<p>Recently discovered neuropeptides orexins (orexin-A and -B) act as endogenous ligands for G-protein-coupled receptors called OX<sub>1</sub> and OX<sub>2</sub> receptors. Our previous studies have established model systems for investigation of the pharmacology and signaling of these receptors in recombinant CHO cells. OX<sub>1</sub> receptor-expressing CHO cells were mainly utilized in this thesis.</p><p>Orexin-A and -B activate both OX<sub>1</sub> and OX<sub>2</sub> receptors. However, orexin-B is less potent in activating OX<sub>1</sub> receptors than orexin-A, whereas the peptides are equipotent on OX<sub>2</sub> receptors. We have performed mutagenesis on orexin-A to investigate the basis for this selectivity. We show that OX<sub>2</sub> receptor is generally less affected by the mutations and thus OX<sub>2</sub><sup> </sup>receptor appears to have less strict requirements for ligand binding, likely explaining the lack of difference in affinity/potency between orexin-A and orexin-B on OX<sub>2</sub> receptor.</p><p>The other studies focus on orexin receptor signaling. OX<sub>1</sub> receptors are shown to regulate adenylyl cyclase both in positive and negative manner, activate different MAP-kinases (ERK1/2 and p38) and induce cell death after long-lasting stimulation. Adenylyl cyclase regulation occurs likely through three different G-protein families, Gi, Gs and Gq. For ERK1/2, several downstream pathways, such as Ras, Src, PI3-kinase and protein kinase C (PKC) are implicated. OX<sub>1</sub> receptor-mediated activation of ERK is suggested to be cytoprotective whereas p38 MAP-kinase induces programmed cell death. </p><p>Three particularly interesting findings were made. Firstly, novel PKC δ (delta) is suggested to regulate adenylyl cyclase, whereas conventional and atypical PKCs are involved in activation of ERK. Secondly, adenylyl cyclase and ERK activation is fully dependent on extracellular Ca<sup>2+</sup>. Further experiments suggest that the previously discovered receptor-operated Ca<sup>2+</sup> influx is not affecting the downstream effectors of orexin receptors but that it instead enables orexin receptors to couple to several signal cascades. Thirdly, upon inhibition of caspases, classical mediators of programmed cell death, OX<sub>1 </sub>receptor-mediated cell death is not reversed, but instead the pathways to death are altered so de novo gene transcription is no longer required for cell death.</p>
| Identifer | oai:union.ndltd.org:UPSALLA/oai:DiVA.org:uu-5890 |
| Date | January 2005 |
| Creators | Ammoun, Sylwia |
| 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 ; 56 |
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