Edgar Schömig

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.

Pharmacokinetics of clopidogrel after administration of a high loading dose

The adenosine diphosphate (ADP) receptor P2Y12 blocking agent clopidogrel is clinically proven to be efficient in preventing thrombotic events. However, its therapeutic value is limited by an, as yet poorly explained, interindividual heterogeneity in platelet inhibition. To evaluate possible pharmacokinetic determinants of this response variability, we developed a sensitive and specific liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for quantification of unmodified inactive clopidogrel, its inactive carboxyl metabolite, and its active thiol metabolite in plasma. Analyte concentrations and platelet aggregation were assessed in ten healthy volunteers receiving an oral load of 600 mg clopidogrel. Subjects showed marked inter-individual differences in maximal platelet inhibition and in plasma pharmacokinetics. Univariate regression revealed linear correlations between maximal antiplatelet effect and peak plasma concentrations (cmax) of unchanged clopidogrel (r=0.76; p=0.01), of the carboxyl metabolite (r=0.70; p=0.03), and of the thiol metabolite (r=0.73; p=0.02), as well as linear correlations between cmax values of clopidogrel and its metabolites. This indicates that the response variability is predominantly caused by individual differences in clopidogrel absorption and that other factors, such as ADP receptor reactivity or differences in bioactivation of clopidogrel, do not play a major role.

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.

The Extraneuronal Transporter for Monoamine Transmitters Exists in Cells Derived from Human Central Nervous System Glia

From studies on sympathetically innervated peripheral tissues it is well known that both neuronal and nonneuronal transport systems contribute to the inactivation of released monoamine transmitters. The close proximity between synapses and glia cell processes in the CNS leads to the so far unresolved question whether non‐neuronal transporters are involved in the inactivation of centrally released monoamine transmitters such as noradrenaline, dopamine and 5‐hydroxytryptamine. 1‐Methyl‐4‐phenylpyridinium (MPP+) is a prototypical substrate of the extraneuronal monoamine transporter (uptake2). [3H]MPP+ was found to accumulate in various human glioma cell lines. [3H]MPP+transport was characterized in more detail in HTZ146 human glioma cells. The Ki values of various compounds for the inhibition of initial rates of [3H]MPP+ transport into HTZ146 cells were closely correlated with the known Ki values for the inhibition of the extraneuronal monoamine transporter (P < 0.001, r= 0.991, n= 7). The rank order of inhibitory potencies was decynium 22 > corticosterone > cyanine 863 > O‐methylisoprenaline > quinine > clonidine > quinidine. [3H]MPP+ accumulation was investigated not only in various CNS tumour cell lines but also in primary cultures of human astrocytes and rat cerebral cortex slices. In all tested experimental systems, accumulation was sensitive to cyanine‐related inhibitors of the extraneuronal monoamine transporter. These findings suggest that the extraneuronal monoamine transporter exists in glia cells. Furthermore, it was shown that MPP+ is able to make use of the extraneuronal monoamine transporter not only to enter but also to leave glia cells. This finding suggests that the extraneuronal monoamine transporter may play a key role in the mechanism of 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) neurotoxicity.

Neuronal sodium homoeostatis and axoplasmic amine concentration determine calcium-independent noradrenaline release in normoxic and ischemic rat heart.

Calcium-independent noradrenaline release was studied in the isolated perfused rat heart under conditions of normoxia, cyanide intoxication, and ischemia. The release of endogenous noradrenaline and dihydroxyphenylglycol were determined by high-performance liquid chromatography. The release of dihydroxyphenylglycol, the main neuronal noradrenaline metabolite, was used as an indicator of the free axoplasmic amine concentration. When storage function of neuronal vesicles was disturbed by Ro 4-1284 or trimethyltin, high dihydroxyphenylglycol release was observed without concomitant overflow of noradrenaline. If, however, these agents were combined with inhibition of Na+K+-ATPase or with veratridine-induced entry of sodium into the neuron, both dihydroxyphenylglycol and noradrenaline were released. Noradrenaline release was independent of extracellular calcium and was suppressed by blockade of neuronal catecholamine uptake (uptake1), indicating nonexocytotic noradrenaline liberation from the sympathetic nerve ending. This release critically depended on two conditions: 1) increased cytoplasmic concentrations of noradrenaline within the sympathetic neuron and 2) intraneuronal sodium accumulation. Both conditions together were required to induce noradrenaline efflux across the plasma membrane using the uptake1 carrier in reverse of its normal transport direction. A disturbed energy status of the sympathetic neuron, induced by cyanide intoxication or ischemia, likewise caused calcium-independent noradrenaline release by interfering with both vesicular storage function and neuronal sodium homoeostatis. Again, release was sensitive to uptake1 blockade. Since neuronal sodium accumulation was the rate-limiting step, release was further accelerated when residual Na+,K+-ATPase activity was inhibited. Na+-H+ exchange was identified as the predominant pathway of sodium entry into the sympathetic nerve ending in ischemia, and its inhibition by amiloride and ethylisopropylamiloride markedly suppressed ischemia-induced noradrenaline release.

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.

Differential regulation of distinct phenotypic features of serotonergic neurons by bone morphogenetic proteins.

Bone morphogenetic proteins (BMPs), growth and differentiation factor 5 (GDF5) and glial cell line‐derived neurotrophic factor (GDNF) are members of the transforming growth factor‐β superfamily that have been implicated in tissue growth and differentiation. Several BMPs are expressed in embryonic and adult brain. We show now that BMP‐2, –6 and –7 and GDF5 are expressed in the embryonic rat hindbrain raphe. To start to define roles for BMPs in the regulation of serotonergic (5‐HT) neuron development, we have generated serum‐free cultures of 5‐HT neurons isolated from the embryonic (E14) rat raphe. Addition of saturating concentrations (10 ng/mL) of BMP‐6 and GDF5 augmented numbers of tryptophan hydroxylase (TpOH) ‐immunoreactive neurons and cells specifically taking up 5,7‐dihydroxytryptamine (5,7‐DHT) by about two‐fold. Alterations in 5‐HT neuron numbers were due to the induction of serotonergic markers rather than increased survival, as shown by the efficacy of short‐term treatments. Importantly, BMP‐7 selectively induced 5,7‐DHT uptake without affecting TpOH immunoreactivity. BMP‐6 and –7 also promoted DNA synthesis and increased numbers of cells immunoreactive for vimentin and glial fibrillary acidic protein (GFAP). Pharmacological suppression of cell proliferation or glial development abolished the induction of serotonergic markers by BMP‐6 and –7, suggesting that BMPs act indirectly by stimulating synthesis or release of glial‐derived serotonergic differentiation factors. Receptor bodies for the neurotrophin receptor trkB, but not trkC, abolished the BMP‐mediated effects on serotonergic development, suggesting that the glia‐derived factor is probably brain‐derived neurotrophic factor (BDNF) or neurotrophin‐4. In support of this notion, we detected increased levels of BDNF mRNA in BMP‐treated cultures. Together, these data suggest both distinct and overlapping roles of several BMPs in regulating 5‐HT neuron development.