Rapid stimulation of presynaptic serotonin transport by A3 adenosine 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 A3 adenosine receptors (A3AR) stimulates SERT activity via both PKG and p38 MAPK (Zhu et al., 2004a). Whether A3ARs regulate SERT in the central nervous system (CNS) is unknown. Here we report that the A3AR agonist N6-(3-iodobenzyl)-N-methyl-5'carbamoyladenosine (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 A3AR antagonist 3-ethyl-5-benzyl-2-methyl-phenylethynyl-6-phenyl-1,4(±)dihydropyridine-3,5-dicarboxylate (MRS1191) and is absent from synaptosomes prepared from A3AR knockout mice. Kinetic analyses demonstrate that IB-MECA induces an increase of 5-HT transport Vmax with no significant change in Km. As in RBL-2H3 cells, IB-MECA stimulation of synaptosomal 5-HT uptake can be blocked by preincubation with PKG antagonists N-[2-(methylamino)ethy]-5-isoquinoline-sulfonamide (H8) and DT-2 (YGRKKRRQRRRPPLRK5H), as well as by the p38 MAPK inhibitor SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole]. Chronoamperometry studies in the anesthetized rat hippocampus support a role for A3ARs in SERT regulation in vivo. Together, these results identify a novel, region-specific action of CNS A3ARs in the modulation of SERT-mediated 5-HT transport that may be relevant for the etiology and/or therapy of 5-HT-linked brain disorders.