<p>The neuropeptides orexin-A and -B act as endogenous ligands for G-protein-coupled receptors (GPCRs) called OX<sub>1</sub> and OX<sub>2</sub> receptors. Previous observations have established that orexin receptors have an ability to couple to different G-proteins and signaling pathways and induce Ca<sup>2+</sup> elevations via both receptor-operated Ca<sup>2+</sup> channels (ROCs) and store-operated Ca<sup>2+</sup> channels (SOCs). This thesis further elucidates the intracellular signaling mechanisms of orexin receptors.</p><p>Orexin receptors were shown to activate ERK (extracellular signal-regulated kinase) via Ras, protein kinase C, phosphatidylinositol-3 kinase and Src. Ca<sup>2+</sup> influx was shown to be obligatory for the activation of ERK and adenylyl cyclase, wherewith a hypothesis was formed that submembrane Ca<sup>2+</sup> elevation is of central importance for the regulation of orexin receptors' coupling to different signaling pathways. This was further investigated with respect to OX<sub>1</sub>R-mediated activation of phospholipase C (PLC) showing that ROC influx was of more central importance for the OX<sub>1</sub>R signaling, but also SOCs amplified PLC activity. A technique to block OX<sub>1</sub>R-induced IP<sub>3 </sub>increase and subsequent Ca<sup>2+</sup> release was devised, leaving ROC influx as the only source of Ca<sup>2+</sup> elevation upon OX<sub>1</sub>R activation. This block had no effect on OX<sub>1</sub>R-mediated activation of ERK, showing that ROC-dependent influx is the most central Ca<sup>2+</sup> elevating process in OX<sub>1</sub>R signaling. OX<sub>1</sub>Rs' coupling to PLC was further investigated by measuring the metabolites generated, inositol phosphates and diacylglycerol (DAG). The results indicate involvement of two different PLC activities with different substrate specificities, which results in, at low orexin-A concentrations, DAG production without concomitant production of IP<sub>3</sub>. At even lower orexin-A concentrations, OX<sub>1</sub>Rs generate DAG by activating phospholipase D. In conclusion, the results strengthen the hypothesis that ROCs have a central role in orexin receptor signaling and DAG may be the signal of preference.</p>
| Identifer | oai:union.ndltd.org:UPSALLA/oai:DiVA.org:uu-8613 |
| Date | January 2008 |
| Creators | Johansson, Lisa |
| Publisher | Uppsala University, Department of Neuroscience, Uppsala : Acta Universitatis Upsaliensis |
| Source Sets | DiVA Archive at Upsalla University |
| Language | English |
| Detected Language | English |
| Type | Doctoral thesis, comprehensive summary, text |
| Relation | Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, 1651-6206 ; 327 |
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