May 2010
Volume 51, Issue 5
Free
Glaucoma  |   May 2010
The Prostaglandin Transporter OATP2A1 Is Expressed in Human Ocular Tissues and Transports the Antiglaucoma Prostanoid Latanoprost
Author Affiliations & Notes
  • Michaela E. Kraft
    From the Institute of Experimental and Clinical Pharmacology and Toxicology, and
  • Hartmut Glaeser
    From the Institute of Experimental and Clinical Pharmacology and Toxicology, and
  • Kathrin Mandery
    From the Institute of Experimental and Clinical Pharmacology and Toxicology, and
  • Jörg König
    From the Institute of Experimental and Clinical Pharmacology and Toxicology, and
  • Daniel Auge
    From the Institute of Experimental and Clinical Pharmacology and Toxicology, and
  • Martin F. Fromm
    From the Institute of Experimental and Clinical Pharmacology and Toxicology, and
  • Ursula Schlötzer-Schrehardt
    the Department of Ophthalmology, Friedrich-Alexander-University of Erlangen-Nuremberg, Erlangen, Germany.
  • Ulrich Welge-Lüssen
    the Department of Ophthalmology, Friedrich-Alexander-University of Erlangen-Nuremberg, Erlangen, Germany.
  • Friedrich E. Kruse
    the Department of Ophthalmology, Friedrich-Alexander-University of Erlangen-Nuremberg, Erlangen, Germany.
  • Oliver Zolk
    From the Institute of Experimental and Clinical Pharmacology and Toxicology, and
  • Corresponding author: Oliver Zolk, Institute of Experimental and Clinical Pharmacology and Toxicology, University of Erlangen-Nuremberg, Fahrstrasse 17, 91054 Erlangen, Germany; zolk@pharmakologie.uni-erlangen.de
Investigative Ophthalmology & Visual Science May 2010, Vol.51, 2504-2511. doi:10.1167/iovs.09-4290
  • Views
  • PDF
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Michaela E. Kraft, Hartmut Glaeser, Kathrin Mandery, Jörg König, Daniel Auge, Martin F. Fromm, Ursula Schlötzer-Schrehardt, Ulrich Welge-Lüssen, Friedrich E. Kruse, Oliver Zolk; The Prostaglandin Transporter OATP2A1 Is Expressed in Human Ocular Tissues and Transports the Antiglaucoma Prostanoid Latanoprost. Invest. Ophthalmol. Vis. Sci. 2010;51(5):2504-2511. doi: 10.1167/iovs.09-4290.

      Download citation file:


      © ARVO (1962-2015); The Authors (2016-present)

      ×
  • Supplements
Abstract

Purpose.: Latanoprost, a prostaglandin F analogue, has become one of the most widely used medications for the treatment of glaucoma. The authors hypothesized that organic anion transporting polypeptides (OATPs) are responsible for the uptake of latanoprost into ocular tissues and, hence, that they contribute to the interindividual differences in drug concentrations and effects.

Methods.: Expression of prostaglandin (PG) transporters (OATP2A1, OATP2B1) in human ocular tissues was determined using real-time RT-PCR and immunofluorescence. The inhibitory interactions between latanoprost and its active metabolite (the free acid) and the uptake of prototypical substrates (PGE2 and bromosulfophthalein) were tested in stably transfected human embryonic kidney cells overexpressing either OATP2A1 or OATP2B1. These cells were also used to investigate whether latanoprost and latanoprost acid are substrates of OATP2A1 or OATP2B1.

Results.: OATP2A1 and OATP2B1 mRNA expression was highest in the choroid/retinal pigment epithelium (RPE) complex and ciliary body. OATP2A1 protein expression was most prominent in the RPE and in epithelial and endothelial cell layers of anterior segment tissues, such as cornea, conjunctiva, iris, and ciliary body, whereas OATP2B1 protein was additionally expressed in trabecular meshwork, Schlemm canal, and choroidal vasculature. Latanoprost and latanoprost acid significantly inhibited both OATP2A1 and OATP2B1. Uptake experiments demonstrated that latanoprost acid is effectively transported by OATP2A1 (affinity constant [K m], 5.4 μM; maximum uptake rate [V max], 21.5 pmol/mg protein/min) and less effectively by OATP2B1.

Conclusions.: The results presented herein suggest that at least OATP2A1 plays a role in the intraocular disposition of the therapeutically used prostanoid latanoprost.

The prostanoids are an established group of ocular hypotensive drugs used for the clinical management of glaucoma. The group includes five chemical compounds that are structurally derived from the naturally occurring prostaglandin (PG) F2: latanoprost, bimatoprost, travoprost, tafluprost, and unoprostone. Latanoprost is the most widely used antiglaucoma prostanoid. Prostanoids increase uveoscleral and trabecular (conventional) outflow through several mechanisms, including changing the cell shape, cytoskeletal alterations, and remodeling of the extracellular matrix. 1,2  
Topical application of the prodrug latanoprost (ester) leads to absorption of approximately 1% of the drug through the cornea, where it is completely hydrolyzed to the biologically active acid form. 3,4 A previous study, as well as our own pilot experiments, demonstrated remarkable interindividual variations in the concentration of latanoprost acid in the aqueous humor after a single 1.5-μg dose of latanoprost, 5 a finding that might be related to interindividual variations in the clearance of the drug. At physiological pH, prostanoids predominate as charged organic anions and diffuse poorly through the lipid bilayer of endothelial cells. 6,7 Therefore, the clearance of prostanoids from the aqueous humor might be dependent on active uptake mechanisms. 
We hypothesized that uptake transporters are involved in the clearance of latanoprost from the aqueous humor, and we focused on two transporters, both of which belong to the organic anion transporting polypeptide (OATP) family 811 : OATP2A1 (gene name SLCO2A1) and OATP2B1 (gene name SLCO2B1). The functional profile of OATP2A1 expressed in vitro strongly suggests a role primarily in PGE2 and PGF uptake and degradation. 1215 Although OATP2A1 mRNA expression has been demonstrated in the human eye, 16 its ocular expression pattern at the protein level has not been investigated. Moreover, it remains to be established whether the antiglaucoma prostanoids that are used clinically are readily transported by OATP2A1. 
OATP2B1 is expressed at significant levels in the human ciliary body epithelium. 17,18 Functionally, OATP2B1 has a relatively narrow substrate spectrum and transports, for example, bromosulfophthalein (BSP), estrone-3-sulfate, and dehydroepiandrosterone sulfate. Although the findings regarding OATP2B1-mediated transport of endogenous PGs such as PGE2 remain controversial, 9,1921 one study has demonstrated low-affinity transport of the PGF2-derived antiglaucoma drug unoprostone carboxylate. 17 Whether other antiglaucoma prostanoids are also transported by OATP2B1 remains unclear. 
In this study we addressed whether OATP2A1 or OATP2B1 is involved in the cellular uptake of latanoprost and, therefore, in the ocular clearance of this widely used antiglaucoma drug. 
Materials and Methods
Aqueous Humor and Human Tissue Samples
Twelve patients (nine women, three men; age range, 60–95 years) received one 30-μL drop of 0.005% latanoprost (Xalatan; Pfizer, New York, NY) into the eye. Solutions were administered 23 to 159 minutes before routine cataract surgery. Aqueous humor samples (50 μL) were withdrawn from treated eyes at surgery initiation. Times from instillation to sampling were recorded. Before entry into the study, the patients signed informed consent forms. The study was approved by the ethics committee of the University of Erlangen-Nuremberg. 
Human whole globes, enucleated within 6 hours of death from human organ donors, were immediately dissected, and the following tissues were collected: choroidea/retinal pigment epithelium (RPE), sensory retina, iris, ciliary body, lens, and cornea. The tissues were snap-frozen in liquid nitrogen and stored at −80°C until processing. All tissues were from donors free of ocular disease and other systemic complications. For comparison of transporter expression, human tissue samples from brain cortex (n = 2), liver (n = 3), placenta (n = 2), heart, and kidney (n = 3) were studied (tissues were frozen within 6 hours of sampling). These human biological materials were rendered anonymous by irreversibly removing identifiers, and their use was approved by the ethics committee of the University of Erlangen-Nuremberg. The studies were conducted in accordance with the tenets of the Declaration of Helsinki regarding the use of human subjects in clinical trials. 
LC/MS/MS Assay of Latanoprost Acid in Aqueous Humor Samples
We added 50 μL internal standard solution (PGE2-d4 10 ng/mL in methanol; Cayman, Ann Arbor, MI), 5 μL methanol, and 2 mL t-butyl-methylether to 50-μL aqueous humor samples. After extraction for 15 minutes and then centrifugation, 1.5 mL of the organic layer was transferred into glass tubes and dried under a stream of nitrogen before reconstitution in 100 μL eluent plus 0.1% formic acid. The injected volume was 40 μL. HPLC was performed with an Agilent system (Series 1100; Agilent Technologies Deutschland GmbH, Böblingen, Germany). An HPLC column (Synergi 4-μm Hydro-RP 80 A 150 × 2.0 ID) with guard column (Phenomenex, Aschaffenburg, Germany) was used for chromatographic separation, a mixture of 40% acetonitrile (LC-MS grade), and 60% water (LC-MS grade) as the mobile phase. Flow rate was set at 0.5 mL/min; retention times were 2.5 minutes for latanoprost acid and 3.0 minutes for internal standard. 
Quantification was performed with a triple quadrupole mass spectrometer (Sciex API 4000; Applied Biosystems, Toronto, ON, Canada) with a turbo ion spray interface using multiple reaction monitoring in the negative ion mode. The transmissions for latanoprost acid were 389.4 m/z (Q1; 355.0 m/z for internal standard) and 345.2 m/z (Q3; 237.0 m/z for internal standard), and main MS parameters were as follows: collision gas, 8; curtain gas, 25; nebulizer gas (GS 1), 30; turbo gas (GS 2), 10; ion spray voltage, −4000 V; and temperature, 500°C. The peak area ratio of latanoprost acid to internal standard was calculated using analysis software (Analyst 1.4.2; Applied Biosystems, Foster City, CA). 
The lower limit of quantification was 1 ng/mL. A calibration curve was constructed using 1/X-weighted linear regression between spiked aqueous humor concentrations and the measured ratios, which were linear over the range 1 to 50 ng/mL (r = 0.9995). Aqueous humor calibration standards (1, 2.5, 5, 10, 25, and 50 ng/mL), quality controls, blank samples, and double-blank samples were prepared in the same manner. Quality controls were routinely assayed at 2500, 312.5, 39.1, and 4.9 μM. Quality control intraday coefficients of variation were 2.8%, 3.8%, 4.9%, and 6.2%, and interday coefficients of variation were 8.0%, 2.2%, 1.5%, and 3.3%. 
Quantitative Real-Time RT-PCR
Total RNA from choroidea, retina, iris, ciliary body, lens, and cornea (n = 5 each) was extracted with an RNA isolation kit (Illustra RNAspin Mini; GE Healthcare, Little Chalfont, Buckinghamshire, UK). Total RNA (500 ng each) was reverse-transcribed (Superscript III; Invitrogen, Carlsbad, CA), and cDNA was subsequently amplified (45 cycles of 50°C for 2 minutes, 95°C for 15 seconds, and 60°C for 1 minute) with a sequence detection system (ABI 7900HT; Applied Biosystems). Predesigned primers and TaqMan probes for SLCO2A1 (OATP2A1), SLCO2B1 (OATP2B1), and the housekeeping genes ACTB (β-actin) and GAPDH (glyceraldehyde 3-phosphate dehydrogenase) were used (Hs00194554_m1, Hs00200670_m1, Hs00357333_g1, Hs00266705_g1; Applied Biosystems). Primers were designed to amplify DNA fragments crossing exon/exon boundaries. For comparison, OATP2A1 and OATP2B1 mRNA levels were determined in human liver, brain, kidney, placenta, and heart cDNA pools under equal conditions. Relative amounts of OATP2A1 and OATP2B1 mRNA were calculated using the δ-delta-CT method 22 by relating the PCR signal of the target transcript in human tissue samples to that in the choroid samples, which served as a calibrator. Target mRNA levels were normalized to the internal control genes ACTB and GAPDH according to the equation: relative target mRNA level = 2−(ΔΔCT,ACTB+ΔΔCT,GAPDH)/2, where ΔΔC T,ACTB = (C T,TargetC T,ACTB)Tissue − (C T,TargetC T,ACTB)Choroid, and ΔΔC T,GAPDH = (C T,TargetC T,GAPDH)Tissue − (C T,TargetC T,GAPDH)Choroid
Immunofluorescence
Five human donor eyes were investigated. Each eye was divided with a razor blade at the equator in an anterior and a posterior part. From each segment for each antibody, at least six sections were cut. PFA-fixed eyes from human organ donors were washed with phosphate-buffered saline (pH 7.4) and placed in embedding medium (Tissue-Tek-OCT; Jung, Nussloch, Germany). Tissues were frozen at −20°C and serial sectioned into 12-μm thin slices with a cryotome (Kryostat CM 3050S; Leica, Bensheim, Germany). Paraffin-embedded tissue was sectioned at 7 μm thin and was deparaffinized in xylol. Sections placed on poly-l-lysine–coated slides were incubated with dry milk solution (Blotto; Santa Cruz Biotechnology, Heidelberg, Germany) for 1 hour at room temperature to prevent nonspecific staining. 
OATP2A1 polyclonal antibody was from Abnova (Heidelberg, Germany). OATP2B1 antiserum was raised in rabbits using a peptide corresponding to the carboxyl-terminal amino acids 690 to 709 of human OATP2B1 (AVEQQLLVSGPGKKPEDSRV; NCBI accession number NP_009187) coupled to keyhole limpet hemocyanin (Peptide Specialty Laboratories, Heidelberg, Germany). The specificity of both antisera was confirmed previously. 23,24 Slides were incubated with diluted (1:100 dilution in blocking buffer: 2% bovine serum albumin and 0.2% Triton X-100 in Tris-buffered saline) antibodies at 4°C overnight. After a wash in Tris-buffered saline, the sections were incubated with goat anti–mouse IgG Cy-2 or swine anti–rabbit IgG Cy-2 (1:100; Dianova, Hamburg, Germany) in blocking buffer for 2 hours at room temperature. 
Stained sections were analyzed by confocal laser microscopy (MRC 600 confocal imaging system; Bio-Rad Laboratories, Richmond, CA) under a microscope (Axioplan; Zeiss, Oberkochen, Germany). 
Cell Lines Overexpressing OATP2A1 and OATP2B1
Human embryonic kidney (HEK) 293 cells were stably transfected with the SLCO2A1 cDNA (GenBank ID, NM_005630) encoding human OATP2A1. The recombinant OATP2A1 protein was integrated into the plasma membrane of HEK-OATP2A1 cells and was functionally active, as demonstrated previously. 23 The HEK-OATP2B1 cell line was established by stable transfection with the SLCO2B1 cDNA (isoform 1; GenBank ID, NM_007256). Transfections were carried out as described. 25 For uptake experiments, the cells (500,000/well) were seeded in 24-well plates coated with 0.1 mg/mL poly-d-lysine and grown to confluence for 2 days. 
Competition Assays
For inhibition studies, the prototypical tracer substrates [3H]PGE2 (Perkin Elmer, Boston, MA; 185.6 Ci/mmol) and [3H]-BSP (Hartmann Analytic, Braunschweig, Germany; 19.5 Ci/mmol) for OATP2A1 and OATP2B1 uptake, respectively, were used. Unlabeled compound (PGE2 [Cayman, Ann Arbor, MI], BSP [Sigma-Aldrich, Taufkirchen, Germany]) was added to reach a final substrate concentration of 1 μM each. Before experiments were started, the cells were washed with prewarmed (37°C) uptake buffer (142 mM NaCl, 5 mM KCl, 1 mM K2HPO4, 1.2 mM MgSO4, 1.5 mM CaCl2, 5 mM glucose, and 12.5 mM HEPES, pH 7.3). Cells were then incubated with tracer substrate in the presence or absence of latanoprost or latanoprost acid (1, 3, 10, 30, 100, 300, 1000 μM) at 37°C. It is well known from previous studies that OATP2A1-mediated uptake of PGE2 is linear for up to 10 minutes. 14,26 In contrast, time- dependant uptake of BSP in HEK-OATP2B1 cells was not previously investigated. Our own results demonstrated linear uptake for at least 10 minutes. To ensure sufficient baseline uptake of the tracer substrate in our inhibition experiments, an incubation period of 10 minutes was chosen. Subsequently, the cells were washed three times with ice-cold uptake buffer and lysed with 5 mM Tris-HCl (pH 7.5) plus 0.1% Triton X-100. The intracellular accumulation of tracer was measured by liquid scintillation counting (Perkin Elmer), and uptake was related to the concentration of cellular proteins, which was determined with bicinconic acid assay (BCA Protein Assay Kit; Thermo Scientific, Rockford, IL). 
Uptake Assay
Cells (HEK-OATP2A1 or HEK-OATP2B1 and the respective vector controls) were incubated with uptake buffer containing either latanoprost or latanoprost acid at 37°C for the indicated periods. Subsequently, cells were washed three times with ice-cold uptake buffer before they were lysed with 5 mM Tris-HCl (pH 7.5) plus 0.1% Triton X-100. Intracellular accumulation of latanoprost or latanoprost acid was determined by LC/MS/MS. Each experiment was repeated at least three times. 
LC/MS/MS Assay for Latanoprost and Latanoprost Acid in Cell Lysates
Ninety microliters of internal standard solution (latanoprost: etoricoxib [i.e., etoricoxib is the internal standard for latanoprost] 5 ng/mL in acetonitril/H2O 40:60 [vol/vol] plus 0.1% formic acid; latanoprost acid: PGE2-d4 10 ng/mL in methanol/H2O 25:75 [vol/vol] plus 0.1% formic acid) were added to 10-μL cell lysate samples. Injected volume was 40 μL. 
Quantification was performed as described for aqueous humor samples with the following modifications for latanoprost: multiple reaction monitoring was used in the positive ion mode, and another HPLC column (Nucleodur C18 5 μm 100 A 30 × 2.0 ID; Macherey-Nagel, Düren, Germany) was used for chromatographic separation. Retention times were 6.1 minuted for latanoprost and 0.9 minute for the internal standard. Transmissions for latanoprost were 433.1 m/z (359.1 m/z for internal standard) for Q1 and 379.3 m/z (280.2 m/z for internal standard) for Q3. The primary MS parameters were as follows: collision gas, 4; curtain gas, 10; nebulizer gas (GS 1), 40; turbo gas (GS 2), 20; ion spray voltage, 4500 V; temperature, 700°C. 
Calibration curves were constructed using 1/X-weighted linear regression (latanoprost) or 1/X2-weighted quadratic regression (latanoprost acid) between spiked cell lysate concentrations and the measured ratios, which were linear over the range 9.8 to 2500 ng/mL (latanoprost) and 1.2 to 2500 ng/mL (latanoprost acid). 
Data and Statistical Analyses
Inhibition dose-response curves were analyzed by curve-fitting and calculation of IC50 values with appropriate software (Prism; GraphPad, La Jolla, CA). All data are presented as mean ± SEM. Differences between two groups were tested by the Student's t-test for unpaired data. 
Results
Latanoprost Acid Levels in the Aqueous Humor
Concentrations of the active drug, latanoprost acid, were determined at 23 to 159 (mean, 91) minutes after the topical application of a single 1.5-μg dose of latanoprost. For ethical reasons, multiple sampling was not possible (e.g., to generate time-concentration curves for each patient). Figure 1 shows the intraocular concentrations of latanoprost acid in patients as a function of time from latanoprost application. Based on these data, a nonlinear regression fit was calculated, representing the average course of latanoprost acid concentrations in the aqueous humor over time. Of note, we observed a marked deviation from the average concentration-time curve in some samples, suggesting a considerable interindividual variation of intraocular latanoprost concentrations. 
Figure 1.
 
Concentration of latanoprost-free acid in the aqueous humor of 12 patients after topical application of 1.5 μg latanoprost.
Figure 1.
 
Concentration of latanoprost-free acid in the aqueous humor of 12 patients after topical application of 1.5 μg latanoprost.
OATP2A1 and OATP2B1 mRNA Expression in Ocular Tissues
Transcript levels of the uptake transporters OATP2A1 and OATP2B1 were determined in human ocular tissues by real-time RT-PCR. For comparison, expression levels were also investigated in human nonocular tissues, such as liver, brain, kidney, placenta, and heart. In these tissues, OATP2A1 and OATP2B1 transcript levels were highest in the placenta and the liver, respectively, which fits well with previous reports. 
In ocular tissues, mRNA expression of both OATP2A1 and OATP2B1 was highest in the choroid/RPE-complex and in the ciliary body and was much lower in the sensory retina, iris, lens, and cornea (Fig. 2). 
Figure 2.
 
OATP2A1 (A) and OATP2B1 (B) mRNA expression in human liver, brain, kidney, placenta, and heart (gray) and in ocular tissues (black). Values are normalized to the housekeeping genes GAPDH and ACTB, and all transcript levels are related to choroid expression.
Figure 2.
 
OATP2A1 (A) and OATP2B1 (B) mRNA expression in human liver, brain, kidney, placenta, and heart (gray) and in ocular tissues (black). Values are normalized to the housekeeping genes GAPDH and ACTB, and all transcript levels are related to choroid expression.
Ocular Localization of OATP2A1 and OATP2B1 Protein
Both OATP2A1 and OATP2B1 proteins were expressed in virtually all tissues of the human eye, showing a similar distribution pattern in anterior segment tissues but differential expression patterns in posterior segment tissues, such as retina and choroid. OATP2A1 immunoreactivity was detected in the corneal epithelium, particularly its superficial layers, in the corneal endothelium, and in stromal keratocytes (Figs. 3A, 3B). Intense staining could also be observed in the suprabasal epithelial cells of the bulbar conjunctiva (Fig. 3C). Expression of OATP2A1 was apparent in the vessels of the iris stroma, both in vascular endothelial and adventitial cells (Fig. 3D). Moreover, OATP2A1 could be immunolocalized to the basal aspects of the nonpigmented and pigmented epithelium of the ciliary body facing the aqueous humor and ciliary stroma, respectively (Fig. 3E). In the posterior part of the human eye, only weak staining was observed in the RPE, whereas the sensory retina and the choroid were essentially negative (Fig. 3F). 
Figure 3.
 
Immunolocalization of OATP2A1 in human ocular tissues (green fluorescence, left) and corresponding phase-contrast images (right). Scale bar, 20 μm. (A) Epithelium (CEP) and stroma of the cornea (CS). (B) Endothelium (CEN) and stroma of the cornea (CS). (C) Conjunctival epithelium (CJE). (D) Stroma (IS), vessels, and pigmented epithelium (IPE) of the iris. (E) Ciliary body with its nonpigmented (CBNPE) and pigmented (CBPE) epithelium. (F) Choroid (CH) and RPE.
Figure 3.
 
Immunolocalization of OATP2A1 in human ocular tissues (green fluorescence, left) and corresponding phase-contrast images (right). Scale bar, 20 μm. (A) Epithelium (CEP) and stroma of the cornea (CS). (B) Endothelium (CEN) and stroma of the cornea (CS). (C) Conjunctival epithelium (CJE). (D) Stroma (IS), vessels, and pigmented epithelium (IPE) of the iris. (E) Ciliary body with its nonpigmented (CBNPE) and pigmented (CBPE) epithelium. (F) Choroid (CH) and RPE.
Immunolocalization of OATP2B1 showed staining patterns in anterior segment tissues similar to those of OATP2A1 (i.e., in basal and superficial cells of the corneal and conjunctival epithelia; Figs. 4A, 4C), the corneal endothelium (Fig. 4B), vascular and perivascular cells of the iris and ciliary stroma (Figs. 4D, 4E), basal aspects of the ciliary epithelia (Fig. 4E), and endothelial cells of the trabecular meshwork and Schlemm canal. However, strong immunofluorescence for OATP2B1 was observed not only in the RPE (Fig. 4F) but also in the vascular endothelia of choroidal vessels (Fig. 4F). 
Figure 4.
 
Immunolocalization of OATP2B1 in human anterior eye tissues (green fluorescence, left) and corresponding phase-contrast images (right). Scale bar, 20 μm. (A) Epithelium (CEP) and stroma of the cornea (CS). (B) Endothelium (CEN) and stroma of the cornea (CS). (C) Conjunctival epithelium (CJE). (D) Stroma (IS), vessels, and pigmented epithelium of the iris. (E) Ciliary body with its nonpigmented (CBNPE) and pigmented (CBPE) epithelium. (F) Choroid (CH) and RPE.
Figure 4.
 
Immunolocalization of OATP2B1 in human anterior eye tissues (green fluorescence, left) and corresponding phase-contrast images (right). Scale bar, 20 μm. (A) Epithelium (CEP) and stroma of the cornea (CS). (B) Endothelium (CEN) and stroma of the cornea (CS). (C) Conjunctival epithelium (CJE). (D) Stroma (IS), vessels, and pigmented epithelium of the iris. (E) Ciliary body with its nonpigmented (CBNPE) and pigmented (CBPE) epithelium. (F) Choroid (CH) and RPE.
OATP2A1- and OATP2B1-Dependent Transport
HEK cells stably overexpressing OATP2A1 or OATP2B1 were generated so that the significance of these transporters could be studied in the uptake of prostanoid antiglaucoma drugs. Figure 5 shows the concentration-dependent effects of latanoprost and latanoprost acid on OATP2A1- and OATP2B1-mediated uptake of their respective prototype substrates. Both latanoprost and latanoprost acid were potent inhibitors of OATP2A1-mediated uptake of PGE2, with IC50 values in the low micromolar range (latanoprost acid IC50, 0.7 μM [95% confidence interval (CI), 0.6–0.8 μM]; latanoprost acid IC50, 3.2 μM [95% CI, 2.8–3.7 μM]). 
Figure 5.
 
(A) Inhibition of OATP2A1-mediated prostaglandin E2 (PGE2, 1 μM) uptake by latanoprost and latanoprost acid. (B) Inhibition of OATP2B1-mediated BSP (1 μM) uptake by latanoprost and latanoprost acid. n = 3 independent experiments conducted in duplicate.
Figure 5.
 
(A) Inhibition of OATP2A1-mediated prostaglandin E2 (PGE2, 1 μM) uptake by latanoprost and latanoprost acid. (B) Inhibition of OATP2B1-mediated BSP (1 μM) uptake by latanoprost and latanoprost acid. n = 3 independent experiments conducted in duplicate.
Prostanoids were less potent inhibitors of OATP2B1-mediated BSP uptake (latanoprost acid IC50, 23 μM [95% CI, 15–38 μM]; latanoprost acid IC50, 88 μM [95% CI, 59–130 μM]). 
Figures 6 and 7 demonstrate that, when expressed in HEK cells, OATP2A1 and OATP2B1 mediate the rapid, time-dependent uptake of latanoprost acid. For both OATP transporters, intracellular accumulation of latanoprost acid, when applied at a concentration of 50 μM, reached equilibrium by 5 minutes To evaluate the binding characteristics (affinity constant, K m) and the maximum uptake rates (V max) of the OATP transporters, the initial uptake (4 minutes' incubation) of latanoprost acid at different concentrations was investigated. In HEK-OATP2A1 cells, net uptake was saturable with K m = 5.4 μM and V max = 21.5 pmol/mg/min (Figs. 6B, 6C). In contrast, OATP2B1-mediated net uptake of latanoprost acid was not saturable up to 125 μM latanoprost acid. Nevertheless, the uptake of latanoprost acid at 15 μM was twofold higher in HEK-OATP2B1 compared with control cells (P < 0.001; Fig. 7B). Altogether, the results indicate that latanoprost acid is readily transported by OATP2A1 but is a weak substrate of OATP2B1. 
Figure 6.
 
(A) Uptake of latanoprost acid (50 μM) as a function of time by OATP2A1 expressed in HEK cells. n = 3 independent experiments conducted in duplicate. (B) Concentration-dependent uptake of latanoprost acid in OATP2A1-transfected HEK cells and control cells (empty vector-transfected, VC) after 4 minutes' incubation. Net uptake was calculated as the difference between total uptake in HEK-OATP2A1 and control cells. n = 8 independent experiments conducted in duplicate. (C) Lineweaver-Burk (double-reciprocal) plot for determination of K m and V max was calculated from the data set shown in (B). n = 8.
Figure 6.
 
(A) Uptake of latanoprost acid (50 μM) as a function of time by OATP2A1 expressed in HEK cells. n = 3 independent experiments conducted in duplicate. (B) Concentration-dependent uptake of latanoprost acid in OATP2A1-transfected HEK cells and control cells (empty vector-transfected, VC) after 4 minutes' incubation. Net uptake was calculated as the difference between total uptake in HEK-OATP2A1 and control cells. n = 8 independent experiments conducted in duplicate. (C) Lineweaver-Burk (double-reciprocal) plot for determination of K m and V max was calculated from the data set shown in (B). n = 8.
Figure 7.
 
(A) Uptake of latanoprost acid (50 μM) as a function of time by OATP2B1 expressed in HEK cells. n = 3 independent experiments conducted in duplicate. (B) Uptake of latanoprost acid (15 μM) in OATP2B1-transfected HEK cells and vector control cells (VC) after 4 minutes' incubation. n = 8 independent experiments conducted in duplicate.
Figure 7.
 
(A) Uptake of latanoprost acid (50 μM) as a function of time by OATP2B1 expressed in HEK cells. n = 3 independent experiments conducted in duplicate. (B) Uptake of latanoprost acid (15 μM) in OATP2B1-transfected HEK cells and vector control cells (VC) after 4 minutes' incubation. n = 8 independent experiments conducted in duplicate.
We also investigated whether latanoprost (the ester prodrug) is transported by OATP2A1 or OATP2B1. Even when the compound was added to the cells at the highest concentration (500 μM), neither latanoprost nor its metabolite, latanoprost acid, was detected within the cells by LC/MS/MS. Taken together, the results suggest that the active acidic metabolite of latanoprost, but not the ester prodrug, is the substrate of the uptake transporters OATP2A1 and OATP2B1. 
Discussion
More than three decades ago, Bito et al. 2729 provided the first evidence that the facilitated removal of PGs from the eye occurs by way of an active transport mechanism. Twenty years later, the molecular mechanisms underlying PG transport were unraveled by cloning the cDNA of potential PG transporters, such as OATP2A1. 12,26 Here we demonstrate for the first time that latanoprost, the most widely used PG for the treatment of elevated intraocular pressure in glaucoma, is a high-affinity substrate of OATP2A1 that is expressed predominantly in the choroid/RPE-complex and in the ciliary body of the human eye. 
The active metabolites of antiglaucoma drugs are negatively charged under physiological pH, and it is generally believed that they cannot cross cell membranes without an active transportation system. Previous work proposes members of the OATP/SLCO family—OATP1A2, OATP2B1, OATP4A1—as candidates for uptake of the active carboxylic (M1) metabolite of unoprostone. 17 Among these transporters, unoprostone M1 had the highest affinity to OATP2B1, followed by OATP1A2 and OATP4A1. Our results extend this previous observation by the demonstration that latanoprost acid is also a substrate of OATP2B1. Moreover, we have demonstrated the expression of OATP2B1 in the choroid/RPE-complex and ciliary body at relevant levels, both on the mRNA level and protein level, whereas expression in other eye tissues was markedly lower (sensory retina, iris, lens, cornea). The rank-order expression levels of OATP2B1 in ocular tissues largely conform to recently reported data from Zhang et al., 18 who investigated three major compartments of the human eye. In that study, the relative expression of OATP2B1 was higher in the iris/ciliary body than in the retina/choroid or cornea. 
Although several studies have implicated OATP family members other than OATP2A1 in PG transport in the human eye, few have focused on the role of OATP2A1 for the ocular disposition of PGs. 16,30,31 In their pioneering work, Schuster et al. 16 demonstrated that OATP2A1 mRNA is expressed in a broad variety of human ocular tissues, though expression levels were not quantified and compared with those of human nonocular tissues. In the present study, we show that OATP2A1 mRNA is relatively abundant in the choroid/RPE-complex and ciliary body compared with other human tissues, such as liver, brain, kidney, and heart. On the protein level, OATP2A1 expression was most prominent in epithelial and endothelial cell layers of anterior segment tissues, whereas its expression in the posterior segment was weak. In contrast, OATP2B1 mRNA and protein were detected primarily in the ciliary body and choroid/RPE complex and could also be localized to epithelial and endothelial cell layers, particularly vascular endothelia of the choroidal stroma. Protein expression of this transporter in blood vessels has been reported previously. 24,32 Furthermore, the protein expression detected in our study largely conforms to the investigations of OATP2B1 in the human ciliary body, 17 demonstrating expression at the inner border of the epithelium facing the aqueous humor. In addition, OATP2B1 expression was observed in the trabecular meshwork and Schlemm's canal. 
Unlike most other organs, the eye has no PG-metabolizing enzymes. 33,34 Therefore, a physiological role of OATP2A1 could be the clearance of endogenous PGs synthesized in the ciliary epithelium and the ciliary body. 35 In addition, OATP2A1 may contribute to the clearance of antiglaucoma prostanoids from the aqueous humor. Indeed, we have demonstrated that latanoprost acid is a substrate of OATP2A1 with a K m in the low micromolar range. A notable finding was that the affinity of latanoprost acid for OATP2A1 was higher than for OATP2B1. We also showed that both latanoprost and its biologically active acid form are potent inhibitors of OATP2A1-mediated PGE2 uptake. It has been demonstrated previously that 17-phenyl PGF, whose structure is related to that of latanoprost acid, potently inhibits OATP2A1-dependent uptake of PGE2 with an inhibitory constant (K i) of 168 nM. 31 For latanoprost acid, a K i of 149 nM was reported for the inhibition of OATP2A1-dependent PGE1 uptake. 30 The reported K i values correspond to the peak concentrations of latanoprost acid detected in aqueous humor samples from patients in our study (approximately 120 nM). Our findings, together with previous observations, raise the possibility that latanoprost acts—in addition to direct activation of the PGF receptor (sensitive to PGF)—by the inhibition of PG uptake, thereby decreasing local clearance and increasing the bioavailability of endogenous PGs. 
In summary, the present study confirms the considerable variability in concentrations of the biologically active acid form of latanoprost in the aqueous humor after topical application of a standard dose of the drug. Protein expression of OATP2A1 and OATP2B1 was demonstrated in virtually all tissues of the anterior and posterior segments of the human eye. Moreover, we have provided experimental evidence that transporters from the OATP family, particularly OATP2A1, are involved in the clearance of latanoprost acid from the aqueous humor and may thereby influence the bioavailability of latanoprost in patients. 
Footnotes
 Supported by Deutsche Forschungsgemeinschaft (Grant DFG GL588/2-1).
Footnotes
 Disclosure: M.E. Kraft, None; H. Glaeser, None; K. Mandery, None; J. König, None; D. Auge, None; M.F. Fromm, None; U. Schlötzer-Schrehardt, None; U. Welge-Lüssen, None; F.E. Kruse, None; O. Zolk, None
The authors thank Evi Hoier and Martin Gillich for excellent technical assistance. 
References
Lütjen-Drecoll E Kruse FE . Primary open angle glaucoma: morphological bases for the understanding of the pathogenesis and effects of antiglaucomatic substances. Ophthalmologe. 2007;104:167–178. [CrossRef] [PubMed]
Bahler CK Howell KG Hann CR Fautsch MP Johnson DH . Prostaglandins increase trabecular meshwork outflow facility in cultured human anterior segments 195. Am J Ophthalmol. 2008;145:114–119. [CrossRef] [PubMed]
Sjöquist B Basu S Byding P Bergh K Stjernschantz J . The pharmacokinetics of a new antiglaucoma drug, latanoprost, in the rabbit. Drug Metab Dispos. 1998;26:745–754. [PubMed]
Sjöquist B Tajallaei S Stjernschantz J . Pharmacokinetics of latanoprost in the cynomolgus monkey: first communication: single intravenous, oral or topical administration on the eye. Arzneimittelforschung. 1999;49:225–233. [PubMed]
Sjöquist B Stjernschantz J . Ocular and systemic pharmacokinetics of latanoprost in humans. Surv Ophthalmol. 2002;47(suppl 1):S6–S12. [CrossRef] [PubMed]
Baroody RA Bito LZ . The impermeability of the basic cell membrane to thromboxane-B2′ prostacyclin and 6-keto-PGF 1 alpha. Prostaglandins. 1981;21:133–142. [CrossRef] [PubMed]
Bito LZ Baroody RA . Impermeability of rabbit erythrocytes to prostaglandins 182. Am J Physiol. 1975;229:1580–1584. [PubMed]
König J Seithel A Gradhand U Fromm MF . Pharmacogenomics of human OATP transporters. Naunyn Schmiedebergs ArchPharmacol. 2006;372:432–443. [CrossRef]
Hagenbuch B Meier PJ . Organic anion transporting polypeptides of the OATP/SLC21 family: phylogenetic classification as OATP/ SLCO superfamily, new nomenclature and molecular/functional properties. Pflugers Arch. 2004;447:653–665. [CrossRef] [PubMed]
Hagenbuch B Gui C . Xenobiotic transporters of the human organic anion transporting polypeptides (OATP) family. Xenobiotica. 2008;38:778–801. [CrossRef] [PubMed]
Zair ZM Eloranta JJ Stieger B Kullak-Ublick GA . Pharmacogenetics of OATP (SLC21/SLCO), OAT and OCT (SLC22) and PEPT (SLC15) transporters in the intestine, liver and kidney. Pharmacogenomics. 2008;9:597–624. [CrossRef] [PubMed]
Kanai N Lu R Satriano JA Bao Y Wolkoff AW Schuster VL . Identification and characterization of a prostaglandin transporter. Science. 1995;268:866–869. [CrossRef] [PubMed]
Chan BS Satriano JA Pucci M Schuster VL . Mechanism of prostaglandin E2 transport across the plasma membrane of HeLa cells and Xenopus oocytes expressing the prostaglandin transporter “PGT”. J Biol Chem. 1998;273:6689–6697. [CrossRef] [PubMed]
Pucci ML Bao Y Chan B . Cloning of mouse prostaglandin transporter PGT cDNA: species-specific substrate affinities. Am J Physiol. 1999;277:R734–R741. [PubMed]
Itoh S Lu R Bao Y Morrow JD Roberts LJ Schuster VL . Structural determinants of substrates for the prostaglandin transporter PGT. Mol Pharmacol. 1996;50:738–742. [PubMed]
Schuster VL Lu R Coca-Prados M . The prostaglandin transporter is widely expressed in ocular tissues. Surv Ophthalmol. 1997;41(suppl 2):S41–S45. [CrossRef] [PubMed]
Gao B Huber RD Wenzel A . Localization of organic anion transporting polypeptides in the rat and human ciliary body epithelium. Exp Eye Res. 2005;80:61–72. [CrossRef] [PubMed]
Zhang T Xiang CD Gale D Carreiro S Wu EY Zhang EY . Drug transporter and cytochrome P450 mRNA expression in human ocular barriers: implications for ocular drug disposition. Drug Metab Dispos. 2008;36:1300–1307. [CrossRef] [PubMed]
Hagenbuch B Meier PJ . The superfamily of organic anion transporting polypeptides 188. Biochim Biophys Acta. 2003;1609:1–18. [CrossRef] [PubMed]
Tamai I Nozawa T Koshida M Nezu J Sai Y Tsuji A . Functional characterization of human organic anion transporting polypeptide B (OATP-B) in comparison with liver-specific OATP-C. Pharm Res. 2001;18:1262–1269. [CrossRef] [PubMed]
Kullak-Ublick GA Ismair MG Stieger B . Organic anion-transporting polypeptide B (OATP-B) and its functional comparison with three other OATPs of human liver. Gastroenterology. 2001;120:525–533. [CrossRef] [PubMed]
Livak KJ Schmittgen TD . Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001;25:402–408. [CrossRef] [PubMed]
Mandery K Bujok K Schmidt I . Influence of cyclooxygenase inhibitors on the function of the prostaglandin transporter OATP2A1 expressed in human gastroduodenal mucosa. J Pharmacol Exp Ther. 2010;332:345–351. [CrossRef] [PubMed]
Bronger H König J Kopplow K . ABCC drug efflux pumps and organic anion uptake transporters in human gliomas and the blood-tumor barrier. Cancer Res. 2005;65:11419–11428. [CrossRef] [PubMed]
Seithel A Eberl S Singer K . The influence of macrolide antibiotics on the uptake of organic anions and drugs mediated by OATP1B1 and OATP1B3. Drug Metab Dispos. 2007;35:779–786. [CrossRef] [PubMed]
Lu R Kanai N Bao Y Schuster VL . Cloning, in vitro expression, and tissue distribution of a human prostaglandin transporter cDNA(hPGT). J Clin Invest. 1996;98:1142–1149. [CrossRef] [PubMed]
Bito LZ Baroody R . Concentrative accumulation of 3H-prostaglandins by some rabbit tissues in vitro: the chemical nature of the accumulated 3H-labelled substances. Prostaglandins. 1974;7:131–140. [CrossRef] [PubMed]
Bito LZ Salvador EV . Intraocular fluid dynamics, 3: the site and mechanism of prostaglandin transfer across the blood intraocular fluid barriers. Exp Eye Res. 1972;14:233–241. [CrossRef] [PubMed]
Wallenstein MC Bito LZ . The effects of intravitreally injected prostaglandin E1 on retinal function and their enhancement by a prostaglandin-transporter inhibitor. Invest Ophthalmol Vis Sci. 1978;17:795–799. [PubMed]
Kashiwagi K Kanai N Tsuchida T . Comparison between isopropyl unoprostone and latanoprost by prostaglandin E(2)induction, affinity to prostaglandin transporter, and intraocular metabolism. Exp Eye Res. 2002;74:41–49. [CrossRef] [PubMed]
Schuster VL Itoh S Andrews SW . Synthetic modification of prostaglandin F(2alpha) indicates different structural determinants for binding to the prostaglandin F receptor versus the prostaglandin transporter. Mol Pharmacol. 2000;58:1511–1516. [PubMed]
Grube M Köck K Oswald S . Organic anion transporting polypeptide 2B1 is a high-affinity transporter for atorvastatin and is expressed in the human heart. Clin Pharmacol Ther. 2006;80:607–620. [CrossRef] [PubMed]
Cheng-Bennett A Poyer J Weinkam RJ Woodward DF . Lack of prostaglandin F2 alpha metabolism by human ocular tissues. Invest Ophthalmol Vis Sci. 1990;31:1389–1393. [PubMed]
Goh Y Urade Y Fujimoto N Hayaishi O . Content and formation of prostaglandins and distribution of prostaglandin-related enzyme activities in the rat ocular system. Biochim Biophys Acta. 1987;921:302–311. [CrossRef] [PubMed]
Maihöfner C Schlötzer-Schrehardt U Gühring H . Expression of cyclooxygenase-1 and −2 in normal and glaucomatous human eyes. Invest Ophthalmol Vis Sci. 2001;42:2616–2624. [PubMed]
Figure 1.
 
Concentration of latanoprost-free acid in the aqueous humor of 12 patients after topical application of 1.5 μg latanoprost.
Figure 1.
 
Concentration of latanoprost-free acid in the aqueous humor of 12 patients after topical application of 1.5 μg latanoprost.
Figure 2.
 
OATP2A1 (A) and OATP2B1 (B) mRNA expression in human liver, brain, kidney, placenta, and heart (gray) and in ocular tissues (black). Values are normalized to the housekeeping genes GAPDH and ACTB, and all transcript levels are related to choroid expression.
Figure 2.
 
OATP2A1 (A) and OATP2B1 (B) mRNA expression in human liver, brain, kidney, placenta, and heart (gray) and in ocular tissues (black). Values are normalized to the housekeeping genes GAPDH and ACTB, and all transcript levels are related to choroid expression.
Figure 3.
 
Immunolocalization of OATP2A1 in human ocular tissues (green fluorescence, left) and corresponding phase-contrast images (right). Scale bar, 20 μm. (A) Epithelium (CEP) and stroma of the cornea (CS). (B) Endothelium (CEN) and stroma of the cornea (CS). (C) Conjunctival epithelium (CJE). (D) Stroma (IS), vessels, and pigmented epithelium (IPE) of the iris. (E) Ciliary body with its nonpigmented (CBNPE) and pigmented (CBPE) epithelium. (F) Choroid (CH) and RPE.
Figure 3.
 
Immunolocalization of OATP2A1 in human ocular tissues (green fluorescence, left) and corresponding phase-contrast images (right). Scale bar, 20 μm. (A) Epithelium (CEP) and stroma of the cornea (CS). (B) Endothelium (CEN) and stroma of the cornea (CS). (C) Conjunctival epithelium (CJE). (D) Stroma (IS), vessels, and pigmented epithelium (IPE) of the iris. (E) Ciliary body with its nonpigmented (CBNPE) and pigmented (CBPE) epithelium. (F) Choroid (CH) and RPE.
Figure 4.
 
Immunolocalization of OATP2B1 in human anterior eye tissues (green fluorescence, left) and corresponding phase-contrast images (right). Scale bar, 20 μm. (A) Epithelium (CEP) and stroma of the cornea (CS). (B) Endothelium (CEN) and stroma of the cornea (CS). (C) Conjunctival epithelium (CJE). (D) Stroma (IS), vessels, and pigmented epithelium of the iris. (E) Ciliary body with its nonpigmented (CBNPE) and pigmented (CBPE) epithelium. (F) Choroid (CH) and RPE.
Figure 4.
 
Immunolocalization of OATP2B1 in human anterior eye tissues (green fluorescence, left) and corresponding phase-contrast images (right). Scale bar, 20 μm. (A) Epithelium (CEP) and stroma of the cornea (CS). (B) Endothelium (CEN) and stroma of the cornea (CS). (C) Conjunctival epithelium (CJE). (D) Stroma (IS), vessels, and pigmented epithelium of the iris. (E) Ciliary body with its nonpigmented (CBNPE) and pigmented (CBPE) epithelium. (F) Choroid (CH) and RPE.
Figure 5.
 
(A) Inhibition of OATP2A1-mediated prostaglandin E2 (PGE2, 1 μM) uptake by latanoprost and latanoprost acid. (B) Inhibition of OATP2B1-mediated BSP (1 μM) uptake by latanoprost and latanoprost acid. n = 3 independent experiments conducted in duplicate.
Figure 5.
 
(A) Inhibition of OATP2A1-mediated prostaglandin E2 (PGE2, 1 μM) uptake by latanoprost and latanoprost acid. (B) Inhibition of OATP2B1-mediated BSP (1 μM) uptake by latanoprost and latanoprost acid. n = 3 independent experiments conducted in duplicate.
Figure 6.
 
(A) Uptake of latanoprost acid (50 μM) as a function of time by OATP2A1 expressed in HEK cells. n = 3 independent experiments conducted in duplicate. (B) Concentration-dependent uptake of latanoprost acid in OATP2A1-transfected HEK cells and control cells (empty vector-transfected, VC) after 4 minutes' incubation. Net uptake was calculated as the difference between total uptake in HEK-OATP2A1 and control cells. n = 8 independent experiments conducted in duplicate. (C) Lineweaver-Burk (double-reciprocal) plot for determination of K m and V max was calculated from the data set shown in (B). n = 8.
Figure 6.
 
(A) Uptake of latanoprost acid (50 μM) as a function of time by OATP2A1 expressed in HEK cells. n = 3 independent experiments conducted in duplicate. (B) Concentration-dependent uptake of latanoprost acid in OATP2A1-transfected HEK cells and control cells (empty vector-transfected, VC) after 4 minutes' incubation. Net uptake was calculated as the difference between total uptake in HEK-OATP2A1 and control cells. n = 8 independent experiments conducted in duplicate. (C) Lineweaver-Burk (double-reciprocal) plot for determination of K m and V max was calculated from the data set shown in (B). n = 8.
Figure 7.
 
(A) Uptake of latanoprost acid (50 μM) as a function of time by OATP2B1 expressed in HEK cells. n = 3 independent experiments conducted in duplicate. (B) Uptake of latanoprost acid (15 μM) in OATP2B1-transfected HEK cells and vector control cells (VC) after 4 minutes' incubation. n = 8 independent experiments conducted in duplicate.
Figure 7.
 
(A) Uptake of latanoprost acid (50 μM) as a function of time by OATP2B1 expressed in HEK cells. n = 3 independent experiments conducted in duplicate. (B) Uptake of latanoprost acid (15 μM) in OATP2B1-transfected HEK cells and vector control cells (VC) after 4 minutes' incubation. n = 8 independent experiments conducted in duplicate.
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×