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W1662 Mast Cell Regulation of Na-Glutamine Co-Transporters B0AT1 in Villus and SN2 in Crypt Cells in the Chronically Inflamed Small Intestine
W1659
of mouse proximal tubules using forskolin and A23187, agents that inhibit Nhe3 activity and short term dexamethasone treatment an agent that causes acute stimulation of Nhe3 activity. Intracellular pH, monitored by SNARF4, was measured according to Murtazina et al J. Biol. Chem. 282(34): 25141, 2007. Thin slices of mouse kidney cortex were superfused, acidified by NH4 then exposed to TMA or Na and initial rates of alkalization were measured in delta pH/min. HOE 694 was added to inhibit Nhe1 and Nhe2 activity. When forskolin and A23187 were applied to wt and Clcn5 ko mouse tubules both groups had inhibition of NHe3 activity but there was no difference between wt and Clcn5 ko. When short tern dexamethasone was given, wt mouse proximal tubules showed an increase in Nhe3 activity from 0.29±0.02 to 0.39±0.03, p<0.005 (34%) whereas the Clcn5 ko mouse had an initial rate of 0.21±0.01 which increased only to 0.25±0.01, p<0.05 (19%). Previously, surface biotinylation experiments in OK cells, an accepted cell culture model of the proximal tubule, showed that there is normally increased exocytosis of Nhe3 cells are treated with dexamethasone. However in OK cells with Clc-5 silenced, using two separate shRNA constructs, there was reduced exocytosis of Nhe3 (76%) compared to control plasmid or a scrambled Clc-5 shRNA construct (180%). These results suggest that stimulation of Nhe3 activity may be dysregulated in both the Clcn5 ko mouse and in OK cells. We propose that there is more Nhe3 inside the cell and less on the plasma membrane because the basal and regulated insertion of Nhe3 onto the plasma membrane is reduced. We conclude that Clc5, located in early and recycling endosomes, is important for outward trafficking of Nhe3.
AGA Abstracts
Peroxynitrite Inhibits Na-Glucose Co-Transport But Not Na/H Exchange in IEC-18 Cells Palanikumar Manoharan, Ramesh Kekuda, Prosenjit Saha, Subha Arthur, Uma Sundaram Background: During chronic intestinal inflammation in rabbits intestinal villus cell brush border membrane (BBM) Na-glucose co-transport (SGLT1), but not Na/H exchange (NHE3) is inhibited. The mechanism of inhibition is secondary to a decrease in the number of BBM co-transporters. Peroxynitrite (OONO), a potent oxidant, is produced in abundance in the mucosa during chronic enteritis. However, the effect of OONO on SGLT1 and NHE3 in intestinal epithelial cells is unknown. Aim: Determine the effect of OONO on SGLT1 and NHE3 in epithelial cells and decipher the mechanism. Methods: Rat small intestinal epithelial cell (IEC-18) monolayers grown on transwell plates were treated with 100μM SIN-1 (OONO donor) for 24 hours. 3H-OMG uptake and pH dependent, amiloride sensitive 22Na uptake were performed for SGLT1 and NHE3 activity respectively. OONO was measured as fluorescence produced from oxidation of dihydrorhodamine 123. Na+/K+ ATPase activity was determined by measuring the inorganic phosphate released. Western blots were performed using rat specific antibodies. Results: SIN-1 treatment inhibited SGLT1 in IEC-18 cells (220±22 pmol/mg protein/2 min in control and 76±14 pmol/mg protein/2 min in SIN-1 cells, n=3, p<0.01). However, NHE3 activity was unaltered in SIN-1 treated IEC-18 cells (data not shown). SIN-1 treated cells produced 40X OONO more fluorescence compared to values of control cells. Uric acid (1mM) a natural scavenger of OONO prevented the OONO mediated SGLT1 inhibition (204±15 pmol/mg protein/2 min). Na+/K+-ATPase which maintains the favorable intracellular Na gradient for SGLT1 was decreased by SIN-1 treatment in IEC-18 cells (22.5±2 nmol Pi/mg protein/min in control and 13.1±1.1 nmol Pi/mg protein/ min in SIN-1 cells, n=3, p<0.05). Kinetics studies demonstrated that the mechanism of inhibition of SGLT1 by SIN-1 was secondary to reduction in the number of co-transporters (Vmax) without an alteration in the affinity (Vmax of SGLT1 is 4.5±0.8 nmol/mg protein/ 30sec in control and 1.21±0.1 nmol/mg protein/30sec in SIN-1 cells, n=3, p<0.05). Western blot analysis of SIN-1 treated IEC-18 cells showed a significant decrease in the SGLT1 protein expression. Conclusion: Peroxynitrite significantly inhibits BBM SGLT1 while it does not alter NHE3 activity. The mechanism of SGLT1 inhibition, secondary decreased cotransporter numbers, is identical to that seen in villus cells during chronic enteritis. Thus, these data indicate that peroxynitite, known to be elevated in the mucosa, likely mediates the inhibition of villus cell BBM SGLT1 In Vivo in the chronically inflamed intestine in rabbits.
W1662 Mast Cell Regulation of Na-Glutamine Co-Transporters B0AT1 in Villus and SN2 in Crypt Cells in the Chronically Inflamed Small Intestine Jamilur R. Talukder, Subha Arthur, Uma Sundaram BACKGROUND: Na-glutamine co-transport (NGcT) B0AT1 is inhibited in villus cells while SN2 is stimulated in crypt cells in the chronically inflamed intestine in which mucosal mast cells are increased. How mast cells may regulate B0AT1 and SN2 in villus and SN2 in crypt cells is unknown AIM: Determine the effect of mast cell membrane stabilizer ketotifen (K) on B0AT1 in villus and SN2 in crypt cells during chronic enteritis. METHODS: Villus and crypt cells were isolated from the rabbit intestine by Ca++ chelation. Brush border membrane vesicles (BBMV) were prepared from cells by Ca++ precipitation and differential centrifugation. Uptake studies were performed by rapid filtration using [3H]-glutamine. K (10mg/kg/ day) was administered to rabbits intramuscularly for 2 days. Mast cell marker enzyme βhexosaminidase activity was measured. Western blots were done with specific antibodies. RESULTS: During chronic enteritis ketotifen reversed the inhibition of BOAT1 in villus cells (150±10 pmol/mg prot. in control; 56±3 in inflamed; 145±11 in inflamed+K, n=3,p<0.01) and the stimulation SN2 in crypt cells (46±4 pmol/mg prot. in control; 120±8 in inflamed; 44±4 in inflamed+K, n=3,p<0.01). It had no effect on NGcT in either cells in the normal intestine (data not shown). In the chronically inflamed intestine ketotifen reversed B0AT1 inhibition in villus cell BBMV (95±8 pmol/mg prot. in control; 20±0.1 in inflamed; 83±3 in inflamed+K, n=3,p<0.01) as well as SN2 stimulation in crypt cell BBMV (32±3 pmol/mg prot. in control; 60±10 in inflamed; 36±3 in inflamed+K, n=3, p<0.01). Another mucosal mast cell membrane stabilizer, doxantrazole, also alleviated the alterations in BOAT1 and SN2 in villus and crypt cell BBMV respectively. Doxantrazole normalized mucosal β-hexosaminidase levels during chronic enteritis. Kinetic studies demonstrated that ketotifen reversed the inhibition of BOAT1 in villus cell BBMV by restoring co-transporter numbers and the stimulation of SN2 in crypt cell BBMV by restoring the affinity of the co-transporter for glutamine. Preliminary Western blot studies showed that during chronic enteritis ketotifen restored immuno reactive BOAT1 levels in villus cell BBM while SN2 levels remained unchanged in crypt cell BBM. CONCLUSION: Mucosal mast cell membrane stabilization in the chronically inflamed intestine reversed the inhibition of B0AT1 in villus cells by restoring co-transporter numbers while the stimulation of SN2 in crypt cells was reversed by restoring its affinity for glutamine. Thus, mast cells likely mediate the unique alterations of B0AT1 in villus and SN2 in crypt cells as a common upstream immune regulator during chronic enteritis.
W1660 Lack of CFTR-Dependent Bicarbonate Secretion Blunts Response of Murine Intestinal Mucus Release to Stimulation Mary Abigail S. Garcia, Ning Yang, Paul M. Quinton Background and Aim: The basis for the development of abnormal mucus accumulation in cystic fibrosis (CF)-affected organs remains unexplained. Defective bicarbonate secretion in CF may be a plausible culprit in the characteristic mucus accumulation in this disease, because 1) mucus and HCO3- appear to be physiologically secreted together in most, if not all, affected organs and 2) the pancreatic bicarbonate defect in CF segregates the severity of this disease. We, therefore, hypothesize that lack of HCO3- in CF adversely affects normal mucin release. Method: HCO3- free luminal perfusates from paired segments of distal small intestines of C57Bl/6 mice were analyzed for mucin content using the Periodic Acid-Schiff and wheat germ agglutinin lectin binding assays in the presence and absence of serosal HCO3-. Prostaglandin E2 (PGE2) and 5-hydroxytryptamine (5-HT) were used as mucin secretagogues. Fluid secretory rates were measured using a Ringers-filled closed intestinal sac with impermeable ions NMDG-gluconate in the lumen in place of NaCl to prevent absorption. Increases in weight were interpreted as fluid secretion. Results: In wild-type (WT) intestine, rates of mucin release were similar at basal conditions with or without HCO3-, but lack of HCO3- in PGE2 and 5-HT stimulated conditions inhibited mucin release by about half. Although bumetanide decreased intestinal mucin release similarly, this inhibition of mucin release was not due to inhibition of fluid secretory rates, as lack of HCO3- did not reduce fluid secretion. Interestingly, mucin release from the intestine of CF mouse model carrying ΔF508, the commonest human CF mutation, did not appear different with and without HCO3- and in fact did not seem to respond to stimulation with either PGE2 or 5-HT. GlyH-101 inhibition of cystic fibrosis transmembrane regulator (CFTR) in WT mice blunted mucin release in response to secretagogue stimulation as well. Conclusion: It appears that a lack of CFTR-dependent HCO3- secretion inhibits mucin release. Lack of functioning CFTR seems to dampen mucin secretory response to stimulation via a decrease in secreted HCO3- and decrease in fluid secretion. HCO3- secreted concurrently with mucins may enhance mucin release by sequestering Ca2+ and H+ cations during the release of condensed mucin granules enabling polyanionic charges to electrostatically expand these huge molecules as they are secreted. We propose that the characteristically aggregated mucus, long observed in mucin secreting organs in CF, may be a consequence of defective HCO3- transport in this disease.
W1663 Farnesoid X Receptor Activation Downregulates Chloride Secretion in Colonic Epithelial Cells Niamh Keating, Stephen J. Keely BACKGROUND: The Farnesoid X receptor (FXR) is a nuclear hormone receptor that is activated by bile acids. FXR is involved in the regulation of bile-acid biosynthesis and FXR agonists have been shown to be beneficial in preventing cholestatic liver damage and in prevention of bacterial overgrowth and resultant epithelial damage in the intestine. However, to date a role for the FXR in regulating epithelial transport processes has not been reported. Our previous work has shown that, in contrast to their well-established prosecretory effects at high concentrations, at lower, physiologically relevant concentrations, bile acids, such as deoxycholic acid (DCA), chronically inhibit colonic Cl- secretion. However, the mechanisms underlying such antisecretory actions remain unknown. AIM: To investigate a potential role for FXR in regulating colonic epithelial secretion. METHODS: Cl- secretory responses to the Ca2+-dependent agonist, carbachol (CCh; 100 μM) or the cAMP-dependent agonist, forskolin (FSK; 10 μM), were measured as changes in short circuit current (ISC) across voltage-clamped monolayers of T84 cells. GW4064 was used as a selective FXR agonist. Protein expression was determined by western blotting. RESULTS: Western blotting revealed that FXR is expressed in T84 cells. Pretreatment of T84 cells with GW4064 (2 μM) for 24 hrs attenuated subsequent secretory responses to CCh and FSK to 54.4 ± 3.3% and 72.3 ± 3.3% of those in control cells, respectively (p < 0.001; n = 14). The effects of GW4064 were both concentration- and time-dependent with antisecretory effects being apparent at 0.1 μM and within 12 hrs of treatment. As previously reported, pretreatment of T84 cells with DCA (50 μM for 24 hrs) also attenuated secretory responses to CCh and FSK. However, the antisecretory effects of GW4064 and DCA were additive, suggesting independent mechanisms of action. Furthermore, the antisecretory effect of GW4064, but not DCA, was inhibited by an FXR antagonist, guggulsterone (20 μM). Finally, CCh-stimulated Na+/K+ ATPase activity, measured in apically-permeabilized monolayers, was inhibited 31.3 ± 6.3% by GW4064
W1661 Reduced Stimulation of NHE3 Activity and Reduced Exocytosis with Knockdown of Clc-5, An Endosomal Chloride Channel Zhihong Lin, Olga Kovbasnjuk, Rakhilya Murtazina, Boyoung Cha, Nicholas C. Zachos, Ann Hubbard, Mark Donowitz, Sandra E. Guggino The regulation of NHE3 activity is critical for sodium balance in the human kidney and intestine. The Clcn5 knockout mouse (Clcn5 ko) has an increased fractional excretion of sodium and water. We hypothesize that there might be a trafficking defect in Nhe3, the major transporter involved in regulation of sodium and water reabsorption in the proximal tubule. Previous work by Piwon et al Nature 408:369, 2000 showed that the Nhe3 protein was reduced on the plasma membrane but increased in an intracellular compartment of the proximal tubule. In preliminary confocal experiments, we confirmed this result. Next, we investigated the effect of Clcn5 knockout on Nhe3 activity, by using two photon microscopy
AGA Abstracts
A-712
of mouse proximal tubules using forskolin and A23187, agents that inhibit Nhe3 activity and short term dexamethasone treatment an agent that causes acute stimulation of Nhe3 activity. Intracellular pH, monitored by SNARF4, was measured according to Murtazina et al J. Biol. Chem. 282(34): 25141, 2007. Thin slices of mouse kidney cortex were superfused, acidified by NH4 then exposed to TMA or Na and initial rates of alkalization were measured in delta pH/min. HOE 694 was added to inhibit Nhe1 and Nhe2 activity. When forskolin and A23187 were applied to wt and Clcn5 ko mouse tubules both groups had inhibition of NHe3 activity but there was no difference between wt and Clcn5 ko. When short tern dexamethasone was given, wt mouse proximal tubules showed an increase in Nhe3 activity from 0.29±0.02 to 0.39±0.03, p<0.005 (34%) whereas the Clcn5 ko mouse had an initial rate of 0.21±0.01 which increased only to 0.25±0.01, p<0.05 (19%). Previously, surface biotinylation experiments in OK cells, an accepted cell culture model of the proximal tubule, showed that there is normally increased exocytosis of Nhe3 cells are treated with dexamethasone. However in OK cells with Clc-5 silenced, using two separate shRNA constructs, there was reduced exocytosis of Nhe3 (76%) compared to control plasmid or a scrambled Clc-5 shRNA construct (180%). These results suggest that stimulation of Nhe3 activity may be dysregulated in both the Clcn5 ko mouse and in OK cells. We propose that there is more Nhe3 inside the cell and less on the plasma membrane because the basal and regulated insertion of Nhe3 onto the plasma membrane is reduced. We conclude that Clc5, located in early and recycling endosomes, is important for outward trafficking of Nhe3.
AGA Abstracts
Peroxynitrite Inhibits Na-Glucose Co-Transport But Not Na/H Exchange in IEC-18 Cells Palanikumar Manoharan, Ramesh Kekuda, Prosenjit Saha, Subha Arthur, Uma Sundaram Background: During chronic intestinal inflammation in rabbits intestinal villus cell brush border membrane (BBM) Na-glucose co-transport (SGLT1), but not Na/H exchange (NHE3) is inhibited. The mechanism of inhibition is secondary to a decrease in the number of BBM co-transporters. Peroxynitrite (OONO), a potent oxidant, is produced in abundance in the mucosa during chronic enteritis. However, the effect of OONO on SGLT1 and NHE3 in intestinal epithelial cells is unknown. Aim: Determine the effect of OONO on SGLT1 and NHE3 in epithelial cells and decipher the mechanism. Methods: Rat small intestinal epithelial cell (IEC-18) monolayers grown on transwell plates were treated with 100μM SIN-1 (OONO donor) for 24 hours. 3H-OMG uptake and pH dependent, amiloride sensitive 22Na uptake were performed for SGLT1 and NHE3 activity respectively. OONO was measured as fluorescence produced from oxidation of dihydrorhodamine 123. Na+/K+ ATPase activity was determined by measuring the inorganic phosphate released. Western blots were performed using rat specific antibodies. Results: SIN-1 treatment inhibited SGLT1 in IEC-18 cells (220±22 pmol/mg protein/2 min in control and 76±14 pmol/mg protein/2 min in SIN-1 cells, n=3, p<0.01). However, NHE3 activity was unaltered in SIN-1 treated IEC-18 cells (data not shown). SIN-1 treated cells produced 40X OONO more fluorescence compared to values of control cells. Uric acid (1mM) a natural scavenger of OONO prevented the OONO mediated SGLT1 inhibition (204±15 pmol/mg protein/2 min). Na+/K+-ATPase which maintains the favorable intracellular Na gradient for SGLT1 was decreased by SIN-1 treatment in IEC-18 cells (22.5±2 nmol Pi/mg protein/min in control and 13.1±1.1 nmol Pi/mg protein/ min in SIN-1 cells, n=3, p<0.05). Kinetics studies demonstrated that the mechanism of inhibition of SGLT1 by SIN-1 was secondary to reduction in the number of co-transporters (Vmax) without an alteration in the affinity (Vmax of SGLT1 is 4.5±0.8 nmol/mg protein/ 30sec in control and 1.21±0.1 nmol/mg protein/30sec in SIN-1 cells, n=3, p<0.05). Western blot analysis of SIN-1 treated IEC-18 cells showed a significant decrease in the SGLT1 protein expression. Conclusion: Peroxynitrite significantly inhibits BBM SGLT1 while it does not alter NHE3 activity. The mechanism of SGLT1 inhibition, secondary decreased cotransporter numbers, is identical to that seen in villus cells during chronic enteritis. Thus, these data indicate that peroxynitite, known to be elevated in the mucosa, likely mediates the inhibition of villus cell BBM SGLT1 In Vivo in the chronically inflamed intestine in rabbits.
W1662 Mast Cell Regulation of Na-Glutamine Co-Transporters B0AT1 in Villus and SN2 in Crypt Cells in the Chronically Inflamed Small Intestine Jamilur R. Talukder, Subha Arthur, Uma Sundaram BACKGROUND: Na-glutamine co-transport (NGcT) B0AT1 is inhibited in villus cells while SN2 is stimulated in crypt cells in the chronically inflamed intestine in which mucosal mast cells are increased. How mast cells may regulate B0AT1 and SN2 in villus and SN2 in crypt cells is unknown AIM: Determine the effect of mast cell membrane stabilizer ketotifen (K) on B0AT1 in villus and SN2 in crypt cells during chronic enteritis. METHODS: Villus and crypt cells were isolated from the rabbit intestine by Ca++ chelation. Brush border membrane vesicles (BBMV) were prepared from cells by Ca++ precipitation and differential centrifugation. Uptake studies were performed by rapid filtration using [3H]-glutamine. K (10mg/kg/ day) was administered to rabbits intramuscularly for 2 days. Mast cell marker enzyme βhexosaminidase activity was measured. Western blots were done with specific antibodies. RESULTS: During chronic enteritis ketotifen reversed the inhibition of BOAT1 in villus cells (150±10 pmol/mg prot. in control; 56±3 in inflamed; 145±11 in inflamed+K, n=3,p<0.01) and the stimulation SN2 in crypt cells (46±4 pmol/mg prot. in control; 120±8 in inflamed; 44±4 in inflamed+K, n=3,p<0.01). It had no effect on NGcT in either cells in the normal intestine (data not shown). In the chronically inflamed intestine ketotifen reversed B0AT1 inhibition in villus cell BBMV (95±8 pmol/mg prot. in control; 20±0.1 in inflamed; 83±3 in inflamed+K, n=3,p<0.01) as well as SN2 stimulation in crypt cell BBMV (32±3 pmol/mg prot. in control; 60±10 in inflamed; 36±3 in inflamed+K, n=3, p<0.01). Another mucosal mast cell membrane stabilizer, doxantrazole, also alleviated the alterations in BOAT1 and SN2 in villus and crypt cell BBMV respectively. Doxantrazole normalized mucosal β-hexosaminidase levels during chronic enteritis. Kinetic studies demonstrated that ketotifen reversed the inhibition of BOAT1 in villus cell BBMV by restoring co-transporter numbers and the stimulation of SN2 in crypt cell BBMV by restoring the affinity of the co-transporter for glutamine. Preliminary Western blot studies showed that during chronic enteritis ketotifen restored immuno reactive BOAT1 levels in villus cell BBM while SN2 levels remained unchanged in crypt cell BBM. CONCLUSION: Mucosal mast cell membrane stabilization in the chronically inflamed intestine reversed the inhibition of B0AT1 in villus cells by restoring co-transporter numbers while the stimulation of SN2 in crypt cells was reversed by restoring its affinity for glutamine. Thus, mast cells likely mediate the unique alterations of B0AT1 in villus and SN2 in crypt cells as a common upstream immune regulator during chronic enteritis.
W1660 Lack of CFTR-Dependent Bicarbonate Secretion Blunts Response of Murine Intestinal Mucus Release to Stimulation Mary Abigail S. Garcia, Ning Yang, Paul M. Quinton Background and Aim: The basis for the development of abnormal mucus accumulation in cystic fibrosis (CF)-affected organs remains unexplained. Defective bicarbonate secretion in CF may be a plausible culprit in the characteristic mucus accumulation in this disease, because 1) mucus and HCO3- appear to be physiologically secreted together in most, if not all, affected organs and 2) the pancreatic bicarbonate defect in CF segregates the severity of this disease. We, therefore, hypothesize that lack of HCO3- in CF adversely affects normal mucin release. Method: HCO3- free luminal perfusates from paired segments of distal small intestines of C57Bl/6 mice were analyzed for mucin content using the Periodic Acid-Schiff and wheat germ agglutinin lectin binding assays in the presence and absence of serosal HCO3-. Prostaglandin E2 (PGE2) and 5-hydroxytryptamine (5-HT) were used as mucin secretagogues. Fluid secretory rates were measured using a Ringers-filled closed intestinal sac with impermeable ions NMDG-gluconate in the lumen in place of NaCl to prevent absorption. Increases in weight were interpreted as fluid secretion. Results: In wild-type (WT) intestine, rates of mucin release were similar at basal conditions with or without HCO3-, but lack of HCO3- in PGE2 and 5-HT stimulated conditions inhibited mucin release by about half. Although bumetanide decreased intestinal mucin release similarly, this inhibition of mucin release was not due to inhibition of fluid secretory rates, as lack of HCO3- did not reduce fluid secretion. Interestingly, mucin release from the intestine of CF mouse model carrying ΔF508, the commonest human CF mutation, did not appear different with and without HCO3- and in fact did not seem to respond to stimulation with either PGE2 or 5-HT. GlyH-101 inhibition of cystic fibrosis transmembrane regulator (CFTR) in WT mice blunted mucin release in response to secretagogue stimulation as well. Conclusion: It appears that a lack of CFTR-dependent HCO3- secretion inhibits mucin release. Lack of functioning CFTR seems to dampen mucin secretory response to stimulation via a decrease in secreted HCO3- and decrease in fluid secretion. HCO3- secreted concurrently with mucins may enhance mucin release by sequestering Ca2+ and H+ cations during the release of condensed mucin granules enabling polyanionic charges to electrostatically expand these huge molecules as they are secreted. We propose that the characteristically aggregated mucus, long observed in mucin secreting organs in CF, may be a consequence of defective HCO3- transport in this disease.
W1663 Farnesoid X Receptor Activation Downregulates Chloride Secretion in Colonic Epithelial Cells Niamh Keating, Stephen J. Keely BACKGROUND: The Farnesoid X receptor (FXR) is a nuclear hormone receptor that is activated by bile acids. FXR is involved in the regulation of bile-acid biosynthesis and FXR agonists have been shown to be beneficial in preventing cholestatic liver damage and in prevention of bacterial overgrowth and resultant epithelial damage in the intestine. However, to date a role for the FXR in regulating epithelial transport processes has not been reported. Our previous work has shown that, in contrast to their well-established prosecretory effects at high concentrations, at lower, physiologically relevant concentrations, bile acids, such as deoxycholic acid (DCA), chronically inhibit colonic Cl- secretion. However, the mechanisms underlying such antisecretory actions remain unknown. AIM: To investigate a potential role for FXR in regulating colonic epithelial secretion. METHODS: Cl- secretory responses to the Ca2+-dependent agonist, carbachol (CCh; 100 μM) or the cAMP-dependent agonist, forskolin (FSK; 10 μM), were measured as changes in short circuit current (ISC) across voltage-clamped monolayers of T84 cells. GW4064 was used as a selective FXR agonist. Protein expression was determined by western blotting. RESULTS: Western blotting revealed that FXR is expressed in T84 cells. Pretreatment of T84 cells with GW4064 (2 μM) for 24 hrs attenuated subsequent secretory responses to CCh and FSK to 54.4 ± 3.3% and 72.3 ± 3.3% of those in control cells, respectively (p < 0.001; n = 14). The effects of GW4064 were both concentration- and time-dependent with antisecretory effects being apparent at 0.1 μM and within 12 hrs of treatment. As previously reported, pretreatment of T84 cells with DCA (50 μM for 24 hrs) also attenuated secretory responses to CCh and FSK. However, the antisecretory effects of GW4064 and DCA were additive, suggesting independent mechanisms of action. Furthermore, the antisecretory effect of GW4064, but not DCA, was inhibited by an FXR antagonist, guggulsterone (20 μM). Finally, CCh-stimulated Na+/K+ ATPase activity, measured in apically-permeabilized monolayers, was inhibited 31.3 ± 6.3% by GW4064
W1661 Reduced Stimulation of NHE3 Activity and Reduced Exocytosis with Knockdown of Clc-5, An Endosomal Chloride Channel Zhihong Lin, Olga Kovbasnjuk, Rakhilya Murtazina, Boyoung Cha, Nicholas C. Zachos, Ann Hubbard, Mark Donowitz, Sandra E. Guggino The regulation of NHE3 activity is critical for sodium balance in the human kidney and intestine. The Clcn5 knockout mouse (Clcn5 ko) has an increased fractional excretion of sodium and water. We hypothesize that there might be a trafficking defect in Nhe3, the major transporter involved in regulation of sodium and water reabsorption in the proximal tubule. Previous work by Piwon et al Nature 408:369, 2000 showed that the Nhe3 protein was reduced on the plasma membrane but increased in an intracellular compartment of the proximal tubule. In preliminary confocal experiments, we confirmed this result. Next, we investigated the effect of Clcn5 knockout on Nhe3 activity, by using two photon microscopy
AGA Abstracts
A-712