Rapid Stimulation of Presynaptic Serotonin Transport by A₃Adenosine Receptors

Abstract

The inactivation of synaptic serotonin (5-hydroxytryptamine, 5-HT) is largely established through the actions of the presynaptic, antidepressant-sensitive 5-HT transporter (SERT, SLC6A4). Recent studies have demonstrated post-translational regulation of SERT mediated by multiple Ser/Thr kinases, including protein kinases C and G (PKC and PKG) and p38 mitogen-activated protein kinase (MAPK), as well as the Ser/ Thr phosphatase PP2A. Less well studied are specific surface receptors that target these signaling pathways to control SERT surface expression and/or catalytic rates. Using rat basophilic leukemia 2H3 cell line (RBL-2H3), we previously established that activation of A 3 adenosine receptors (A 3 AR) stimulates SERT activity via both PKG and p38 MAPK (Zhu et al., 2004a). Whether A 3 ARs regulate SERT in the central nervous system (CNS) is unknown. Here we report that the A 3 AR agonist N 6 -(3-iodobenzyl)-N-methyl-5Јcarbamoyladenosine(IB-MECA) rapidly (10 min) and selectively stimulates 5-HT transport in mouse midbrain, hippocampal, and cortical synaptosomes. IB-MECA-induced stimulation of 5-HT uptake is blocked by the selective A 3 AR antagonist 3-ethyl-5-benzyl-2-methyl-phenylethynyl-6-phenyl-1,4(Ϯ)dihydropyridine-3,5-dicarboxylate (MRS1191) and is absent from synaptosomes prepared from Serotonin (5-hydroxytryptamine, 5-HT) signaling is critical to thermoregulation, appetite, sexual drive, and mood. Multiple mechanisms contribute to the presynaptic control of serotonergic signaling, including the synthesis, storage, release, and inactivation of 5-HT. 5-HT inactivation after release is accomplished via the antidepressant-sensitive 5-HT transporter (SERT), a presynaptic membrane protein. SERT is a member of the Na ϩ /Cl Ϫ -dependent solute transporter family (SLC6A4) (Blakely et al., 1991;Ramamoorthy et al., 1993) and is related most closely to dopamine and norepinephrine transporters. Targeted disruption of the murine SERT gene leads to disruption of presynaptic 5-HT homeostasis (Murphy et al., 2004) and is accompanied by anxiety-related behavioral changes (Jennings et al., 2006). Human SERT gene variants have been linked to mental disorders, including autism, depression, anxiety, and obses- Article, publication date, and citation information can be found at

Regulation of serotonin transporter activity by adenosine in intestinal epithelial cells

“…Recent results obtained in transfected cells and in mouse brain synaptosomes showed that adenosine yielded an increase in SERT activity; however, in this case, the effect was mediated by A 3 adenosine receptors [38,39]. These results are in contrast to ours; nevertheless, the tissue studied, the adenosine receptor and the signalling pathway involved were different.…”

Lipopolysaccharide-induced serotonin transporter up-regulation involves PKG-I and p38MAPK activation partially through A3 adenosine receptor

“…PKA, PKC and PKG activation have also been demonstrated to impose post-translational regulation on SERT by phosphorylation, possibly reflecting the heterologous context of SERT expression. Zhu et al have reported that involvement of A3AR in serotonin transporter trafficking and activation in RBL-2H3 cells and SERT stimulation by A3AR requires activation of PKG by a phospholipase C, Ca 2+ , NOS and cGMP-dependent mechanism (14,27). In the present study, we provided evidence that Ca 2+ -CaMK-II/iNOS/PKG-I signaling pathways contribute to up-regulation of SERT in RBL-2H3 cells by LPS.…”

“…Many studies have demonstrated RBL-2H3 cells expressing serotonin synthesis and transporter systems as an ideal model for elucidating the regulatory mechanisms relating to SERT (14)(15)(16). Consistent with previous reports, results obtained in our laboratory have shown that the fluorescent styryl dye ASP assay enables the detection of dynamic transport activities of SERT and is amenable for measuring 5-HT uptake (17)(18)(19)(20)(21).The results obtained from the present study showed that LPS treatment enhances 5-HT uptake in RBL-2H3 cells in a dosedependent manner.…”

Transgenic elimination of high-affinity antidepressant and cocaine sensitivity in the presynaptic serotonin transporter

“…Synaptosomes were prepared as described previously (52) with 5-HT transport activity assessed at 37°C as previously described (53). For competition uptake assays, samples containing 25 to 100 μg were incubated in duplicate with 20 nM Electrophysiologic Slice Preparation and Recordings.…”