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Glutamate release mechanisms from megakaryocytes

McGrath, Catherine Jane (2007) Glutamate release mechanisms from megakaryocytes. PhD thesis, University of York.

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Abstract

Cardiovascular disease (CVD) is one of the main causes of death in western society. Platelet activation, thrombus formation and plaque rupture are all central events in the pathogenesis of acute coronary syndromes, therefore therapies targeted at controlling platelet numbers and aggregation are likely to be beneficial in the treatment of CVD. Megakaryocytes (MKs) which are the precursors to platelets are an ideal target for these therapies, however the intrinsic factors that regulate the production and shedding of platelet precursors are poorly understood. Recent studies identified that MKs express functional NMDA-type glutamate receptors similar to those found in the CNS and that antagonism of these receptors prevents normal MK differentiation and platelet function. This thesis investigates glutamate signalling within MKs further, focusing on the glutamate release capability of MK cells and the mechanisms involved. Using molecular and cellular techniques it was demonstrated that MK cells expressed numerous regulatory proteins required for vesicular glutamate release, including core SNARE proteins, VAMP, SNAP-23 and syntaxin; specific glutamate-loading vesicle proteins, VGLUTI and VGLUT2; and glutamate transporters, EAATI and EAAT2. Active vesicle recycling was observed in MK cells using a fluorescent reporter and an enzyme-linked fluorimetric assay confirmed that MK cells constitutively released glutamate and that glutamate release levels increased significantly following MK differentiation. Transient transfection of the human cell line MEG-Ol with tetanus toxin, which disables vesicle recycling, induced a 30% decrease (P<O.OOI) in released glutamate compared to empty vector controls. In contrast, over-expressing VGLUTI caused a 41% increase (P<O.OO1) in glutamate release activity of MEG-O1 cells compared to controls. These data demonstrate that MK cells regulate glutamate exocytosis through specific vesicular proteins, indicating that glutamate signalling may be a potential target for CVD therapies. Also the observations that MKs both release and recycle glutamate indicates an important role for glutamate signalling from these cells in autocrine and paracrine interactions within the bone marrow microenvironment.

Item Type: Thesis (PhD)
Academic Units: The University of York > Biology (York)
Identification Number/EthosID: uk.bl.ethos.485836
Depositing User: EThOS Import (York)
Date Deposited: 24 Sep 2015 14:34
Last Modified: 24 Sep 2015 14:34
URI: http://etheses.whiterose.ac.uk/id/eprint/9950

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