Dirk Gründemann

Molecular identification of the corticosterone-sensitive extraneuronal catecholamine transporter.

Catecholaminergic signaling regulates various physiological functions, such as blood pressure and is implicated in drug dependence, affective disorders and male aggressive behavior. The actions of released catecholamines are terminated by sodium-driven, high-affinity transporters in the plasma membrane of the releasing neurons and by a corticosterone-sensitive, low-affinity, high-capacity extraneuronal transport system, originally named uptake2, found in sympathetically innervated tissues and in central nervous system glia. Here we report the molecular identification and pharmacological characterization of the extraneuronal catecholamine transporter, which is unrelated to the family of sodium-driven neuronal monoamine transporters.

Transport of Monoamine Transmitters by the Organic Cation Transporter Type 2, OCT2

The recently cloned apical renal transport system for organic cations (OCT2) exists in dopamine-rich tissues such as kidney and some brain areas (Gründemann, D., Babin-Ebell, J., Martel, F., Örding, N., Schmidt, A., and Schömig, E. (1997)J. Biol. Chem. 272, 10408–10413). The study at hand was performed to answer the question of whether OCT2 accepts dopamine and other monoamine transmitters as substrate. 293 cells were stably transfected with the OCT2r cDNA resulting in the 293OCT2r cell line. Expression of OCT2r in 293 cells induces specific transport of tritiated dopamine, noradrenaline, adrenaline, and 5-hydroxytryptamine (5-HT). Initial rates of specific3H-dopamine, 3H-noradrenaline,3H-adrenaline, and 3H-5-HT transport were saturable, the K m values being 2.1, 4.4, 1.9, and 3.6 mmol/liter. The corresponding V max values were 3.9, 1.0, 0.59, and 2.5 nmol min−1·mg of protein−1, respectively. 1,1′-diisopropyl-2,4′-cyanine (disprocynium24), a known inhibitor of OCT2 with a potent eukaliuric diuretic activity, inhibited 3H-dopamine uptake into 293OCT2r cells with an K i of 5.1 (2.6, 9.9) nmol/liter. In situ hybridization reveals that, within the kidney, the OCT2r mRNA is restricted to the outer medulla and deep portions of the medullary rays indicating selective expression in the S3 segment of the proximal tubule. These findings open the possibility that OCT2r plays a role in renal dopamine handling.

Molecular cloning and characterization of two novel transport proteins from rat kidney

The recent cloning of renal transport systems for organic anions and cations (OAT1, OCT1, and OCT2) opened the possibility to search, via polymerase chain reaction (PCR) homology screening, for novel transport proteins. Two integral membrane proteins, UST1 and UST2, were cloned from rat kidney. RT‐PCR revealed that UST1 is confined to the kidney whereas UST2 mRNA was detected in all tested tissues. Sequence analyses suggest that UST1 and UST2, together with four related transporters, comprise, within the major facilitator superfamily, a so far unrecognized transporter family, termed amphiphilic solute facilitator (ASF) family. Characteristic signatures for the ASF family were identified.

Catecholamine transport by the organic cation transporter type 1 (OCT1)

1 Liver and kidney extract adrenaline and noradrenaline from the circulation by a mechanism which does not seem to be one of the classical catecholamine transporters. The hypothesis that OCT1 is involved–the organic cation transporter type 1 which exists in rat kidney and liver–was tested. 2 Based on human embryonic kidney cells (293), we constructed a cell line which stably expresses OCT1r (293OCT1r cells). Transfection with OCT1 resulted in a transport activity not only for prototypical known substrates of OCT1 such as 3H‐1‐methyl‐4‐phenylpyridinium and 14C‐tetraethylammonium but also for the catecholamines 3H‐adrenaline, 3H‐noradrenaline (3H‐NA) and 3H‐dopamine (3H‐DA), the indoleamine 3H‐5‐hydroxytryptamine (3H‐5HT) as well as the indirect sympathomimetic 14C‐tyramine. 3 For 3H‐DA, 3H‐5HT and 3H‐NA, at non‐saturating concentrations, the rate constants for inwardly directed substrate flux (kin) were 6.9±0.8, 3.1±0.2, and 1.2±0.1 μl min−1 mg protein−1. In wild type cells (293WT) the corresponding kins were considerably lower, being 0.94±0.40, 0.47±0.08 and 0.23±0.05 μl min−1 mg protein−1 (n=12). The indirectly determined half‐saturating concentrations of DA, 5HT, and NA were 1.1 (95% c.i.: 0.8, 1.4), 0.65 (0.49, 0.86), and 2.8 (2.1, 3.7) mmol l−1 (n=3). 4 Specific 3H‐DA uptake in 293OCT1r cells was resistant to cocaine (1 μmol l−1), 3H‐5HT uptake was resistant to citalopram (300 nmol l−1) and 3H‐NA uptake was resistant to desipramine (100 nmoll−1), corticosterone (1 μmol l−1), and reserpine (10 nmol l−1) which rules out the involvement of classical transporters for biogenic amines. 5 The findings demonstrate that OCT1 efficiently transports catecholamines and other biogenic amines and support the hypothesis that OCT1 is responsible for hepatic and renal inactivation of circulating catecholamines.

The Carnitine Transporter SLC22A5 Is Not a General Drug Transporter, but It Efficiently Translocates Mildronate

In addition to its function as carnitine transporter, novel organic cation transporter type 2 (OCTN2; human gene symbol SLC22A5) is widely recognized as a transporter of drugs. This notion is based on several reports of direct measurement of drug accumulation. However, a rigorous, comparative, and comprehensive analysis of transport efficiency of OCTN2 has not been available so far. In the present study, OCTN2 orthologs from human, rat, and chicken were expressed in 293 cells using an inducible expression system. Uptake of trans-4-[4-(dimethylamino)styryl]-1-methylpyridinium iodide (ASP+), cephaloridine, ergothioneine, gabapentin, mildronate, pyrilamine, quinidine, spironolactone, tetraethylammonium, verapamil, and vigabatrin was determined by liquid chromatography/mass spectrometry. For reference, uptake of carnitine was measured in parallel. Our results indicate that OCTN2-mediated uptake of drugs was not significantly different from zero or, with tetraethylammonium and ergothioneine, was minute relative to carnitine. The carnitine congener mildronate, by contrast, was transported very efficiently. Thus, OCTN2 is not a general drug transporter but a highly specific carrier for carnitine and closely related molecules. Transport parameters (cellular accumulation, transporter affinity, sodium dependence) were similar for mildronate and carnitine. Efficiency of transport of mildronate was even higher than that of carnitine. Hence, our results establish that OCTN2 is a key target of the cardioprotective agent mildronate because it controls, as integral protein of the plasma membrane, cellular entry of mildronate and enables efficient access to intracellular targets. The highest levels of human OCTN2 mRNA were detected by real-time reverse transcription-polymerase chain reaction in kidney, ileum, breast, small intestine, skeletal muscle, and ovary but also in some heart and central nervous system tissues.

Apical uptake of organic cations by human intestinal Caco-2 cells: putative involvement of ASF transporters

Abstract. The aim of this work was to characterise the intestinal absorption of organic cations, by testing the possibility of involvement of known members of the amphiphilic solute facilitator (ASF) family in this process. For that purpose, the characteristics of the uptake of 1-methyl-4-phenylpyridinium, a model organic cation, at the brush-border membrane of Caco-2 cells were compared with those of the extraneuronal monoamine transporter (EMT)-mediated transport.Uptake of [3H]MPP+ by Caco-2 and 293hEMT cells showed pH-dependence: it was significantly reduced (to 86% and 62% of control, respectively) when the pH of the extracellular medium was decreased to 6.2, and increased (to 116% and 136% of control, respectively) when the extracellular pH was increased to 8.2. Uptake of [3H]MPP+ by Caco-2 cells and 293hEMT cells showed potential-dependence: substitution of KCl for NaCl in the incubation medium resulted in a reduction in the inward transport of [3H]MPP+ (to 70% and 40% of control, respectively). Uptake of [3H]MPP+ by Caco-2 and 293hEMT cells showed only little dependence on Na+: substitution of NaCl of the incubation media with LiCl resulted in a small decrease (of 19% and 14%, respectively) in [3H]MPP+ uptake. However, when NaCl was substituted with choline chloride, a significant reduction in [3H]MPP+ uptake by Caco-2 and 293hEMT cells (of 56% and 68%, respectively) was observed. The effect of various compounds on initial rates of [3H]MPP+ uptake into Caco-2 and 293hEMT cells was tested. All compounds tested interacted with the specific [3H]MPP+ uptake in both cell lines. There was no correlation between the IC50s in relation to inhibition of [3H]MPP+ uptake into Caco-2 cells and into 293hEMT cells. Reverse transcriptase-polymerase chain reaction indicates that mRNA of hEMT and of the human organic cation transporter 1 (hOCT1) are present in Caco-2 cells.In conclusion, our results suggest that uptake of organic cations at the brush-border membrane of Caco-2 cells may occur through two distinct Na+-independent transporters belonging to the ASF family: hEMT and hOCT1.

Transport of small organic cations in the rat liver

The kidneys and the liver are the principal organs for the inactivation of circulating organic cations. Recently, an organic cation transporter (OCT1) has been cloned from rat kidney. In order to answer the question whether OCT1 is involved also in hepatic uptake of organic cations, the pharmacological characteristics of organic cation transport in hepatocytes were compared to the characteristics of transiently expressed OCT1.Primary cultures of rat hepatocytes avidly accumulated the small organic cation 3H-1-methyl-4-phenylpyridinium (3H-MPP+). At equilibrium, the hepatocytes accumulated 3H-MPP+ 56-fold. Initial rates of specific 3H-MPP+ transport in hepatocytes were saturable. The half-saturating concentration was 13 μmol/l. 3H-MPP+ transport was sensitive to quinine (Ki = 0.79 μmol/l) and cyanine863 (Ki = 0.097 µmol/l). Quinine and cyanine863 are known inhibitors of type I hepatic transport of cationic drugs and of renal excretion of organic cations, respectively. To compare the functional characteristics of 3H-MPP+ transport in hepatocytes with those of OCT1, OCT1 has been heterologously expressed and characterized in a mammalian cell line (293 cells). Initial rates of 3H-MPP+ transport were saturable, the Km being 13 μmol/l. The rank order of inhibitory potencies of various inhibitors was almost identical in hepatocytes and 293 cells transiently transfected with OCT1. There was a positive correlation between the Kis for the inhibition of 3H-MPP+ transport in isolated hepatocytes and transfected 293 cells (r = 0.85; P<0.01; n = 8).The results indicate that OCT1 is functionally expressed not only in the kidney but also in hepatocytes where it is responsible for the transport of small organic cations which, in the past, have been classified as type I substrates.

The Extraneuronal Monoamine Transporter Exists in Human Central Nervous System Glia

Publisher Summary Research shows that the extraneuronal monoamine transporter exists in cells that stem from human central nervous system (CNS) glia. The physiological role of the extraneuronal monoamine transporter in the CNS may be characterized by a second line of defense. It inactivates that fraction of monoamines that escapes neuronal re-uptake and thus prevents uncontrolled spreading of the signal. Also, from various studies on sympathetically innervated peripheral organs, it is well known that both the neuronal type of noradrenaline transporter (uptake1) and the extraneuronal monoamine transporter (uptake2) are important for the inactivation of released catecholamines. The extraneuronal monoamine transporter operates independently of the Na+ gradient. It is, at least partially, driven by the membrane potential and is sensitive to corticosterone. In the CNS, the close proximity between catecholaminergic neurons and glia cells raises the question whether the extraneuronal monoamine transporter contributes to the inactivation of centrally released catecholamines. There is strong evidence in support of the view that glia cells accumulate monoamines by a saturable transport system; the underlying mechanism, however, is still under research. Two developments have opened the possibility to readdress this question. These are: the isocyanines and pseudoisocyanines represent a novel class of selective and extremely potent inhibitors of the extraneuronal monoamine transporter, and the neurotoxin 1-methyl-4-phenylpyridinium (MPP+) turned out to be an excellent substrate of the extraneuronal monoamine transporter that is superior to noradrenaline for in vitro studies.

Contribution of allelic variations in transporters to the phenotype of drug response

Pharmacogenomics seeks to explain the variability in drug response. Neurotransmitter transporters from the SLCA6 family are direct or indirect targets for psychotropic drugs, and their genetic variations may directly influence response to antidepressant or antipsychotic drugs. Furthermore, drug transporters located in natural barriers, such as the blood brain barrier, may influence response to psychoactive substrates. In the 5′-upstream regulatory region of the neuronal serotonin transporter lays a 44-base pair insertion/deletion polymorphism resulting in a long and a short variant. Several studies have reported a better response to selective serotonin reuptake inhibitors in individuals carrying two long alleles, however, some studies report contradictory results. Moreover, several genetic variants are known in the human norepinephrine transporter gene, and though one study reports differences in antidepressant response due to the NET G1287A polymorphism, results should be replicated by others before conclusions can be drawn. Dopamine transporters play an important role in psychotropic drug response, and a variable number of tandem repeats polymorphism in the 3′-untranslated region of the dopamine transporter gene has been studied in regards to possible correlation with antipsychotic drug response but without showing an association. P-glycoprotein has been shown to influence drug concentrations in CNS but so far, the studies on genetic polymorphisms did not show effects on the phenotype of response. Thus, several studies have looked at the influence of genetic polymorphisms on psychotropic drug response gaining different results. Best evidence exists for the serotonin transporter polymorphism influencing the response to selective serotonin reuptake inhibitors but the effects are relatively small. So far, transporter genotypes are not yet eligible for individual prediction of drug response.

Uptake of mIBG and catecholamines in noradrenaline- and organic cation transporter-expressing cells: potential use of corticosterone for a preferred uptake in neuroblastoma- and pheochromocytoma cells

For imaging of neuroblastoma and phaeochromocytoma, [(123)I]meta-iodobenzylguanidine ([(123)I]mIBG) is routinely used, whereas [(18)F]6-fluorodopamine ([(18)F]6-FDA) is sporadically applied for positron emission tomography in pheochromocytoma. Both substances are taken up by catecholamine transporters (CATs). In competition, some other cell types are able to take up catecholamines and related compounds probably by organic cation (OCT) [extraneuronal monoamine (EMT)] transporters (OCT1, OCT2, OCT3=EMT). In this study, we investigated the uptake of radioiodine-labeled meta-iodobenzylguanidine (mIBG) as well as [(3)H]dopamine (mimicring 6-fluorodopamine) and [(3)H]noradrenaline. SK-N-SH (neuroblastoma) and PC-12 (phaeochromocytoma) cells were used and compared with HEK-293 cells transfected with OCT1, OCT2 and OCT3, respectively. In order to gain a more selective uptake in CAT expressing tumor cells, different specific inhibitors were measured. Uptake of mIBG into OCT-expressing cells was similar or even better as into both CAT-expressing cell lines, whereas dopamine and noradrenaline uptake was much lower in OCT-expressing cells. In presence of corticosterone (f.c. 10(-4) M], catecholamine and mIBG uptake into SK-N-SH and PC-12 cells was only slightly reduced. In contrast, this process was significantly inhibited in OCT2 and OCT3 transfected HEK-293 as well as in Caki-1 cells, which naturally express OCT3. We conclude that the well-known corticosteroid corticosterone might be used in combination with [(18)F]6-FDA or [(123)I]mIBG to improve specific imaging of neuroblastoma and pheochromocytoma and to reduce irradiation dose to nontarget organs in [(131)I]mIBG treatment.