uu.seUppsala University Publications
Change search
ReferencesLink to record
Permanent link

Direct link
Arachidonic acid release mediated by OX1 orexin receptors
University of Helsinki.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Physiology. (Kukkonen)
University of Helsinki.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Physiology. (Kukkonen)
2010 (English)In: British Journal of Pharmacology, ISSN 0007-1188, E-ISSN 1476-5381, Vol. 159, no 1, 212-221 p.Article in journal (Refereed) Published
Abstract [en]

Background and purpose: We have previously shown that lipid mediators, produced by phospholipase D and C, are generated in OX1 orexin receptor signalling with high potency, and presumably mediate some of the physiological responses to orexin. In this study, we investigated whether the ubiquitous phospholipase A2 (PLA2) signalling system is also involved in orexin receptor signalling.Experimental approach:  Recombinant Chinese hamster ovary-K1 cells, expressing human OX1 receptors, were used as a model system. Arachidonic acid (AA) release was measured from 3H-AA-labelled cells. Ca2+ signalling was assessed using single-cell imaging.Key results:  Orexins strongly stimulated [3H]-AA release (maximally 4.4-fold). Orexin-A was somewhat more potent than orexin-B (pEC50= 8.90 and 8.38 respectively). The concentration2013response curves appeared biphasic. The release was fully inhibited by the potent cPLA2 and iPLA2 inhibitor, methyl arachidonyl fluorophosphonate, whereas the iPLA2 inhibitors, R- and S-bromoenol lactone, caused only a partial inhibition. The response was also fully dependent on Ca2+ influx, and the inhibitor studies suggested involvement of the receptor-operated influx pathway. The receptor-operated pathway, on the other hand, was partially dependent on PLA2 activity. The extracellular signal-regulated kinase, but not protein kinase C, were involved in the PLA2 activation at low orexin concentrations.Conclusions and implications:  Activation of OX1 orexin receptors induced a strong, high-potency AA release, possibly via multiple PLA2 species, and this response may be important for the receptor-operated Ca2+ influx. The response coincided with other high-potency lipid messenger responses, and may interact with these signals.

Place, publisher, year, edition, pages
2010. Vol. 159, no 1, 212-221 p.
Keyword [en]
orexin, hypocretin, OX1 receptor, phospholipase A2, arachidonic acid, PLA2, iPLA2, cPLA2, Ca2+ influx
National Category
Medical and Health Sciences
URN: urn:nbn:se:uu:diva-111369DOI: 10.1111/j.1476-5381.2009.00535.xISI: 000273766600022OAI: oai:DiVA.org:uu-111369DiVA: diva2:280814
Available from: 2009-12-11 Created: 2009-12-11 Last updated: 2010-12-21Bibliographically approved
In thesis
1. 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.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 508
orexin, phospholipase, Calcium, cell signalling, G protein coupled receptor, ERK, live-cell imaging, arachidonic acid, diacylglycerol
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)
Available from: 2010-01-27 Created: 2009-12-03 Last updated: 2010-06-09Bibliographically approved

Open Access in DiVA

No full text

Other links

Publisher's full text

Search in DiVA

By author/editor
Kukkonen, Jyrki P.
By organisation
In the same journal
British Journal of Pharmacology
Medical and Health Sciences

Search outside of DiVA

GoogleGoogle Scholar
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

Altmetric score

Total: 221 hits
ReferencesLink to record
Permanent link

Direct link