The Human Organic Anion Transport Protein SLC21A6 Is Not Sufficient for Bilirubin Transport

Abstract

Bilirubin is a yellow pigment derived from the degradation of heme. Because of intramolecular hydrogen bonding, it is water insoluble (1). Following formation in various cells throughout the body, it is released into the circulation where it binds avidly to albumin (2, 3). Under normal circumstances, it is extracted from albumin and taken up rapidly by hepatocytes (4, 5). Although the kinetics of uptake suggests carrier mediation, the nature of this carrier has remained elusive (6, 7). The fact that bilirubin can pass rapidly through lipid bilayers has led some investigators to question the necessity of postulating the existence of a carrier (8, 9). A recent report (10) indicates that the human organic anion transport protein SLC21A6 (also known as OATP2, 1 LST-1, and OATP-C) mediates high affinity transport of bilirubin. That study was performed using HEK293 cells that had been stably transfected with OATP2. Cells were harvested from poly-D-lysine-coated plastic dishes, and bilirubin uptake studies were performed on cells in suspension. Nonspecific binding was not reported although it was used to correct uptake data, and we were concerned that the harvesting procedure could result in cell permeabilization. To validate the results in that report, we devised a system in which we could test the ability of OATP2-transfected cells to transport bilirubin without the necessity of detaching the cells from the culture dishes. Initially, we established an assay to quantify bilirubin transport in overnight cultured rat hepatocytes. This assay was then used to examine bilirubin transport by HeLa cells that had been stably transfected with OATP2 under the regulation of a metallothionein promoter. OATP2 expression was induced by incubation of these cells in zinc for 48 h. Uninduced cells did not express OATP2 and were used as controls. These studies showed that cultured rat hepatocytes transported bilirubin with kinetics virtually identical to transport of sulfobromophthalein (BSP). In contrast, OATP2-expressing HeLa cells transported BSP but not bilirubin. In further studies, OATP2-transfected HEK293 cells that were obtained from Cui et al. (10) were also studied using the methodology that they described. We were unable to reproduce their results with respect to bilirubin transport, although these cells transported BSP as they described. The existence of a bilirubin transporter has been an important field of study for many years. The current studies using a newly established assay for bilirubin transport indicate that a role for OATP2 in hepatocyte bilirubin transport is unlikely. EXPERIMENTAL PROCEDURES Preparation of Radiolabeled Ligands

Risk Factors for Severe Hyperbilirubinemia in Neonates

“…In this study, the prevalence of variant 388 in the OATP 2 gene in the case and control groups was 37.5 and 22%, respectively, the former higher than the latter, and the OR for severe hyperbilirubinemia of this variation in the case group was statistically significant. Although it has been reported that OATP 2 is involved in the transportation of both conjugated and unconjugated bilirubins (1), the results of a more recent study showed that a role for OATP 2 in hepatocyte bilirubin transport is unlikely (36). However, the results of this study show that the change of amino acid (aspartic acid to asparagine, encoded by nucleotides 388 -390) at codon 130 of OATP 2 may reduce the function in unconjugated bilirubin elimination, and the mechanism is worthy of further investigation.…”

UGT1A1, SLCO1B1, and SLCO1B3 polymorphisms vs. neonatal hyperbilirubinemia: is there an association?

“…Kang et al (26) did not observe any association of these same polymorphisms with bilirubin levels in a Korean sample. For instance, although Cui et al inferred that SLCO1B1 could be the hepatic transporter for bilirubin (7), Wang et al (27) concluded the unlikelihood of this function, and Zucker et al (28) suggested that bilirubin uptake could occur through passive diffusion. Watchko et al (3) reported that G6PD-deficient infants who were homozygous for the G allele of rs2306283 were more common in the icteric group as compared with controls (87.5% vs. 20%, P = 0.015), respectively.…”

Cytochrome P450 2A5 and bilirubin: Mechanisms of gene regulation and cytoprotection

“…A member of the organic anion transporter protein family, termed OATP2/8/C, has been suggested to mediate BR uptake, but it still remains questionable and requires confirmation (Briz et al, 2003;Wang et al, 2003b;Steeg et al, 2010;de Graaf et al, 2011). After entering the hepatocytes, BR binds to glutathione-S-transferases (GSTs), a major group of detoxification enzymes (Levi et al, 1969;Pickett, 1989;Strange et al, 2001).…”