Differential pharmacological in vitro properties of organic cation transporters and regional distribution in rat brain
Introduction
Monoaminergic neurotransmission pathways control major central physiological and behavioral functions, such as locomotor activity, attention, mood and affect. A crucial step in the tight regulation of monoaminergic pathways is the rapid removal of released neurotransmitters from the synaptic cleft, which is principally ensured by the high-affinity Na+- and Cl−-dependent transporters for dopamine (DA), serotonin (5-HT), or norepinephrine (NE). These high-affinity neuronal monoamine transporters are targets for psychoactive compounds, including addictive drugs and antidepressants (Bönisch and Brüss, 1994, Giros and Caron, 1993).
Recently, attention has been focused on another category of transporters, the organic cation transporters (OCTs), which is also potentially involved in the control of monoaminergic neurotransmission. OCTs belong to a family of polyspecific transporters, SLC22, endowed with detoxification functions in peripheral organs. Initiated in 1994 with the molecular cloning of rat OCT1, the isolation of OCTs from liver, kidney or placenta of several animal species allowed their pharmacological characterization in heterologous systems, providing evidence that these transporters accept as substrates a variety of positively charged endogenous substances and xenobiotics, ranging from hormone-like compounds to antiviral and antihistaminergic agents (Koepsell et al., 2003). These in vitro studies also showed that the three main OCT subtypes (OCT1, OCT2 and OCT3) transport DA, 5-HT, NE or histamine, albeit with much lower affinity than the neuronal monoamine transporters (Breidert et al., 1998, Busch et al., 1998, Gründemann et al., 1998a, Gründemann et al., 1998b, Gründemann et al., 1999, Wu et al., 1998). OCTs display the low-affinity kinetics and overall characteristics of “uptake2”, an extraneuronal catecholamine uptake system described much earlier in peripheral tissues with sympathetic innervation (Bönisch, 1980, Iversen, 1965, Trendelenburg, 1988). As uptake2, transport of the neurotoxin 1-methyl-4-phenyl pyridinium (MPP+) by all three OCTs was found to be Na+- and Cl−-independent and sensitive to nanomolar concentrations of cyanine dye derivatives. The OCT3 transporter, in particular, was shown to reflect most accurately all the properties of uptake2, including selective inhibition by corticosteroids and especially by O-methylated catecholamines (Busch et al., 1996, Gründemann et al., 1997, Gründemann et al., 1998a, Gründemann et al., 1998b, Hayer-Zillgen et al., 2002, Wu et al., 1998).
Although predominantly expressed in peripheral tissues, all three OCTs were also detected in the brain (Busch et al., 1998, Gründemann et al., 1997, Haag et al., 2004, Vialou et al., 2004, Wu et al., 1998), in agreement with evidence of a low-affinity transport system with properties similar to peripheral uptake2 (Hendley et al., 1970, Wilson et al., 1988). The relative importance and role of organic cation transporters in the central nervous system remain, however, unclear. Recently, part of the answer was brought forth by investigation of mice lacking OCT3, which showed alterations of the neural and behavioral responses to environmentally induced variations in osmolarity (Vialou et al., 2004).
Interestingly, OCTs were shown to interact in vitro with diverse neuroactive compounds, including psychostimulants and antiparkinson agents (Koepsell et al., 2003). In an earlier report (Wu et al., 1998), an inhibitory effect of d-amphetamine on the rat OCT3 subtype could be demonstrated. Using different in vitro model systems, two other studies showed uptake of the glutamatergic N-methyl d-aspartate (NMDA) receptor antagonists amantadine and memantine by OCT1 and OCT2 (Busch et al., 1998, Goralski et al., 2002).
No study beside that of Hayer-Zillgen et al. (2002) has hitherto simultaneously examined monoamine transport by all three OCTs of one species in a given expression system. Moreover, discrepancies in the properties between rat and human orthologs have been reported, for instance in transport of the model substrate tetraethylammonium by OCT3 (Gründemann et al., 1998a, Gründemann et al., 1999, Wu et al., 1998) and in sensitivity to β-estradiol of OCT1 (Hayer-Zillgen et al., 2002, Wu et al., 1998). These observations raised the question of the fundamental pharmacological properties of each of these OCT subtypes and their functional specialization in monoamine transport in the brain, as well as of their potentially differential implication in the response to psychoactive agents. In the present study, we evaluated human and rat OCT1, OCT2 and OCT3 in stably transfected HEK293 cells for monoamine transport efficacy and sensitivity toward several neuroactive compounds, including psychostimulants and NMDA receptor antagonists, and investigated by comparative in situ hybridization analysis their regional distribution in rat brain.
Section snippets
Cell culture and generation of stable transfectants
All cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum, 100 UI/ml penicillin and 100 μg/ml streptomycin (Invitrogen, Gaithersburg, MD) at 37 °C in a humidified atmosphere (5% CO2). The cells were subcultured every 5 days to maintain exponential growth and medium was replaced every 2–3 days. For generation of cells stably expressing rat OCTs, HEK293 (ATCC-CRL-1573) cells were transfected with constructs containing rat OCT cDNAs in a pCR3 vector
Differential distribution of organic cation transporters in rat brain
Investigation of the regional distribution of OCT transcripts by comparative in situ hybridization analysis revealed a distinct expression pattern for each subtype. In our experimental conditions, OCT3 transcripts were found to be the most easily detected of the three subtypes in rat brain. OCT3 mRNA was very highly expressed in the subfornical organ (Fig. 1a). Lower yet substantial labeling was observed in the dorsal raphe (Fig. 1b), throughout the thalamus, in particular in the anterodorsal
Discussion
Several transporters have been proposed to mediate low-affinity catecholamine transport in peripheral tissues and in brain, including OCTs and PMAT, a newly described transporter with affinity constants for DA and 5-HT falling in an intermediate range between high-affinity transporters and OCTs (Engel et al., 2004, Engel and Wang, 2005).
The present study provides novel information on the basis for this low-affinity transport in brain and underscores the importance in the rat of OCT2 and OCT3,
Acknowledgments
The authors wish to thank E Schömig and D Gründemann (University of Cologne, Germany) and V. Ganapathy (Medical College of Georgia, Augusta, USA) for the kind gift of rat OCT1 and OCT2, and OCT3 cDNAs, respectively, S. El Mestikawy (INSERM U513) and S. Dumas (Helios, Paris) for advice concerning in situ hybridization. A.A. and V.V. are recipients of fellowships from Institut de Recherches Servier and the French Ministry for Research, respectively. Financial support was provided by Institut
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