membrane surface (as determined by accessibility to cell surface biotinylation) was reduced following 24 hrs of hypoxia (57.7% decrease from normoxic control). Conclusions: NKCCI function is inhibited by hypoxia, independent of the loss of apical CI- channel function and likely due to downregulation of NKCCI gene expression. This event may contribute to the inhibition of net cAMP-dependent CI- secretion observed under hypoxic conditions.
Results (mean±SEM)
healthy controls DM patients Pvalue
ka
peak30MG (mM)
limeof peak (min)
AUCal30 min
0,056±0,01 0,085±0,01 004
0,64±0.08 0,88±0,07 0,05
17±2 19±2 0,47
2,66±033 3.51±0,25 0,09
3135 EFFECTS OF ADENOSINE (ADO) ON HUMAN COLONIC MU· COSA IN VITRO. Martin Riegler, Charalabos Pothoulakis, Tacettin Sogukoglu, Maria Prettenhofer, Edwin C. Mun, Jeffrey B. Matthews, Michael Wlk, Etienne Wenzl, Univ Clin of Surg, Vienna, Austria; Beth Israel Deaconess Med Ctr, Boston, MA. INTRODUCTION:Adenosine has a broad but discrete localization throughout the digestive tract. A growing body of evidence indicates that adenosine is involved in the mediation of colonic secretion. For example, adenosine (ADO) participates in the mediation of neurotensin stimulated chloride secretion in human colonic mucosa in vitro and induces chloride secretion in monolayers of cultured colonic epithelial (T84) cells. However, the effect of ADO on human colonic mucosa is not known. Therefore this study was designed to investigate the effect of ADO on human colonic mucosa in vitro and to explore the mechanism by which ADO mediates colonic responses. METHODS:We tested the effect of ADO on short circuit current (Isc, an electrophysiologic measure for transepithelial ion movement) of Ussing chambered human colonic mucosa in CI-free and Cl-containing buffer. Drugs (10. 8 to 10-4) for pharmacologic characterization of ADO-induced Isc-changes were added serosally 30 min prior to serosal administration of ADO (10-4M). RESULTS:Serosal administration of adenosine caused a dose-dependent Isc increase (/lIsc 47.63::':3.4, 30.25::':4, 16.75::':0.67, 2.88::':0.35 MAJcm 2 , following administration of 10-3 , 10.4, 10-5, and 10-6 M of ADO, respectively, n=6), which was completely blocked in CI-free buffer and restored in CI-containing buffer. ADO-induced Isc increase was inhibited by NalK/2CL-cotransporter inhibitor burnetanide, prostaglandin synthesis inhibitor indomethacin, nerve cell blocker tetrodotoxin, the nicotinic and muscarinic receptor antagonist hexamethonium and atropine, respectively. Unspecific ADO-receptor (ADOR)inhibitor 8-phenyltheophyllin, specific ADOR2 inhibitor 3,7-dimethyl-l-propargylxanthine, and specific ADORI inihibitor 1,3-dipropyl8-(2-amino-4-chlorophenyl)xanthine,inhibited ADO response. Adenosine-2 receptor inhibitor was 10 times more potent than Adenosine-I receptor inhibitor in reducing adenosine-induced Isc increase. Preincubation with ADO-uptake inhibitors S-(4-nitrobenzyl)-6-thio-guanosine and -inosine prolonged Isc response by 30%, without impairing Isc-increase. CONCLUSION: Adenosine (ADO)stimulates chloride secretion in human colonic mucosa by a mechanism involving specific receptors.mucosal nerves, and the secretagogues acethylcholine and prostaglandins, Our data indicate that adenosine may play an important role in the mediation of chloride secretion in human colon.
GASTROENTEROLOGY Vol. 118, No.4
3132
3134
LEVAMISOLE INHmITS INTESTINAL EPITHELIAL CHLORIDE SECRETION: MULTIPLE ACTIONS AT APICAL AND BASOLATERAL MEMBRANE SITES. Edward C. Mun, Celina M. Hanson, Isabel Calvo, Jaekyung C. Song, Amanda Hillman, Jeffrey B. Matthews, Beth Israel Deaconess Med Ctr & Harvard Med Sch, Boston, MA.
SMALL INTESTINAL GLUCOSE ABSORPTION IS INCREASED IN TYPE 1 DIABETES MELLITUS. Christopher Keith Rayner, Matthijs P. Schwartz, P. Sytze van Dam, Willem Renooij, Martin de Smet, Michael Horowitz, Andre 1. Smout, Melvin Samsom, Royal Adelaide Hosp, Adelaide, Australia; Univ Hosp Utrecht, Utrecht, Netherlands. Intestinal glucose absorption is increased in animal models of diabetes mellitus, but little data is available in humans. Disordered small intestinal motility in diabetes potentially affects glucose absorption. Our aim was to evaluate duodenal motility and glucose absorption in patients with type I diabetes mellitus (DM). 9 healthy volunteers (median 26 yrs, 22-54) and 6 patients with type I DM (37 yrs, 30-46), 2 of whom had evidence of cardiac autonomic neuropathy, were studied. A manometric catheter with 12 duodenal sideholes was positioned across the pylorus. During euglycemia, a nutrient liquid was infused intraduodenally for 30 min (Nutridrink, 3 kcallmin), followed by 4 g/25 mL 3-0-methylglucose (30MG) over 2 minutes. Blood was sampled every 5 min for the first hour, and then every 30 min; 30MG concentrations were measured by gas-liquid chromatography. Automated analysis of duodenal manometry was performed. Comparisons were made using Student's t test. Both the absorption rate coefficient (ka) (P<0.05) and the peak concentration of 30MG (P=0.05) were greater in DM patients than controls, with no difference in the timing of the peak. The area under the 30MG curve in the first 30 minutes tended to be greater (P=0.09) in the patients. There was no overall difference in the frequency of duodenal pressure waves and propagated sequences between patients and controls, although there was a non-significant trend for more duodenal waves (785::':127 v 536::':59, P=0.08) and antegrade duodenal sequences (167::':34 v 100::':12, P=0.06) in DM patients in the 30 minute period after 3-0MG infusion. We conclude that absorption of glucose in the small intestine is increased in patients with type I DM when compared with healthy controls, which may potentially relate to increased duodenal motility.
Levamisole (LEV) weakly activates CFfR, the apical Cl channel of cAMP-mediated Cl secretion, via effects on an alkaline phosphatase. Despite activating CFrR, we found that LEV inhibits cAMP-sensitive basolateral K+ channels. We now examine LEV action on the major alternative (Ca2+) pathway of CI- secretion and on Ca2+-regulated K+ channels. Methods: In T84 intestinal epithelial cells grown on permeable supports, CI- secretion was measured as short-circuit current (Ise> by voltage clamp. Basolateral K+ and apical Cl currents (IK and Id were measured after nystatin permeabilization of the contralateral membrane. Data are mean ::': SEM, *=p<.05 by ANOV A. Results: LEV inhibited the I se response to the muscarinic M 3 receptor (M3R) Ca 2+ -agonist carbachol with IC50~300MM (Ise=94::':6 vs. 47::':6MAJcm2 for control vs. 300MM LEV, n = 120*). LEV right-shifted the dose-response to carbachol, consistent with competitive blockade of M 3R (total n>IOO*). In contrast, LEV enhanced the I se to two Ca2+-agonists acting distal to M 3R (Isc = 156::':13 and 187::': 23 percent control for A23187 and thapsigargin respectively, n= 13*). In apically permeabilized cells, LEV abolished the I K elicited by carbachol (*) but not by A23187 or by thapsigargin (n=4). In basolaterally permeabilized cells, LEV did not augment Iel in the presence of A23187 or thapsigargin (n=4). A brominated D-isomer derivative of LEV (D-bromotetramisole, inactive against alkaline phosphatase) blocked I se with greater potency than either the L-isomer or LEV (n=72*). Conclusions: LEV inhibits cholinergic secretion via competitive M 3R blockade. LEV does not inhibit Ca 2+ -regulated K+ channels when activated distal to M 3R activation, but instead enhances net Cl secretion due to LEV action at apical CFfR. Our data illustrate that CI- secretion involves integrated events at both apical and basolateral membranes, and that single agents may exert multiple and even opposing actions on these sites. Because LEV and its D-brominated derivatives inhibit both cAMP and cholinergic Cl secretory responses (albeit by different mechanisms), these agents hold promise as novel antisecretory therapy.
3133 HYPOXIA DOWN·REGULATES NA-K·2CL COTRANSPORTER (NKCC1) GENE EXPRESSION AND FUNCTION. Edward C. Mun, G.D. Vivek Sagar, Celina M. Hanson, Jaekyung C. Song, Jeffrey B. Matthews, Beth Israel Deaconess Med Ctr & Harvard Med Sch, Boston, MA. A variety of intestinal disorders are associated with tissue hypoxia, a stimulus that is increasingly recognized to alter patterns of gene expression and protein function that favor cell adaptation and survival. Taylor et al (J Pharmacol Exp Ther, 1998) showed that hypoxia inhibits cAMP-mediated cr secretion in cultured intestinal epithelial cells, an effect that at least in part involves down-regulation of apical CFfR CI- channels. We have shown that NKCC I, the transport site responsible for basolateral chloride entry, is a major determinant of the rate of transepithelial Cl secretion, and that NKCCI gene expression may be regulated independently of apical CFrR. Aim: To examine whether hypoxia-mediated inhibition of Cl secretion is regulated by changes in NKCCI function and gene expression. Methods: T84 intestinal epithelial cells grown on flasks or permeable supports were subjected to low (1%) O2 in hypoxic chambers. NKCCI activity was measured by bumetanide-sensitive 86Rb+ trans-flux across monolayers that were apically permeabilized with nystatin (allowing NKCCI function to be assessed independent of changes in driving force and other transport sites). NKCCI mRNA expression was assessed by Northern assay, and NKCCI protein levels were assayed by irnmunoprecipitationlWestern blotting. Surface expression of NKCCI was examined by selective basolateral membrane biotinylation. Relative amounts of mRNA and protein bands were quantified by densitometry. Results: Hypoxia inhibited cAMP-elicited cr secretion in a time-dependent manner (~40% reduction at 12 hrs, p<.05, n=3), consistent with earlier reports. Following 24 hr hypoxia, both basal and stimulated 86Rb+ transflux were decreased (27.0% inhibition for basal activity, p<.Ol, and 37.5% reduction for stimulated NKCCI activity, p<.05, n=3 each). Hypoxia decreased NKCCI mRNA in a time-dependent fashion (-75% decrease at 24 hrs). Total cellular NKCCI protein level was also decerased by hypoxia (57% inhibition at 24 hrs). Additionally, the relative amount of NKCCI expressed in the basolateral membrane surface (as determined by accessibility to cell surface biotinylation) was reduced following 24 hrs of hypoxia (57.7% decrease from normoxic control). Conclusions: NKCCI function is inhibited by hypoxia, independent of the loss of apical CI- channel function and likely due to downregulation of NKCCI gene expression. This event may contribute to the inhibition of net cAMP-dependent CI- secretion observed under hypoxic conditions.
Results (mean±SEM)
healthy controls DM patients Pvalue
ka
peak30MG (mM)
limeof peak (min)
AUCal30 min
0,056±0,01 0,085±0,01 004
0,64±0.08 0,88±0,07 0,05
17±2 19±2 0,47
2,66±033 3.51±0,25 0,09
3135 EFFECTS OF ADENOSINE (ADO) ON HUMAN COLONIC MU· COSA IN VITRO. Martin Riegler, Charalabos Pothoulakis, Tacettin Sogukoglu, Maria Prettenhofer, Edwin C. Mun, Jeffrey B. Matthews, Michael Wlk, Etienne Wenzl, Univ Clin of Surg, Vienna, Austria; Beth Israel Deaconess Med Ctr, Boston, MA. INTRODUCTION:Adenosine has a broad but discrete localization throughout the digestive tract. A growing body of evidence indicates that adenosine is involved in the mediation of colonic secretion. For example, adenosine (ADO) participates in the mediation of neurotensin stimulated chloride secretion in human colonic mucosa in vitro and induces chloride secretion in monolayers of cultured colonic epithelial (T84) cells. However, the effect of ADO on human colonic mucosa is not known. Therefore this study was designed to investigate the effect of ADO on human colonic mucosa in vitro and to explore the mechanism by which ADO mediates colonic responses. METHODS:We tested the effect of ADO on short circuit current (Isc, an electrophysiologic measure for transepithelial ion movement) of Ussing chambered human colonic mucosa in CI-free and Cl-containing buffer. Drugs (10. 8 to 10-4) for pharmacologic characterization of ADO-induced Isc-changes were added serosally 30 min prior to serosal administration of ADO (10-4M). RESULTS:Serosal administration of adenosine caused a dose-dependent Isc increase (/lIsc 47.63::':3.4, 30.25::':4, 16.75::':0.67, 2.88::':0.35 MAJcm 2 , following administration of 10-3 , 10.4, 10-5, and 10-6 M of ADO, respectively, n=6), which was completely blocked in CI-free buffer and restored in CI-containing buffer. ADO-induced Isc increase was inhibited by NalK/2CL-cotransporter inhibitor burnetanide, prostaglandin synthesis inhibitor indomethacin, nerve cell blocker tetrodotoxin, the nicotinic and muscarinic receptor antagonist hexamethonium and atropine, respectively. Unspecific ADO-receptor (ADOR)inhibitor 8-phenyltheophyllin, specific ADOR2 inhibitor 3,7-dimethyl-l-propargylxanthine, and specific ADORI inihibitor 1,3-dipropyl8-(2-amino-4-chlorophenyl)xanthine,inhibited ADO response. Adenosine-2 receptor inhibitor was 10 times more potent than Adenosine-I receptor inhibitor in reducing adenosine-induced Isc increase. Preincubation with ADO-uptake inhibitors S-(4-nitrobenzyl)-6-thio-guanosine and -inosine prolonged Isc response by 30%, without impairing Isc-increase. CONCLUSION: Adenosine (ADO)stimulates chloride secretion in human colonic mucosa by a mechanism involving specific receptors.mucosal nerves, and the secretagogues acethylcholine and prostaglandins, Our data indicate that adenosine may play an important role in the mediation of chloride secretion in human colon.