Calcium and Phospholipases in Orexin Receptor Signaling

Abstract: The neuropeptides orexin-A and -B act as endogenous ligands for G-protein-coupled receptors (GPCRs) called OX1 and OX2 receptors. Previous observations have established that orexin receptors have an ability to couple to different G-proteins and signaling pathways and induce Ca2+ elevations via both receptor-operated Ca2+ channels (ROCs) and store-operated Ca2+ channels (SOCs). This thesis further elucidates the intracellular signaling mechanisms of orexin receptors.Orexin receptors were shown to activate ERK (extracellular signal-regulated kinase) via Ras, protein kinase C, phosphatidylinositol-3 kinase and Src. Ca2+ influx was shown to be obligatory for the activation of ERK and adenylyl cyclase, wherewith a hypothesis was formed that submembrane Ca2+ elevation is of central importance for the regulation of orexin receptors' coupling to different signaling pathways. This was further investigated with respect to OX1R-mediated activation of phospholipase C (PLC) showing that ROC influx was of more central importance for the OX1R signaling, but also SOCs amplified PLC activity. A technique to block OX1R-induced IP3 increase and subsequent Ca2+ release was devised, leaving ROC influx as the only source of Ca2+ elevation upon OX1R activation. This block had no effect on OX1R-mediated activation of ERK, showing that ROC-dependent influx is the most central Ca2+ elevating process in OX1R signaling. OX1Rs' 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 IP3. At even lower orexin-A concentrations, OX1Rs 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.