Bilirubin (Br), the product of heme catabolism, is formed in all cells and shed into the blood, where it is transported by albumin to the liver. At this level, it is taken up, glucurono-conjugated and excreted into the bile by an ATP-dependent efflux pump. Uptake of Br from the blood into the liver has been proposed to be mediated by OATP1B1 (Cui Y et al 2001). Later, this Br transport function has been questioned (Wang Y et al 2003). Bilitranslocase (BTL) is a plasma membrane organic anion carrier that binds Br with high affinity (Kd=2nM) (Battiston L et al 1998). The aim of this work was to test directly the Br transport capacity of BTL in liver cells. A cell transport assay was set up, based on the measurement of the time-dependent disappearance of Br from a medium bathing a monolayer of cultured human liver cells (HepG2). The involvement of BTL was investigated by testing the effect of an anti-sequence antibody on the kinetics of Br disappearance. The medium containing Br was a simple phosphate buffered saline solution (pH 7.4). Under these conditions, its solubility is <70nM, i.e. high enough to saturate BTL, but far too low to be detected by radioactive counting or conventional UV-Vis spectrometry. The samples were therefore assayed by thermal lens spectrometry (Franko M 2001), a technique that enabled to measure Br in the range 2-50nM, avoiding the confounding presence of albumin. Br uptake was found to be a quite fast phenomenon, that was abolished not only by the anti- sequence anti-BTL antibody, but also by nicotinic acid, that binds to BTL with high affinity (Kd=11 nM) at the same level as Br. The serine reagent PMSF, that binds to the Br- and nicotinic acid binding site of BTL (Passamonti S et al 1997), abolished Br uptake into cells as well. Taurocholate and digoxin, two OATP reference substrates, did not affect Br uptake in our assay. Thus, BTL is a Br carrier, as also accepted by the Transport Classification Database (http://tcdb.ucsd.edu/).
BILIRUBIN ANALYSIS BY THERMAL LENS SPECTROMETRY: A TOOL TO INVESTIGATE THE MOLECULAR MECHANISM OF ITS MEMBRANE TRANSPORT
PASSAMONTI, SABINA;DECORTI, GIULIANA;
2005-01-01
Abstract
Bilirubin (Br), the product of heme catabolism, is formed in all cells and shed into the blood, where it is transported by albumin to the liver. At this level, it is taken up, glucurono-conjugated and excreted into the bile by an ATP-dependent efflux pump. Uptake of Br from the blood into the liver has been proposed to be mediated by OATP1B1 (Cui Y et al 2001). Later, this Br transport function has been questioned (Wang Y et al 2003). Bilitranslocase (BTL) is a plasma membrane organic anion carrier that binds Br with high affinity (Kd=2nM) (Battiston L et al 1998). The aim of this work was to test directly the Br transport capacity of BTL in liver cells. A cell transport assay was set up, based on the measurement of the time-dependent disappearance of Br from a medium bathing a monolayer of cultured human liver cells (HepG2). The involvement of BTL was investigated by testing the effect of an anti-sequence antibody on the kinetics of Br disappearance. The medium containing Br was a simple phosphate buffered saline solution (pH 7.4). Under these conditions, its solubility is <70nM, i.e. high enough to saturate BTL, but far too low to be detected by radioactive counting or conventional UV-Vis spectrometry. The samples were therefore assayed by thermal lens spectrometry (Franko M 2001), a technique that enabled to measure Br in the range 2-50nM, avoiding the confounding presence of albumin. Br uptake was found to be a quite fast phenomenon, that was abolished not only by the anti- sequence anti-BTL antibody, but also by nicotinic acid, that binds to BTL with high affinity (Kd=11 nM) at the same level as Br. The serine reagent PMSF, that binds to the Br- and nicotinic acid binding site of BTL (Passamonti S et al 1997), abolished Br uptake into cells as well. Taurocholate and digoxin, two OATP reference substrates, did not affect Br uptake in our assay. Thus, BTL is a Br carrier, as also accepted by the Transport Classification Database (http://tcdb.ucsd.edu/).Pubblicazioni consigliate
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