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Diurnal variation in SGLT1 induction and function
AGIO AGAABSTRACTS
GASTROENTEROLOGY Vol. 118, No.4
3140
3142
MOUSE GUANYLIN AND UROGUANYLIN ARE PARADOXICALLY INCREASED IN TISSUE AND SERUM IN RESPONSE TO OSMOTIC DIARRHEA. Kris A. Steinbrecher, James T. Knapmeyer, Mitchell B. Cohen, Acad Hosp Med Ctr, Cincinnati, OH. Guanylin (Gn) and uroguanylin (Ugn) are endogenous peptide hormones that are highly homologous to the diarrhea-causing E. coli enterotoxins. These putative secretagogues are released from intestinal epithelia into the intestinal lumen and systemic circulation. Once secreted, Gn and Ugn bind their transmembrane receptor Guanylate Cyclase C (GC-C), initiating signal transduction pathways that lead to fluid secretion in the intestine. However, there is little evidence to suggest that this pathway plays a significant role in intestinal or systemic fluid homeostasis in the whole animal. In an effort to determine whether Gn or Ugn respond to changes in intestinal fluid balance, we hypothesized that a diet rich in lactose would osmotically draw water into the intestinal lumen of mice and cause a compensatory downregulation of GnlUgn. Mice were fed either a standard control diet or a 75% lactose/25% control diet for either 2 days or 4 days. Mice were sacrificed and gut and carcass weights were obtained at each time point. Gn and Ugn levels were determined by Northern and Western analysis in intestine, kidney and serum. Animals fed the lactose diet for 2 days developed overt diarrhea. weight loss and significantly increased gut-to-carcass ratios. GC-C RNA levels were unchanged. Surprisingly. 2 days on the lactose diet resulted in significantly increased Gn and Ugn RNA and protein levels throughout the intestine and increased Ugn RNA and protein levels in the kidney. In addition, circulating levels of Ugn were also increased at 2 days. These increases were specific to the lactose diet. No change was seen in GnlUgn RNA after 2 days of fasting or 2 days of fluid deprivation. By 4 days, when the diarrhea had resolved, RNA levels of both GnIUgn had returned to baseline. Protein levels remained elevated only in cecum and kidney. We conclude that this model of osmotic diarrhea results in paradoxically increased levels of these peptide ligands in epithelia of the intestine and kidney as well as an increased release of Ugn into the blood. These data are contradictory to current thinking concerning the physiological role of these ligands in fluid homeostasis. We therefore speculate that Gn and Ugn, or their prohormones, have a major function distinct from their potential roles as intestinal secretagogues.
DIURNAL VARIATION IN SGLTI INDUCTION AND FUNCTION. Ali Tavakkolizadeh, Urs V. Berger, David B. Rhoads, Robert Shen, Lynne L. Levitsky, Michael J. Zinner, Stanley W. Ashley, Edward E. Whang, Brigham & Women's Hosp, Harvard Med Sch, Boston, MA; Harvard Med Sch, Boston, MA; MA Gen Hosp, Harvard Med Sch, Boston, MA.
3141 THE OXIDANT MONOCHLORAMINE POTENTIATES AGONIST·STIMULATED CL SECRETION BY DIRECT ACTIVATION OF BASOLATERAL MEMBRANE K CONDUCTANCE IN HUMAN INTESTINAL T84 CELLS. Kazunori Sugi, Mark W. Musch, Anca Di, Deborah J. Nelson, Eugene B. Chang, Univ of Chicago, Chicago, IL. Background: Monochloramine (MC), a physiologically important and cell permeant oxidant, potentiates the secretory effect of both calcium- and cAMP-mediated secretagogues in T84 cells. This effect would significantly amplify the effects of mucosal inflammation in causing net secretion and watery diarrhea. Through indirect measures, we determined that the oxidant was affecting a Rb efflux pathway and hypothesized that was a result of direct activation andlor alteration of the calcium-sensitivity of this K channel. In this study. we tested this hypothesis by performing whole cell electrophysiological measurements in T84 cells under Ca-clamped conditions. Methods: Whole cell recordings of T84 cells were performed at room temperature in a nominally Ca++-free NMDG-aspartate bath solution. Current/voltage (IIV) relationships were determined in the cells prior to and following exposure to O.lmM MC in solutions in which K+ was the only permeant species and its theoretical reversal potential being 81 mV. Internal solutions were buffered to either 0,40, or 100 nM Ca++ using BAPTA or EGT A. Results: The addition of MC stimulated a current potential which reached a plateau after 2-5 min under Ca-free conditions where the mean current at 180 mV increased 28 fold. At 40 nM Ca++, an increase of 31 fold was observed. Interestingly, the TEA-sensitivity of the K conductance was lost following addition of MC, whereas it continued to be sensitive to the inhibitor quinine (lmM), possibly due to covalent modification of the transporter. The electrophysiological finding thus confirm previous observations made with measurement of Rb efflux. [Conclusions] The oxidant monochloramine potentiates the secretory effects of both Ca++ - and cAMP-stimulated secretion by a direct effect on calciumactivated basolateral K channel conductance. lowering its Ca-activation threshold. This resulting increase in K recycling combined with agoniststimulated increases in apical membrane anion conductance would markedly increase the magnitude of active secretion. This effect may play an important role in amplifying and prolonging the secretory response of inflamed intestinal mucosa and enhancing the severity of diarrhea.
Background: Diurnal variation in intestinal sugar absorption has been described, but its underlying mechanisms are unknown. We hypothesized that this phenomenon maybe due to periodicity in SGLTI transcription and activity. Materials and Methods: Rats in a 12 hour light-dark cycle (07: 00-19:00) were sacrificed at 10:00, 16:00, 22:00, or 04:00. Proximal jejunum was mounted in Ussing chambers and ~I sc in response to addition of 30mM 3-0-Methylglucose (3-0-MG) was recorded. 3H_Ia_ belled 3-0-MG fluxes were calculated before and after addition of lOOIJ.M phloridzin. SGLTl mRNA expression was evaluated using in-situ hybridization. Results: 3-0-MG induced ~I sc and absorptive fluxes were significantly greater at 16:00 and 22:00 (Table I). These increases were phloridzin inhibitable. There was increased mRNA expression along the entire crypt-villus axes, including the upper third of the villi, at the above time points. At 04:00 and 10:00, SGLTl mRNA was confined to the crypt and lower villi. Conclusions: The diurnal variation in sugar absorption may be due to periodicity in SGLTl transcription and function.
Table 1 TIme
M..l!iAlcm2)
Jm-.
J~m
J...
10:00 16:00 22:00
844±7.2 1374±13.8· 136,4±12.8· 1167±8.5
2.83±O.16 4.22±O.25' 3.36±O.28. 3.01±0.26
1.3?±O.30 1.3?±O.15 1.10±O.15 0.8?±0.0?
1,4?±O,41 2.86±O.33· 2.26±O22 2.14±O22
04:00
• p<0.05 compared to 10:00 using ANOVA and post-hoc Tukey test Jm-', J.-m, Jne. measured in!lmole.cm'.hr'
3143 EVIDENCE FOR TWO DISTINCT TRANSPORTERS INVOLVED IN CHLORIDE ABSORPTION IN THE HUMAN COLONIC APICAL MEMBRANE VESICLES. Sangeeta Tyagi, Reena J. Kavilaveettil, Shadwan Alsafwah, Krishnamurthy Ramaswamy, Pradeep K. Dudeja, Univ of Illinois at Chicago, Chicago, IL. Recent studies have indicated that mutations in human DRA (Down Regulated in Adenoma) gene might be related to Congenital Chloride Diarrhea and that DRA may be responsible for intestinal apical Cl'-HC0:i exchange. The cDNA sequence for DRA is homologous to S04' transporters but not to anion exchangers such as AEs. Also, in-vitro functional studies of DRA have shown it to be capable of 504'. oxalate and CI' transport. Our earlier studies have established the presence of a distinct CI'-OH' (HC0:i) exchanger with minimal affinity for S04' . However, to date, the mechanism(s) of S04' transport across the human colonic apical membrane and the possible role of this transporter in oxalate and cr transport were not elucidated. The aim of our current studies was to elucidate the mechanism of S04' uptake in human proximal colonic apical membrane vesicles (AMV) and to define its interactions with oxalate and cr transport. Our results demonstrate the presence of a pH-gradient driven carrier-mediated S04'-OH' exchange process in these membranes, based on the following: i) a marked increase in S04' uptake in the presence of an outwardly directed OR gradient, ii) significant inhibition of S04' uptake by the anion exchange inhibitor DIDS, and iii) demonstration of saturation kinetics (Km for S04' : 0.80 ± 0.17 mM and Vmax: 649 ± 74 pmoVmg proteinll 0 sec). To elucidate the role of S04'-OH' exchanger in oxalate and CI transport, the effects of oxalate and CI- on S04' uptake were examined. The OH' - gradient driven S04' uptake was significantly inhibited by increasing concentrations of Cl' (1-10 mM) in the incubation media with a K; for Cl of9.3 ± 1.4 mM, indicating lower affinity for chloride. Also, the S04'-OH- exchange was competitively inhibited by oxalate with a Ki lower than for Cl. In contrast, the pH and HC0:i gradient-stimulated Cl uptake was not inhibited by the presence of increasing concentrations of S04' (5-50 mM). Also, S04'-OH- exchange activity was highly sensitive to inhibition by DIDS compared to Cl'-OH' (HC0:i) exchange with a low sensitivity. These data strongly suggest that the S04'-OH'and CI'-HC0:i exchangers are distinct transporters. Based on inhibitor profile and substrate affinities for various anions, we speculate that two distinct transporters may be involved in Cl' transport in the human intestinal luminal membranes: i) an anion exchanger with high affinity for S04' and oxalate but low affinity for Cl; ii) a distinct Cl'-OH' (HCO;) exchanger with low affinity for S04'.
GASTROENTEROLOGY Vol. 118, No.4
3140
3142
MOUSE GUANYLIN AND UROGUANYLIN ARE PARADOXICALLY INCREASED IN TISSUE AND SERUM IN RESPONSE TO OSMOTIC DIARRHEA. Kris A. Steinbrecher, James T. Knapmeyer, Mitchell B. Cohen, Acad Hosp Med Ctr, Cincinnati, OH. Guanylin (Gn) and uroguanylin (Ugn) are endogenous peptide hormones that are highly homologous to the diarrhea-causing E. coli enterotoxins. These putative secretagogues are released from intestinal epithelia into the intestinal lumen and systemic circulation. Once secreted, Gn and Ugn bind their transmembrane receptor Guanylate Cyclase C (GC-C), initiating signal transduction pathways that lead to fluid secretion in the intestine. However, there is little evidence to suggest that this pathway plays a significant role in intestinal or systemic fluid homeostasis in the whole animal. In an effort to determine whether Gn or Ugn respond to changes in intestinal fluid balance, we hypothesized that a diet rich in lactose would osmotically draw water into the intestinal lumen of mice and cause a compensatory downregulation of GnlUgn. Mice were fed either a standard control diet or a 75% lactose/25% control diet for either 2 days or 4 days. Mice were sacrificed and gut and carcass weights were obtained at each time point. Gn and Ugn levels were determined by Northern and Western analysis in intestine, kidney and serum. Animals fed the lactose diet for 2 days developed overt diarrhea. weight loss and significantly increased gut-to-carcass ratios. GC-C RNA levels were unchanged. Surprisingly. 2 days on the lactose diet resulted in significantly increased Gn and Ugn RNA and protein levels throughout the intestine and increased Ugn RNA and protein levels in the kidney. In addition, circulating levels of Ugn were also increased at 2 days. These increases were specific to the lactose diet. No change was seen in GnlUgn RNA after 2 days of fasting or 2 days of fluid deprivation. By 4 days, when the diarrhea had resolved, RNA levels of both GnIUgn had returned to baseline. Protein levels remained elevated only in cecum and kidney. We conclude that this model of osmotic diarrhea results in paradoxically increased levels of these peptide ligands in epithelia of the intestine and kidney as well as an increased release of Ugn into the blood. These data are contradictory to current thinking concerning the physiological role of these ligands in fluid homeostasis. We therefore speculate that Gn and Ugn, or their prohormones, have a major function distinct from their potential roles as intestinal secretagogues.
DIURNAL VARIATION IN SGLTI INDUCTION AND FUNCTION. Ali Tavakkolizadeh, Urs V. Berger, David B. Rhoads, Robert Shen, Lynne L. Levitsky, Michael J. Zinner, Stanley W. Ashley, Edward E. Whang, Brigham & Women's Hosp, Harvard Med Sch, Boston, MA; Harvard Med Sch, Boston, MA; MA Gen Hosp, Harvard Med Sch, Boston, MA.
3141 THE OXIDANT MONOCHLORAMINE POTENTIATES AGONIST·STIMULATED CL SECRETION BY DIRECT ACTIVATION OF BASOLATERAL MEMBRANE K CONDUCTANCE IN HUMAN INTESTINAL T84 CELLS. Kazunori Sugi, Mark W. Musch, Anca Di, Deborah J. Nelson, Eugene B. Chang, Univ of Chicago, Chicago, IL. Background: Monochloramine (MC), a physiologically important and cell permeant oxidant, potentiates the secretory effect of both calcium- and cAMP-mediated secretagogues in T84 cells. This effect would significantly amplify the effects of mucosal inflammation in causing net secretion and watery diarrhea. Through indirect measures, we determined that the oxidant was affecting a Rb efflux pathway and hypothesized that was a result of direct activation andlor alteration of the calcium-sensitivity of this K channel. In this study. we tested this hypothesis by performing whole cell electrophysiological measurements in T84 cells under Ca-clamped conditions. Methods: Whole cell recordings of T84 cells were performed at room temperature in a nominally Ca++-free NMDG-aspartate bath solution. Current/voltage (IIV) relationships were determined in the cells prior to and following exposure to O.lmM MC in solutions in which K+ was the only permeant species and its theoretical reversal potential being 81 mV. Internal solutions were buffered to either 0,40, or 100 nM Ca++ using BAPTA or EGT A. Results: The addition of MC stimulated a current potential which reached a plateau after 2-5 min under Ca-free conditions where the mean current at 180 mV increased 28 fold. At 40 nM Ca++, an increase of 31 fold was observed. Interestingly, the TEA-sensitivity of the K conductance was lost following addition of MC, whereas it continued to be sensitive to the inhibitor quinine (lmM), possibly due to covalent modification of the transporter. The electrophysiological finding thus confirm previous observations made with measurement of Rb efflux. [Conclusions] The oxidant monochloramine potentiates the secretory effects of both Ca++ - and cAMP-stimulated secretion by a direct effect on calciumactivated basolateral K channel conductance. lowering its Ca-activation threshold. This resulting increase in K recycling combined with agoniststimulated increases in apical membrane anion conductance would markedly increase the magnitude of active secretion. This effect may play an important role in amplifying and prolonging the secretory response of inflamed intestinal mucosa and enhancing the severity of diarrhea.
Background: Diurnal variation in intestinal sugar absorption has been described, but its underlying mechanisms are unknown. We hypothesized that this phenomenon maybe due to periodicity in SGLTI transcription and activity. Materials and Methods: Rats in a 12 hour light-dark cycle (07: 00-19:00) were sacrificed at 10:00, 16:00, 22:00, or 04:00. Proximal jejunum was mounted in Ussing chambers and ~I sc in response to addition of 30mM 3-0-Methylglucose (3-0-MG) was recorded. 3H_Ia_ belled 3-0-MG fluxes were calculated before and after addition of lOOIJ.M phloridzin. SGLTl mRNA expression was evaluated using in-situ hybridization. Results: 3-0-MG induced ~I sc and absorptive fluxes were significantly greater at 16:00 and 22:00 (Table I). These increases were phloridzin inhibitable. There was increased mRNA expression along the entire crypt-villus axes, including the upper third of the villi, at the above time points. At 04:00 and 10:00, SGLTl mRNA was confined to the crypt and lower villi. Conclusions: The diurnal variation in sugar absorption may be due to periodicity in SGLTl transcription and function.
Table 1 TIme
M..l!iAlcm2)
Jm-.
J~m
J...
10:00 16:00 22:00
844±7.2 1374±13.8· 136,4±12.8· 1167±8.5
2.83±O.16 4.22±O.25' 3.36±O.28. 3.01±0.26
1.3?±O.30 1.3?±O.15 1.10±O.15 0.8?±0.0?
1,4?±O,41 2.86±O.33· 2.26±O22 2.14±O22
04:00
• p<0.05 compared to 10:00 using ANOVA and post-hoc Tukey test Jm-', J.-m, Jne. measured in!lmole.cm'.hr'
3143 EVIDENCE FOR TWO DISTINCT TRANSPORTERS INVOLVED IN CHLORIDE ABSORPTION IN THE HUMAN COLONIC APICAL MEMBRANE VESICLES. Sangeeta Tyagi, Reena J. Kavilaveettil, Shadwan Alsafwah, Krishnamurthy Ramaswamy, Pradeep K. Dudeja, Univ of Illinois at Chicago, Chicago, IL. Recent studies have indicated that mutations in human DRA (Down Regulated in Adenoma) gene might be related to Congenital Chloride Diarrhea and that DRA may be responsible for intestinal apical Cl'-HC0:i exchange. The cDNA sequence for DRA is homologous to S04' transporters but not to anion exchangers such as AEs. Also, in-vitro functional studies of DRA have shown it to be capable of 504'. oxalate and CI' transport. Our earlier studies have established the presence of a distinct CI'-OH' (HC0:i) exchanger with minimal affinity for S04' . However, to date, the mechanism(s) of S04' transport across the human colonic apical membrane and the possible role of this transporter in oxalate and cr transport were not elucidated. The aim of our current studies was to elucidate the mechanism of S04' uptake in human proximal colonic apical membrane vesicles (AMV) and to define its interactions with oxalate and cr transport. Our results demonstrate the presence of a pH-gradient driven carrier-mediated S04'-OH' exchange process in these membranes, based on the following: i) a marked increase in S04' uptake in the presence of an outwardly directed OR gradient, ii) significant inhibition of S04' uptake by the anion exchange inhibitor DIDS, and iii) demonstration of saturation kinetics (Km for S04' : 0.80 ± 0.17 mM and Vmax: 649 ± 74 pmoVmg proteinll 0 sec). To elucidate the role of S04'-OH' exchanger in oxalate and CI transport, the effects of oxalate and CI- on S04' uptake were examined. The OH' - gradient driven S04' uptake was significantly inhibited by increasing concentrations of Cl' (1-10 mM) in the incubation media with a K; for Cl of9.3 ± 1.4 mM, indicating lower affinity for chloride. Also, the S04'-OH- exchange was competitively inhibited by oxalate with a Ki lower than for Cl. In contrast, the pH and HC0:i gradient-stimulated Cl uptake was not inhibited by the presence of increasing concentrations of S04' (5-50 mM). Also, S04'-OH- exchange activity was highly sensitive to inhibition by DIDS compared to Cl'-OH' (HC0:i) exchange with a low sensitivity. These data strongly suggest that the S04'-OH'and CI'-HC0:i exchangers are distinct transporters. Based on inhibitor profile and substrate affinities for various anions, we speculate that two distinct transporters may be involved in Cl' transport in the human intestinal luminal membranes: i) an anion exchanger with high affinity for S04' and oxalate but low affinity for Cl; ii) a distinct Cl'-OH' (HCO;) exchanger with low affinity for S04'.