Presynaptic, plasma membrane serotonin (5-hydroxytryptamine; 5-HT) transporters (SERTs) clear 5-HT following vesicular release and are regulated through trafficking-dependent pathways. Recently, we provided evidence for a trafficking-independent mode of SERT regulation downstream of adenosine receptor (AR) activation that is sensitive to p38 MAPK inhibitors. Here, we probe this pathway in greater detail, demonstrating elevation of 5-HT transport by multiple p38 MAPK activators (anisomycin, H(2)O(2), and UV radiation), in parallel with p38 MAPK phosphorylation, as well as suppression of anisomycin stimulation by p38 MAPK siRNA treatments. Studies with transporter-transfected Chinese hamster ovary cells reveal that SERT stimulation is shared with the human norepinephrine transporter but not the human dopamine transporter. Saturation kinetic analyses of anisomycin-SERT activity reveal a selective reduction in 5-HT K(m) supported by a commensurate increase in 5-HT potency (K(i)) for displacing surface antagonist binding. Anisomycin treatments that stimulate SERT activity do not elevate surface SERT surface density whereas stimulation is lost with preexposure of cells to the surface-SERT inactivating reagent, 2-(trimethylammonium)ethyl methane thiosulfonate. Guanylyl cyclase (1H-(1,2,4)-oxadiazolo[4,3-a]-quinoxalin-1-one) and protein kinase G inhibitors (H8, DT-2) block AR stimulation of SERT yet fail to antagonize SERT stimulation by anisomycin. We thus place p38 MAPK activation downstream of protein kinase G in a SERT-catalytic regulatory pathway, distinct from events controlling SERT surface density. In contrast, the activity of protein phosphatase 2A inhibitors (fostriecin and calyculin A) to attenuate anisomycin stimulation of 5-HT transport suggests that protein phosphatase 2A is a critical component of the pathway responsible for p38 MAPK up-regulation of SERT catalytic activity.
"More important for the control of anxiety and arousal responses, A 3 ARs are physically associated with SERT, and the activation of these receptors rapidly and transiently increases this association and the surface trafficking of SERT (Zhu et al. 2011). The activation of A 3 ARs has been shown to increase serotonin uptake in an cGMP-and p38 mitogen-activated protein kinase (MAPK)-dependent way (Okada et al. 1997, 1999; Zhu et al. 2007), a response that has also been observed in stable expression systems, including RBL2H3 cells (Miller and Hoffman 1994; Zhu et al. 2004), CHO cells (Zhu et al. 2005), and the immortalized serotonergic cell line RN46A (Chang et al. 2012). "
[Show abstract][Hide abstract] ABSTRACT: Rationale The adenosine A3 receptor and the nitric oxide (NO) pathway regulate the function and localization of serotonin transporters (SERTs). These transporters regulate extracellular serotonin levels, which are correlated with defensive behavior. Objective The purpose of this study was to understand the role of the A3AR on anxiety and arousal models in zebrafish, and whether this role is mediated by the nitrergic modulation of serotonin uptake. Methods The effects of IB-MECA (0.01 and 0.1 mg/kg) were assessed in a series of behavioral tasks in adult zebrafish, as well as on extracellular serotonin levels in vivo and serotonin uptake in brain homogenates. Finally, the interaction between IB-MECA and drugs blocking voltage-dependent calcium channels (VDCCs), NO synthase, and SERT was analyzed. Results At the lowest dose, IB-MECA decreased bottom dwelling and scototaxis, while at the highest dose, it also decreased shoaling, startle probability, and melanophore responses. These effects were accompanied by an increase in brain extracellular serotonin levels. IB-MECA also concentration-dependently increased serotonin uptake in vitro. The effects of IB-MECA on extracellular 5-HT, scototaxis, and geotaxis were blocked by l-NAME, while only the effects on 5-HT and scototaxis were blocked by verapamil. In vitro, the increase in 5-HT uptake was dependent on VDCCs and NO. Finally, fluoxetine blocked the effect of IB-MECA on scototaxis, but not geotaxis. Conclusion These results suggest that the effect of IB-MECA on scototaxis are mediated by a VDCC-NO-SERT pathway. While NO seems to mediate the effects of IB-MECA on geotaxis, neither VDCCs nor SERT seems to be involved in this process.
"Deletion of p38a Produces Stress Resilience that controls SERT protects against the depressive-like effects of stress. Although regulation of SERT by p38 had been implicated based on in vitro studies (Zhu et al., 2005; Samuvel et al., 2005), the demonstration that stress-induced p38a MAPK causes translocation of SERT to the plasma membrane in brain provides a clear molecular explanation for stressinduced dysphoria. The data presented here show that in serotonin neurons, p38a MAPK acts to directly influence SERT trafficking and ultimately to increase the rate of serotonin reuptake . "
[Show abstract][Hide abstract] ABSTRACT: Maladaptive responses to stress adversely affect human behavior, yet the signaling mechanisms underlying stress-responsive behaviors remain poorly understood. Using a conditional gene knockout approach, the α isoform of p38 mitogen-activated protein kinase (MAPK) was selectively inactivated by AAV1-Cre-recombinase infection in specific brain regions or by promoter-driven excision of p38α MAPK in serotonergic neurons (by Slc6a4-Cre or ePet1-Cre) or astrocytes (by Gfap-CreERT2). Social defeat stress produced social avoidance (a model of depression-like behaviors) and reinstatement of cocaine preference (a measure of addiction risk) in wild-type mice, but not in mice having p38α MAPK selectively deleted in serotonin-producing neurons of the dorsal raphe nucleus. Stress-induced activation of p38α MAPK translocated the serotonin transporter to the plasma membrane and increased the rate of transmitter uptake at serotonergic nerve terminals. These findings suggest that stress initiates a cascade of molecular and cellular events in which p38α MAPK induces a hyposerotonergic state underlying depression-like and drug-seeking behaviors.
"Multiple signaling pathways seem to contribute to the regulation of SERT-mediated 5-HT clearance (Blakely et al., 2005). With respect to SERT stimulation, G-protein coupled receptor (GPCR) stimulation can activate protein kinase GI (PKGI)-linked pathways that rapidly up-regulate SERT activity via increased SERT surface expression (Steiner et al., 2008) and via a p38 mitogen-activated protein kinase (MAPK)-linked pathway that induces a catalytic activation of SERT (Zhu et al., 2004, 2005). This latter pathway can be independently activated through stimulation of proinflammatory cytokine receptors (Blakely et al., 2005; Zhu et al., 2006, 2007). "
[Show abstract][Hide abstract] ABSTRACT: Activation of A₃ adenosine receptors (A₃ARs) rapidly enhances the activity of antidepressant-sensitive serotonin (5-HT) transporters (SERTs) in vitro, ex vivo, and in vivo. A₃AR agonist stimulation of SERT activity is lost in A₃AR knockout mice. A₃AR-stimulated SERT activity is mediated by protein kinase G1 (PKGI)- and p38 mitogen-activated protein kinase (MAPK)-linked pathways that support, respectively, enhanced SERT surface expression and catalytic activation. The mechanisms by which A₃ARs target SERTs among other potential effectors is unknown. Here we present evidence that A₃ARs are coexpressed with SERT in midbrain serotonergic neurons and form a physical complex in A₃AR/hSERT cotransfected cells. Treatment of A₃AR/SERT-cotransfected Chinese hamster ovary cells with the A₃AR agonist N⁶-(3-iodobenzyl)-N-methyl-5'-carbamoyladenosine (1 μM, 10 min), conditions previously reported to increase SERT surface expression and 5-HT uptake activity, enhanced the abundance of A₃AR/SERT complexes in a PKGI-dependent manner. Cotransfection of SERT with L90V-A₃AR, a hyperfunctional coding variant identified in subjects with autism spectrum disorder, resulted in a prolonged recovery of receptor/transporter complexes after A₃AR activation. Because PKGI and nitric-oxide synthetase are required for A₃AR stimulation of SERT activity, and proteins PKGI and NOS both form complexes with SERT, our findings suggest a mechanism by which signaling pathways coordinating A₃AR signaling to SERT can be spatially restricted and regulated, as well as compromised by neuropsychiatric disorders.
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