Mechanisms of Hepatocyte Organic Anion Transport

42.2.1.1

Influence of Albumin Binding

Studies performed in the isolated perfused rat liver revealed that as much as 50% of bilirubin or sulfobromophthalein (BSP) is removed from perfusate in a single pass. Despite the fact that these compounds are bound avidly to albumin, it is the free organic anion rather than the albumin-organic anion complex that is taken up. Subsequent investigations performed with overnight cultured rat hepatocytes revealed saturable BSP uptake that was inhibited by bilirubin. Similar to findings in the isolated perfused rat liver, ligand but not albumin was taken up by cultured hepatocytes; that is, albumin remained extracellular. The lack of specificity for albumin as a carrier was noted in studies in the isolated perfused rat liver in which there was no enhancement of bilirubin uptake when it was presented as a complex with albumin as compared to its presentation as a complex with its normally cytosolic binding protein ligandin or in the absence of albumin.

42.2.1.2

Driving Forces

The driving forces for nonbile acid organic anion uptake have also been the subject of a great deal of study. Early experiments performed in isolated rat hepatocytes indicated saturable uptake of BSP without a requirement for cellular energy, as determined by the lack of effect of preincubation of cells in antimycin A, rotenone, or carbonylcyanide m -chlorophenylhydrazone. However, subsequent studies using different metabolic inhibitors (DNP, KCN, or sodium azide/2-deoxyglucose) clearly showed that cellular energy was required for BSP uptake, and a similar difference in sensitivity of the uptake process to various metabolic inhibitors was also seen in studies of GSH-BSP uptake by isolated rat hepatocytes. The physiologic significance of this differential sensitivity remains unknown.

The development of a short-term cultured rat hepatocyte system facilitated studies in which the environment could be manipulated, and transport characteristics and driving forces could be examined. In particular, isosmotic substitution of NaCl in the medium by sucrose resulted in an 80% fall in uptake of BSP or bilirubin by cultured rat hepatocytes. Although this result might have signified a requirement for extracellular Na ⁺ , this was not the case. There was no significant effect on uptake by substitution of Na ⁺ in medium by K ⁺ , Li ⁺ , or choline. In contrast, when Cl ⁻ was replaced by HCO ₃ ⁻ or gluconate, ligand uptake was markedly reduced. Similar Cl ⁻ dependence has been described for hepatocyte uptake of bilirubin by cultured rat hepatocytes and by isolated perfused rat liver. The mechanism for this effect remains unclear. There was no stimulation of BSP uptake by unidirectional Cl ⁻ gradients, although the affinity of BSP for the surface of hepatocytes was approximately 10-fold higher in the presence of Cl ⁻ than in its absence. It is interesting to note that Cl ⁻ -dependent BSP uptake is seen in differentiated hepatocytes and is lost with time in culture and in hepatoma cell lines. As this Cl ⁻ -dependent extraction of BSP from albumin appeared to be characteristic of the high-affinity hepatocyte organic anion uptake mechanism, it was subsequently utilized to develop an assay to clone the transporter using a functional expression strategy in Xenopus laevis oocytes (see below). The influence of unidirectional pH gradients on BSP transport by cultured rat hepatocytes was also examined. These studies showed that BSP transport was stimulated by an inside-to-outside OH ⁻ gradient, consistent with OH ⁻ exchange or H ⁺ cotransport.

42.2.1.3

Physiologic Modulation of Transport

As noted above, it is possible for lipophilic molecules to cross a lipid bilayer without requiring protein facilitation. However, the specificity of the hepatocyte for uptake of many of these compounds and their reduced uptake during physiologic perturbations, as noted below, suggest that simple passage through lipid bilayers represents at best a minor part of their uptake. One example is the process of liver regeneration following two-thirds partial hepatectomy in the rat. Within 6 hours of the regenerative stimulus, influx of bilirubin or BSP is reduced by 50%, when calculated independently of liver mass, returning to normal levels by 4 days. Similar studies were performed with the hypolipidemic agent nafenopin, which when administered to rats for 2 days, results in liver hypertrophy and hyperplasia that morphologically resembles regeneration. Of note is the fact that influx of BSP and bilirubin glucuronides was reduced by 50% in these rats, while bilirubin influx was normal. This dissociation of uptake for these subclasses of organic anions suggests that they may have partially independent uptake mechanisms. Extracellular ATP also downregulates hepatocyte uptake of BSP, and this has been attributed to signal transduction mediated by a purinergic receptor that recognizes ATP ^{− 4.}

42.2.2.1

Biochemical Studies

The studies described above are consistent with the existence of an organic anion transporter(s) that is present on the basolateral (sinusoidal) plasma membrane of hepatocytes. The nature of this transporter has been the subject of investigation for several years. Initial studies described high-affinity binding of BSP to preparations of liver cell plasma membrane and a 55 kDa protein termed BSP/bilirubin binding protein was identified. Little has been done subsequently to characterize the potential physiologic role of this protein, and it must be borne in mind that functional studies were performed at high concentrations of ligand in the absence of albumin, suggesting that low-affinity transport was examined. In addition, this protein was shown to be present on the surface of HepG2 cells, a cell line that lacks the high-affinity transporter and is unable to take up BSP in the presence of albumin. Other studies isolated a 170 kDa protein termed bilitranslocase on the basis of its ability to transport BSP and bilirubin when incorporated into liposomes. However, this protein is not hepatocyte specific and is also present in HepG2 and vascular endothelial cells. Bilitranslocase was cloned from a rat liver expression library utilizing a monoclonal antibody. The derived protein sequence is unique and has been used in a number of functional studies. However, analysis of the nucleotide sequence (Genbank accession number Y12178) reveals that it is 94% identical to the sequence of the inverse strand of ceruloplasmin, suggesting that a cloning artifact likely occurred. A third group of studies identified a 55 kDa organic anion-binding protein (oabp) following photoaffinity labeling of rat liver plasma membrane preparations by ³⁵ S-BSP. However, this protein was shown to be identical to the ß-subunit of F ₁ -ATPase, an inner mitochondrial membrane protein. Although the protein appeared to have a form that could be identified on the plasma membrane of hepatocytes, its potential function in organic anion transport remains unknown.

42.2.2.2

Molecular Biology Studies

As discussed above, Cl ⁻ -dependent extraction of BSP from albumin is characteristic of the high-affinity hepatocyte organic anion uptake mechanism. This unique transport characteristic was used to clone the transporter using a functional expression strategy in X. laevis oocytes. A single cRNA was isolated which when injected into oocytes resulted in a substantial signal of BSP uptake activity that was suppressed by Cl ⁻ substitution. The corresponding cDNA encodes a rat liver protein that was named organic anion transporting polypeptide (oatp, now known as oatp1a1), the first member of the solute carrier organic anion transporter gene family Slco. Since the initial description of oatp1a1, over 20 additional members of the oatp family have been described. These proteins have a relatively high degree of amino acid similarity as well as overlap of transported substrates, although their tissue distributions are varied. In subsequent studies, we used oatp1a1 as a well-characterized, prototypical member of the oatp family. It mediates high-affinity transport of a diverse group of compounds including organic anions, such as sulfobromophthalein (BSP) and taurocholate, peptide drugs including the thrombin inhibitor CRC220 and the angiotensin-converting enzyme inhibitor enalapril, and various steroid hormones such as estradiol 17β-glucuronide. There is no clear common structural similarity of compounds that are substrates for any of the oatps, although pharmacophore modeling has provided some insights into substrate requirements. Transport by oatps is Na ⁺ -independent, involving exchange with an intracellular anion such as bicarbonate or glutathione. Computer analysis of oatp1a1 cDNA reveals an open reading frame of 2010 nucleotides, encoding a protein with a high degree of hydrophobicity. Although computer modeling predicts 12- as well as 10-transmembrane domain structures, glycosylation mapping and antigenic epitope accessibility in protein expressed in transfected cells and rat hepatocytes indicate that the 12-transmembrane domain model is correct and that the second and fifth extracellular loops are glycosylated at asparagines 124, 136, and 492. Oatp1a1 expression appears to be limited to hepatocytes and epithelial cells of the choroid plexus and the S3 segment of the proximal tubule.

As illustrated in Table 42.1 , several members of the oatp family are highly expressed in hepatocytes from rats, mice, and humans. These hepatocyte oatps are distributed on the basolateral plasma membrane and have overlapping substrate specificities. They are of similar size and have similar predicted membrane topologies and biochemical characteristics. Interestingly, multiple sequence alignment reveals that these oatps have 15 cysteine residues in common ( Fig. 42.1 ) that are highly conserved over the entire oatp family. It can be hypothesized that these cysteines provide an important component of transporter function through the formation of intramolecular and possibly intermolecular disulfide bonds.

42.2.3.1

Biochemistry

Oatps in the liver are localized normally to the basolateral (sinusoidal) plasma membrane of the hepatocyte. As predicted by computer modeling, they are hydrophobic proteins, remaining membrane bound even after extraction with 0.1 M Na ₂ CO _3. On immunoblot, oatp1a1 migrates with a molecular mass of 80 kDa. Following treatment with N-glycanase, the apparent molecular mass decreases to 65 kDa, consistent with N-linked glycosylation. It has been suggested that glycosylation of oatp1a1 is a determinant of its trafficking to the plasma membrane as well as possibly its transport activity.

42.2.3.2

Function

The function of oatps, including oatp1a1, have been examined in several systems including cDNA injected Xenopus oocytes, transiently transfected cells in which transporter was functionally expressed, and in permanently transfected cells in which oatp1a1 expression is under control of a metallothionein promoter. While these studies have provided a good deal of information regarding compounds that OATPs can transport in vitro, their function in vivo has been less clear. Recently, there have been several functional studies with a targeted disruption of the gene encoding oatp1b2 in mice. This targeted disruption was not liver specific and of interest is the fact that there was no alteration of fertility, development, or viability of these mice. The effects of disruption of Oatp1b2 on its transport function were somewhat subtle. The knockout mice had an approximately fourfold reduction in the liver-to-plasma ratio of rifampicin, smaller differences (less than twofold) for cerivastatin and lovastatin, and no difference for pravastatin or simvastatin. They also had reduced plasma clearance of dibromosulfophthalein (DBSP) and were protected from the toxic effects of microcystin and phalloidin. In further studies, mice in which expression of all members of the Oatp1a and Oatp1b families were knocked out were prepared. These mice had reduced plasma clearance of a number of drugs including methotrexate and fexofenadine. They also had elevated levels of unconjugated bile acids as well as conjugated hyperbilirubinemia. There was no change in plasma levels of conjugated bile acids.

It should be noted that there is no true homolog of murine Oatp1b2 in human liver. Although OATP1B1 and OATP1B3 have been suggested as homologs of the murine protein, their amino acid sequences are only 64% and 66% identical, respectively, to that of mouse Oatp1b2. In addition, the mouse protein has a PDZ consensus-binding site at its C-terminus, while these two human proteins do not. However, in a recent large population-based study of patients taking simvastatin, 85 subjects who developed signs of myopathy, a known side effect of this drug, had a polymorphism in the gene encoding OATP1B1. Several previous smaller studies also suggested that polymorphisms in OATP1B1 were associated with altered statin pharmacokinetics. Coincidentally, the elevation of conjugated bilirubin levels in the double knockout mice is similar to that has been described in patients with the Rotor syndrome, a rare disorder in which there is chronic elevation of conjugated bilirubin in plasma with otherwise normal routine liver function tests. These individuals have recently been found to have simultaneous null mutations in the genes encoding OATP1B1 and OATP1B3. It was suggested that after conjugation with glucuronic acid in the hepatocyte, bilirubin glucuronides are effluxed from the cell into the circulation subsequently undergoing OATP-mediated reuptake. In the absence of both OATP1B1 and OATP1B3, such as that occurs in Rotor syndrome patients, levels of conjugated bilirubin in the circulation rise. It has been suggested that after the formation in the hepatocyte, bilirubin glucuronides are pumped out of the cell back into the sinusoidal circulation by the basolateral plasma membrane protein, mrp3 (AbcMRP3 (ABCC3)c3). These conjugates are then subjected to reuptake by neighboring hepatocytes, mediated by the basolateral plasma membrane proteins OATP1B1 and OATP1B3.

42.2.3.3

Regulation of Oatp Function