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

Direct link
Rapid and easy semi-quantitative evaluation method for diacylglycerol and inositol-1,4,5-trisphosphate generation in orexin receptor signalling
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)
2010 (English)In: Acta Physiologica, ISSN 1748-1708, E-ISSN 1748-1716, Vol. 198, no 3, 387-392 p.Article in journal (Refereed) Published
Abstract [en]

Aim: Fluorescent protein-based indicators have enabled measurement of intracellular signals previously nearly inaccessible for studies. However, indicators showing intracellular translocation upon response suffer from serious limitations, especially the very time-consuming data collection. We therefore set out in this study to evaluate whether fixing and counting cells showing translocation could mend this issue.

Methods: Altogether three different genetically encoded indicators for diacylglycerol and inositol-1,4,5-trisphosphate were transiently expressed in Chinese hamster ovary cells stably expressing human OX1 orexin receptors. Upon stimulation with orexin-A, the cells were fixed with six different protocols.

Results: Different protocols showed clear differences in their ability to preserve the indicator’s localization (i.e. translocation after stimulus) and its fluorescence, and the best results for each indicator were obtained with a different protocol. The concentration

Conclusion: The counting method, as used here, works at single time point and looses the single-cell-quantitative aspect. However, it also has some useful properties. First, it easily allows processing of a 100- to 1000-fold higher cell numbers than real-time imaging producing statistically consistent population-quantitative data much faster. Secondly, it does not require expensive real-time imaging equipment. Fluorescence in fixed cells can also be quantitated, though this analysis would be more time-consuming than cell counting. Thirdly, in addition to the quantitative data collection, the method could be applied for identifying responsive cells. This might be very useful in identification of e.g. orexin-responding neurones in a large population of non-responsive cells in primary cultures.

Place, publisher, year, edition, pages
Wiley , 2010. Vol. 198, no 3, 387-392 p.
National Category
URN: urn:nbn:se:uu:diva-111132DOI: 10.1111/j.1748-1716.2009.02017.xISI: 000274147900021PubMedID: 19583704OAI: oai:DiVA.org:uu-111132DiVA: diva2:279462
Available from: 2009-12-03 Created: 2009-12-03 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 textPubMed

Search in DiVA

By author/editor
Ekholm, MarieKukkonen, Jyrki P.
By organisation
In the same journal
Acta Physiologica

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: 152 hits
ReferencesLink to record
Permanent link

Direct link