β-Phenylethylamine requires the dopamine transporter to increase extracellular dopamine in Caenorhabditis elegans dopaminergic neurons

https://doi.org/10.1016/j.neuint.2013.10.010Get rights and content

Highlights

  • βPEA increases the extracellular concentrations of DA in differentiated C. elegans neurons.

  • The dopamine transporter (DAT) blocker RTI-55 partly blocks the βPEA-induced increase of extracellular DA.

  • The vesicular monoamine transporter is not required to generate the βPEA-induced increase of extracellular DA.

  • βPEA generates DA efflux in a subset of dopaminergic neurons βPEA does not generate DA efflux in DAT knockout neurons.

Abstract

β-Phenylethylamine (βPEA) is an endogenous amine that has been shown to increase the synaptic levels of dopamine (DA). A number of in vitro and behavioral studies suggest the dopamine transporter (DAT) plays a role in the effects generated by βPEA, however the mechanism through which βPEA affects DAT has not yet been elucidated. Here, we used Caenorhabditis (C.) elegans DAT (DAT-1) expressing LLC-pk1 cells and neuronal cultures to investigate whether the βPEA-induced increase of extracellular DA required DAT-1. Our data show that βPEA increases extracellular dopamine both in DAT-1 transfected cells and cultures of differentiated neurons. RTI-55, a cocaine homologue and DAT inhibitor, completely blocked the βPEA-induced effect in transfected cells. However in neuronal cultures, RTI-55 only partly inhibited the increase of extracellular DA generated by βPEA. These results suggest that βPEA requires DAT-1 and other, not yet identified proteins, to increase extracellular DA when tested in a native system. Furthermore, our results suggest that βPEA-induced increase of extracellular DA does not require functional monoamine vesicles as genetic ablation of the C. elegans homologue vesicular monoamine transporter, cat-1, did not compromise the ability of βPEA to increase extracellular DA. Finally, our electrophysiology data show that βPEA caused fast-rising and self-inactivating amperometric currents in a subset of wild-type DA neurons but not in neurons isolated from dat-1 knockout animals. Taken together, these data demonstrate that in both DA neurons and heterogeneous cultures of differentiated C. elegans neurons, βPEA releases cytoplasmic DA through DAT-1 to ultimately increase the extracellular concentration of DA.

Introduction

β-Phenylethylamine (βPEA) is an endogenous trace amine present in the central nervous system, however, its role in the mammalian physiology is still unknown. Previous studies demonstrated that βPEA is synthesized in neurons that also contain tyrosine hydroxylase and coexists with dopamine (DA) in the nigrostriatal brain regions (Juorio et al., 1991). In striatal tissue, βPEA synthesis occurs with a rate similar to that of DA, but since it is more efficiently metabolized by monoamine oxidase enzymes, striatal βPEA concentrations are about three orders of magnitude lower than DA levels (Paterson et al., 1990). These data suggest that it is crucial the DA neurons keep low concentrations of βPEA to guarantee a proper physiological activity. In fact, changes in urinary βPEA levels have been documented in various human disorders including schizophrenia, attention deficit hyperactive disorder (ADHD) and depression (Baker et al., 1991, O’Reilly and Davis, 1994, Sandler et al., 1980). A direct interaction between dopaminergic neurons and βPEA has also been demonstrated in in vivo and in vitro experiments showing that βPEA induces DA release (Bailey et al., 1987, Ishida et al., 2005, Kuroki et al., 1990, Nakamura et al., 1998, Sotnikova et al., 2004, Yamada et al., 1998), and inhibits DA uptake (Liang and Rutledge, 1982, Raiteri et al., 1976). Moreover, in vivo studies showed that physiological βPEA concentrations directly and transiently inhibit the firing rate of the DA neurons through the activation of the DA D2 autoreceptors (Ishida et al., 2005, Mercuri et al., 1997, Rodriguez and Barroso, 1995). Interestingly, the firing inhibition caused by βPEA as well as βPEA-induced behaviors (Barroso and Rodriguez, 1996) were not affected by pretreatment with the vesicular monoamine transporter (VMAT) blocker reserpine. These data suggested that βPEA stimulates the release of DA from a non-vesicular cytoplasmic pool.

In this study, we investigated the effect of βPEA on extracellular levels of DA in Caenorhabditis elegans cultured neurons. We found that in isolated DA neurons, βPEA requires DAT to induce transient DA efflux. Furthermore, our data suggest that βPEA-induced DA efflux utilizes cytosolic DA since genetic ablation of VMAT did not affect the increase of extracellular DA induced by βPEA.

Section snippets

C. elegans husbandry and transgenic animals

Wild-type animals (N2) and knockout animals for DAT-1 (dat-1(ok157)III) and the C. elegans VMAT homologue CAT-1 (cat-1(ok411) were obtained from the Caenorhabditis Genetic Center (University of Minnesota, Minneapolis, MN, USA). All C. elegans strains were grown on bacteria lawns of NA22 and maintained at 22–24 °C using standard methods (Brenner, 1974). For amperometry recordings, we used the BY250 strain (gift from Dr. Blakely, Vanderbilt University) expressing cytosolic GFP under the control of

β-Phenylethylamine increases extracellular levels of DA in DAT-1 transfected cells and C. elegans cultured embryonic cells

Previous studies showed that βPEA induces DA release and/or inhibits DA uptake. We investigated whether βPEA increased extracellular DA in cultures of LLC-pk1 cells expressing C. elegans DAT (DAT-1). After preloading with 20 nM [3H]DA, cells were treated with 100 μM βPEA for 1 min. As shown in Fig. 1, βPEA caused a statistically significant increase (120 ± 20%) of extracellular [3H]DA with respect to control treated samples (one-way ANOVA test; p = 0.0001), whereas no change in extracellular [3H]DA

Conclusion

In the brain, the amount of extracellular DA is controlled by a tight balance between DA release and uptake events. In vitro and in vivo studies have showed that βPEA enhances the extracellular levels of DA by inducing DA release and/or preventing DA uptake through mammalian DAT. Here we found that in C. elegans neuronal cultures, the βPEA-induced increase of extracellular DA was partly inhibited by the DAT blocker RTI-55, whereas RTI-55 did not have any effect when applied alone (Fig. 2).

Conflict of interest

The authors declare no conflict of interest.

Acknowledgment

The authors thank the support from NIH Grant R21 DA024797 and the NIH funded COBRE P20 GM103329.

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    Present address: Department of Biomedical Sciences, University of Creighton, 2500 California Plaza, Omaha, NE 68178, United States.

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