McGrath, Catherine Jane (2007) Glutamate release mechanisms from megakaryocytes. PhD thesis, University of York.
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.
Metadata
Awarding institution: | University of York |
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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 |
Open Archives Initiative ID (OAI ID): | oai:etheses.whiterose.ac.uk:9950 |
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