Year: 2011
Type: dissertation
Abstract: The orexin-1 receptor (OX1R) is a member of a superfamily of G-protein coupled receptors (GPCRs).Two hypothalamic neuropeptides, orexin-A/hypocretin-1 (Ox-A) and orexin-B/hypocretin-2, bind to and activate OX1R and a related GPCR, the orexin-2 receptor.The physiological effects of the orexin system include regulation of feeding, energy metabolism, endocrine and autonomic systems, and sleep/wake cycle.At the cellular level, OX1R stimulation leads to neuronal excitation, activation of the adenylyl cyclase pathway and several protein kinases, and an increase in the intracellular Ca 2+ concentration ([Ca 2+ ]i).OX1R couples to Gq/11-proteins and activates phospholipase C (PLC -pathway leading to release of Ca 2+ from the intracellular Ca 2+ stores and the subsequent capacitative Ca 2+ entry (CCE) aiming to refill depleted stores.This is the main pathway utilized when high nanomolar Ox-A concentrations are evaluated, but at lower, and probably physiologically more relevant, concentrations there was a predominance of receptor-operated Ca 2+ influx without any detectable release of stores.The aim of this study was to elucidate the poorly characterized signalling mechanisms used by the OX1R leading to and regulating Ca 2+ responses.The investigations with the patch clamp and Ca 2+ imaging techniques in recombinant cell models demonstrated that even a low nanomolar Ox-A concentration was able to evoke a highly voltage-dependent depolarization of the membrane, an increased ion current, and a robust elevation of [Ca 2+ ]i.It was found that Ca 2+ responses at low Ox-A concentrations were acutely dependent on voltage and extracellular Ca 2+ concentration as well as being sensitive to blockers of receptor-operated Ca 2+ entry.Protein kinase C (PKC) was found to be a crucial regulator of these responses.Based on the pharmacological profile of the responses and the experiments with dominant negative canonical transient receptor potential channel (TRPC) subtypes, the most probable ion channel involved in the Ca 2+ influx is the diacylglycerol-activated and PKC-regulated TRPC3.At higher Ox-A concentrations, Ca 2+ responses occurred also in the absence of extracellular Ca 2+ and were sensitive to inhibitors of store release and CCE, pointing to involvement of the PLC -pathway.In addition to TRPC3 channel, phospholipase A2 and protein kinase D1/3 were identified as novel targets of OX1R mediated activation and as prominent modulators of Ca 2+ oscillations induced by low nanomolar Ox-A concentrations.In conclusion, it was possible to confirm the present view of Ca 2+ signalling of OX1R and to identify novel players in the pathways activated by the receptor.It is hoped that these results will be useful in constructing a more comprehensive picture of OX1R signalling and in developing efficient therapies for OX1R -related disorders.
Author: Hanna Peltonen
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W3095178777