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Multiple phospholipase activation by OX(1) orexin/hypocretin receptors
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience. (Kukkonen)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience. (Kukkonen)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience. (Kukkonen)
2008 (English)In: Cellular and Molecular Life Sciences (CMLS), ISSN 1420-682X, E-ISSN 1420-9071, Vol. 65, no 12, 1948-1956 p.Article in journal (Refereed) Published
Abstract [en]

We investigated coupling of OX(1) receptors to phospholipase activation and diacylglycerol generation in Chinese hamster ovary (CHO) cells using both biochemical and fluorescence "real-time" methods. The results indicate that at lowest orexin-A concentrations (highest potency), diacylglycerol generated results from phospholipase D activity. At 10-100-fold higher orexin-A concentrations, phospholipase C is activated, likely hydrolyzing phosphatidylinositol (PI) or phosphatidylinositol monophosphate (PIP) but not phosphatidylinositol bisphosphate (PIP(2)). At further 7-fold higher orexin-A concentrations, PIP(2) is hydrolyzed, releasing both diacylglycerol and inositol-1,4,5-trisphosphate. Thus, OX(1) orexin receptors connect to multiple phospholipase activities, apparently composed of at least one phospholipase D and two different phospholipase C activities. At low agonist concentrations, diacylglycerol and phosphatidic acid are the preferred products, and interestingly, it seems that even the primarily activated phospholipase C mainly works to increase diacylglycerol and not inositol-1,4,5-trisphosphate.

Place, publisher, year, edition, pages
2008. Vol. 65, no 12, 1948-1956 p.
Keyword [en]
Orexin, hypocretin, phospholipase C, phospholipase D, phosphoinositides, phosphatidylcholine, diacylglycerol, live cell imaging
National Category
Medical and Health Sciences
URN: urn:nbn:se:uu:diva-111131DOI: 10.1007/s00018-008-8206-zISI: 000256927400017PubMedID: 18488139OAI: oai:DiVA.org:uu-111131DiVA: diva2:279465
Available from: 2009-12-03 Created: 2009-12-03 Last updated: 2010-06-07Bibliographically 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

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