Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.555259
Title: Role of the cannabinoid signalling system in microglial cell function
Author: El Deeb, Khalil Mohamed Khalil
Awarding Body: University of Nottingham
Current Institution: University of Nottingham
Date of Award: 2011
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Abstract:
Cannabinoids have well known immunomodulatory properties, through actions mainly on CB2 receptors, which are abundantly expressed in microglia, and or other yet as unidentified receptors. The primary objective of this study, therefore, was to investigate the possibility of modulating particular microglial functions (nitric oxide (NO) release, endocannabinoid hydrolysis and calcium mobilisation) with cannabinoids using an immortalized cell lines, specifically, mouse-derived BV -2 cells and rat-derived Highly Aggressive Proliferating Immortalized (HAPI) cells to overcome the challenges of having enough primary microglia for experimental use. NO release was stimulated with lipopolysaccharide (LPS) and or interferon y (IFN-y) and quantified using the Greiss reaction. While the selective CB2 agonist, JWH133, a number of endocannabinoids and the CBI and CB2 receptor antagonists, rimonabant and SR144528, all failed to significantly alter LPS-stimulated NO formation in RAPI or BV -2 cells, the non-selective CBI/CB2 agonist CP55940 (only at 10 ~M) significantly attenuated NO formation in both HAPI and BV -2 cells. Medium from ischaemic neuroblastoma cells, SHSY5Y cells, and the PPARy agonist, rosiglitazone, were also able to attenuate LPS-induced NO release via CB2 receptor-independent mechanisms. Although IFN-y enhanced LPS-induced NO release in BV-2 cells without affecting basal levels, WIN55-212-2 and JWH133 ( 1 uM) were unable to affect LPS/IFN-y induced NO release. HAPI and BV-2 cells appeared to express CB] and CB2 receptor protein using immunoblotting, but RT-PCR revealed a lack of mRNA expression of either receptor in both cell lines. Endocannabinoid, AEA and PEA, hydrolysis was assessed in BV-2 cells by monitoring liberation of [3H]-ethanolamine from labelled AEA or PEA. In intact BV -2 cells, marked differences in the time courses for PEA and AEA hydrolysis were observed, raising the possibility of a distinct microglial enzyme responsible for PEA hydrolysis. Expression of GPR55, in BV-2 cells was confirmed using qRT-PCR analysis. LPI, evoked calcium elevations in a concentration-dependent manner. Although cannabinoids were unable to produce a significant calcium response in BV-2 cells, CBD and i19-THC produced attenuations of LPI-induced calcium elevation while CP55940 and rimonabant, showed less significant reductions. In order to evaluate the role of GPR55, two batches of GPR55- HEK293 cells from different sources were employed; however neither appeared to express GPR55 mRNA. CBD and ∆9-THC were also able to attenuate calcium responses to the P2 receptor agonists, ATP and ADP, in BV- 2 cells indicating a non-selective antagonist activity, as well as the possibility of P2_ receptor expression in BV -2 cells. Although GPR55 agonists were unable to stimulate microglial migration in an in vitro assay, ATP (100 u M) significantly enhanced microglial migration which was inhibited by both CBD and ∆9 - THC (10uM). In conclusion, in the absence of conventional cannabinoid receptor expression in microglial cell lines, cannabinoids appear to affect microglia function through acting on the potential cannabinoid-like receptor, GPR55, and through P2 receptors, both of which may present new avenues in the treatment of neurodegenerative and neuroinflammatory illnesses.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.555259  DOI: Not available
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