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Regulation of OX1 orexin/hypocretin receptor-coupling to phospholipase C by Ca2+ influx
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Physiology. (Kukkonen)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Physiology. (Kukkonen)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Physiology. (Kukkonen)
2007 (English)In: British Journal of Pharmacology, ISSN 0007-1188, E-ISSN 1476-5381, Vol. 150, no 1, 97-104 p.Article in journal (Refereed) Published
Abstract [en]

Background and purpose: Orexin (OX) receptors induce Ca2+ elevations via both receptor-operated Ca2+ channels (ROCs) and the "conventional" phospholipase C (PLC)-Ca2+ release-store-operated Ca2+ channel (SOC) pathways. In this study we assessed the ability of these different Ca2+ influx pathways to amplify OX, receptor signalling to PLC in response to stimulation with the physiological ligand orexin-A.

Experimental approach: PLC activity was assessed in CHO cells stably expressing human OX1 receptors.

Key results: Inhibition of total Ca2+ influx by reduction of the extracellular [Ca2+] to 1 mu M effectively inhibited the receptor-stimulated PLC activity at low orexin-A concentrations (by 93% at 1 nM), and this effect was gradually reduced by higher orexin-A concentrations. A similar but weaker inhibitory effect (84% at 1 nM) was obtained on depolarization to similar to 0 mV, which disrupts most of the driving force for Ca2+ entry. The inhibitor of the OX, receptor-activated ROCs, tetraethylammonium chloride (TEA), was somewhat less effective than the reduction in extracellular [Ca2+] at inhibiting PLC activation, probably because it only partially blocks ROCs. The partial inhibitor of both ROCs and SOCs, Mg2+, and the SOC inhibitors, dextromethorphan, SKF-96365 (1-[beta-(3-(4-methoxyphenyl)propoxy)-4-methoxyphenethyl]-1H-imidazole HCl) and 2-APB (2-aminoethoxydiphenyl borate), inhibited PLC activity at low concentrations of orexin-A, but were not as effective as TEA.

Conclusions and implications: Both ROCs and SOCs markedly amplify the OX1 receptor-induced PLC response, but ROCS are more central for this response. These data indicate the crucial role of ROCs in orexin receptor signalling.

Place, publisher, year, edition, pages
2007. Vol. 150, no 1, 97-104 p.
Keyword [en]
Orexin, hypocretin, receptor, G-protein-coupled receptor, calcium, calcium influx, phospholipase C
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-96994DOI: 10.1038/sj.bjp.0706959ISI: 000243528000013PubMedID: 17115071OAI: oai:DiVA.org:uu-96994DiVA: diva2:171753
Available from: 2008-04-11 Created: 2008-04-11 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Calcium and Phospholipases in Orexin Receptor Signaling
Open this publication in new window or tab >>Calcium and Phospholipases in Orexin Receptor Signaling
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2008. 58 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 327
Keyword
Physiology, orexin, receptor, calcium, phospholipase, cell signaling, GPCR, Fysiologi
Identifiers
urn:nbn:se:uu:diva-8613 (URN)978-91-554-7151-4 (ISBN)
Public defence
2008-05-02, B22, BMC, Husargatan 3, Uppsala, 13:15
Opponent
Supervisors
Available from: 2008-04-11 Created: 2008-04-11 Last updated: 2013-09-20Bibliographically approved
2. OX1 Orexin Receptor Signalling to Phospholipases
Open this publication in new window or tab >>OX1 Orexin Receptor Signalling to Phospholipases
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The neuropeptides orexin-A and orexin-B were discovered in 1998 and were first described as regulators of feeding behaviour. Later research has shown that they have an important role in the regulation of sleep. Two G protein-coupled receptors, OX1 and OX2 orexin receptors, mediate the cellular responses to orexins. The overall aim of this thesis was to investigate the OX1 orexin receptors signalling to phospholipases.

Previous investigations have determined that orexin receptors induce Ca2+ elevations through both receptor-operated Ca2+ channels (ROCs) and store-operated Ca2+ channels (SOCs). In this thesis we investigated the importance of these influxpathways on orexin-mediated phospholipase (PLC) activation. The results demonstrate that ROC influx is enough to fully support orexin-stimulated PLC activation but that SOC influx has a further amplifying role. We also investigated the metabolites generated after PLC activation, inositolphosphates and diacylglycerol (DAG). The results indicate involvement of two different PLC activities with different substrate specificities one of them leading to DAG production without co-occurring IP3 production at low orexin receptor stimulation. The results also suggest that at even lower orexin receptor stimulation DAG is produced via the activation of phospholipase D.

In this thesis we also investigated if the ubiquitous phospholipase A2 (PLA2) signalling system is involved in orexin receptor signalling. The results demonstrate that stimulation of the OX1 orexin receptors leads to arachidonic acid (AA) release. This release is fully dependent on Ca2+ influx, probably through ROC, and at the same time the studies demonstrate that ROC influx is partly dependent on PLA2 activation. At low orexin receptor activation the AA release seemed to in part rely on extracellular signal-regulated kinase.

We also devised two methods to aid in these investigations. The first method enabled studies of the receptor-operated Ca2+ influx without interference of the co-occurring store-operated Ca2+ influx. This was done by the expression of IP3-metabolising enzymes IP3-3-kinase-A and IP3-5-phosphatase-I. The second method enables quantification of DAG and IP3 signalling in fixed cells using GFP-fused indicators, leading to a semi-quantitative but easily applicable pharmacological assay.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2010. 63 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 508
Keyword
orexin, phospholipase, Calcium, cell signalling, G protein coupled receptor, ERK, live-cell imaging, arachidonic acid, diacylglycerol
Identifiers
urn:nbn:se:uu:diva-111138 (URN)978-91-554-7686-1 (ISBN)
Public defence
2010-02-19, B21, Biomedicinskt centrum (BMC), Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2010-01-27 Created: 2009-12-03 Last updated: 2010-06-09Bibliographically approved

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Johansson, LisaEkholm, Marie E.Kukkonen, Jyrki P.

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