Active Pharmaceutical Ingredient Uptake by Zebrafish (Danio rerio) Oct2 (slc22a2) Transporter Expressed in Xenopus laevis Oocytes.

Uptake of active pharmaceutical ingredients (APIs) across the gill epithelium of fish is via either a passive or facilitated transport process, with the latter being more important at the lower concentrations more readily observed in the environment. The solute carrier (SLC) 22A family, which includes the organic cation transporter OCT2 (SLC22A2), has been shown in mammals to transport several endogenous chemicals and APIs. Zebrafish oct2 was expressed in Xenopus oocytes and the uptake of ranitidine, propranolol, and tetraethylammonium characterized. Uptake of ranitidine and propranolol was time- and concentration-dependent with a km and Vmax for ranitidine of 246 µM and 45 pmol/(oocyte × min) and for propranolol of 409 µM and 190 pmol/(oocyte × min), respectively. Uptake of tetraethylammonium (TEA) was inhibited by propranolol, amantadine, and cimetidine, known to be human OCT2 substrates, but not quinidine or ranitidine. At external media pH 7 and 8 propranolol uptake was 100-fold greater than at pH 6; pH did not affect ranitidine or TEA uptake. It is likely that cation uptake is driven by the electrochemical gradient across the oocyte. Uptake kinetics parameters, such as those derived in the present study, coupled with knowledge of transporter localization and abundance and API metabolism, can help derive pharmacokinetic models. Environ Toxicol Chem 2022;41:2993-2998. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Involvement of Human Multidrug and Toxic Compound Extrusion (MATE) Transporters in Testosterone Transport.

Multidrug and toxic compound extrusion (MATE) transporters are primarily expressed in the kidneys and liver, where they contribute to the excretion of organic cations. Our previous study suggested that pig MATE2 (class III) participates in testosterone secretion from Leydig cells. In humans, it is unclear which MATE class is involved in testosterone transport. In this study, we aimed to clarify whether human MATE1 (hMATE1) or human MATE2K (hMATE2K) mediates testosterone transport. To confirm that testosterone inhibits transporter-mediated tetraethylammonium (TEA) uptake, a cis-inhibition assay was performed using cells that stably expressed hMATE1 or hMATE2K. Docking simulations were performed to characterize differences in the binding of hMATE1 and hMATE2K to testosterone. Transport experiments in LLC-PK1 cells that stably expressed hMATE1 were used to test whether hMATE1 mediates testosterone transport. We detected differences between the amino acid sequences of the substrate-binding sites of hMATE1 and hMATE2K that could potentially be involved in testosterone binding. Testosterone and estradiol inhibited TEA uptake mediated by hMATE1 but not that mediated by hMATE2K. Transport experiments in LLC-PK1 cells indicated that testosterone might be transported via hMATE1. This study suggested that hMATE1, but not hMATE2K, is involved in human testosterone transport.

Carbon nanoparticles enhance potassium uptake via upregulating potassium channel expression and imitating biological ion channels in BY-2 cells.

BACKGROUND: Carbon nanoparticles (CNPs) have been reported to boost plant growth, while the mechanism that CNPs enhanced potassium uptake for plant growth has not been reported so far. RESULTS: In this study, the function that CNPs promoted potassium uptake in BY-2 cells was established and the potassium accumulated in cells had a significant correlation with the fresh biomass of BY-2 cells. The K+ accumulation in cells increased with the increasing concentration of CNPs. The K+ influx reached high level after treatment with CNPs and was significantly higher than that of the control group and the negative group treated with K+ channels blocker, tetraethylammonium chloride (TEA+). The K+ accumulation was not reduced in the presence of CNPs inhibitors. In the presence of potassium channel blocker TEA+ or CNPs inhibitors, the NKT1 gene expression was changed compared with the control group. The CNPs were found to preferentially transport K+ than other cations determined by rectification of ion current assay (RIC) in a conical nanocapillary. CONCLUSIONS: These results indicated that CNPs upregulated potassium gene expression to enhance K+ accumulation in BY-2 cells. Moreover, it was speculated that the CNPs simulated protein of ion channels via bulk of carboxyl for K+ permeating. These findings will provide support for improving plant growth by carbon nanoparticles.

Purification and reconstitution of polyspecific H+/organic cation antiporter human MATE1.

Human MATE1 (multidrug and toxin extrusion 1, hMATE1) is a H+/organic cation (OC) exchanger responsible for the final step of toxic organic cation excretion in the kidney and liver. To investigate the mechanism of transport, we have established an in vitro assay procedure that includes its expression in insect cells, solubilization with octyl glucoside, purification, and reconstitution into liposomes. The resultant proteoliposomes containing hMATE1 as the sole protein component took up radiolabeled tetraethylammonium (TEA) in a ∆pH-dependent and electroneutral fashion. Furthermore, lipid-detergent micelle containing hMATE1 showed ∆pH-dependent TEA binding similar to transport. Mutated hMATE1 with replacement E273Q completely lacked these TEA binding and transport. In the case of divalent substrates, transport was electrogenic. These observations indicate that the stoichiometry of OC/H+ exchange is independent of substrate charge. Purification and reconstitution of hMATE1 is considered to be suitable for understanding the detailed molecular mechanisms of hMATE1. The results suggest that Glu273 of hMATE1 plays essential roles in substrate binding and transport.

Bacterial production and reconstitution in proteoliposomes of Solanum lycopersicum CAT2: a transporter of basic amino acids and organic cations.

KEY MESSAGE: The vacuolar SlCAT2 was cloned, over-produced in E. coli and reconstituted in proteoliposomes. Arg, Ornithine and Lys were identified as substrates. Unexpectedly, also the organic cations Tetraethylammonium and Acetylcholine were transported indicating involvement of SlCAT2 in signaling. In land plants several transporters are involved in ion and metabolite flux across membranes of cells or intracellular organelles. The vacuolar amino acid transporter CAT2 from Solanum lycopersicum was investigated in this work. SlCAT2 was cloned from tomato flower cDNA, over-produced in Escherichia coli and purified by Nichel-chelating chromatography. For functional studies, the transporter was reconstituted in proteoliposomes. Competence of SlCAT2 for Arg transport was demonstrated measuring uptake of [3H]Arg in proteoliposomes which was trans-stimulated by internal Arg or ornithine. Uptake of [3H]Ornithine and [3H]Lys was also detected at lower efficiency with respect to [3H]Arg. Transport was activated by the presence of intraliposomal ATP suggesting regulation by the nucleotide. The prototype for organic cations tetraethylammonium (TEA) was also transported by SlCAT2. However, scarce reciprocal inhibition between TEA and Arg was found, while the biguanide metformin was able to strongly inhibit uptake of both substrates. These findings suggest that amino acids and organic cations may interact with the transporter through different functional groups some of which are common for the two types of substrates. Interestingly, reconstituted SlCAT2 showed competence for acetylcholine transport, which was also inhibited by metformin. Kinetics of Arg and Ach transport were performed from which Km values of 0.29 and 0.79 mM were derived, respectively.

Possible Role of Organic Cation Transporters in the Distribution of [11C]Sulpiride, a Dopamine D2 Receptor Antagonist.

We synthesized [11C]sulpiride as a positron emission tomography probe for investigating the drug distribution in the human body. [11C]Sulpiride was injected to healthy male subjects in either tracer dose of [11C]sulpiride (approximately 222 MBq) or with therapeutic dose of sulpiride (500 mg, peroral) 3 h before the injection in a crossover fashion. Whole-body positron emission tomography imaging demonstrated that [11C]sulpiride accumulated exceedingly in the bladder, followed by liver, gall bladder, and kidney, respectively, at 30 min after the injection, whereas scarcely in the brain. Oral dose of sulpiride decreased the hepatic accumulation of the radioactivity by 60%. From in vitro experiments, we found that sulpiride is a substrate of hOCT1 (Km 2.6 µM), hOCT2 (Km 68 µM), hMATE1 (Km 40 µM), and hMATE2-K (Km 60 µM). Moreover, the uptake of sulpiride by human hepatocytes was diminished by tetraethylammonium, and saturable with Km of 18 µM. Oct1/2 double knockout mice and wild-type mice received Mate1 inhibitors (pyrimethamine/cimetidine) manifested reduced renal clearance of sulpiride, accompanied with its accumulation in the plasma. In conclusion, we found that sulpiride is a substrate of OCT1, OCT2, MATE1, and MATE2-K, and this suggests that [11C]sulpiride would be a useful radioligand to investigate the organic cation transporters in humans.

Involvement of Organic Cation Transporters in the Kinetics of Trimethylamine N-oxide.

Recent studies suggest that trimethylamine N-oxide (TMAO) is associated with the development of chronic kidney disease and heart failure. In this study, we investigated the importance of organic cation transporters (OCTs) in the clearance and tissue distribution of TMAO. The low-affinity and high-capacity transport of TMAO by mouse and human OCT1 and OCT2 was observed. Uptake and efflux of TMAO by the mouse hepatocytes as well as TMAO uptake into mouse kidney slices were significantly decreased by the addition of tetraethylammonium or Oct1/2 double knockout (dKO). Plasma concentrations of endogenous TMAO and TMAO-d9 given by intravenous infusion was 2-fold higher in Oct1/2 dKO than in wild-type mice due to significant decrease in its renal clearance. These results indicate that OCTs have a crucial role in the kinetics of TMAO in mice. In human, however, the OCT2-mediated tubular secretion in the urinary excretion of TMAO was insignificant because the renal clearance of TMAO was similar to that of creatinine in both young and elderly subjects, suggesting the species difference in the urinary excretion mechanisms of TMAO between mouse and human.

Protein Kinase C-Independent Inhibition of Organic Cation Transporter 1 Activity by the Bisindolylmaleimide Ro 31-8220.

Ro 31-8220 is a potent protein kinase C (PKC) inhibitor belonging to the chemical class of bisindolylmaleimides (BIMs). Various PKC-independent effects of Ro 31-8220 have however been demonstrated, including inhibition of the ATP-binding cassette drug transporter breast cancer resistance protein. In the present study, we reported that the BIM also blocks activity of the solute carrier organic cation transporter (OCT) 1, involved in uptake of marketed drugs in the liver, in a PKC-independent manner. Ro 31-8220, in contrast to other pan-PKC inhibitors such as staurosporine and chelerythrine, was thus shown to cis-inhibit uptake of the reference OCT1 substrate tetraethylammonium in OCT1-transfected HEK293 cells in a concentration-dependent manner (IC50 = 0.18 µM) and without altering membrane expression of OCT1. This blockage of OCT1 was also observed in human hepatic HepaRG cells that constitutionally express OCT1. It likely occurred through a mixed mechanism of inhibition. Ro 31-8220 additionally trans-inhibited TEA uptake in OCT1-transfected HEK293 cells, which likely discards a transport of Ro 31-8220 by OCT1. Besides Ro 31-8220, 7 additional BIMs, including the PKC inhibitor LY 333531, inhibited OCT1 activity, whereas 4 other BIMs were without effect. In silico analysis of structure-activity relationships next revealed that various molecular descriptors, especially 3D-WHIM descriptors related to total size, correspond to key physico-chemical parameters for inhibition of OCT1 activity by BIMs. In addition to activity of OCT1, Ro 31-8220 inhibited those of other organic cation transporters such as multidrug and toxin extrusion protein (MATE) 1 and MATE2-K, whereas, by contrast, it stimulated that of OCT2. Taken together, these data extend the nature of cellular off-targets of the BIM Ro 31-8220 to OCT1 and other organic cation transporters, which has likely to be kept in mind when using Ro 31-8220 and other BIMs as PKC inhibitors in experimental or clinical studies.

Memristors: Memory elements in potato tubers.

A memristor is a nonlinear element because its current-voltage characteristic is similar to that of a Lissajous pattern for nonlinear systems. This element was postulated recently and researchers are looking for it in different biosystems. We investigated electrical circuitry of red Irish potato tubers (Solanum tuberosum L.). The goal was to discover if potato tubers might have a new electrical component - a resistor with memory. The analysis was based on a cyclic current-voltage characteristic where the resistor with memory should manifest itself. We found that the electrostimulation by bipolar sinusoidal or triangle periodic waves induces electrical responses in the potato tubers with fingerprints of memristors. Tetraethylammonium chloride, an inhibitor of voltage gated K(+) channels, transforms a memristor to a resistor in potato tubers. Our results demonstrate that a voltage gated K(+) channel in the excitable tissue of potato tubers has properties of a memristor. Uncoupler carbonylcyanide-4-trifluoromethoxy-phenyl hydrazone decreases the amplitude of electrical responses at low and high frequencies of bipolar periodic sinusoidal or triangle electrostimulating waves. The discovery of memristors in plants creates a new direction in the understanding of electrical phenomena in plants.

Organic cation transporter function in different in vitro models of human lung epithelium.

Organic cation transporters (OCT) encoded by members of the solute carrier (SLC) 22 family of genes are involved in the disposition of physiological substrates and xenobiotics, including drugs used in the treatment of chronic obstructive lung diseases and asthma. The aim of this work was to identify continuously growing epithelial cell lines that closely mimic the organic cation transport of freshly isolated human alveolar type I-like epithelial cells (ATI) in primary culture, and which consequently, can be utilised as in vitro models for the study of organic cation transport at the air-blood barrier. OCT activity was investigated by measuring [(14)C]-tetraethylammonium (TEA) uptake into monolayers of Calu-3, NCI-H441 and A549 lung epithelial cell lines in comparison to ATI-like cell monolayers in primary culture. Levels of time-dependent TEA uptake were highest in A549 and ATI-like cells. In A549 cells, TEA uptake had a saturable and a non-saturable component with Km=528.5±373.1µM, Vmax=0.3±0.1nmol/min/mg protein and Kd=0.02µl/min/mg protein. TEA uptake into Calu-3 and NCI-H441 cells did not reach saturation within the concentration range studied. RNAi experiments in A549 cells confirmed that TEA uptake was mainly facilitated by OCT1 and OCT2. Co-incubation studies using pharmacological OCT modulators suggested that organic cation uptake pathways share several similarities between ATI-like primary cells and the NCI-H441 cell line, whereas more pronounced differences exist between primary cells and the A549 and Calu-3 cell lines.

Mechanisms involved in the vasorelaxant effects produced by the acute application of amfepramone in vitro to rat aortic rings

Amfepramone (diethylpropion) is an appetite-suppressant drug used for the treatment of overweight and obesity. It has been suggested that the systemic and central activity of amfepramone produces cardiovascular effects such as transient ischemic attacks and primary pulmonary hypertension. However, it is not known whether amfepramone produces immediate vascular effects when applied in vitro to rat aortic rings and, if so, what mechanisms may be involved. We analyzed the effect of amfepramone on phenylephrine-precontracted rat aortic rings with or without endothelium and the influence of inhibitors or blockers on this effect. Amfepramone produced a concentration-dependent vasorelaxation in phenylephrine-precontracted rat aortic rings that was not affected by the vehicle, atropine, 4-AP, glibenclamide, indomethacin, clotrimazole, or cycloheximide. The vasorelaxant effect of amfepramone was significantly attenuated by NG-nitro-L-arginine methyl ester (L-NAME) and tetraethylammonium (TEA), and was blocked by removal of the vascular endothelium. These results suggest that amfepramone had a direct vasorelaxant effect on phenylephrine-precontracted rat aortic rings, and that inhibition of endothelial nitric oxide synthase and the opening of Ca2+-activated K+ channels were involved in this effect.

Ionic Selectivity and Permeation Properties of Human PIEZO1 Channels.

Members of the eukaryotic PIEZO family (the human orthologs are noted hPIEZO1 and hPIEZO2) form cation-selective mechanically-gated channels. We characterized the selectivity of human PIEZO1 (hPIEZO1) for alkali ions: K+, Na+, Cs+ and Li+; organic cations: TMA and TEA, and divalents: Ba2+, Ca2+, Mg2+ and Mn2+. All monovalent ions permeated the channel. At a membrane potential of -100 mV, Cs+, Na+ and K+ had chord conductances in the range of 35-55 pS with the exception of Li+, which had a significantly lower conductance of ~ 23 pS. The divalents decreased the single-channel permeability of K+, presumably because the divalents permeated slowly and occupied the open channel for a significant fraction of the time. In cell-attached mode, 90 mM extracellular divalents had a conductance for inward currents carried by the divalents of: 25 pS for Ba2+ and 15 pS for Ca2+ at -80 mV and 10 pS for Mg2+ at -50 mV. The organic cations, TMA and TEA, permeated slowly and attenuated K+ currents much like the divalents. As expected, the channel K+ conductance increased with K+ concentration saturating at ~ 45 pS and the KD of K+ for the channel was 32 mM. Pure divalent ion currents were of lower amplitude than those with alkali ions and the channel opening rate was lower in the presence of divalents than in the presence of monovalents. Exposing cells to the actin disrupting reagent cytochalasin D increased the frequency of openings in cell-attached patches probably by reducing mechanoprotection.

Mechanisms involved in the vasorelaxant effects produced by the acute application of amfepramone in vitro to rat aortic rings.

Amfepramone (diethylpropion) is an appetite-suppressant drug used for the treatment of overweight and obesity. It has been suggested that the systemic and central activity of amfepramone produces cardiovascular effects such as transient ischemic attacks and primary pulmonary hypertension. However, it is not known whether amfepramone produces immediate vascular effects when applied in vitro to rat aortic rings and, if so, what mechanisms may be involved. We analyzed the effect of amfepramone on phenylephrine-precontracted rat aortic rings with or without endothelium and the influence of inhibitors or blockers on this effect. Amfepramone produced a concentration-dependent vasorelaxation in phenylephrine-precontracted rat aortic rings that was not affected by the vehicle, atropine, 4-AP, glibenclamide, indomethacin, clotrimazole, or cycloheximide. The vasorelaxant effect of amfepramone was significantly attenuated by NG-nitro-L-arginine methyl ester (L-NAME) and tetraethylammonium (TEA), and was blocked by removal of the vascular endothelium. These results suggest that amfepramone had a direct vasorelaxant effect on phenylephrine-precontracted rat aortic rings, and that inhibition of endothelial nitric oxide synthase and the opening of Ca2+-activated K+ channels were involved in this effect.

Unstirred Water Layers and the Kinetics of Organic Cation Transport.

PURPOSE: Unstirred water layers (UWLs) present an unavoidable complication to the measurement of transport kinetics in cultured cells, and the high rates of transport achieved by overexpressing heterologous transporters exacerbate the UWL effect. This study examined the correlation between measured Jmax and Kt values and the effect of manipulating UWL thickness or transport Jmax on the accuracy of experimentally determined kinetics of the multidrug transporters, OCT2 and MATE1. METHODS: Transport of TEA and MPP was measured in CHO cells that stably expressed human OCT2 or MATE1. UWL thickness was manipulated by vigorous reciprocal shaking. Several methods were used to manipulate maximal transport rates. RESULTS: Vigorous stirring stimulated uptake of OCT2-mediated transport by decreasing apparent Kt (Ktapp) values. Systematic reduction in transport rates was correlated with reduction in Ktapp values. The slope of these relationships indicated a 1500 µm UWL in multiwell plates. Reducing the influence of UWLs (by decreasing either their thickness or the Jmax of substrate transport) reduced Ktapp by 2-fold to >10-fold. CONCLUSIONS: Failure to take into account the presence of UWLs in experiments using cultured cells to measure transport kinetics can result in significant underestimates of the apparent affinity of multidrug transporters for substrates.

Plasmodium falciparum chloroquine resistance transporter is a H+-coupled polyspecific nutrient and drug exporter.

Extrusion of chloroquine (CQ) from digestive vacuoles through the Plasmodium falciparum CQ resistance transporter (PfCRT) is essential to establish CQ resistance of the malaria parasite. However, the physiological relevance of PfCRT and how CQ-resistant PfCRT gains the ability to transport CQ remain unknown. We prepared proteoliposomes containing purified CQ-sensitive and CQ-resistant PfCRTs and measured their transport activities. All PfCRTs tested actively took up tetraethylammonium, verapamil, CQ, basic amino acids, polypeptides, and polyamines at the expense of an electrochemical proton gradient. CQ-resistant PfCRT exhibited decreased affinity for CQ, resulting in increased CQ uptake. Furthermore, CQ competitively inhibited amino acid transport. Thus, PfCRT is a H(+)-coupled polyspecific nutrient and drug exporter.

Functional and molecular effects of mercury compounds on the human OCTN1 cation transporter: C50 and C136 are the targets for potent inhibition.

The effect of mercury compounds has been tested on the organic cation transporter, hOCTN1. MeHg(+), Hg(2+), or Cd(2+) caused strong inhibition of transport. 1,4-Dithioerythritol (DTE), cysteine (Cys), and N-acetyl-l-cysteine reversed (NAC) the inhibition at different extents. 2-Aminoethyl methanethiosulfonate hydrobromide (MTSEA), a prototype SH reagent, exerted inhibition of transport similar to that observed for the mercurial agents. To investigate the mechanism of action of mercurials, mutants of hOCTN1 in which each of the Cys residues was substituted by Ala have been constructed, over-expressed in Escherichia coli, and purified. Tetraethylammonium chloride (TEA) uptake mediated by each mutant in proteoliposomes was comparable to that of wild type (WT). IC50 values of the WT and mutants for the mercury compounds were derived from dose-response analyses. The mutants C50A and C136A showed significant increase of IC50 indicating that the 2 Cys residues were involved in the interaction with the mercury compounds and inhibition of the transporter. The double mutant C50A/C136A was constructed; the lack of inhibition confirmed that the 2 Cys residues are the targets of mercury compounds. MTSEA showed similar behavior with respect to the mercurial reagents with the difference that increased IC50 was observed also in the C81A mutant. Similar results were obtained when transport was measured as acetylcholine uptake. Ethyl mercury (Thimerosal) inhibited hOCTN1 as well. C50A, C50A/C136A and, at very lower extent, C136A showed increased IC50 indicating that C50 was the major target of this mercury compound. The homology model of hOCTN1 was built using as template PiPT and validated by the experimental data on mutant proteins.

Components of foods inhibit a drug exporter, human multidrug and toxin extrusion transporter 1.

Human multidrug and toxic compounds extrusion transporter 1 (hMATE1/SLC47A1) is a H(+)-coupled organic cation exporter responsible for the final step of excretion of various xenobiotics at the kidney and liver. In this study, effects of dietary constituents on hMATE1 mediated drug transport were examined to evaluate possible food-drug interactions. Bergamottin inhibited hMATE1 mediated tetraethyl ammonium transport activity, with a Ki of 98.7 µM. Coumarins, flavonols, and catechin inhibited hMATE1 activity. Among 23 compounds tested, isorhamnetin was the strongest inhibitor of hMATE1 with the Ki of 0.32 µM in a competitive manner. Since isorhamnetin is abundant in Ginkgo biloba that is widely used for herbal supplements, the findings suggest the potential hMATE1 related food-drug interactions.

The mitochondrial fluorescent dye rhodamine 123 is a high-affinity substrate for organic cation transporters (OCTs) 1 and 2.

Rhodamine 123 is a fluorescent cationic dye commonly used as a mitochondrial probe and known or suspected to be transported by certain drug membrane transporters. The present study was designed to characterize the putative interactions of rhodamine 123 with human organic cation transporter (OCT) 1 and OCT2. Intracellular uptake of the dye was demonstrated to be enhanced in both hOCT1- and hOCT2-overexpressing HEK293 cells when compared with control HEK293 cells. This increase of rhodamine 123 influxes was found to be a saturable carrier-mediated process, with low K(m) values (K(m) = 0.54 µm and K(m) = 0.61 µm for transport of the dye in hOCT1- and hOCT2-positive HEK293 cells, respectively). Known inhibitors of hOCT1 and hOCT2 activities such as verapamil, amitriptyline, prazosin, and quinine were next demonstrated to decrease rhodamine 123 accumulation in hOCT1- and hOCT2-overexpressing HEK293 cells. In addition, the dye was found to inhibit hOCT1- and hOCT2-mediated uptake of tetraethylammonium (TEA), a model substrate for both hOCT1 and hOCT2; rhodamine 123 appeared nevertheless to be a more potent inhibitor of hOCT1-mediated TEA transport (IC50 = 0.37 µm) than of that mediated by hOCT2 (IC50 = 61.5 µm). Taken together, these data demonstrate that rhodamine 123 is a high-affinity substrate for both hOCT1 and hOCT2. This dye may be therefore useful for fluorimetrically investigating cellular hOCT1 or hOCT2 activity, knowing, however, that other factors potentially contributing to cellular accumulation of rhodamine 123, including mitochondrial membrane potential or expression of the efflux transporter P-glycoprotein, have also to be considered.

TREK-1 currents in smooth muscle cells from pregnant human myometrium.

The mechanisms governing maintenance of quiescence during pregnancy remain largely unknown. The current study characterizes a stretch-activated, tetraethylammonium-insensitive K(+) current in smooth muscle cells isolated from pregnant human myometrium. This study hypothesizes that these K(+) currents can be attributed to TREK-1 and that upregulation of this channel during pregnancy assists with the maintenance of a negative cell membrane potential, conceivably contributing to uterine quiescence until full term. The results of this study demonstrate that, in pregnant human myometrial cells, outward currents at 80 mV increased from 4.8 ± 1.5 to 19.4 ± 7.5 pA/pF and from 3.0 ± 0.8 to 11.8 ± 2.7 pA/pF with application of arachidonic acid (AA) and NaHCO3, respectively, causing intracellular acidification. Similarly, outward currents were inhibited following application of 10 µM fluphenazine by 51.2 ± 9.8% after activation by AA and by 73.9 ± 4.2% after activation by NaHCO3. In human embryonic kidney (HEK-293) cells stably expressing TREK-1, outward currents at 80 mV increased from 91.0 ± 23.8 to 247.5 ± 73.3 pA/pF and from 34.8 ± 8.9 to 218.6 ± 45.0 pA/pF with application of AA and NaHCO3, respectively. Correspondingly, outward currents were inhibited 89.5 ± 2.3% by 10 µM fluphenazine following activation by AA and by 91.6 ± 3.4% following activation by NaHCO3. Moreover, currents in human myometrial cells were activated by stretch and were reduced by transfection with small interfering RNA or extracellular acidification. Understanding gestational regulation of expression and gating of TREK-1 channels could be important in determining appropriate maintenance of uterine quiescence during pregnancy.

Multiple mechanisms of ligand interaction with the human organic cation transporter, OCT2.

OCT2 is the entry step for organic cation (OC) secretion by renal proximal tubules. Although many drugs inhibit OCT2 activity, neither the mechanistic basis of their inhibition nor their transport status is generally known. Using representatives of several structural classes of OCT2-inhibitory ligands described recently (Kido Y, Matsson P, Giacomini KM. J Med Chem 54: 4548-4558, 2011), we determined the kinetic basis of their inhibition of 1-methyl-4-phenylpyridinium (MPP) transport into Chinese hamster ovary cells that stably expressed hOCT2. The "cluster II" inhibitors (which contain known OCT2 substrates) metformin and cimetidine interacted competitively with MPP. However, other cluster II compounds, including tetraethylammonium (TEA), diphenidol and phenyltoloxamine, were mixed-type inhibitors of MPP transport (i.e., decreasing J(max) and increasing K(t)). A cluster III (neutral steroid) representative, adrenosterone, and a cluster I (large, flexible cation) representative, carvedilol, displayed noncompetitive inhibitory profiles. Competitive counterflow (CCF) was used to determine whether the inhibitory ligands served as substrates of hOCT2. Carvedilol (cluster I) and adrenosterone (cluster III) did not support CCF, consistent with the prediction that members of these structural classes are likely to be nontransported inhibitors of OCT2. The cluster II representatives MPP, metformin, cimetidine, and TEA all supported CCF, consistent with independent assessments of their OCT2-mediated transport. However, the other cluster II representatives, diphenidol and phenyltoloxamine, failed to support CCF, suggesting that neither compound is transported by OCT2. An independent assessment of diphenidol transport (using liquid chromatography with tandem mass spectroscopy) confirmed this observation. The results underscore the caution required for development of predictive models of ligand interaction with multidrug transporters.