Uptake of bilirubin into HepG2 cells assayed by thermal lens spectroscopy. Function of bilitranslocase.
Bilitranslocase is a carrier protein localized at the basolateral domain of the hepatocyte plasma membrane. It transports various organic anions, including bromosulfophthalein and anthocyanins. Functional studies in subcellular fractions enriched in plasma membrane revealed a high-affinity binding site for bilirubin, associated with bilitranslocase. The aim of this work was to test whether the liver uptake of bilirubin depends on the activity of bilitranslocase. To this purpose, an assay of bilirubin uptake into HepG2 cell cultures was set up. The transport assay medium contained bilirubin at a concentration of approximately 50 nm in the absence of albumin. To analyse the relative changes in bilirubin concentration in the medium throughout the uptake experiment, a highly sensitive thermal lens spectrometry method was used. The mechanism of bilirubin uptake into HepG2 cells was investigated by using inhibitors such as anti-sequence bilitranslocase antibodies, the protein-modifying reagent phenylmethanesulfonyl fluoride and diverse organic anions, including nicotinic acid, taurocholate and digoxin. To validate the assay further, both bromosulfophthalein and indocyanine green uptake in HepG2 cells was also characterized. The results obtained show that bilitranslocase is a carrier with specificity for both bilirubin and bromosulfophthalein, but not for indocyanine green.
Passamonti
Sabina
S
Dipartimento di Biochimica, Biofisica e Chimica delle Macromolecole, Università di Trieste, Italy. passamonti@bbcm.units.it
Terdoslavich
Michela
M
Margon
Alja
A
Cocolo
Alessandra
A
Medic
Nevenka
N
Micali
Fulvio
F
Decorti
Giuliana
G
Franko
Mladen
M
D000838:Anions / Q000737:chemistry; D000906:Antibodies / Q000276:immunology; D001663:Bilirubin / Q000378:metabolism*; D001692:Biological Transport; D045744:Cell Line, Tumor; D002570:Ceruloplasmin; D004077:Digoxin / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D007208:Indocyanine Green; D008565:Membrane Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D009525:Niacin / Q000494:pharmacology; D027361:Organic Anion Transporters / Q000378:metabolism; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D013057:Spectrum Analysis / Q000379:methods*; D013379:Substrate Specificity; D013448:Sulfobromophthalein; D013656:Taurocholic Acid / Q000494:pharmacology; D013696:Temperature
D000838:Anions; D000906:Antibodies; D008565:Membrane Proteins; D027361:Organic Anion Transporters; D013448:Sulfobromophthalein; D009525:Niacin; D010664:Phenylmethylsulfonyl Fluoride; D013656:Taurocholic Acid; D004077:Digoxin; D002570:Ceruloplasmin; C033838:bilitranslocase; D007208:Indocyanine Green; D001663:Bilirubin