Expression of bilitranslocase in the vascular endothelium and its function as a flavonoid transporter

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Determination of bilirubin by thermal lens spectrometry and studies of its transport into hepatic cells

The liver is responsible for clearance of bilirubin, the end product of heme catabolism, from the bloodstream. The main aim of our investigation was to determine the role of the carrier protein bilitranslocase in bilirubin uptake into the liver. Our experiments consisted of exposing cell cultures to bilirubin solutions under different conditions and measuring the uptake of bilirubin into the cells. However, since bilirubin is only slightly soluble in aqueous solution (< 70 nM at pH 7.4), we had to use bilirubin concentrations that are far below the limit of detection of the commonly used techniques (e.g. LOD for HPLC with UV-Vis detection $\cong 10$ $\mu$M). TLS showed up to be a suitable technique for investigation of bilirubin uptake with an LOD of 2 nM. Under basal conditions, bilirubin uptake did not occur. However, increase of cytosolic NADH due to catabolism of specific substrates (e.g. lactate or ethanol) seemed to trigger bilirubin uptake. Furthermore, bilirubin uptake was completely inhibited by addition of specific anti-bilitranslocase antibodies. We can thus infer that, under these conditions, bilitranslocase is the main bilirubin transporter

Uptake of grape anthocyanins into the rat kidney and the involvement of bilitranslocase

6Anthocyanins are among the most common flavonoids in the human diet. In spite of their very low bioavailability, anthocyanins are indicated as active in preventing the progress of cardiovascular and neurodegenerative diseases, obesity, inflammation, and cancer. Any piece of knowledge concerning absorption, tissue distribution, metabolism, and excretion of dietary anthocyanins is expected to help understanding the apparent paradox between their low concentrations in cells and their bioactivity. The aim of this work was to investigate the renal uptake of dietary anthocyanins and the underlying molecular mechanism. A solution containing anthocyanins extracted from grape (Vitis vinifera) was introduced into the isolated stomach of anesthetized rats; after both 10 and 30 min, plasma, liver, and kidney were analyzed for their anthocyanin contents. While anthocyanins in the liver were at apparent equilibrium with plasma both after 10 and 30 min, kidney anthocyanins were 3- and 2.3-fold higher than in plasma, after 10 and 30 min, respectively. Since the transport activity of the bilitranslocase in kidney basolateral membrane vesicles was competitively inhibited by malvidin 3-glucoside (K(i) = 4.8 +/- 0.2 microM), the anthocyanin uptake from blood into kidney tubular cells is likely to be mediated by the kidney isoform of this organic anion membrane transporter.nonemixedVANZO; A; TERDOSLAVICH; M; BRANDONI; A; TORRES; A. M; VRHOVSEK; U; PASSAMONTI S.Vanzo, A; Terdoslavich, M; Brandoni, A; Torres, A. M; Vrhovsek, U; Passamonti, Sabin

BILIRUBIN ANALYSIS BY THERMAL LENS SPECTROMETRY: A TOOL TO INVESTIGATE THE MOLECULAR MECHANISM OF ITS MEMBRANE TRANSPORT

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/)

Bioavailability of flavonoids: a review of their membrane transport and the function of bilitranslocase in animal and plant organisms.

Fruits and vegetables are rich in flavonoids, and ample epidemiological data show that diets rich in fruits and vegetables confer protection against cardiovascular, neurodegenerative and inflammatory diseases, and cancer. However, flavonoid bioavailability is reportedly very low in mammals and the molecular mechanisms of their action are still poorly known. This review focuses on membrane transport of flavonoids, a critical determinant of their bioavailability. Cellular influx and efflux transporters are reviewed for their involvement in the absorption of flavonoids from the gastro-intestinal tract and their subsequent tissue distribution. A focus on the mammalian bilirubin transporter bilitranslocase (TCDB 2.A.65.1.1) provides further insight into flavonoid bioavailability and its relationship with plasma bilirubin (an endogenous antioxidant). The general function of bilitranslocase as a flavonoid membrane transporter is further demonstrated by the occurrence of a plant homologue in organs (petals, berries) where flavonoid biosynthesis is most active. Bilitranslocase appears associated with sub-cellular membrane compartments and operates as a flavonoid membrane transporter