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Hepatocyte canalicular transport of organic anions in bile duct-ligated rats
Hepatdoy cornmunlcations
International Hepatology Communications 5 (1996) 326-330
ELSEVIER
Hepatocyte canalicular transport of organic anions in bile duct-ligated rats Yukihiko “Second
Adachi”,*, Hirohisa Miya”, Yoshifumi Okuyamab, Toshinori Kamisakoa, Toshio Yamamotob
Department
bLzye Science
Research
of Internal Institute,
Medicine, Kinki Osakasayama, Kinki University,
University School Osaka 589, Japan 377-2 Ohnohigashi,
of Medicine, Osakasayama.
377-2
Ohnohigashi,
Osaka
589, Japan
Received 16 May 1996; revised 10 July 1996; accepted 12 July 1996
Abstract
Transport of various intrinsic and extrinsic organic anions acrossthe canalicular membrane of hepatocytes has been confirmed to occur via ATP-dependent primary active transport. To investigatetheseprocesses in the presenceof obstructivejaundice, the uptake of radiolabeledtaurocholate and pravastatin, a non-bile acid organic anion, by isolated hepatocytecanalicular membranevesicleswas observedafter bile duct ligation in rats. The Mg + + -ATPase and Na + , K+ -ATPase activity did not differ betweenmembranevesicle preparationsfrom sham-operatedand bile duct-ligated rats. Taurocholate uptake decreased significantly to 45% and 29%,while pravastatin uptake decreasedto 35%and 35%of that in sham-operatedrats at 2 and 7 days after bile duct ligation. Theseresultsindicate that the ATP-dependent transport systemsfor both bile saltsand non-bile acid organic anions are impairedsoon after the onsetof obstructive jaundice. Obstructive jaundice; Bile duct ligation; Hepatic excretion; Bile acid; Organic anion; Membrane transport; Membrane vesicles Keywords:
* Corresponding author. Tel: + 81 723 660221; fax: + 81 723 660206. 0928-4346/96/$12.00 0 1996 Elsevier Science Ireland Ltd. All rights reserved PIZ SO928-4346(96)00317-9
Y. Adachi et al. / International Hepatology Communications 5 (1996) 326-330
327
1. Introduction Hepatic excretion of bile acids and non-bile acid organic anions (NBAOAs) has been confirmed to occur mainly via ATP-dependent primary active transport [1,2]. Alteration of these transport systems is reported to be involved in the pathogenesis of cyclosporin C- [3], ethinylestradiol[4] and endotoxin-induced [5] cholestasis in rats. In the present study, changes of these transport systems were investigated in rats with bile duct ligation.
2. Materials
and methods
2.1. Animals and treatment Male SD rats weighing 230-280 g were obtained from Japan SLC Co., Ltd. (Hamamatsu, Japan). The animals were allowed free access to food and were housed at a constant temperature and humidity with a 12-h light (07:00-19:00 h), 12-h dark cycle. Rats were either subjected to bile duct ligation or to a sham operation under ether anesthesia, and their livers were removed before operation or at 2 or 7 days afterwards. 2.2. Preparation
of rat hepatocyte canalicular
CMV were isolated and purified method of Kobayashi et al. [6].
from
membrane vesicles (CMV) Sprague-Dawley
rat livers by the
2.3. Chemicals [3H]Taurocholate sodium salt ([3H]TC; 185 MBq/umol) was obtained from New England Nuclear Corp. (Boston, MA) and [14C]pravastatin sodium salt ([14C]PV; 0.42 MBq/unrol) was a kind gift from Dr Komai (Sankyo Co., Ltd., Tokyo, Japan). The other chemicals were purchased from Sigma Chemical Co. (St Louis, MO) or Wako Pure Chemical industries (Osaka, Japan) and were all of analytical grade. 2.4. CMV uptake of rH]TC
or [‘“C]PV
Uptake of [3H]TC (concentration in the medium: 50 umol/l) was determined by a rapid filtration assay in the presence of 1 mmol/l ATP [I], while [‘4C]PV (300 umol/l) uptake was determined in the presence of 5 mmol/l ATP and a pH gradient (from 5.2 outside to 6.7 inside the CMV) [7]. An ATP-regenerating system was added to the medium in both of the uptake studies [1,7]. ATP-dependent uptake was calculated by subtracting the uptake in the absence of ATP from that in its presence.
328
Y. Ada&
2.5. Statistical
et al. / Intermtional
Hepntology
Conmunications
5 (1996)
326-330
analysis
Five determinations of uptake (each in duplicate) were performed using at least two different CMV preparations in each transport experiment. Data were expressed as the mean _+SD. Statistical comparison was made by the Student’s t-test,
3. Results
3.1. Purity of CMV The increase of Mg+ + -ATPase, alkaline phosphatase, and Na+, K+-ATPase activity [l] relative to that of liver homogenate in control (preoperative) rat CMV was 15.0 + 3.4-fold, 51 .O + 9.2-fold, and 7.4 _+1.8-fold (mean f S.D.), respectively. The activities of these enzymes in CMV from sham-operated and bile duct-ligated rat livers (after 2 and 7 days) were not significantly different from the control levels. 3.2. Changes of rH]TC
and [‘“C]PV
uptake by CMV after bile duct ligation
ATP-dependent [‘H]TC uptake decreased progressively being respectively about 45% and 20% of the control level at 2 and 7 days after bile duct ligation (Fig. 1A). [14C]PV uptake decreased significantly to 35% of the control level at both 2 and 7 days after bile duct ligation (Fig. 1B).
4. Discussion
Primary active transport is involved in the biliary excretion of bile salts and NBAOAs by the canalicular bile salt transporter and multispecific organic anion transporter [1,2,8]. The present findings indicated that ATP-dependent transport systems for both bile salts and NBAOA (as represented by pravastatin) are equally damaged in the early stage of obstructive jaundice. The &s for the uptake of [3H]TC and [14C]PV are 47 and 300 nmol/l, respectively [1,7]. The concentrations of these substrates used here were near their K,s and it is difficult to tell which of V,,, and Z& is more affected by bile duct obstruction. In the presence of experimental cholestasis, varying degrees of suppression of organic anion transporters have been reported. For example, cyclosporin C initially decreases TC transport by CMV [3], ethinylestradiol decreases both TC and glutathione conjugate (NBAOA) transport to a similar extent [4], and endotoxin initially decreases biliary excretion of 4-dinitrophenyl-glutathione (NBAOA) in the perfused rat liver [5]. Schrenk et al. [9] reported induction of multidrug resistance (mdr) gene expression of plasma membrane proteins by
Y. Adachi er al. / International Hepatology Communications 5 (1996) 326-330
low,
(4
329
(8)
Fig. 1. Changes of [3H]TC and [r4C]PV uptake by CMV after bile duct ligation in rats. (A) [7H]TC uptake, (B) [‘“C]PV uptake. Solid circles indicate control values obtained before operation or after sham operation, while open circles indicate values obtained after bile duct ligation. Mean k S.D., n = 5. *P < 0.05 vs. both the sham operation group and the control group before operation.
alpha-naphthylisothiocyanate treatment or bile duct ligation in rats, which contrasts with the above reports as well as with the results reported here. After the onset of obstructive jaundice, extensive changes of various canalicular excretory processes for organic anions and cations may occur. The mechanism of these changes and their interrelationship should be studied in future.
References [l] Adachi Y. Kobayashi H, Kurumi Y, et al. ATP-dependent taurocholate transport by rat liver canalicular membrane vesicles. Hepatology 1991; 14: 655-659. [2] Nishida T, Gatmaitan Z, Roy-Chowdhury .I, Arias IM. Two distinct mechanisms for bilirubin glucuronide transport by rat bile canalicular membrane vesicles. Demonstration of defective ATP-dependent transport in rats (TR-) with inherited conjugated hyperbilirubinemia. J Clin Invest 1992: 90: 2130-2135. [3] Kadmon M. Kltinemann C, Bbhme M, et al. Cholestasis caused by inhibition of the adenosine triphosphate-dependent transport from the hepatocyte into bile. Gastroenterology 1993; 104: 1507.-1514. [4] Bossard R, Stieger B, O’Neill B, Fricker G, Maier PJ. Ethinylestradiol treatment induces multiple canalicular membrane transport alteration in rat liver. J Clin Invest 1993; 91: 271442720. [5] Roelofsen H, Schoemaker B, Bakker C. Ottenhoff R, Jansen PLM, Oude Elferink RPJ. Impaired hepatocanalicular organic anion transport in endotoxemic rats. Am J Physiol 1995; 269: 642776434. [6] Kobayashi K, Sogame Y, Hara H. Hayashi K. Mechanism of glutathione S-conjugate transport in canalicular and basolateral rat liver plasma membranes. J Biol Chem 1990: 265: 77377741.
330
Y. Ada&i
et ul. / International
Heptolog~~
Communications
5 (1996)
326-330
[7] Adachi Y. Okuyama Y, Miya H, et al. Pravastatin transport across the hepatocyte canalicular membrane requires both ATP and a transmembrane pH gradient. J Gastroenterol Hepatol 1996; 11: 580-585. [8] Oude Elferink RPJ, Meijer DKF, Kuipers F, Jansen PLM, Groen AK, Groothuis GMM. Hepatobiliary secretion of organic compounds; molecular mechanisms of membrane transport, Biochim Biophys Acta 1995: 1241: 2155268. [9] Schrenk D, Gant TW, Preisegger K-W, Silverman JA. Marino PA, Thorgeirsson SS. Induction of multidrug resistance gene expression during cholestasis in rats and nonhuman primates. Hepatology 1993: 17: 854-860.
International Hepatology Communications 5 (1996) 326-330
ELSEVIER
Hepatocyte canalicular transport of organic anions in bile duct-ligated rats Yukihiko “Second
Adachi”,*, Hirohisa Miya”, Yoshifumi Okuyamab, Toshinori Kamisakoa, Toshio Yamamotob
Department
bLzye Science
Research
of Internal Institute,
Medicine, Kinki Osakasayama, Kinki University,
University School Osaka 589, Japan 377-2 Ohnohigashi,
of Medicine, Osakasayama.
377-2
Ohnohigashi,
Osaka
589, Japan
Received 16 May 1996; revised 10 July 1996; accepted 12 July 1996
Abstract
Transport of various intrinsic and extrinsic organic anions acrossthe canalicular membrane of hepatocytes has been confirmed to occur via ATP-dependent primary active transport. To investigatetheseprocesses in the presenceof obstructivejaundice, the uptake of radiolabeledtaurocholate and pravastatin, a non-bile acid organic anion, by isolated hepatocytecanalicular membranevesicleswas observedafter bile duct ligation in rats. The Mg + + -ATPase and Na + , K+ -ATPase activity did not differ betweenmembranevesicle preparationsfrom sham-operatedand bile duct-ligated rats. Taurocholate uptake decreased significantly to 45% and 29%,while pravastatin uptake decreasedto 35%and 35%of that in sham-operatedrats at 2 and 7 days after bile duct ligation. Theseresultsindicate that the ATP-dependent transport systemsfor both bile saltsand non-bile acid organic anions are impairedsoon after the onsetof obstructive jaundice. Obstructive jaundice; Bile duct ligation; Hepatic excretion; Bile acid; Organic anion; Membrane transport; Membrane vesicles Keywords:
* Corresponding author. Tel: + 81 723 660221; fax: + 81 723 660206. 0928-4346/96/$12.00 0 1996 Elsevier Science Ireland Ltd. All rights reserved PIZ SO928-4346(96)00317-9
Y. Adachi et al. / International Hepatology Communications 5 (1996) 326-330
327
1. Introduction Hepatic excretion of bile acids and non-bile acid organic anions (NBAOAs) has been confirmed to occur mainly via ATP-dependent primary active transport [1,2]. Alteration of these transport systems is reported to be involved in the pathogenesis of cyclosporin C- [3], ethinylestradiol[4] and endotoxin-induced [5] cholestasis in rats. In the present study, changes of these transport systems were investigated in rats with bile duct ligation.
2. Materials
and methods
2.1. Animals and treatment Male SD rats weighing 230-280 g were obtained from Japan SLC Co., Ltd. (Hamamatsu, Japan). The animals were allowed free access to food and were housed at a constant temperature and humidity with a 12-h light (07:00-19:00 h), 12-h dark cycle. Rats were either subjected to bile duct ligation or to a sham operation under ether anesthesia, and their livers were removed before operation or at 2 or 7 days afterwards. 2.2. Preparation
of rat hepatocyte canalicular
CMV were isolated and purified method of Kobayashi et al. [6].
from
membrane vesicles (CMV) Sprague-Dawley
rat livers by the
2.3. Chemicals [3H]Taurocholate sodium salt ([3H]TC; 185 MBq/umol) was obtained from New England Nuclear Corp. (Boston, MA) and [14C]pravastatin sodium salt ([14C]PV; 0.42 MBq/unrol) was a kind gift from Dr Komai (Sankyo Co., Ltd., Tokyo, Japan). The other chemicals were purchased from Sigma Chemical Co. (St Louis, MO) or Wako Pure Chemical industries (Osaka, Japan) and were all of analytical grade. 2.4. CMV uptake of rH]TC
or [‘“C]PV
Uptake of [3H]TC (concentration in the medium: 50 umol/l) was determined by a rapid filtration assay in the presence of 1 mmol/l ATP [I], while [‘4C]PV (300 umol/l) uptake was determined in the presence of 5 mmol/l ATP and a pH gradient (from 5.2 outside to 6.7 inside the CMV) [7]. An ATP-regenerating system was added to the medium in both of the uptake studies [1,7]. ATP-dependent uptake was calculated by subtracting the uptake in the absence of ATP from that in its presence.
328
Y. Ada&
2.5. Statistical
et al. / Intermtional
Hepntology
Conmunications
5 (1996)
326-330
analysis
Five determinations of uptake (each in duplicate) were performed using at least two different CMV preparations in each transport experiment. Data were expressed as the mean _+SD. Statistical comparison was made by the Student’s t-test,
3. Results
3.1. Purity of CMV The increase of Mg+ + -ATPase, alkaline phosphatase, and Na+, K+-ATPase activity [l] relative to that of liver homogenate in control (preoperative) rat CMV was 15.0 + 3.4-fold, 51 .O + 9.2-fold, and 7.4 _+1.8-fold (mean f S.D.), respectively. The activities of these enzymes in CMV from sham-operated and bile duct-ligated rat livers (after 2 and 7 days) were not significantly different from the control levels. 3.2. Changes of rH]TC
and [‘“C]PV
uptake by CMV after bile duct ligation
ATP-dependent [‘H]TC uptake decreased progressively being respectively about 45% and 20% of the control level at 2 and 7 days after bile duct ligation (Fig. 1A). [14C]PV uptake decreased significantly to 35% of the control level at both 2 and 7 days after bile duct ligation (Fig. 1B).
4. Discussion
Primary active transport is involved in the biliary excretion of bile salts and NBAOAs by the canalicular bile salt transporter and multispecific organic anion transporter [1,2,8]. The present findings indicated that ATP-dependent transport systems for both bile salts and NBAOA (as represented by pravastatin) are equally damaged in the early stage of obstructive jaundice. The &s for the uptake of [3H]TC and [14C]PV are 47 and 300 nmol/l, respectively [1,7]. The concentrations of these substrates used here were near their K,s and it is difficult to tell which of V,,, and Z& is more affected by bile duct obstruction. In the presence of experimental cholestasis, varying degrees of suppression of organic anion transporters have been reported. For example, cyclosporin C initially decreases TC transport by CMV [3], ethinylestradiol decreases both TC and glutathione conjugate (NBAOA) transport to a similar extent [4], and endotoxin initially decreases biliary excretion of 4-dinitrophenyl-glutathione (NBAOA) in the perfused rat liver [5]. Schrenk et al. [9] reported induction of multidrug resistance (mdr) gene expression of plasma membrane proteins by
Y. Adachi er al. / International Hepatology Communications 5 (1996) 326-330
low,
(4
329
(8)
Fig. 1. Changes of [3H]TC and [r4C]PV uptake by CMV after bile duct ligation in rats. (A) [7H]TC uptake, (B) [‘“C]PV uptake. Solid circles indicate control values obtained before operation or after sham operation, while open circles indicate values obtained after bile duct ligation. Mean k S.D., n = 5. *P < 0.05 vs. both the sham operation group and the control group before operation.
alpha-naphthylisothiocyanate treatment or bile duct ligation in rats, which contrasts with the above reports as well as with the results reported here. After the onset of obstructive jaundice, extensive changes of various canalicular excretory processes for organic anions and cations may occur. The mechanism of these changes and their interrelationship should be studied in future.
References [l] Adachi Y. Kobayashi H, Kurumi Y, et al. ATP-dependent taurocholate transport by rat liver canalicular membrane vesicles. Hepatology 1991; 14: 655-659. [2] Nishida T, Gatmaitan Z, Roy-Chowdhury .I, Arias IM. Two distinct mechanisms for bilirubin glucuronide transport by rat bile canalicular membrane vesicles. Demonstration of defective ATP-dependent transport in rats (TR-) with inherited conjugated hyperbilirubinemia. J Clin Invest 1992: 90: 2130-2135. [3] Kadmon M. Kltinemann C, Bbhme M, et al. Cholestasis caused by inhibition of the adenosine triphosphate-dependent transport from the hepatocyte into bile. Gastroenterology 1993; 104: 1507.-1514. [4] Bossard R, Stieger B, O’Neill B, Fricker G, Maier PJ. Ethinylestradiol treatment induces multiple canalicular membrane transport alteration in rat liver. J Clin Invest 1993; 91: 271442720. [5] Roelofsen H, Schoemaker B, Bakker C. Ottenhoff R, Jansen PLM, Oude Elferink RPJ. Impaired hepatocanalicular organic anion transport in endotoxemic rats. Am J Physiol 1995; 269: 642776434. [6] Kobayashi K, Sogame Y, Hara H. Hayashi K. Mechanism of glutathione S-conjugate transport in canalicular and basolateral rat liver plasma membranes. J Biol Chem 1990: 265: 77377741.
330
Y. Ada&i
et ul. / International
Heptolog~~
Communications
5 (1996)
326-330
[7] Adachi Y. Okuyama Y, Miya H, et al. Pravastatin transport across the hepatocyte canalicular membrane requires both ATP and a transmembrane pH gradient. J Gastroenterol Hepatol 1996; 11: 580-585. [8] Oude Elferink RPJ, Meijer DKF, Kuipers F, Jansen PLM, Groen AK, Groothuis GMM. Hepatobiliary secretion of organic compounds; molecular mechanisms of membrane transport, Biochim Biophys Acta 1995: 1241: 2155268. [9] Schrenk D, Gant TW, Preisegger K-W, Silverman JA. Marino PA, Thorgeirsson SS. Induction of multidrug resistance gene expression during cholestasis in rats and nonhuman primates. Hepatology 1993: 17: 854-860.