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

2021 ◽  
Vol 22 (17) ◽  
pp. 9658
Author(s):  
Tim N. Koepp ◽  
Alexander Tokaj ◽  
Pavel I. Nedvetsky ◽  
Ana Carolina Conchon Costa ◽  
Beatrice Snieder ◽  
...  
Keyword(s):  
Organic Cation ◽  
Basolateral Membrane ◽  
Three Dimensional ◽  
Organic Cation Transporter ◽  
Organic Cations ◽  
Membrane Domain ◽  
Cell Culture Model ◽  
Hek Cells ◽  
Culture Model ◽  
Organic Cation Transporter 2

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 (Km) of 3.6 ± 1.2 µM, similar to what was measured in the hOCT2-HEK cells (Km = 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 p56lck 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.

scholarly journals Regulation Mechanisms of Expression and Function of Organic Cation Transporter 1

2021 ◽  
Vol 11 ◽  
Author(s):  
Giuliano Ciarimboli
Keyword(s):  
Organic Cation ◽  
Basolateral Membrane ◽  
Organic Cation Transporter ◽  
Organic Cations ◽  
Regulatory Processes ◽  
Organic Cation Transporter 1 ◽  
Exogenous Origin ◽  
And Function

The organic cation transporter 1 (OCT1) belongs together with OCT2 and OCT3 to the solute carrier family 22 (SLC22). OCTs are involved in the movement of organic cations through the plasma membrane. In humans, OCT1 is mainly expressed in the sinusoidal membrane of hepatocytes, while in rodents, OCT1 is strongly represented also in the basolateral membrane of renal proximal tubule cells. Considering that organic cations of endogenous origin are important neurotransmitters and that those of exogenous origin are important drugs, these transporters have significant physiological and pharmacological implications. Because of the high expression of OCTs in excretory organs, their activity has the potential to significantly impact not only local but also systemic concentration of their substrates. Even though many aspects governing OCT function, interaction with substrates, and pharmacological role have been extensively investigated, less is known about regulation of OCTs. Possible mechanisms of regulation include genetic and epigenetic modifications, rapid regulation processes induced by kinases, regulation caused by protein–protein interaction, and long-term regulation induced by specific metabolic and pathological situations. In this mini-review, the known regulatory processes of OCT1 expression and function obtained from in vitro and in vivo studies are summarized. Further research should be addressed to integrate this knowledge to known aspects of OCT1 physiology and pharmacology.

scholarly journals O32 Ontogeny of human kidney OCT2 expression across the paediatric age range

2019 ◽  
Vol 104 (6) ◽  
pp. e14.2-e14
Author(s):  
N Smeets ◽  
B van Groen ◽  
J Pertijs ◽  
M Wilmer ◽  
B Smeets ◽  
...  
Keyword(s):  
Organic Cation ◽  
Basolateral Membrane ◽  
Age Groups ◽  
Kidney Tissue ◽  
Human Kidney ◽  
Organic Cation Transporter ◽  
Age Related ◽  
Paediatric Age ◽  
Organic Cation Transporter 2 ◽  
Related Pattern

BackgroundIn adults, the organic cation transporter 2 (protein name OCT2, gene name SLC22A2) is localised in the kidney proximal tubules where it mediates organic cation secretion. Hence, the transporter plays a role in the disposition and excretion of several drugs and drug-drug interactions. To better understand the disposition of OCT2 substrate drugs in children, we studied OCT2 localisation and expression in paediatric kidney tissue.MethodsThe expression of OCT2 was visualised in tissue using immunohistochemical staining. Tissues were derived post-mortem from children aged 0 -14 years. Gestational age varied between 24 and 40 weeks. Intensity of the staining at the basolateral membrane was scored by two individual observers using three categories; negative, detectible and high. Agreement between two observers was determined using Cohen’s kappa.Results44 kidney samples (n=17 neonates, n=17 infants, n=7 children, n=3 adolescent) were analysed and scored. There was substantial agreement between two judgements with a kappa of 0.773 (p< 0.005). No age related pattern was observed in the expression of OCT2. Even in the youngest age group, the expression of OCT2 was clearly visible.ConclusionThe kidney expression of OCT2 did not show an age-related pattern. In all age groups, expression levels were similar and OCT2 was properly localised at the basolateral membrane. These findings suggest that, with increasing age, OCT2 will not influence the renal excretion of its substrates.Disclosure(s)Nothing to disclose

scholarly journals The Role of NF-kB in the Downregulation of Organic Cation Transporter 2 Expression and Renal Cation Secretion in Kidney Disease

2022 ◽  
Vol 8 ◽  
Author(s):  
Chao Han ◽  
Juan Zheng ◽  
Fengyi Wang ◽  
Qingyang Lu ◽  
Qingfa Chen ◽  
...  
Keyword(s):  
Kidney Disease ◽  
Organic Cation ◽  
Rat Kidney ◽  
Basolateral Membrane ◽  
Kidney Injury ◽  
Organic Cation Transporter ◽  
Tubular Secretion ◽  
Gene Reporter Assay ◽  
Organic Cation Transporter 2

Organic cation transporter 2 (OCT2), encoded by the SLC22A2 gene, is the main cation transporter on the basolateral membrane of proximal tubular cells. OCT2 facilitates the entry step of the vectorial transport of most cations from the peritubular space into the urine. OCT2 downregulation in kidney disease models is apparent, yet not clear from a mechanistic vantage point. The aim of this study was to explore the role of inflammation, a common thread in kidney disease, and NF-kB in OCT2 modulation and tubular secretion. Among the OCTs, OCT2 was found consistently downregulated in the kidney of rats with chronic kidney disease (CKD) or acute kidney injury (AKI) and in patients diagnosed with CKD, and it was associated with the upregulation of TNFα renal expression. Exposure to TNFα reduced the expression and function of OCT2 in primary renal proximal tubule epithelial cells (RPTEC). Silencing or pharmacological inhibition of NF-kB rescued the expression of OCT2 in the presence of TNFα, indicating that OCT2 repression was NF-kB-dependent. In silico prediction coupled to gene reporter assay demonstrated the presence of at least one functional NF-kB cis-element upstream the transcription starting site of the SLC22A2 gene. Acute inflammation triggered by lipopolysaccharide injection induced TNFα expression and the downregulation of OCT2 in rat kidney. The inflammation did reduce the active secretion of the cation Rhodamine 123, with no impairment of the glomerular filtration. In conclusion, the NF-kB pathway plays a major role in the transcriptional regulation of OCT2 and, in turn, in the overall renal secretory capacity.

Charge-to-substrate ratio during organic cation uptake by rat OCT2 is voltage dependent and altered by exchange of glutamate 448 with glutamine

2009 ◽  
Vol 296 (4) ◽  
pp. F709-F722 ◽  
Author(s):  
Bernhard M. Schmitt ◽  
Dmitry Gorbunov ◽  
Peter Schlachtbauer ◽  
Brigitte Egenberger ◽  
Valentin Gorboulev ◽  
...  
Keyword(s):  
Organic Cation ◽  
Three Dimensional ◽  
Organic Cation Transporter ◽  
Tubular Damage ◽  
Xenopus Laevis Oocytes ◽  
Lactose Permease ◽  
Cation Uptake ◽  
Ion Substitution ◽  
Charge Excess ◽  
Organic Cation Transporter 2

Uptake of substrate and electric charge was measured simultaneously in voltage-clamped Xenopus laevis oocytes expressing rat organic cation transporter 2 (rOCT2). At 0 mV, saturating substrate concentrations induced uptake of more positive elementary charges than monovalent organic cations, with charge-to-substrate ratios of 1.5 for guanidinium+, 3.5 for tetraethylammonium+, and 4.0 for 1-methyl-4-phenylpyridinium+. At negative holding potentials, the charge-to-substrate ratios decreased toward unity. At 0 mV, charge-to-substrate ratios higher than unity were observed at different extracellular pH and after replacement of extracellular Na+, K+, Ca2+, Mg2+, and/or Cl−. Charge-to-substrate ratios were not influenced by intracellular succinate2− or glutarate2−. The effects of membrane potential and ion substitution strongly suggest that the surplus of transported positive charge is not generated by passive ion permeabilities. Rather, we hypothetize that small cations are taken up together with organic cation substrates whereas the outward reorientation of the empty transporter is electroneutral. Nonselective cotransport of small cations was supported by the three-dimensional structures of rOCT2 in its inward-facing and outward-facing conformations, which we determined by homology modeling based on known corresponding structures of H+-lactose permease of E. coli, and by functional analysis of OCT mutants. In our model, the innermost cavity of the outward-open binding cleft is negatively charged by Glu448 and Asp475, whereas the inward-open innermost cavity is electroneutral, containing Asp379, Asp475, Lys215, and Arg440. Substitution of Glu448 by glutamine reduced the charge-to-TEA+ ratio at 0 mV to unity. The observed charge excess associated with organic cation uptake into depolarized cells may contribute to tubular damage in renal failure.

scholarly journals Reduced Hepatic Uptake and Intestinal Excretion of Organic Cations in Mice with a Targeted Disruption of the Organic Cation Transporter 1 (Oct1 [Slc22a1]) Gene

2001 ◽  
Vol 21 (16) ◽  
pp. 5471-5477 ◽  
Author(s):  
Johan W. Jonker ◽  
Els Wagenaar ◽  
Carla A. A. M. Mol ◽  
Marije Buitelaar ◽  
Hermann Koepsell ◽  
...  
Keyword(s):  
Organic Cation ◽  
Basolateral Membrane ◽  
Organic Cation Transporter ◽  
Hepatic Uptake ◽  
Organic Cations ◽  
Wild Type ◽  
Targeted Disruption ◽  
Intestinal Excretion ◽  
Organic Cation Transporter 1

ABSTRACT The polyspecific organic cation transporter 1 (OCT1 [SLC22A1]) mediates facilitated transport of small (hydrophilic) organic cations. OCT1 is localized at the basolateral membrane of epithelial cells in the liver, kidney, and intestine and could therefore be involved in the elimination of endogenous amines and xenobiotics via these organs. To investigate the pharmacologic and physiologic role of this transport protein, we generated Oct1 knockout (Oct1 −/−) mice.Oct1 −/− mice appeared to be viable, healthy, and fertile and displayed no obvious phenotypic abnormalities. The role of Oct1 in the pharmacology of substrate drugs was studied by comparing the distribution and excretion of the model substrate tetraethylammonium (TEA) after intravenous administration to wild-type and Oct1 −/− mice. InOct1 −/− mice, accumulation of TEA in liver was four to sixfold lower than in wild-type mice, whereas direct intestinal excretion of TEA was reduced about twofold. Excretion of TEA into urine over 1 h was 53% of the dose in wild-type mice, compared to 80% in knockout mice, probably because inOct1 −/− mice less TEA accumulates in the liver and thus more is available for rapid excretion by the kidney. In addition, we found that absence of Oct1 leads to decreased liver accumulation of the anticancer drug metaiodobenzylguanidine and the neurotoxin 1-methyl-4-phenylpyridium. In conclusion, our data show that Oct1 plays an important role in the uptake of organic cations into the liver and in their direct excretion into the lumen of the small intestine.

Characterization of regulatory mechanisms and states of human organic cation transporter 2

2006 ◽  
Vol 290 (6) ◽  
pp. C1521-C1531 ◽  
Author(s):  
Jürgen Biermann ◽  
Detlef Lang ◽  
Valentin Gorboulev ◽  
Hermann Koepsell ◽  
Aleksandra Sindic ◽  
...  
Keyword(s):  
Organic Cation ◽  
Organic Cation Transporter ◽  
Binding Pocket ◽  
Organic Cations ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Organic Cation Transporter 2 ◽  
Interaction Domains ◽  
Specific Uptake

Polyspecific organic cation transporters (OCTs) have a large substrate binding pocket with different interaction domains. To determine whether OCT regulation is substrate specific, suitable fluorescent organic cations were selected by comparing their uptake in wild-type (WT) human embryonic kidney (HEK)-293 cells and in HEK-293 cells stably transfected with hOCT2. N-amidino-3,5-diamino-6-chloropyrazine-carboxamide (amiloride) and 4-[4-(dimethylamino)-styryl]- N-methylpyridinium (ASP) showed concentration-dependent uptake in hOCT2 at 37°C. After subtraction of unspecific uptake determined in WT at 37°C or in hOCT2 at 8°C saturable specific uptake of both substrates was measured. Km values of hOCT2-mediated uptake of 95 μM amiloride and 24 μM ASP were calculated. Inhibition of amiloride and ASP uptake by several organic cations was also measured [IC50 (in μM) for amiloride and ASP, respectively, tetraethylammonium (TEA) 98 and 30, cimetidine 14 and 26, and tetrapentylammonium (TPA) 7 and 2]. Amiloride and ASP uptake were significantly reduced by inhibition of Ca2+/CaM complex (−55 ± 5%, n = 10 and −63 ± 2%, n = 15, for amiloride and ASP, respectively) and stimulation of PKC (−54 ± 5%, n = 14, and −31 ± 6%, n = 26) and PKA (−16 ± 5%, n = 16, and −18 ± 4%, n = 40), and they were increased by inhibition of phosphatidylinositol 3-kinase (+28 ± 6%, n = 8, and +55 ± 17%, n = 16). Inhibition of Ca2+/CaM complex resulted in a significant decrease of Vmax (160–99 photons/s) that can be explained in part by a reduction of the membrane-associated hOCT2 (−22 ± 6%, n = 9) as determined using FACScan flow cytometry. The data indicate that saturable transport by hOCT2 can be measured by the fluorescent substrates amiloride and ASP and that transport activity for both substrates is regulated similarly. Inhibition of the Ca2+/CaM complex causes changes in transport capacity via hOCT2 trafficking.

scholarly journals Molecular and cellular physiology of organic cation transporter 2

2019 ◽  
Vol 317 (6) ◽  
pp. F1669-F1679 ◽  
Author(s):  
Stephen H. Wright
Keyword(s):  
Organic Cation ◽  
Critical Role ◽  
Organic Cation Transporter ◽  
Initial Step ◽  
Organic Cations ◽  
Molecular Characteristics ◽  
Ligand Interaction ◽  
Organic Cation Transporter 2 ◽  
Influence Of Substrate ◽  
Inhibitory Potential

Organic cation transporters play a critical role in mediating the distribution of cationic pharmaceuticals. Indeed, organic cation transporter (OCT)2 is the initial step in the renal secretion of organic cations and consequently plays a defining role in establishing the pharmacokinetics of many cationic drugs. Although a hallmark of OCTs is their broad selectivity, this characteristic also makes them targets for unwanted, adverse drug-drug interactions (DDIs), making them a focus for efforts to develop models of ligand interaction that could predict and preempt these adverse interactions. This review discusses the molecular characteristics of these transporters as well as the evidence that established the OCTs as key players in the distribution of organic cations. However, the primary focus is the present understanding of the complexity of ligand interaction with OCTs, particularly OCT2, including evidence for the presence of multiple ligand-binding sites and the influence of substrate structure on the affinity of the transporter for inhibitory ligands. This leads to a discussion of the complexities associated with the development of protocols for assessing the inhibitory potential of new molecular entities to perpetrate unwanted DDIs, the criteria that should be considered in the interpretation of the results of such protocols, and the challenges associated with development of models capable of predicting unwanted DDIs.

Sign in / Sign up

Export Citation Format

Share Document