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Regulation of intestinal epithelial Na-K-2Cl cotransporter expression by protein kinase C
GASTROENTEROLOGY Vol. 114, No. 4
A368 AGA ABSTRACTS
G1500 IGMESINE INHIBITION OF VIP-INDUCED JEJUNAL HYPERSECRETION IN RATS; INVOLVEMENT OF ENDOGENOUS SOMATOSTATIN AND NERVOUS PATHWAYS. H. Fargeau, P.J.M. Rivi~re**, J.L. Junien*, M. Chovet. Jouveinal Parke-Davis, Fresnes, France. *Present adress: Laboratoire Ferring, Gentilly, France, **Present adress: Ferring Research Institute Inc, San Diego, USA The sigma ligand igmesine (JO 1784) has been shown to act on nerves to inhibit intestinal ion transport in the isolated mouse jejunumL The present study evaluates the ability of igmesine to inhibit the VIP-induced jejunal hypersecretion in rats and investigates the possibility of the involvement of endogenous somatostatin in the response to igmesine. Methods: In anesthetized (pentobarbital, 60 mg/kg i.p.) fasted Sprague-Dawley rats (160-180 g), a jejunal loop was isolated by two ligations at 5 and 25 cm distal to the ligament of Treitz. The loop was filled with saline (2 ml, 37°C). Jejunal secretion was stimulated by a 30 min intraarterial infusion of VIP. At the end of the VIP infusion, the loop was collected, measured and weighed before and after fluid removal to determine water net flux (mg/cm). Intravenous (i.v.) bolus injections of igmesine or octreotide were performed 15 min before starting VIP infusion. Tetrodotoxin (TTX) at 5 mg/kg, the somatosatin antagonist, cyclosomatostatin (CSS) at 1 mg/kg, or the sigma antagonist, BMY-14802 (BMY) at 1 mg/kg were given by i.v. route 5 min before igmesine or octreotide. Results: In the basal state the net water flux was positive (+3.13 ±3.9 mg/cm). VIP (0.03-0.33 mg.min-1) induced a dose-related inversion of net flux. The submaximal effect (- 34.1 -+7.1 mg/cm) was obtained at the dose of 0.1 mg.min-1. VIP (0.1 mg.min-1) induced jejunal hypersecretion was inhibited in a doserelated manner by igmesine and octreotide (EDso: 178 and 0.132 mg/kg i.v., respectively). Igmesine (1 mg/kg) and octreotide (1 mg/kg) responses were inhibited by TTX (-96% and -45%, respectively) and CSS (-88% and -100%, respectively). BMY abolished the igmesine response but did not alter the octreotide response. TI'X, CSS and BMY did not alterper se the VIP response. Conclusion: The VIP response was blocked by Igmesine in a TTX-, CSS- and BMY-sensitive manner, suggesting an indirect action on the enterocyte through sigma receptors, nerve- and somatostatin-pathways. Octreotide response was BMY-insensitive indicating that somatostatin receptors activated during the Igmesine response are likely to be distal to the sigma receptors. [1] Rivi~re et al. J Pharm Exp Ther 1993; 264: 1268. G1501 PLASMA LATHOSTEROL AS A SCREENING TEST FOR BILE A C I D MALABSORIrrION: CORRELATION WITH 7SSeHCAT TEST. F~irkkil~i ___,Taavitsainen M, Kairemo K, Miettinen T. Helsinki University Hospital, Finland Background: Idiopathic bile acid malabsorption is not uncommon cause of chronic diarrhea. Several methods have been used to detect bile acid malabsorption, most frequently the 75SeHCAT test. Retention times both for three and seven days has been used. We have previously shown (1) a close correlation with SeHCAT T 1/2 h, fecal bile acid excretion and plasma lathosterol, a cholesterol precursor, which has been shown to rise markedly as a response to increased bile acid synthesis. Aims: To evaluate the sensitivity and specificity of plasma lathosterol determination as a screening test using 75SeHCAT 72 h retention % (SeHCAT 72%) and SeHCAT T 1/2 as a standards. Patients and methods: 181 patients with suspected bile acid malabsorption underwent routine 75SeHCAT test. After an overnight fast study subjects were given a capsule containig 5-9 pCi of 75SeHCAT (Amersham Inc, UK). Measurements were done by Siemens Scintiview type II camera. Plasma cholesterol and lathosterols were analyzed by gas liquid chromatography from saponified plasma samples. Results: Plasma lathosterol pg/ml cholesterol (P-LS/C) and also LN(P-LS/C) correlated closely inversely with 75SeHCAT 72%, r=-0.360, p < 0.001 and r=-0.285, p<0.001 and with SeHCAT T 1/2, r=-0.309, p<0.001 and r=-0.267, p < 0.001. Using the mean ± 2SD LN(LS/C) value (=5.87 pg/ml cholesterol) of normal controls (1) as the cut-off value the sensitivity was 79% and specificity 71%, respectively. Conclusion: Plasma lathosterol determination offers a relatively sensitive and specific method for a simple and rapid screening of bile acid malabsorption without need of isotopes. [t] F~irkkil~tM, et al. Clin Science 90;315-19, 1996. • G1502 REGULATION OF INTESTINAL EPITHELIAL Na-K-2CL COTRANSPORTER EXPRESSION BY PROTEIN KINASE C. OC Farokzhad, EC Mun, JA Smith, JK Sicklick, JC Song, JB Matthews. Dept. of Surgery, Beth Israel Deaconess Medical Center, Boston MA. Epithelial cells lining the intestinal crypts express the Na-K-2CI cotransporter (NKCC) on the basolateral plasma membrane, where it serves as an important regulatory site for water and electrolyte secretion and, hence, diarrheal disorders. We showed that activation of protein kinase C (PKC) by the phorbol ester PMA progressively decreases the number of NKCC units on the plasma membrane (as measured by specific binding of the loop diuretic bumetanide). AIM: to examine whether prolonged activation of PKC by PMA alters NKCC gene expression. METHODS: NKCC activity was studied in model human intestinal epithelial T84 ceils grown on permeable supports by the bumetanidesensitive uptake of 86Rb. NKCC protein and steady-state
mRNA levels were determined by immunoprecipitation and Northern analysis. Data: mean ± SEM for at least n=3; statistics: ANOVA. RESULTS: PMA but not its PKC-inactive stereoisomer 4et-PMA, decreased basal and cAMP-stimulated N K C C function, with peak effects at 100 nM. Inhibition of NKCC function by PMA was evident by 45 min (peak cAMP-stimulated NKCC activity=66.7±6.5% control), profound by 4 hrs (18.1±1.8% control), and abolished within 24 hrs. PMA but not 4et-PMA time- and dosedependently decreased NKCC steady state mRNA levels, an effect that only became apparent after 4 hrs exposure to 100 nM PMA. Treatment of ceils with cyclohexamide did not affect the ability of PMA to decrease NKCC mRNA, and thus this effect does not require de novo protein synthesis. PMA also decreased NKCC protein levels after 8 hrs with 100 nM PMA, and these levels corresponded tightly to mRNA abundance. Long-term (48-72 hr) exposure to PMA, which is known to selectively down-regulate some but not all PKC isoforms, was associated with the return of NKCC mRNA and protein expression by 48 and 72 hrs, respectively. However, at 72 hrs, cAMPstimulated NKCC activity (bumetanide-sensitive S6Rb uptake) remained essentially undetectable. CONCLUSIONS: PKC activation rapidly inhibits NKCC function, an effect which precedes the inhibition of NKCC mRNA expression. Longterm exposure to PMA (with likely downregulation of as-yet-unspecified PKC isoforms) was associated with a return of NKCC mRNA and protein expression, but not NKCC function. Taken together, our data suggests that distinct PKC isoforms may regulate the expression and function of NKCC. Supported by DK48010 and ADHF student fellowship. • G1503 HEAT SHOCK PROTEINS PROTECT THE BARRIER FUNCTION OF EPITHELIAL CELL LINES. H. Feldhaar, J. Ries, W.F. Caspary, J. Stein. II Department of Medicine, J. W. Goethe University, Frankfurt, Germany Sublethal heat stress (HS) leads to an increase in resistance of cellmonolayers during a second HS or chemically induced damage. The objective of this study was to assess possible protective effects of HSPs on the epithelial barrier function. Method: Caco-2 and MDCK-celIs were grown under standard conditions. Transepithelial resistance (TER) was measured in Ussing-chambers as a parameter for the integrity of confluency of cellmonolayers. Ceils were pretreated with HS (10 min, 45°C) 18 h prior to the experiments and then TER was measured in Ussing-chambers. After a 30 rain equilibration-period ceils were treated with different concentrations of Latrunculin B (Lat. B) a known inhibitor of actin-polymerisation. Results: Caco-2 cells showed a significantly reduced decrease of TER after HS (14.9 ± 5.1%) when exposed to a second HS in Ussingchambers than controls (22.6-+ 5.3%). Experiments with MDCK cells showed a dose-dependent decrease in TER when treated with Lat. B over a 60 rain period. Heatpretreated cells showed a significantly lesser decrease of TER when treated with intermediate concentrations of Lat. B. Treatment with 0.05pg/ml Lat. B was followed by a decrease of 1.7 ± 0.9% after 60 min compared to 0 _+1.4% in heat-pretreated ceils. 0.075pg/ml Lat. B led to a decrease of 24.8 + 2.9% in controls and 16.9- 2.6% in heat-pretreated cells. Western blot analysis showed a temperature dependent rise in expression of HSP 27 and HSP 70. Northern blot analysis showed a maximum of mRNA of both HSPs after 3 h after HS. Discussion: A higher level of HSPs after a HS correlates with a less decreased TER during a second HS in Caco-2 ceils. Experiments with Lat. B show that the cytoskeleton, including the tight junctions, seems to be a target for protection of HSPs so that they help to maintain the barrier function of epithelial cell lines. 1.05
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tlme (ran) -.e.- co~'ol --*- cor~. O,OSr~gLat B ~ con. 0,075pg Lat B con. 0.75pg Lat B
-o-- HS con. -a-- HS 0,051~ Lat B --o-'- HS 0,075pg Lat. B -.q-- HS 0,75pg tat B
• G1504
THE ROLE OF SUBSTANCE P AND NEUROKININ RECEPTORS IN ACID-INDUCED HYPEREMIA IN THE OPOSSUM ESOPHAGUS. M, J. Feldman. G.P. Morris, and W.G. Paterson. GI Diseases Research Unit, Hotel Dieu Hospital and Queen's University, Kingston, Canada. Both mast ceils and neuropeptides have been shown to play a role in mediating reactive hyperemia during acute esophageal luminal acid exposure in a number of animal models. The tachykinin substance P (SP) is known to
A368 AGA ABSTRACTS
G1500 IGMESINE INHIBITION OF VIP-INDUCED JEJUNAL HYPERSECRETION IN RATS; INVOLVEMENT OF ENDOGENOUS SOMATOSTATIN AND NERVOUS PATHWAYS. H. Fargeau, P.J.M. Rivi~re**, J.L. Junien*, M. Chovet. Jouveinal Parke-Davis, Fresnes, France. *Present adress: Laboratoire Ferring, Gentilly, France, **Present adress: Ferring Research Institute Inc, San Diego, USA The sigma ligand igmesine (JO 1784) has been shown to act on nerves to inhibit intestinal ion transport in the isolated mouse jejunumL The present study evaluates the ability of igmesine to inhibit the VIP-induced jejunal hypersecretion in rats and investigates the possibility of the involvement of endogenous somatostatin in the response to igmesine. Methods: In anesthetized (pentobarbital, 60 mg/kg i.p.) fasted Sprague-Dawley rats (160-180 g), a jejunal loop was isolated by two ligations at 5 and 25 cm distal to the ligament of Treitz. The loop was filled with saline (2 ml, 37°C). Jejunal secretion was stimulated by a 30 min intraarterial infusion of VIP. At the end of the VIP infusion, the loop was collected, measured and weighed before and after fluid removal to determine water net flux (mg/cm). Intravenous (i.v.) bolus injections of igmesine or octreotide were performed 15 min before starting VIP infusion. Tetrodotoxin (TTX) at 5 mg/kg, the somatosatin antagonist, cyclosomatostatin (CSS) at 1 mg/kg, or the sigma antagonist, BMY-14802 (BMY) at 1 mg/kg were given by i.v. route 5 min before igmesine or octreotide. Results: In the basal state the net water flux was positive (+3.13 ±3.9 mg/cm). VIP (0.03-0.33 mg.min-1) induced a dose-related inversion of net flux. The submaximal effect (- 34.1 -+7.1 mg/cm) was obtained at the dose of 0.1 mg.min-1. VIP (0.1 mg.min-1) induced jejunal hypersecretion was inhibited in a doserelated manner by igmesine and octreotide (EDso: 178 and 0.132 mg/kg i.v., respectively). Igmesine (1 mg/kg) and octreotide (1 mg/kg) responses were inhibited by TTX (-96% and -45%, respectively) and CSS (-88% and -100%, respectively). BMY abolished the igmesine response but did not alter the octreotide response. TI'X, CSS and BMY did not alterper se the VIP response. Conclusion: The VIP response was blocked by Igmesine in a TTX-, CSS- and BMY-sensitive manner, suggesting an indirect action on the enterocyte through sigma receptors, nerve- and somatostatin-pathways. Octreotide response was BMY-insensitive indicating that somatostatin receptors activated during the Igmesine response are likely to be distal to the sigma receptors. [1] Rivi~re et al. J Pharm Exp Ther 1993; 264: 1268. G1501 PLASMA LATHOSTEROL AS A SCREENING TEST FOR BILE A C I D MALABSORIrrION: CORRELATION WITH 7SSeHCAT TEST. F~irkkil~i ___,Taavitsainen M, Kairemo K, Miettinen T. Helsinki University Hospital, Finland Background: Idiopathic bile acid malabsorption is not uncommon cause of chronic diarrhea. Several methods have been used to detect bile acid malabsorption, most frequently the 75SeHCAT test. Retention times both for three and seven days has been used. We have previously shown (1) a close correlation with SeHCAT T 1/2 h, fecal bile acid excretion and plasma lathosterol, a cholesterol precursor, which has been shown to rise markedly as a response to increased bile acid synthesis. Aims: To evaluate the sensitivity and specificity of plasma lathosterol determination as a screening test using 75SeHCAT 72 h retention % (SeHCAT 72%) and SeHCAT T 1/2 as a standards. Patients and methods: 181 patients with suspected bile acid malabsorption underwent routine 75SeHCAT test. After an overnight fast study subjects were given a capsule containig 5-9 pCi of 75SeHCAT (Amersham Inc, UK). Measurements were done by Siemens Scintiview type II camera. Plasma cholesterol and lathosterols were analyzed by gas liquid chromatography from saponified plasma samples. Results: Plasma lathosterol pg/ml cholesterol (P-LS/C) and also LN(P-LS/C) correlated closely inversely with 75SeHCAT 72%, r=-0.360, p < 0.001 and r=-0.285, p<0.001 and with SeHCAT T 1/2, r=-0.309, p<0.001 and r=-0.267, p < 0.001. Using the mean ± 2SD LN(LS/C) value (=5.87 pg/ml cholesterol) of normal controls (1) as the cut-off value the sensitivity was 79% and specificity 71%, respectively. Conclusion: Plasma lathosterol determination offers a relatively sensitive and specific method for a simple and rapid screening of bile acid malabsorption without need of isotopes. [t] F~irkkil~tM, et al. Clin Science 90;315-19, 1996. • G1502 REGULATION OF INTESTINAL EPITHELIAL Na-K-2CL COTRANSPORTER EXPRESSION BY PROTEIN KINASE C. OC Farokzhad, EC Mun, JA Smith, JK Sicklick, JC Song, JB Matthews. Dept. of Surgery, Beth Israel Deaconess Medical Center, Boston MA. Epithelial cells lining the intestinal crypts express the Na-K-2CI cotransporter (NKCC) on the basolateral plasma membrane, where it serves as an important regulatory site for water and electrolyte secretion and, hence, diarrheal disorders. We showed that activation of protein kinase C (PKC) by the phorbol ester PMA progressively decreases the number of NKCC units on the plasma membrane (as measured by specific binding of the loop diuretic bumetanide). AIM: to examine whether prolonged activation of PKC by PMA alters NKCC gene expression. METHODS: NKCC activity was studied in model human intestinal epithelial T84 ceils grown on permeable supports by the bumetanidesensitive uptake of 86Rb. NKCC protein and steady-state
mRNA levels were determined by immunoprecipitation and Northern analysis. Data: mean ± SEM for at least n=3; statistics: ANOVA. RESULTS: PMA but not its PKC-inactive stereoisomer 4et-PMA, decreased basal and cAMP-stimulated N K C C function, with peak effects at 100 nM. Inhibition of NKCC function by PMA was evident by 45 min (peak cAMP-stimulated NKCC activity=66.7±6.5% control), profound by 4 hrs (18.1±1.8% control), and abolished within 24 hrs. PMA but not 4et-PMA time- and dosedependently decreased NKCC steady state mRNA levels, an effect that only became apparent after 4 hrs exposure to 100 nM PMA. Treatment of ceils with cyclohexamide did not affect the ability of PMA to decrease NKCC mRNA, and thus this effect does not require de novo protein synthesis. PMA also decreased NKCC protein levels after 8 hrs with 100 nM PMA, and these levels corresponded tightly to mRNA abundance. Long-term (48-72 hr) exposure to PMA, which is known to selectively down-regulate some but not all PKC isoforms, was associated with the return of NKCC mRNA and protein expression by 48 and 72 hrs, respectively. However, at 72 hrs, cAMPstimulated NKCC activity (bumetanide-sensitive S6Rb uptake) remained essentially undetectable. CONCLUSIONS: PKC activation rapidly inhibits NKCC function, an effect which precedes the inhibition of NKCC mRNA expression. Longterm exposure to PMA (with likely downregulation of as-yet-unspecified PKC isoforms) was associated with a return of NKCC mRNA and protein expression, but not NKCC function. Taken together, our data suggests that distinct PKC isoforms may regulate the expression and function of NKCC. Supported by DK48010 and ADHF student fellowship. • G1503 HEAT SHOCK PROTEINS PROTECT THE BARRIER FUNCTION OF EPITHELIAL CELL LINES. H. Feldhaar, J. Ries, W.F. Caspary, J. Stein. II Department of Medicine, J. W. Goethe University, Frankfurt, Germany Sublethal heat stress (HS) leads to an increase in resistance of cellmonolayers during a second HS or chemically induced damage. The objective of this study was to assess possible protective effects of HSPs on the epithelial barrier function. Method: Caco-2 and MDCK-celIs were grown under standard conditions. Transepithelial resistance (TER) was measured in Ussing-chambers as a parameter for the integrity of confluency of cellmonolayers. Ceils were pretreated with HS (10 min, 45°C) 18 h prior to the experiments and then TER was measured in Ussing-chambers. After a 30 rain equilibration-period ceils were treated with different concentrations of Latrunculin B (Lat. B) a known inhibitor of actin-polymerisation. Results: Caco-2 cells showed a significantly reduced decrease of TER after HS (14.9 ± 5.1%) when exposed to a second HS in Ussingchambers than controls (22.6-+ 5.3%). Experiments with MDCK cells showed a dose-dependent decrease in TER when treated with Lat. B over a 60 rain period. Heatpretreated cells showed a significantly lesser decrease of TER when treated with intermediate concentrations of Lat. B. Treatment with 0.05pg/ml Lat. B was followed by a decrease of 1.7 ± 0.9% after 60 min compared to 0 _+1.4% in heat-pretreated ceils. 0.075pg/ml Lat. B led to a decrease of 24.8 + 2.9% in controls and 16.9- 2.6% in heat-pretreated cells. Western blot analysis showed a temperature dependent rise in expression of HSP 27 and HSP 70. Northern blot analysis showed a maximum of mRNA of both HSPs after 3 h after HS. Discussion: A higher level of HSPs after a HS correlates with a less decreased TER during a second HS in Caco-2 ceils. Experiments with Lat. B show that the cytoskeleton, including the tight junctions, seems to be a target for protection of HSPs so that they help to maintain the barrier function of epithelial cell lines. 1.05
,o0
•
.
0.85-
0.75-
0.60
lo
io
;o
20
5'o
e'o
7'o
~o
io
tlme (ran) -.e.- co~'ol --*- cor~. O,OSr~gLat B ~ con. 0,075pg Lat B con. 0.75pg Lat B
-o-- HS con. -a-- HS 0,051~ Lat B --o-'- HS 0,075pg Lat. B -.q-- HS 0,75pg tat B
• G1504
THE ROLE OF SUBSTANCE P AND NEUROKININ RECEPTORS IN ACID-INDUCED HYPEREMIA IN THE OPOSSUM ESOPHAGUS. M, J. Feldman. G.P. Morris, and W.G. Paterson. GI Diseases Research Unit, Hotel Dieu Hospital and Queen's University, Kingston, Canada. Both mast ceils and neuropeptides have been shown to play a role in mediating reactive hyperemia during acute esophageal luminal acid exposure in a number of animal models. The tachykinin substance P (SP) is known to