Abstract
The transport of highly purified 3H-labelled unconjugated bilirubin (UCB) was investigated in rat liver plasma membrane vesicles enriched in the canalicular domain and found to be stimulated (more than 5-fold) by the addition of ATP. Other nucleotides, such as AMP, ADP, GTP and a non-hydrolysable ATP analogue (adenosine 5'-[α,β-methylene] triphosphate), did not stimulate [3H]UCB transport, indicating that ATP hydrolysis was necessary for the stimulatory effect. [3H]UCB uptake occurred into an osmotically sensitive space. At an unbound bilirubin concentration ([Bf]) below saturation of the aqueous phase (no more than 70 nM UCB), the ATP-dependent transport followed saturation kinetics with respect to [Bf], with a K(m) of 26 ± 8 nM and a V(max) of 117 ± 11 pmol per 15 s per mg of protein. Unlabelled UCB inhibited the uptake of [3H]UCB, indicating that UCB was the transported species. Inhibitors of ATPase activity such as vanadate or diethyl pyrocarbonate decreased the ATP effect (59 ± 11% and 100% respectively). Daunomycin, a known substrate for multidrug resistance protein-1, and taurocholate did not inhibit the ATP-dependent [3H]UCB transport, suggesting that neither mdr-1 nor the canalicular bile acid transporter is involved in the canalicular transport of UCB. [3H]UCB uptake (both with and without ATP) in canalicular vesicles obtained from TR- rats was comparable to that in vesicles obtained from Wistar rats, indicating that the canalicular multispecific organic anion transporter, cMOAT, does not account for UCB transport. These results indicate that UCB is transported across the canalicular membrane of the liver cell by an ATP-dependent mechanism involving an as yet unidentified transporter.
| Original language | English |
|---|---|
| Pages (from-to) | 99-103 |
| Number of pages | 5 |
| Journal | Biochemical Journal |
| Volume | 331 |
| Issue number | 1 |
| Publication status | Published - Apr 1 1998 |
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- Biochemistry
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ATP-dependent transport of unconjugated bilirubin by rat liver canalicular plasma membrane vesicles. / Pascolo, L.; Bayon, E. J.; Cupelli, F.; Ostrow, J. D.; Tribelli, C.
In: Biochemical Journal, Vol. 331, No. 1, 01.04.1998, p. 99-103.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - ATP-dependent transport of unconjugated bilirubin by rat liver canalicular plasma membrane vesicles
AU - Pascolo, L.
AU - Bayon, E. J.
AU - Cupelli, F.
AU - Ostrow, J. D.
AU - Tribelli, C.
PY - 1998/4/1
Y1 - 1998/4/1
N2 - The transport of highly purified 3H-labelled unconjugated bilirubin (UCB) was investigated in rat liver plasma membrane vesicles enriched in the canalicular domain and found to be stimulated (more than 5-fold) by the addition of ATP. Other nucleotides, such as AMP, ADP, GTP and a non-hydrolysable ATP analogue (adenosine 5'-[α,β-methylene] triphosphate), did not stimulate [3H]UCB transport, indicating that ATP hydrolysis was necessary for the stimulatory effect. [3H]UCB uptake occurred into an osmotically sensitive space. At an unbound bilirubin concentration ([Bf]) below saturation of the aqueous phase (no more than 70 nM UCB), the ATP-dependent transport followed saturation kinetics with respect to [Bf], with a K(m) of 26 ± 8 nM and a V(max) of 117 ± 11 pmol per 15 s per mg of protein. Unlabelled UCB inhibited the uptake of [3H]UCB, indicating that UCB was the transported species. Inhibitors of ATPase activity such as vanadate or diethyl pyrocarbonate decreased the ATP effect (59 ± 11% and 100% respectively). Daunomycin, a known substrate for multidrug resistance protein-1, and taurocholate did not inhibit the ATP-dependent [3H]UCB transport, suggesting that neither mdr-1 nor the canalicular bile acid transporter is involved in the canalicular transport of UCB. [3H]UCB uptake (both with and without ATP) in canalicular vesicles obtained from TR- rats was comparable to that in vesicles obtained from Wistar rats, indicating that the canalicular multispecific organic anion transporter, cMOAT, does not account for UCB transport. These results indicate that UCB is transported across the canalicular membrane of the liver cell by an ATP-dependent mechanism involving an as yet unidentified transporter.
AB - The transport of highly purified 3H-labelled unconjugated bilirubin (UCB) was investigated in rat liver plasma membrane vesicles enriched in the canalicular domain and found to be stimulated (more than 5-fold) by the addition of ATP. Other nucleotides, such as AMP, ADP, GTP and a non-hydrolysable ATP analogue (adenosine 5'-[α,β-methylene] triphosphate), did not stimulate [3H]UCB transport, indicating that ATP hydrolysis was necessary for the stimulatory effect. [3H]UCB uptake occurred into an osmotically sensitive space. At an unbound bilirubin concentration ([Bf]) below saturation of the aqueous phase (no more than 70 nM UCB), the ATP-dependent transport followed saturation kinetics with respect to [Bf], with a K(m) of 26 ± 8 nM and a V(max) of 117 ± 11 pmol per 15 s per mg of protein. Unlabelled UCB inhibited the uptake of [3H]UCB, indicating that UCB was the transported species. Inhibitors of ATPase activity such as vanadate or diethyl pyrocarbonate decreased the ATP effect (59 ± 11% and 100% respectively). Daunomycin, a known substrate for multidrug resistance protein-1, and taurocholate did not inhibit the ATP-dependent [3H]UCB transport, suggesting that neither mdr-1 nor the canalicular bile acid transporter is involved in the canalicular transport of UCB. [3H]UCB uptake (both with and without ATP) in canalicular vesicles obtained from TR- rats was comparable to that in vesicles obtained from Wistar rats, indicating that the canalicular multispecific organic anion transporter, cMOAT, does not account for UCB transport. These results indicate that UCB is transported across the canalicular membrane of the liver cell by an ATP-dependent mechanism involving an as yet unidentified transporter.
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UR - http://www.scopus.com/inward/citedby.url?scp=0032055775&partnerID=8YFLogxK
M3 - Article
C2 - 9512466
AN - SCOPUS:0032055775
VL - 331
SP - 99
EP - 103
JO - Biochemical Journal
JF - Biochemical Journal
SN - 0264-6021
IS - 1
ER -