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