Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.486848
Title: Investigating the roles of the cardiomyocyte V- ATPase and NHE1, and their response to insulin stimulation
Author: Preedy, Amelia Louise
ISNI:       0000 0001 3498 9513
Awarding Body: University of Bath
Current Institution: University of Bath
Date of Award: 2007
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Abstract:
The aims of this thesis were to investigate the effects of insulin on the cellular distribution and activity of the Na+/It exchanger (NHEI) and vacuolar proton pumps (VATPases) in cardiomyocytes and to evaluate their association with GLUT4 vesicles. To address these issues, a highly specific anti-NHEI antibody was raised, which enabled immunofluorescence, immunoprecipitation and co-immunoprecipitation studies into the effects of insulin on NHE1 distribution to be carried out. These studies provided evidence for an insulin-stimulated increase in NHEl at the plasma membrane and a decrease in intracellular NHEI, even though the overall amount cellular NHEI remained constant. A cell free acidity assay (CFAA) was developed to measure NHEI activity within subcellular fractions. An insulin-stimulated increase in plasma membrane NHEI activity was identified in accordance with the increase in plasma membrane NHEI. The CFAA also identified an inhibitable portion of V-ATPase activity within the low density microsomes (LDM) from basal cardiomyocytes, which was absent from LDM of insulinstimulated cells. The insulin-stimulated decrease in LDM V-ATPase activity implied that in the presence of insulin, V-ATPases translocate, as do GLUT4 vesicles. Structural associations between two complexes of V-ATPase (namely complexes Vo and VI) with GLUT4 vesicles were identified by co-immunoprecipitation of GLUT4 vesicles and subsequent Western blotting for the V-ATPase protein. The studies generated data that was consistent with the hypothesis that the insulin-stimulated change in V-ATPase activity in LDM samples may partly occur as a result of the insulinstimulated decrease in general vesicle trafficking and fusion and in part to GLUT4 vesicle translocation and fusion. The thesis identifies further investigations, that are needed to determine whether (a) V-ATPases are necessary for vesicle acidification to occur prior to fusion; (b) whether V-ATPases are involved in the formation of a Yo-Yo transcomplex necessary for docking ofthe GLUT4 vesicles with the PM and finally (c) whether insulin affects the distribution ofNHEI either by stimulating NHEI trafficking or their turnover.
Supervisor: Not available Sponsor: Not available
Qualification Name: University of Bath, 2007 Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.486848  DOI: Not available
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