Figure 1. The sinusoidal endothelium of the liver is fenestrated. Large and small fenestrae in the sinusoidal endothelium are observed in these scanning electron micrographs of sections of rat liver. (A) The portal end of a sinusoid is lined with endothelium containing many large fenestrae. Clusters of small fenestrae are also present. x 4200. (B) The hepatic venous end of a sinusoid contains endothelium with only small fenestrae. x 5250. (C) Sinusoidal endothelium showing both large and small fenestrae. (D) Microvilli on the sinusoidal surface of underlying hepatocytes (arrows) are visible through some fenestrae. x 19,600. Reprinted, with permission, from (57) Grisham JW, Nopanitaya W, Compagno J, and Nagel AE. Scanning electron microscopy of normal rat liver: the surface structure of its cells and tissue components. Am J Anat 144: 295‐321, 1975.

Figure 2. Representative indicator dilution curves from an isolated perfused rat liver. A rat liver was perfused without recirculation at approximately 15 mL/min at 37°C in situ with oxygenated perfusate consisting of 20% (vol/vol) washed bovine erythrocytes in Krebs‐Ringer buffer containing 2 g/dL bovine albumin and 100 mg/dL glucose. At time zero, a small bolus containing ⁵¹Cr labeled red cells (RBC), ¹²⁵I‐albumin (BSA), and ³H‐bilirubin (BR) was injected into the portal vein and all outflow was collected in aliquots approximately 2‐s apart. In this study, recovery of red cells and albumin was essentially identical to what was injected (101% and 106% of injected), indicating that there was no removal during this single pass through the liver. In contrast, only 53% of bilirubin was recovered, indicating that 47% was taken up by the liver. Also of note is the comparison of the shapes of the red cell and albumin curves. Red cells remain in the sinusoids and come out faster, while albumin distributes into the space of Disse and has a more attenuated curve due to its larger volume of distribution.

Figure 3. Functional expression of oatp cRNA in Xenopus laevis oocytes. Oocytes were either not injected or injected with 25 ng of total rat liver mRNA or 0.5 ng of oatp cRNA. Following culture for 3 days, uptake of ³⁵S‐BSP was determined over 2 h at 25°C in 100 mmol/L NaCl containing 7.4 μmol/L BSA (BSA/BSP molar ratio, 3.7) or medium in which NaCl was replaced isosmotically by choline chloride, Na gluconate, or sucrose as indicate. Bars represent means ± SD of 12 to 24 determinations in two separate oocyte preparations. Reprinted, with permission, from (84) Jacquemin E, Hagenbuch B, Stieger B, Wolkoff AW, and Meier PJ. Expression cloning of a rat liver Na⁺‐independent organic anion transporter. Proc Natl Acad Sci U S A 91: 133‐137, 1994. Copyright (1994) National Academy of Sciences, U.S.A.

Figure 4. Diagram of the computer‐generated 12‐transmembrane domain model of oatp1a1. Intracellular and extracellular domains are indicated. The four potential N‐linked glycosylation sites are indicated by the arrows. Although it is in a consensus site for N‐glycosylation, asparagine 62 lies in a transmembrane domain and does not undergo N‐glycosylation whereas asparagines 124, 135, and 492 are extracellular and are N‐glycosylated. Reprinted, with permission, from (189) Wang P, Hata S, Xiao Y, Murray JW, and Wolkoff AW. Topological assessment of oatp1a1: a 12 transmembrane domain integral membrane protein with three N‐linked carbohydrate chains. Am J Physiol Gastrointest Liver Physiol 294: G1052‐G1059, 2008.

Figure 5. Models for the seven transmembrane domain organization of ntcp. Data are consistent with two models. Panel A shows the seven membrane inserted sequences, H1, H2, H3, H4, H5, H6, and H9, with H7 and H8 represented as extending away from the membrane. Panel B shows these latter segments as membrane‐associated or as re‐entrant loops. CHO is an N‐linked carbohydrate. Reprinted, with permission, from (112) Mareninova O, Shin JM, Vagin O, Turdikulova S, Hallen S, and Sachs G. Topography of the membrane domain of the liver Na+‐dependent bile acid transporter. Biochem 44: 13702‐13712, 2005. Copyright (2005) American Chemical Society.

Figure 6. Importance of transporter subcellular distribution on ligand clearance. (A) Transporters such as oatps and ntcp can cycle in vesicles between the plasma membrane and intracellular locations. Recruitment and retrieval mechanisms utilize microtubule‐based motility of vesicles and regulatory elements that may be unique for each transporter. If all transporter were to be sequestered within the cell, as in panel B, ligand (e.g., drug) in the sinusoidal (portal venous) blood, would bypass the hepatocyte uptake mechanism. This could result in high, potentially toxic, levels of this ligand in the peripheral circulation.

Figure 7. Characterization of bidirectional motility of ntcp‐containing vesicles on microtubules (MTs) in vitro. (A) Polarity‐marked fluorescent MTs (red) were attached to glass chambers, incubated with endocytic vesicles prepared from rat liver and washed. The ntcp‐containing, MT‐bound vesicles were then visualized with primary and fluorescent secondary antibodies, and 50 μmol/L ATP was added to initiate motility. Time‐lapse digital fluorescence images were captured in Cy2 (to detect ntcp) and Cy3 (to detect MTs) channels. Seconds after addition of ATP are indicated at the upper left. The arrows follow two motile ntcp‐containing vesicles (bright green dots) moving in opposite directions. Arrowheads show the original location of the vesicles. “+” and “–” indicate the plus and minus ends of the MTs. Bar, 5 μm. (B) Quantification shows that ntcp‐containing vesicles moved with approximately equal frequency toward the plus and minus ends. Parentheses indicate the number of vesicles moving in each direction. Reprinted, with permission, from (149) Sarkar S, Bananis E, Nath S, Anwer MS, Wolkoff AW, and Murray JW. PKCzeta is required for microtubule‐based motility of vesicles containing the ntcp transporter. Traffic 7: 1078‐1091, 2006.

Figure 8. Involvement of the Phosphoinositide 3‐Kinase/Protein Kinase Cζ (PI3K‐PKCζ ) pathway in ntcp‐containing vesicle motility on microtubules (MTs). (A) A simplified PI3K‐PKCζ pathway for regulation of ntcp‐containing vesicle motility. PI3K converts PIP2 to PIP3 that then activates PKCζ, leading to vesicle motility through unknown substrates. LY294002 and PKCζ PS are inhibitors of PI3K and PKCζ, respectively. (B) Motility of ntcp‐containing vesicles on microtubules (MTs) was scored following incubation with buffer, 50, 100, or 200 μmol/L LY294002. (C) Microtubule‐based motility of ntcp‐containing vesicles was scored following incubation with buffer, 10 mmol/L PIP3, 50 mmol/L LY294002 with and without 10 mmol/L PIP3, or PIP3 along with 50 mmol/L PKCζ PS. ^* P < 0.005, ^** P < 0.0001 compared to buffer control. Parentheses indicate the number of vesicles scored. Reprinted, with permission, from (149) Sarkar S, Bananis E, Nath S, Anwer MS, Wolkoff AW, and Murray JW. PKCzeta is required for microtubule‐based motility of vesicles containing the ntcp transporter. Traffic 7: 1078‐1091, 2006.

Figure 9. Influence of phosphorylation on internalization of cell surface oatp1a1 in HuH7 cells stably transfected with GFP‐oatp1a1and in overnight‐cultured rat hepatocytes. (A and B) HuH7‐derived cell lines stably expressing nonphosphorylatable (GFP‐oatp1a1^AA) or phosphomimetic (GFP‐oatp1a1^EE) oatp1a1 constructs were prepared. These cells constitutively express PDZK1. Cells were surface biotinylated with membrane‐impermeant sulfo‐NHS‐SS‐biotin for 30 min at 4°C and then incubated at 37°C for up to 120 min to allow internalization. After removal of residual biotin from the cell surface by reduction, internalized biotinylated GFP‐oatp1a1 was collected on streptavidin‐agarose beads and subjected to immunoblot for oatp1a1. (A) Representative experiment. (B) Results of densitometric quantitation of four individual experiments. Data were normalized to total starting cell surface biotinylated oatp1a1. Lines are drawn through means at each time. Open symbols, oatp1a1^AA; filled symbols, oatp1a1^EE. (C and D) Hepatocytes isolated from rat liver and cultured overnight were surface biotinylated with membrane‐impermeant sulfo‐NHS‐SS‐biotin for 30 min at 4°C, and then incubated at 37°C for 10 or 30 min in the absence (−) or presence (+) of 1 mmol/L ATP. Previous studies showed that this short incubation of rat hepatocytes in extracellular ATP stimulates serine phosphorylation of oatp1a1 via activity of a purinergic receptor. After removal of residual biotin from the cell surface by reduction, internalized biotinylated oatp1a1 was collected on streptavidin‐agarose beads and subjected to immunoblot for oatp1a1. (A) Representative study. (B) Densitometric quantitation of three experiments. Data were normalized to total starting cell surface biotinylated oatp1a1. Values are means ± SE. Reprinted, with permission, from (23) Choi JH, Murray JW, and Wolkoff AW. PDZK1 binding and serine phosphorylation regulate subcellular trafficking of organic anion transport protein 1a1. Am J Physiol Gastrointest Liver Physiol 300: G384‐G393, 2011.

Figure 10. Influence of phosphorylation and interaction with PDZK1 on subcellular distribution of oatp1a1. (A) Human embryonic kidney (HEK) 293T cells were transfected with plasmids encoding nonphosphorylatable (AA) or phosphomimetic (EE) oatp1a1 (oatp1a1^AA and oatp1a1^EE, respectively) linked to green fluorescence protein (GFP) at the NH₂ terminus. Experiments were performed without or with coexpression of PDZK1. After transfection, cells were cultured for 2 days, fixed and permeabilized with 0.1% Triton X‐100 in PBS, and incubated with primary antibody to PDZK1 and Cy3‐labeled secondary antibody. Distribution of fluorescence was examined by confocal microscopy. Scale bar, 8 μm. B. HEK 293T cells were transfected with plasmids encoding FLAG‐PDZK1 as well as full‐length or truncated oatp1a1^AA or oatp1a1^EE linked to GFP at the NH₂ terminus. Truncated plasmids, indicated as −4, encoded oatp1a1 without its last 4 amino acids (KTKL), which define its PDZ‐binding motif. After transfection, cells were cultured for 2 days, fixed and permeabilized with 0.1% Triton X‐100 in PBS, and incubated with primary antibody to PDZK1 and Cy3‐labeled secondary antibody. Distribution of fluorescence was determined by confocal microscopy. These studies indicate that an intact PDZ‐binding motif is required for cell surface expression of oatp1a1 independent of its phosphorylation state. Scale bar, 8 μm. Reprinted, with permission, from (23) Choi JH, Murray JW, and Wolkoff AW. PDZK1 binding and serine phosphorylation regulate subcellular trafficking of organic anion transport protein 1a1. Am J Physiol Gastrointest Liver Physiol 300: G384‐G393, 2011.

Figure 11. Microtubule‐based motility of Oatp1a1‐ associated endocytic vesicles. Oatp1a1‐containing endocytic vesicles were prepared from livers of wild type (WT) and PDZK1 knockout (KO) mice and flowed into microchambers that had been coated with polarity‐marked fluorescent microtubules. After the binding of vesicles to microtubules, motility was initiated with the addition of 50 μmol/L ATP. (A) Representative images demonstrating minus‐end directed movement of an Oatp1a1‐containing vesicle prepared from PDZK1 knockout mouse liver. A red microtubule with attached green Oatp1a1‐labeled vesicles runs horizontally and contains markings for microtubule polarity. The polarity marks were generated by polymerizing brightly fluorescent tubulin from short, dimly fluorescent microtubule seeds, allowing the growth of long microtubule plus ends. Visible from left to right is the microtubule minus end, a dimly fluorescent seed, and the microtubule plus end (+) to which a green, motile vesicle is bound. The white arrow follows this vesicle as it moves toward the minus end of the microtubule. The yellow arrowhead indicates the starting point for the vesicle. Seconds after addition of ATP are indicated at the top left of each panel. In the 34 seconds of this study, the vesicle moved approximately 18 μm (approximately 0.5 μm/s). Scale bar = 10 μm. (B) The percentage of microtubule‐bound vesicles that moved following ATP addition is indicated by the bars for vesicles prepared from wild‐type (open bars) and PDZK1 knockout (solid bars) mice. (C) The percentage of motile vesicles from the studies in (B) moving toward the plus (closed bars) or minus (open bars) ends of microtubules is indicated. Numbers in parentheses represent the number of motile vesicles that were examined. Error bars represent the mean ± SEM ^* P < 0.0001 as compared with plus‐end motility of wild‐type vesicles; ^** P < 0.0001 as compared with minus‐end motility of wild‐type vesicles. Reprinted with permission from (192) Wang WJ, Murray JW, and Wolkoff AW. Oatp1a1 requires PDZK1 to traffic to the plasma membrane by selective recruitment of microtubule‐based motor proteins. Drug Metab Dispos 42: 62‐69, 2013.

Figure 12. Colocalization of motor proteins and PDZK1 with Oatp1a1‐associated vesicles. Endocytic vesicles isolated from wild‐type (WT) and PDZK1 knockout (KO) mouse livers were attached to the glass surface of microchambers and immunostained for Oatp1a1 and motor proteins or PDZK1. (A) Representative images are shown in which Oatp1a1 is in red and PDZK1 or motor proteins are in green. Vesicles in yellow represent colocalization of the two. Scale bar = 10 μm. (B) Quantification of protein colocalization with Oatp1a1‐containing vesicles. The percentage of Oatp1a1‐containing vesicles that colocalized with each of the proteins indicated in the figure is represented by filled (wild‐type) or open (PDZK1 knockout) bars. The number of Oatp1a1‐associated vesicles examined is in parentheses. Error bars represent the mean ± S.E.M. ^* P < 0.0001 as compared with colocalization in wild‐type vesicles. Reprinted, with permission, from (192) Wang WJ, Murray JW, and Wolkoff AW. Oatp1a1 requires PDZK1 to traffic to the plasma membrane by selective recruitment of microtubule‐based motor proteins. Drug Metab Dispos 42: 62‐69, 2013.