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Functional Autoradiography as a Pharmacological Approach for Studying G Protein-Coupled Lipid Receptor Signalling
| Content Provider | Semantic Scholar |
|---|---|
| Author | Aaltonen, Niina |
| Copyright Year | 2013 |
| Abstract | Lipids have recently been recognized as an important class of signalling molecules both in the central nervous system and the periphery. Bioactive lipids are produced by multistep enzymatic pathways from their membrane phospholipid precursors. After they exert their effect by activating their specific receptors, they are rapidly enzymatically degraded. Lysophospholipids and endocannabinoids (eCBs) represent two groups of bioactive lipids. Lysophosphatidic acid (LPA) is a structurally simple lysophospholipid that mainly mediates its actions through six G protein-coupled receptors (GPCRs) (LPA1 6). Endocannabinoids, such as anandamide and 2-arachidonoylglycerol (2-AG), are the body’s natural agonists for the two GPCRs (CB1 and CB2) that also recognize ∆9tetrahydrocannabinol, the psychoactive component present in marijuana. Both LPA and eCBs are involved in the development and function of many organ systems as well as in the pathology of several serious diseases, such as atherosclerosis and cancer. The main objectives of this study were to devise and optimize the methodology used in studying lipid-GPCR signalling and to characterize the enzymatic pathways responsible for lipid messenger synthesis and degradation. The principal method used in the current study was guanosine-5’-O-(3-[35S]thio)-triphosphate ([35S]GTPγS) autoradiography which is applied in a novel way to examine the enzymatic pathways that synthesize and degrade signalling lipids in brain sections. In the first part of the study, the [35S]GTPγS autoradiography method was characterized by mapping rat brain regions with prominent [35S]GTPγS binding under basal conditions. A liquid chromatography-tandem mass spectrometric method was developed to permit the quantitative determination of LPA species from brain tissue samples. Further studies revealed that the enzymatic systems synthesizing and metabolizing lipid mediators were well preserved in rodent brain cryosections. When LPA/2-AG degradation was pharmacologically blocked, brain sections generated endogenous lipids which were able to activate their cognate GPCRs during the autoradiography incubations. It was concluded that lipid phosphate phosphatases (LPPs) degrade the signalling pool of LPA in brain sections but in addition to LPPs, there seems to be alternative phosphatases present in the brain that degrade LPA at the whole brain level. The CB1 receptor-dependent Gi protein activity remained unaltered in several brain regions of diacylglycerol lipase (DAGL) deficient mice when compared to wild-type mice. Alternative enzymes in addition to DAGLs seem to be responsible for synthesizing 2-AG in brain sections. It appears that there are separate enzymes in the brain that synthesize/degrade the signalling and non-signalling lipid pools. Especially when combined with sensitive analytical methods, [35S]GTPγS autoradiography represents a valuable tool for studying the life cycle of bioactive lipids. National Library of Medicine Classification: QU 85.6, QU 93 Medical Subject Headings: Lipids; Receptors; Lysophosphatidic Acid; Lysophospholipids; Endocannabinoids; Guanosine 5’-O-(3-Thiotriphosphate)/metabolism; Enzyme inhibitors/pharmacology; Autoradiography; Tandem Mass Spectrometry; Brain/metabolism; Brain Mapping/methods; Animals |
| File Format | PDF HTM / HTML |
| Alternate Webpage(s) | http://epublications.uef.fi/pub/urn_isbn_978-952-61-1281-7/urn_isbn_978-952-61-1281-7.pdf |
| Language | English |
| Access Restriction | Open |
| Content Type | Text |
| Resource Type | Article |
