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Nitric oxide stimulates bile flow and glutathione excretion independent of cyclic GMP-induced HCO3- excretion
HEPATOLOGY Vol. 22, No. 4, Pt. 2, 1995
AASLD ABSTRACTS
833 BOMBESIN S T I M U L A T E S B I C A R B O N A T E S E C R E T I O N FROM BILE DUCTS, WK Cho, SA Rydberg. and JL Boyer. Liver Center, Yale University School of Medicine, New Haven, CT. Bombesin stimulates bile ductular secretion by activating CI'/HCO3" exchange in isolated intrahepatic bile duct units(Gastroenterology 1995;108:A1049). In order to further study this process, novel functional intrahepatic bile duct units(IBDU) were prepared as descdbed(PNAS in press) and cultured for 24-48 hours. The enclosed lumen of the IBDU were microinjected with cell-impermeable BCECFDextran(70K) to study luminal pH changes. Luminal pH increased with infusion of 10 nM bombesin from 7.59 + 0.06 to 7.70 + 0.09 pHU(n=9)(p<0.01), consistent with stimulation of bicarbonate secretion. The isolated perfused rat liver model was then used to further examine the effects of bombesin on bile flow and bicarbonate secretion. Bile samples were collected at 15 minute intervals and analyzed for bile flow and bicarbonate secretion. One-pass portal perfusion with constant 100 nM bombesin infusion in KRB(without bile acid supplementation) did not produce significant changes in bile flow. However, pH, bicarbonate concentration, and total bicarbonate output of bile increased from 7.59 + 0.03 to 7.65 + 0.03(p<0.05; n=6), 21.7 + 1,0 to 25.6 + 2.1 raM(p<0.01), and 24.3 + 3.6 to 28.1 + 6.1 pmole/min/g liver (p<0.05) respectively. Two week bile duct ligated rats were then used to further amplify the bile ductular secretory response. One-pass portal perfusion of 2 week bile duct ligated rat liver with 100 nM bombesin infusion showed only a mild increase in bile flow from 0.86 + 0.15 to 0.90 + 0.14 Id/min/g liver(n=4). However, pH, bicarbonate concentration, and bicarbonate output of bile increased from 7.52 + 0.05 to 7.64 + 0.09(p<0.05), 17.7 + 1.82 to 24.4 + 4.25 raM(p<0.01), and 15.2 + 3.32 to 21.9 + 5.42 pmole/min/g liver(p<0.05) respectively. Conclusion: Although bombesin is known to release other biliary secretagogues including secret±n, bombesin can directly stimulate bicarbonate secretion in rat bile acting at the level of bile duct.
835
AMILORIDE, BUT NOT DIDS AND NPPB, INHIBIT SECRETIN (SEC)-INDUCED CHOLERESlS: EVIDENCE FOR A ROLE OF Ne+/H+, BUT NOT CI-/HCO3- EXCHANGE AND CI- CHANNELS, IN DUCTULAR BILE FORMATION. E.L.Renner & M.Hevdtmann. Dept. of Clio. PharmacoL, Univ. of Bern, Swltzedand. While several H+IHC03- transporters and CI- channels have been identified in chotanglcoytes, the cellular mechanisms mediating SEC-Induced duotular secretion of HCO3- and water remain III understood. AIM: To explore the role of Na+/H+ and CI-IHCO3- exchange, and of CI- channels In SEC-induced ductular cholerasis. METHODS: Male Sprague-Dawley rats were fed 0.06% (w/w) ANIT for 6 weeks to produce hyperplasle of cecretin-rasponsive bile ductules. Rat livers were perfuced In sltu with Krebs buffer containing vehicle (0.2% DMSO), the Na+l H+exchango inhibitor arnUoride (lmM), the CI-JHCO3-exchange Inhibitor D!DS (100 laM) or the CI-channel blocker NPPB (lO011M). Following 3Omln of equilibration, basal bile was collected for 2xl0mln; SEC (25UJh; or vehicle-0.9% NaCI) was then infused foe 60rnin Into the portal vein and bile collected in 10min For±ode, the last two col±sot±onewhen SEC effects had plateaued being taken as experimental samples. Bile volume was assessed by gravlmetry, blllew [HC03-] as total C02 and blllery [DIDSl end INPPBI bv ohotomotrv. RESULTS (mean ± SEM. n>5 rats/orou,~: . Bile Flow Billery [HC03-] Billary HC03- Output (Id/min*a liver) (mMI (nmol/mln*u liver) . BASAL PERIOD: Control 1.65 ± 0.11 22.7 + 1.7 41.4 ± 5.7 Amlioride 0.92 ± 0.05* 15.9 ± 0.4* 14.7 ± 0.9* DIDS 1.,54 ± 0.19 18.5 ± 1.7 30.7 ± 7.6 NPPB 1.54+0.23 ~?,.1 ±2.5 36.5± 9.5 EXPERIMENTAL PERIOD:. Controt+Vehide 1.24±0.17 18.9±2.3 25.7± 6.6 Controb.SEC 2.36±0.15§ 38.2±2.1§ 91.7± 8.8§ SEC-induced 1.13 ± 0.15 19.3 ± 2.1 65.9 ± 8.9 Arniiodde+Vehlcle 0.75 ± 0.10 15.5 ± 1.4 t2.0 ± 2.5 Amllorlde+SEC 0.94 ± 0.30* 32.8 ± 1.8§ 29.2 ± 8.5* SEC-induced 0.20±0.30" 17.3± 1.8 17.1 ± 8.5* DIDS+Vehide 0.80+0.16 14.2± 1.2 11.5¢ 2.5 DIDS+SEC 1.72±0.17§ 37.1 ±2.0§ 64.2+ 8.1§ SEC-Induced 0.91 ± 0.17 22.9 ± 2.0 52.7 + 8.1 NPPB*Vehlcle 1.22 ± 0.31 16.6 ± 1.7 21.2 ± 7,1 NPFB+SEC 2.64 ± 0.34~ 34.1 ± 1.7§ 90.6 + 13.6~ SEC-Induoed 1A2 ± 0.34 17.5 ± 1.7 69~3 ± 13.6 * p Vs. r e ~ Control <0.05; § p vs. ~ Vehicle <0.05 (ANOVA). Bi[ialy [DIDS] and INPPB] averaged 7±3 and 1OJ:3 mM which Is sufflclant to Inhibit CI-/HCO3exchango and CI- channels, respectively. CONCLUSION: Amlloride, but not DIDS and NPPB, virtually abonshed SEC-Induced and profoundly Inhibited basal bile forrtmtion and blII~yHCO3- secretion. Provided the ANIT fedrat can selve as mode[ for ductular choleresle, this suggests a role for Na+IH+, but not C I - I H ~ exchango and CI- channels, In blllery cecretion of HC:O3- end water by cholengloC,ytas.
315A
834 N I T R I C
OXIDE S T I M U L A T E S B I L E F L O W AND GLUTATHIONE EXCRETION INDEPENDENT OF CYCLIC GMP-INDUCED HCO3EXCRETION. M Trauner. MH Nathanson. A Mennone. SA Rvdber~. R Bruck*. and JL Bover. Dept. Medicine and Liver Center, Yaie University School of Medicine, New Haven, CT and *Dept. Gastroenterology, Edith Wolfson Medical Center, Holon, Israel. Nitric oxide (NO) may affect bile secretion in several ways. NO stimulates the production of cyclic GMP (cGMP), which increases bile flow and HCO3- excretion in isolated perfused rat livers (IPRL) (Hepatology 18:135A,1993). However, NO also impairs bile canalicular contraction (Gastroenterology 108:841,1995) and increases paracellnlar permeability (AJP in press, 1995), both of which would be expected to decrease bile flow. To clarify the role of NO, we examined the effects of the NO donor sodium nitroprusside (SNP) and of dibutyryl cGMP (DBcGMP) on bile acid-independent bile flow in IPRL and isolated rat hepatocyte couplets (IRHC). SNP stimulated bile flow in IPRL in a dose dependent manner (+20% at 1001.tM; +63% at lmM) and increased both HCO3- and glutathione (GSH) output (1.6-fold and 6-fold, respectively at lmM). This SNP-induced choleresis was accompanied by an increase in cGMP concentration in perfusate (75fold) and bile (12-fold). The choleretic effect of SNP was partially inhibited by methylene blue (25~M), an inhibtor of guanyl cyclase. DBcGMP also stimulated bile flow and HCO3- output in a dose dependent manner (from +65% at 25j.tM to +93% at 1001.tM), but did not increase GSH excretion. In addition, DBcGMP (100~tM) stimulated secretion in IRHC (+15%; p<0.01) in HCO3- containing media. Finally, DBcGMP (1001xM)-induced choleresis in IPRL was nearly abolished by DIDS (101.tM). C o n c l u s i o n s : These findings indicate that NO increases canalicular bile secretion by two different mechanisms: (I) stimulation of cGMP-dependent HCO3- excretion via the C1- / HCO3- exchanger, and (2) stimulation of cGMP-independent GSH excretion.
836 NITRIC OXIDE ISOLATED
- INDUCED CHOLERESIS IS IMPAIRED IN P E P . F U S E D LIVERS OF ENDOTOXEMIC R A T S .
M Trauner. MH Nathanson. SA Rvdhem. C Gamma. WC Sessa*. and JL Bover. Dept. Medicine and Liver Center, *De!at. Pharmacology, Yale University School of Medicine, New Haven, CT. Endotoxin produces cholestasis and induces hepatic NO synthesis. Since exogenous NO donors are choleretic in normal rat liver, stimulation of endogenous NO synthesis could represent a protective mechanism against endotoxin-induced cholestasis. To test this hypothesis we studied the effects of NO on bile secretion in isolated perfused rat livers (IPRL) of endotoxin (LPS)-treated rats (16h after LPS from S. typhimurium, 4mg/kg ip). Portal and systemic NO2- + NO3- plasma levels were increased 57-fold in LPS-treated rats. In IPRL from these rats, NO2- + NO3- levels were also elevated in perfusate (8fold) and bile (9-fold), Bile flow, HCO3- and glutathione (GSH) output were decreased in endotoxemic IPRL (-35%, -36%, and -82% vs controls, respectively). Inhibition of NO synthesis with Nto-nitro-Larginine methyl ester (L-NAME, lmM) or stimulation of NO synthesis with L-arginine (lmM) did not change bile flow in these IPRL. Infusion of the NO donor sodium nitroprusside (SNP, lmM) showed a markedly reduced choleretic effect in endotoxemic IPRL compared to controls (+9% vs +65%). NO-induced HCO3- and GSH excretion were also reduced in endotoxemic livers (-89% and -75% vs controls, respectively), further indicating a desensitization to NO-induced choleresis. Perfusate cyclic GMP levels during SNP infusion were 3fold lower than in controls. Although the choleretic effect of 100 I.tM dibutyryl cyclic GMP (DBcGMP) was similar in LPS-treated rats and controls (+89% vs +96%; n.s.), the effect of 25 IXM DBcGMP was slightly reduced in endotoxemic rats (+45% vs +66%; p<0.05). C o n c l u s i o n s : Endotoxemia abolishes the choleretic effect of NO. Thus, increased synthesis of NO may not be able to protect against cholestasis of sepsis. Rather, these abnormalities may contribute to its pathogenesis.
AASLD ABSTRACTS
833 BOMBESIN S T I M U L A T E S B I C A R B O N A T E S E C R E T I O N FROM BILE DUCTS, WK Cho, SA Rydberg. and JL Boyer. Liver Center, Yale University School of Medicine, New Haven, CT. Bombesin stimulates bile ductular secretion by activating CI'/HCO3" exchange in isolated intrahepatic bile duct units(Gastroenterology 1995;108:A1049). In order to further study this process, novel functional intrahepatic bile duct units(IBDU) were prepared as descdbed(PNAS in press) and cultured for 24-48 hours. The enclosed lumen of the IBDU were microinjected with cell-impermeable BCECFDextran(70K) to study luminal pH changes. Luminal pH increased with infusion of 10 nM bombesin from 7.59 + 0.06 to 7.70 + 0.09 pHU(n=9)(p<0.01), consistent with stimulation of bicarbonate secretion. The isolated perfused rat liver model was then used to further examine the effects of bombesin on bile flow and bicarbonate secretion. Bile samples were collected at 15 minute intervals and analyzed for bile flow and bicarbonate secretion. One-pass portal perfusion with constant 100 nM bombesin infusion in KRB(without bile acid supplementation) did not produce significant changes in bile flow. However, pH, bicarbonate concentration, and total bicarbonate output of bile increased from 7.59 + 0.03 to 7.65 + 0.03(p<0.05; n=6), 21.7 + 1,0 to 25.6 + 2.1 raM(p<0.01), and 24.3 + 3.6 to 28.1 + 6.1 pmole/min/g liver (p<0.05) respectively. Two week bile duct ligated rats were then used to further amplify the bile ductular secretory response. One-pass portal perfusion of 2 week bile duct ligated rat liver with 100 nM bombesin infusion showed only a mild increase in bile flow from 0.86 + 0.15 to 0.90 + 0.14 Id/min/g liver(n=4). However, pH, bicarbonate concentration, and bicarbonate output of bile increased from 7.52 + 0.05 to 7.64 + 0.09(p<0.05), 17.7 + 1.82 to 24.4 + 4.25 raM(p<0.01), and 15.2 + 3.32 to 21.9 + 5.42 pmole/min/g liver(p<0.05) respectively. Conclusion: Although bombesin is known to release other biliary secretagogues including secret±n, bombesin can directly stimulate bicarbonate secretion in rat bile acting at the level of bile duct.
835
AMILORIDE, BUT NOT DIDS AND NPPB, INHIBIT SECRETIN (SEC)-INDUCED CHOLERESlS: EVIDENCE FOR A ROLE OF Ne+/H+, BUT NOT CI-/HCO3- EXCHANGE AND CI- CHANNELS, IN DUCTULAR BILE FORMATION. E.L.Renner & M.Hevdtmann. Dept. of Clio. PharmacoL, Univ. of Bern, Swltzedand. While several H+IHC03- transporters and CI- channels have been identified in chotanglcoytes, the cellular mechanisms mediating SEC-Induced duotular secretion of HCO3- and water remain III understood. AIM: To explore the role of Na+/H+ and CI-IHCO3- exchange, and of CI- channels In SEC-induced ductular cholerasis. METHODS: Male Sprague-Dawley rats were fed 0.06% (w/w) ANIT for 6 weeks to produce hyperplasle of cecretin-rasponsive bile ductules. Rat livers were perfuced In sltu with Krebs buffer containing vehicle (0.2% DMSO), the Na+l H+exchango inhibitor arnUoride (lmM), the CI-JHCO3-exchange Inhibitor D!DS (100 laM) or the CI-channel blocker NPPB (lO011M). Following 3Omln of equilibration, basal bile was collected for 2xl0mln; SEC (25UJh; or vehicle-0.9% NaCI) was then infused foe 60rnin Into the portal vein and bile collected in 10min For±ode, the last two col±sot±onewhen SEC effects had plateaued being taken as experimental samples. Bile volume was assessed by gravlmetry, blllew [HC03-] as total C02 and blllery [DIDSl end INPPBI bv ohotomotrv. RESULTS (mean ± SEM. n>5 rats/orou,~: . Bile Flow Billery [HC03-] Billary HC03- Output (Id/min*a liver) (mMI (nmol/mln*u liver) . BASAL PERIOD: Control 1.65 ± 0.11 22.7 + 1.7 41.4 ± 5.7 Amlioride 0.92 ± 0.05* 15.9 ± 0.4* 14.7 ± 0.9* DIDS 1.,54 ± 0.19 18.5 ± 1.7 30.7 ± 7.6 NPPB 1.54+0.23 ~?,.1 ±2.5 36.5± 9.5 EXPERIMENTAL PERIOD:. Controt+Vehide 1.24±0.17 18.9±2.3 25.7± 6.6 Controb.SEC 2.36±0.15§ 38.2±2.1§ 91.7± 8.8§ SEC-induced 1.13 ± 0.15 19.3 ± 2.1 65.9 ± 8.9 Arniiodde+Vehlcle 0.75 ± 0.10 15.5 ± 1.4 t2.0 ± 2.5 Amllorlde+SEC 0.94 ± 0.30* 32.8 ± 1.8§ 29.2 ± 8.5* SEC-induced 0.20±0.30" 17.3± 1.8 17.1 ± 8.5* DIDS+Vehide 0.80+0.16 14.2± 1.2 11.5¢ 2.5 DIDS+SEC 1.72±0.17§ 37.1 ±2.0§ 64.2+ 8.1§ SEC-Induced 0.91 ± 0.17 22.9 ± 2.0 52.7 + 8.1 NPPB*Vehlcle 1.22 ± 0.31 16.6 ± 1.7 21.2 ± 7,1 NPFB+SEC 2.64 ± 0.34~ 34.1 ± 1.7§ 90.6 + 13.6~ SEC-Induoed 1A2 ± 0.34 17.5 ± 1.7 69~3 ± 13.6 * p Vs. r e ~ Control <0.05; § p vs. ~ Vehicle <0.05 (ANOVA). Bi[ialy [DIDS] and INPPB] averaged 7±3 and 1OJ:3 mM which Is sufflclant to Inhibit CI-/HCO3exchango and CI- channels, respectively. CONCLUSION: Amlloride, but not DIDS and NPPB, virtually abonshed SEC-Induced and profoundly Inhibited basal bile forrtmtion and blII~yHCO3- secretion. Provided the ANIT fedrat can selve as mode[ for ductular choleresle, this suggests a role for Na+IH+, but not C I - I H ~ exchango and CI- channels, In blllery cecretion of HC:O3- end water by cholengloC,ytas.
315A
834 N I T R I C
OXIDE S T I M U L A T E S B I L E F L O W AND GLUTATHIONE EXCRETION INDEPENDENT OF CYCLIC GMP-INDUCED HCO3EXCRETION. M Trauner. MH Nathanson. A Mennone. SA Rvdber~. R Bruck*. and JL Bover. Dept. Medicine and Liver Center, Yaie University School of Medicine, New Haven, CT and *Dept. Gastroenterology, Edith Wolfson Medical Center, Holon, Israel. Nitric oxide (NO) may affect bile secretion in several ways. NO stimulates the production of cyclic GMP (cGMP), which increases bile flow and HCO3- excretion in isolated perfused rat livers (IPRL) (Hepatology 18:135A,1993). However, NO also impairs bile canalicular contraction (Gastroenterology 108:841,1995) and increases paracellnlar permeability (AJP in press, 1995), both of which would be expected to decrease bile flow. To clarify the role of NO, we examined the effects of the NO donor sodium nitroprusside (SNP) and of dibutyryl cGMP (DBcGMP) on bile acid-independent bile flow in IPRL and isolated rat hepatocyte couplets (IRHC). SNP stimulated bile flow in IPRL in a dose dependent manner (+20% at 1001.tM; +63% at lmM) and increased both HCO3- and glutathione (GSH) output (1.6-fold and 6-fold, respectively at lmM). This SNP-induced choleresis was accompanied by an increase in cGMP concentration in perfusate (75fold) and bile (12-fold). The choleretic effect of SNP was partially inhibited by methylene blue (25~M), an inhibtor of guanyl cyclase. DBcGMP also stimulated bile flow and HCO3- output in a dose dependent manner (from +65% at 25j.tM to +93% at 1001.tM), but did not increase GSH excretion. In addition, DBcGMP (100~tM) stimulated secretion in IRHC (+15%; p<0.01) in HCO3- containing media. Finally, DBcGMP (1001xM)-induced choleresis in IPRL was nearly abolished by DIDS (101.tM). C o n c l u s i o n s : These findings indicate that NO increases canalicular bile secretion by two different mechanisms: (I) stimulation of cGMP-dependent HCO3- excretion via the C1- / HCO3- exchanger, and (2) stimulation of cGMP-independent GSH excretion.
836 NITRIC OXIDE ISOLATED
- INDUCED CHOLERESIS IS IMPAIRED IN P E P . F U S E D LIVERS OF ENDOTOXEMIC R A T S .
M Trauner. MH Nathanson. SA Rvdhem. C Gamma. WC Sessa*. and JL Bover. Dept. Medicine and Liver Center, *De!at. Pharmacology, Yale University School of Medicine, New Haven, CT. Endotoxin produces cholestasis and induces hepatic NO synthesis. Since exogenous NO donors are choleretic in normal rat liver, stimulation of endogenous NO synthesis could represent a protective mechanism against endotoxin-induced cholestasis. To test this hypothesis we studied the effects of NO on bile secretion in isolated perfused rat livers (IPRL) of endotoxin (LPS)-treated rats (16h after LPS from S. typhimurium, 4mg/kg ip). Portal and systemic NO2- + NO3- plasma levels were increased 57-fold in LPS-treated rats. In IPRL from these rats, NO2- + NO3- levels were also elevated in perfusate (8fold) and bile (9-fold), Bile flow, HCO3- and glutathione (GSH) output were decreased in endotoxemic IPRL (-35%, -36%, and -82% vs controls, respectively). Inhibition of NO synthesis with Nto-nitro-Larginine methyl ester (L-NAME, lmM) or stimulation of NO synthesis with L-arginine (lmM) did not change bile flow in these IPRL. Infusion of the NO donor sodium nitroprusside (SNP, lmM) showed a markedly reduced choleretic effect in endotoxemic IPRL compared to controls (+9% vs +65%). NO-induced HCO3- and GSH excretion were also reduced in endotoxemic livers (-89% and -75% vs controls, respectively), further indicating a desensitization to NO-induced choleresis. Perfusate cyclic GMP levels during SNP infusion were 3fold lower than in controls. Although the choleretic effect of 100 I.tM dibutyryl cyclic GMP (DBcGMP) was similar in LPS-treated rats and controls (+89% vs +96%; n.s.), the effect of 25 IXM DBcGMP was slightly reduced in endotoxemic rats (+45% vs +66%; p<0.05). C o n c l u s i o n s : Endotoxemia abolishes the choleretic effect of NO. Thus, increased synthesis of NO may not be able to protect against cholestasis of sepsis. Rather, these abnormalities may contribute to its pathogenesis.