| Abstract No.: | B-B2074 |
| Country: | Canada |
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| Title: | BIPHASIC EFFECT OF CHRONIC AND ACUTE DOPAMINE D2 AUTORECEPTOR ACTIVATION ON P70-S6 KINASE ACTIVITY IN HEK293 CELLS. |
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| Authors/Affiliations: | 1 Caroline Fasano*; 1 Dominic Thibeault; 1 Louis-Eric Trudeau;
1 Département de Pharmacologie, Faculté de Médecine, Université de Montréal, QC, Canada
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| Content: | OBJECTIVES: We recently demonstrated that chronic activation of the dopamine (DA) D2 autoreceptor with a specific agonist decreases synapse formation by mesencephalic DA neurons in primary culture. This phenomenon was dependent on the mTOR (mammalian Target Of Rapamycin) pathway involved in regulation of protein translation. This was accompanied by inhibition of the activity of p70-S6 kinase, a well-known mTOR target. The heterogeneity of cell populations in mesencephalic cultures complicates further analysis of the mechanisms linking D2 receptor activation to regulation of p70-S6 kinase activity. Moreover our observations were in relative contradiction with the work of Welsh and colleagues (1998) who demonstrated that an acute activation of DA D2 receptor increased p70-S6 kinase activity in CHO cells. The aim of the present study was thus to use a heterologous expression system to determine whether and how activity of p70-S6 kinase can be regulated by DA D2 receptor activation. METHODS: a GFP-tagged short isoform of DA D2 receptor (D2S) was expressed in HEK293 cells. After transfection, cells were treated chronically once per day during three consecutive days with drug or vehicle. For acute treatments, cells were treated for 30 min with drug or vehicle after the third day in culture in order to have the same culture conditions as for the chronically-treated group. Relative activity of p70-S6 kinase was evaluated by western blot by measuring the ratio of Phospho(Thr389)- p70-S6 kinase to total p70-S6 kinase. RESULTS: Chronic activation of D2S with quinpirole, a specific D2 receptor agonist, produced a potent 36 ± 13 % (n = 5) inhibition of p70-S6 kinase phosphorylation. Acute activation of D2S produced the opposite effect, increasing relative phosphorylation by 18 ± 8 % (n = 4). We next investigated the signalling pathways linking D2S activation to p70-S6 kinase. We first evaluated the involvement of protein kinase A, a well known effector of D2 receptors. Both chronic and acute inhibition of protein kinase A with H-89 increased phosphorylation of the p70-S6 kinase. We then investigated involvement of the AKT pathway which has been demonstrated to be strongly inhibited by DA D2 receptors in mice. Preliminary data showed that both chronic and acute stimulation of D2S with quinpirole had no effect on AKT phosphorylation. CONCLUSIONS: The DA D2 autoreceptor regulates p70-S6 kinase in a biphasic fashion, with acute activation causing an increase in activity, while chronic activation causing potent inhibition. These results suggest that the DA D2 receptor can act as a sensitive gating mechanism of this kinase. Inhibition of protein kinase A by the D2 receptor could potentially explain acute p70-S6 kinase activation, but the negative regulation induced by chronic D2 receptor activation appears to be mediated through a separate pathway that still remains to be determined. This study provides support for the hypothesis that the decrease in synapse formation in DA neurons induced by chronic activation of the D2 receptor is mediated through inhibition of the mTOR- p70-S6 kinase pathway. Work supported by the CIHR. |
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