MPP+-Induced Changes in Cellular Impedance as a Measure for Organic Cation Transporter (SLC22A1-3) Activity and Inhibition

The organic cation transporters OCT1-3 (SLC22A1-3) facilitate the transport of cationic endo- and xenobiotics and are important mediators of drug distribution and elimination. Their polyspecific nature makes OCTs highly susceptible to drug–drug interactions (DDIs). Currently, screening of OCT inhibitors depends on uptake assays that require labeled substrates to detect transport activity. However, these uptake assays have several limitations. Hence, there is a need to develop novel assays to study OCT activity in a physiological relevant environment without the need to label the substrate. Here, a label-free impedance-based transport assay is established that detects OCT-mediated transport activity and inhibition utilizing the neurotoxin MPP⁺. Uptake of MPP⁺ by OCTs induced concentration-dependent changes in cellular impedance that were inhibited by decynium-22, corticosterone, and Tyrosine Kinase inhibitors. OCT-mediated MPP⁺ transport activity and inhibition were quantified on both OCT1-3 overexpressing cells and HeLa cells endogenously expressing OCT3. Moreover, the method presented here is a valuable tool to identify novel inhibitors and potential DDI partners for MPP⁺ transporting solute carrier proteins (SLCs) in general.     

Substrates of the Human Brain Proton-Organic Cation Antiporter and Comparison with Organic Cation Transporter 1 Activities

Many organic cations (OCs) may be transported through membranes by a genetically still uncharacterized proton-organic cation (H + OC) antiporter. Here, we characterized an extended substrate spectrum of this antiporter. We studied the uptake of 72 drugs in hCMEC/D3 cells as a model of the human blood–brain barrier. All 72 drugs were tested with exchange transport assays and the transport of 26 of the drugs was studied in more detail concerning concentration-dependent uptake and susceptibility to specific inhibitors. According to exchange transport assays, 37 (51%) drugs were good substrates of the H + OC antiporter. From 26 drugs characterized in more detail, 23 were consistently identified as substrates of the H + OC antiporter in six different assays and transport kinetic constants could be identified with intrinsic clearances between 0.2 (ephedrine) and 201 (imipramine) mL × minute⁻¹ × g protein⁻¹. Excellent substrates of the H + OC antiporter were no substrates of organic cation transporter OCT1 and vice versa. Good substrates of the H + OC antiporter were more hydrophobic and had a lower topological polar surface area than non-substrates or OCT1 substrates. These data and further research on the H + OC antiporter may result in a better understanding of pharmacokinetics, drug–drug interactions and variations in pharmacokinetics.     

Expression and Functional Contribution of Different Organic Cation Transporters to the Cellular Uptake of Doxorubicin into Human Breast Cancer and Cardiac Tissue

Doxorubicin is a frequently used anticancer drug to treat many types of tumors, such as breast cancer or bronchial carcinoma. The clinical use of doxorubicin is limited by its poorly predictable cardiotoxicity, the reasons of which are so far not fully understood. The drug is a substrate of several efflux transporters such as P-gp or BCRP and was recently reported to be a substrate of cation uptake transporters. To evaluate the potential role of transporter proteins in the accumulation of doxorubicin at its site of action (e.g., mammary carcinoma cells) or adverse effects (e.g., heart muscle cells), we studied the expression of important uptake and efflux transporters in human breast cancer and cardiac tissue, and investigated the affinity of doxorubicin to the identified transporters. The cellular uptake studies on doxorubicin were performed with OATP1A2*1, OATP1A2*2, and OATP1A2*3-overexpressing HEK293 cells, as well as OCT1-, OCT2-, and OCT3- overexpressing MDCKII cells. To assess the contribution of transporters to the cytotoxic effect of doxorubicin, we determined the cell viability in the presence and absence of transporter inhibitors in different cell lines. Several transporters, including P-gp, BCRP, OCT1, OCT3, and OATP1A2 were expressed in human heart and/or breast cancer tissue. Doxorubicin could be identified as a substrate of OCT1, OCT2, OCT3, and OATP1A2. The cellular uptake into cells expressing genetic OATP1A2 variants was markedly reduced and correlated well with the increased cellular viability. Inhibition of OATP1A2 (naringin) and OCT transporters (1-methyl-4-phenylpyridinium) resulted in a significant decrease of doxorubicin-mediated cytotoxicity in cell lines expressing the respective transporters. Similarly, the excipient Cremophor EL significantly inhibited the OCT1-3- and OATP1A2-mediated cellular uptake and attenuated the cytotoxicity of doxorubicin. In conclusion, genetic and environmental-related variability in the expression and function of these transporters may contribute to the substantial variability seen in terms of doxorubicin efficacy and toxicity.     

Overlap and Specificity in the Substrate Spectra of Human Monoamine Transporters and Organic Cation Transporters 1, 2, and 3

Human monoamine transporters (MATs) are cation transporters critically involved in neuronal signal transmission. While inhibitors of MATs have been intensively studied, their substrate spectra have received far less attention. Polyspecific organic cation transporters (OCTs), predominantly known for their role in hepatic and renal drug elimination, are also expressed in the central nervous system and might modulate monoaminergic signaling. Using HEK293 cells overexpressing MATs or OCTs, we compared uptake of 48 compounds, mainly phenethylamine and tryptamine derivatives including matched molecular pairs, across noradrenaline, dopamine and serotonin transporters and OCTs (1, 2, and 3). Generally, MATs showed surprisingly high transport activities for numerous analogs of neurotransmitters, but their substrate spectra were limited by molar mass. Human OCT2 showed the broadest substrate spectrum, and also the highest overlap with MATs substrates. Comparative kinetic analyses revealed that the radiotracer meta-iodobenzylguanidine had the most balanced uptake across all six transporters. Matched molecular pair analyses comparing MAT and OCT uptake using the same methodology could provide a better understanding of structural determinants for high cell uptake by MATs or OCTs. The data may result in a better understanding of pharmacokinetics and toxicokinetics of small molecular organic cations and, possibly, in the development of more specific radiotracers for MATs.     

Active Transport of Hepatotoxic Pyrrolizidine Alkaloids in HepaRG Cells

1,2-unsaturated pyrrolizidine alkaloids (PAs) are secondary plant metabolites occurring as food contaminants that can cause severe liver damage upon metabolic activation in hepatocytes. However, it is yet unknown how these contaminants enter the cells. The role of hepatic transporters is only at the beginning of being recognized as a key determinant of PA toxicity. Therefore, this study concentrated on assessing the general mode of action of PA transport in the human hepatoma cell line HepaRG using seven structurally different PAs. Furthermore, several hepatic uptake and efflux transporters were targeted with pharmacological inhibitors to identify their role in the uptake of the PAs retrorsine and senecionine and in the disposition of their N-oxides (PANO). For this purpose, PA and PANO content was measured in the supernatant using LC-MS/MS. Also, PA-mediated cytotoxicity was analyzed after transport inhibition. It was found that PAs are taken up into HepaRG cells in a predominantly active and structure-dependent manner. This pattern correlates with other experimental endpoints such as cytotoxicity. Pharmacological inhibition of the influx transporters Na⁺/taurocholate co-transporting polypeptide (SLC10A1) and organic cation transporter 1 (SLC22A1) led to a reduced uptake of retrorsine and senecionine into HepaRG cells, emphasizing the relevance of these transporters for PA toxicokinetics.     

In Vitro Inhibition of Renal OCT2 and MATE1 Secretion by Antiemetic Drugs

The organic cation transporter 2 (OCT2) and multidrug and toxin extrusion protein 1 (MATE1) mediate the renal secretion of drugs. Recent studies suggest that ondansetron, a 5-HT₃ antagonist drug used to prevent nausea and vomiting, can inhibit OCT2- and MATE1-mediated transport. The purpose of this study was to test the ability of five 5-HT₃ antagonist drugs to inhibit the OCT2 and MATE1 transporters. The transport of the OCT2/MATE1 probe substrate ASP⁺ was assessed using two models: (1) HEK293 kidney cells overexpressing human OCT2 or MATE1, and (2) MDCK cells transfected with human OCT2 and MATE1. In HEK293 cells, the inhibition of ASP⁺ uptake by OCT2 listed in order of potency was palonosetron (IC₅₀: 2.6 μM) > ondansetron > granisetron > tropisetron > dolasetron (IC₅₀: 85.4 μM) and the inhibition of ASP⁺ uptake by MATE1 in order of potency was ondansetron (IC₅₀: 0.1 μM) > palonosetron = tropisetron > granisetron > dolasetron (IC₅₀: 27.4 μM). Ondansetron (0.5–20 μM) inhibited the basolateral-to-apical transcellular transport of ASP⁺ up to 64%. Higher concentrations (10 and 20 μM) of palonosetron, tropisetron, and dolasetron similarly reduced the transcellular transport of ASP⁺. In double-transfected OCT2-MATE1 MDCK cells, ondansetron at concentrations of 0.5 and 2.5 μM caused significant intracellular accumulation of ASP⁺. Taken together, these data suggest that 5-HT₃ antagonist drugs may inhibit the renal secretion of cationic drugs by interfering with OCT2 and/or MATE1 function.     

Transport of free and peptide-bound glycated amino acids: synthesis, transepithelial flux at Caco-2 cell monolayers, and interaction with apical membrane transport proteins

In glycation reactions, the side chains of protein-bound nucleophilic amino acids such as lysine and arginine are post-translationally modified to a variety of derivatives also known as Maillard reaction products (MRPs). Considerable amounts of MRPs are taken up in food. Here we have studied the interactions of free and dipeptide-bound MRPs with intestinal transport systems. Free and dipeptide-bound derivatives of N(6)-(1-fructosyl)lysine (FL), N(6)-(carboxymethyl)lysine (CML), N(6)-(1-carboxyethyl)lysine (CEL), formyline, argpyrimidine, and methylglyoxal-derived hydroimidazolone 1 (MG-H1) were synthesized. The inhibition of L-[(3)H]lysine and [(14) C]glycylsarcosine uptakes was measured in Caco-2 cells which express the H(+)/peptide transporter PEPT1 and lysine transport system(s). Glycated amino acids always displayed lower affinities than their unmodified analogues towards the L-[(3)H]lysine transporter(s). In contrast, all glycated dipeptides except Ala-FL were medium- to high-affinity inhibitors of [(14)C]Gly-Sar uptake. The transepithelial flux of the derivatives across Caco-2 cell monolayers was determined. Free amino acids and intact peptides derived from CML and CEL were translocated to very small extents. Application of peptide-bound MRPs, however, led to elevation (up to 80-fold) of the net flux and intracellular accumulation of glycated amino acids, which were hydrolyzed from the dipeptides inside the cells. We conclude 1) that free MRPs are not substrates for the intestinal lysine transporter(s), and 2) that dietary MRPs are absorbed into intestinal cells in the form of dipeptides, most likely by the peptide transporter PEPT1. After hydrolysis, hydrophobic glycated amino acids such as pyrraline, formyline, maltosine, and argpyrimidine undergo basolateral efflux, most likely by simple diffusion down their concentration gradients.     

Cloning and Functional Characterization of Dog OCT1 and OCT2: Another Step in Exploring Species Differences in Organic Cation Transporters

OCT1 and OCT2 are polyspecific membrane transporters that are involved in hepatic and renal drug clearance in humans and mice. In this study, we cloned dog OCT1 and OCT2 and compared their function to the human and mouse orthologs. We used liver and kidney RNA to clone dog OCT1 and OCT2. The cloned and the publicly available RNA-Seq sequences differed from the annotated exon-intron structure of OCT1 in the dog genome CanFam3.1. An additional exon between exons 2 and 3 was identified and confirmed by sequencing in six additional dog breeds. Next, dog OCT1 and OCT2 were stably overexpressed in HEK293 cells and the transport kinetics of five drugs were analyzed. We observed strong differences in the transport kinetics between dog and human orthologs. Dog OCT1 transported fenoterol with 12.9-fold higher capacity but 14.3-fold lower affinity (higher K_M) than human OCT1. Human OCT1 transported ipratropium with 5.2-fold higher capacity but 8.4-fold lower affinity than dog OCT1. Compared to human OCT2, dog OCT2 showed 10-fold lower transport of fenoterol and butylscopolamine. In conclusion, the functional characterization of dog OCT1 and OCT2 reported here may have implications when using dogs as pre-clinical models as well as for drug therapy in dogs.     

A Combination Strategy to Inhibit Pim‐1: Synergism between Noncompetitive and ATP‐Competitive Inhibitors

Pim‐1 is a serine/threonine kinase critically involved in the initiation and progression of various types of cancer, especially leukemia, lymphomas and solid tumors such as prostate, pancreas and colon, and is considered a potential drug target against these malignancies. In an effort to discover new potent Pim‐1 inhibitors, a previously identified ATP‐competitive indolyl‐pyrrolone scaffold was expanded to derive structure–activity relationship data. A virtual screening campaign was also performed, which led to the discovery of additional ATP‐competitive inhibitors as well as a series of 2‐aminothiazole derivatives, which are noncompetitive with respect to both ATP and peptide substrate. This mechanism of action, which resembles allosteric inhibition, has not previously been characterized for Pim‐1. Notably, further evaluation of the 2‐aminothiazoles indicated a synergistic inhibitory effect in enzymatic assays when tested in combination with ATP‐competitive inhibitors. A synergistic effect in the inhibition of cell proliferation by ATP‐competitive and ATP‐noncompetitive compounds was also observed in prostate cancer cell lines (PC3), where all Pim‐1 inhibitors tested in showed synergism with the known anticancer agent, paclitaxel. These results further establish Pim‐1 as a target in cancer therapy, and highlight the potential of these agents for use as adjuvant agents in the treatment of cancer diseases in which Pim‐1 is associated with chemotherapeutic resistance.     

Transport Turnover Rates for Human OCT2 and MATE1 Expressed in Chinese Hamster Ovary Cells

MATE1 (multidrug and toxin extruder 1) and OCT2 (organic cation transporter 2) play critical roles in organic cation excretion by the human kidney. The transporter turnover rate (TOR) is relevant to understanding both their transport mechanisms and interpreting the in vitro–in vivo extrapolation (IVIVE) required for physiologically-based pharmacokinetic (PBPK) modeling. Here, we use a quantitative western blot method to determine TORs for MATE1 and OCT2 proteins expressed in CHO cells. MATE1 and OCT2, each with a C-terminal V-5 epitope tag, were cell surface biotinylated and the amount of cell surface MATE1 and OCT2 protein was quantified by western analysis, using standard curves for the V5 epitope. Cell surface MATE1 and OCT2 protein represented 25% and 24%, respectively, of the total expression of these proteins in CHO cells. The number of cell surface transporters was ~55 fmol cm⁻² for MATE1 and ~510 fmol cm⁻² for OCT2. Dividing these values into the different J_max values for transport of MPP, metformin, and atenolol mediated by MATE1 and OCT2 resulted in calculated TOR values (±SE, n = 4) of 84.0 ± 22.0 s⁻¹ and 2.9 ± 0.6 s⁻¹; metformin, 461.0 ± 121.0 s⁻¹ and 12.6 ± 2.4 s⁻¹; atenolol, 118.0 ± 31.0 s⁻¹, respectively. These values are consistent with the TOR values determined for a variety of exchangers (NHEs), cotransporters (SGLTs, Lac permease), and uniporters (GLUTs, ENTs).     

LC/MS/MS and radioisotope method combined for recognizing the affinity between catalpol and OCT2 transporter

More and more herbal medicines are found to be the substrates of drug transporters. In this paper, chromatography/tandem mass spectrometry (LC/MS/MS) combined with radioisotope method was used for the quantification of catalpol, a traditional Chinese medicine, to study the affinity relationship between herbal medicines and transporters. Catalpol uptake experiment was carried out by using several transporters (OAT1, OCT2, OAT3, OATP1B1 and OATP2B1). And samples were precipitated with methanol and quantified with LC/MS/MS. The results show that catalpol has a good affinity with OCT2-transfected S2 cells. After studying drug-drug interaction between catalpol and ¹⁴C-tetraethylammonium (TEA), we found that catalpol is able to facilitate TEA transport mediated by OCT2, suggesting that catalpol could probably be a new promoter of OCT2.     

Structures and Mechanisms of a Novel Bacterial Transport System for Fatty Acids

Bacterial acquisition of metabolites is largely facilitated by transporters with unique substrate scopes. The tripartite ATP-independent periplasmic (TRAP) transporters comprise a large family of bacterial proteins that facilitate the uptake of a variety of small molecules. It has been reported that some TRAP systems encode a fourth protein, the T component. The T-component, or TatT, is predicted to be a periplasmic-facing lipoprotein that enables the uptake of metabolites from the outer membrane. However, no substrates were revealed for any TatT and their functional role(s) remained enigmatic. We recently identified a homolog in Methylococcus capsulatus that binds to sterols, and herein, we report two additional homologs that demonstrate a preference for long-chain fatty acids. Our bioinformatics, quantitative analyses of protein-ligand interactions, and high-resolution crystal structures suggest that TatTs might facilitate the trafficking of hydrophobic or lipophilic substrates and represent a new class of bacterial lipid and fatty acid transporters.     

PTPRD and DCC Are Novel BACE1 Substrates Differentially Expressed in Alzheimer’s Disease: A Data Mining and Bioinformatics Study

The β-site Amyloid precursor protein Cleaving Enzyme 1 (BACE1) is an extensively studied therapeutic target for Alzheimer’s disease (AD), owing to its role in the production of neurotoxic amyloid beta (Aβ) peptides. However, despite numerous BACE1 inhibitors entering clinical trials, none have successfully improved AD pathogenesis, despite effectively lowering Aβ concentrations. This can, in part, be attributed to an incomplete understanding of BACE1, including its physiological functions and substrate specificity. We propose that BACE1 has additional important physiological functions, mediated through substrates still to be identified. Thus, to address this, we computationally analysed a list of 533 BACE1 dependent proteins, identified from the literature, for potential BACE1 substrates, and compared them against proteins differentially expressed in AD. We identified 15 novel BACE1 substrates that were specifically altered in AD. To confirm our analysis, we validated Protein tyrosine phosphatase receptor type D (PTPRD) and Netrin receptor DCC (DCC) using Western blotting. These findings shed light on the BACE1 inhibitor failings and could enable the design of substrate-specific inhibitors to target alternative BACE1 substrates. Furthermore, it gives us a greater understanding of the roles of BACE1 and its dysfunction in AD.     

Novel Symmetrical Cage Compounds as Inhibitors of the Symmetrical MRP4-Efflux Pump for Anticancer Therapy

Within the last decades cancer treatment improved by the availability of more specifically acting drugs that address molecular target structures in cancer cells. However, those target-sensitive drugs suffer from ongoing resistances resulting from mutations and moreover they are affected by the cancer phenomenon of multidrug resistance. A multidrug resistant cancer can hardly be treated with the common drugs, so that there have been long efforts to develop drugs to combat that resistance. Transmembrane efflux pumps are the main cause of the multidrug resistance in cancer. Early inhibitors disappointed in cancer treatment without a proof of expression of a respective efflux pump. Recent studies in efflux pump expressing cancer show convincing effects of those inhibitors. Based on the molecular symmetry of the efflux pump multidrug resistant protein (MRP) 4 we synthesized symmetric inhibitors with varied substitution patterns. They were evaluated in a MRP4-overexpressing cancer cell line model to prove structure-dependent effects on the inhibition of the efflux pump activity in an uptake assay of a fluorescent MRP4 substrate. The most active compound was tested to resentisize the MRP4-overexpressing cell line towards a clinically relevant anticancer drug as proof-of-principle to encourage for further preclinical studies.     

Pyrrolo[1,2-a]quinoxalines: Insulin Mimetics that Exhibit Potent and Selective Inhibition against Protein Tyrosine Phosphatase 1B

PTP1B dephosphorylates insulin receptor and substrates to modulate glucose metabolism. This enzyme is a validated therapeutic target for type 2 diabetes, but no current drug candidates have completed clinical trials. Pyrrolo[1,2-a]quinoxalines substituted at positions C1–C4 and/or C7–C8 were found to be nontoxic to cells and good inhibitors in the low- to sub-micromolar range, with the 4-benzyl derivative being the most potent inhibitor (0.24 μm ). Some analogues bearing chlorine atoms at C7 and/or C8 kept potency and showed good selectivity compared to TCPTP (selectivity index >40). The most potent inhibitors behaved as insulin mimetics by increasing glucose uptake. The 4-benzyl derivative inhibited insulin receptor substrate 1 and AKT phosphorylation. Molecular docking and molecular dynamics simulations supported a putative binding mode for these compounds to the allosteric α3/α6/α7 pocket, but inconsistent results in enzyme inhibition kinetics were obtained due to the high tendency of these inhibitors to form stable aggregates. Computational calculations supported the druggability of inhibitors.     

Trospium Chloride Transport by Mouse Drug Carriers of the Slc22 and Slc47 Families

Background: The muscarinic receptor antagonist trospium chloride (TCl) is used for pharmacotherapy of the overactive bladder syndrome. TCl is a hydrophilic positively charged drug. Therefore, it has low permeability through biomembranes and requires drug transporters for distribution and excretion. In humans, the organic cation transporters OCT1 and OCT2 and the multidrug and toxin extrusion MATE1 and MATE2-K carriers showed TCl transport. However, their individual role for distribution and excretion of TCl is unclear. Knockout mouse models lacking mOct1/mOct2 or mMate1 might help to clarify their role for the overall pharmacokinetics of TCl. Method: In preparation of such experiments, TCl transport was analyzed in HEK293 cells stably transfected with the mouse carriers mOct1, mOct2, mMate1, and mMate2, respectively. Results: Mouse mOct1, mOct2, and mMate1 showed significant TCl transport with Km values of 58.7, 78.5, and 29.3 µM, respectively. In contrast, mMate2 did not transport TCl but showed MPP⁺ transport with Km of 60.0 µM that was inhibited by the drugs topotecan, acyclovir, and levofloxacin. Conclusion: TCl transport behavior as well as expression pattern were quite similar for the mouse carriers mOct1, mOct2, and mMate1 compared to their human counterparts.     

Luseogliflozin,a SGLT2 Inhibitor,Does Not Affect Glucose Uptake Kinetics in Renal Proximal Tubules of Live Mice

Proximal tubules (PTs) take up most of the glucose in the glomerular filtrate and return it to peritubular capillary blood. Sodium-glucose cotransporter 2 (SGLT2) at the apical membrane takes up glucose into the cell. Glucose then flows across the cells and is transported to the interstitium via glucose transporter 2 (GLUT2) at the basolateral membrane. However, glucose transport under SGLT2 inhibition remains poorly understood. In this study, we evaluated the dynamics of a fluorescent glucose analog, 2-NBDG, in the PTs of live mice treated with or without the SGLT2 inhibitor, luseogliflozin. We employed real-time multiphoton microscopy, in which insulin enhanced 2-NBDG uptake in skeletal muscle. Influx and efflux of 2-NBDG in PT cells were compared under hypo-, normo-, and hyperglycemic conditions. Luseogliflozin did not exert significant effects on glucose influx parameters under any level of blood glucose. Our results suggest that blood glucose level per se does not alter glucose influx or efflux kinetics in PTs. In conclusion, neither SGLT2 inhibition nor blood glucose level affect glucose uptake kinetics in PTs. The former was because of glucose influx through basolateral GLUT2, which is an established bidirectional transporter.     

Linoleic acid uptake by isolated enterocytes: Influence of α-linolenic acid on absorption

In a previous study we showed that intestinal uptake of α-linolenic acid (18∶3n−3) was carrier-mediated and we suggested that a plasma membrane fatty acid protein was involved in the transport of long-chain fatty acids. To further test this hypothesis, the mechanism of linoleic acid (18∶2n−6) uptake by isolated intestinal cells was examined using a rapid filtration method and 20 mM sodium taurocholate as solubilizing agent. Under these experimental conditions transport of [1-¹⁴C]linoleic acid monomers in the concentration range of 2 to 2220 nM was saturable with a V_m of 5.1±0.6 nmol/mg protein/min and a K_m of 183±7 nM. Experiments carried out in the presence of metabolic inhibitors, such as 2,4-dinitrophenol and antimycin A, suggested that an active, carriermediated mechanism was involved in the intestinal uptake of this essential fatty acid. The addition of excess unlabeled linoleic acid to the incubation medium led to a 89% decrease in the uptake of [1-¹⁴C]linoleic acid, whiled-glucose did not compete for transport into the cell. Other long-chain polyunsaturated fatty acids added to the incubation mixture inhibited linoleic acid uptake by more than 80%. The presence of α-linolenic acid (18∶3n−3) in the incubation medium caused the competitive inhibition (K_i=353 nM) of linoleic acid uptake. The data are compatible with the hypothesis that intestinal uptake of both linoleic, and α-linolenic acid is mediated by a membrane carrier common to long-chain fatty acids.     

Properties of Transport Mediated by the Human Organic Cation Transporter 2 Studied in a Polarized Three-Dimensional Epithelial Cell Culture Model

The renal secretory clearance for organic cations (neurotransmitters, metabolism products and drugs) is mediated by transporters specifically expressed in the basolateral and apical plasma membrane domains of proximal tubule cells. Here, human organic cation transporter 2 (hOCT2) is the main transporter for organic cations in the basolateral membrane domain. In this study, we stably expressed hOCT2 in Madin-Darby Canine Kidney (MDCK) cells and cultivated these cells in the presence of an extracellular matrix to obtain three-dimensional (3D) structures (cysts). The transport properties of hOCT2 expressed in MDCK cysts were compared with those measured using human embryonic kidney cells (HEK293) stably transfected with hOCT2 (hOCT2-HEK cells). In the MDCK cysts, hOCT2 was expressed in the basolateral membrane domain and showed a significant uptake of the fluorescent organic cation 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP⁺) with an affinity (K_m) of 3.6 ± 1.2 µM, similar to what was measured in the hOCT2-HEK cells (K_m = 3.1 ± 0.2 µM). ASP⁺ uptake was inhibited by tetraethylammonium (TEA⁺), tetrapentylammonium (TPA⁺), metformin and baricitinib both in the hOCT2-HEK cells and the hOCT2- MDCK cysts, even though the apparent affinities of TEA⁺ and baricitinib were dependent on the expression system. Then, hOCT2 was subjected to the same rapid regulation by inhibition of p56^lck tyrosine kinase or calmodulin in the hOCT2-HEK cells and hOCT2- MDCK cysts. However, inhibition of casein kinase II regulated only activity of hOCT2 expressed in MDCK cysts and not in HEK cells. Taken together, these results suggest that the 3D cell culture model is a suitable tool for the functional analysis of hOCT2 transport properties, depending on cell polarization.