Transport of sulfobromophthalein and taurocholate in the HepG2 cell line in relation to the expression of membrane carrier proteins.
The transport of two different classes of organic anions (cholephilic dyes; the sulfobromophthalein, BSP, and bile acids; taurocholate, TC) was investigated in the HepG2 cell line. At 37 degrees C, BSP uptake was found to be biphasic with an apparent saturative curve in the concentration range between 0-6 microM followed by a linear component up to 18 microM. Kinetic constant determination showed an apparent Km of 26.6 +/- 3.1 microM and a Vmax of 5.64 +/- 0.82 nmol BSP.min-1.mg prot-1. At 4 degrees C, uptake was linear. By subtracting this latter component from the total uptake, a saturable, carrier mediated uptake was found with an apparent Km of 3.6 +/- 1.0 microM BSP and a Vmax of 0.37 +/- 0.04 nmol BSP.min-1.mg prot-1 (m +/- SEM, n = 6). These values were fully comparable with those found in freshly isolated male hepatocyte. Immunoblot analysis of HepG2 cell plasma membrane revealed the presence of bilitranslocase when tested against a monospecific antibody against this carrier molecule. On the contrary, TC uptake was linear up to concentration of 100 microM TC. No difference was observed in the presence or absence of Na+. Immunoprecipitation analysis showed the absence of the putative carrier of TC. These data indicate that the HepG2 cell line expresses a functioning bilitranslocase-mediated system. Conversely, carrier mediated bile acid uptake is absent in line with the lack of expression of the carrier protein.
Marchegiano
P
P
Cattedra Gastroenterologia, University of Modena, Italy.
Carubbi
F
F
Tiribelli
C
C
Amarri
S
S
Stebel
M
M
Lunazzi
G C
GC
Levy
D
D
Bellentani
S
S
D018528:ATP-Binding Cassette Transporters; D001693:Biological Transport, Active; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D006801:Humans; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D013448:Sulfobromophthalein / Q000378:metabolism*; D013656:Taurocholic Acid / Q000378:metabolism*
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; C029783:bilirubin binding protein, human; C488927:bilirubin binding protein, rat; C038659:taurocholate carrier protein, rat; D013448:Sulfobromophthalein; D013656:Taurocholic Acid