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Title: Role of extracellular ATP and adenosine in the human lens and retinal pigment epithelium
Author: Eldred, Julie Ann.
ISNI:       0000 0001 3443 9466
Awarding Body: University of East Anglia
Current Institution: University of East Anglia
Date of Award: 2004
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Purpose: To detennine the potential impact through signalling, that A TP and its metabolites have on lenticular and retinal ceJJs. To achieve this, I aimed to detennine the levels of ATP and its metabolites in human ocular fluids and to examine the etTect of environmental stress on release of A TP and its metabolites into the extracellular medium. I also aimed to identify functional roles for A TP and its metabolites and for this end I studied growth control and activation of intraceJJular signalling pathways Methods: Two cell-lines were routinely cultured and used for these studies: FHL-124 a human lens epithelial cell-line and hTERT RPE a telomerase transfected human retinal pigment epithelial cell-line. In addition native lens and retinal pigment epithelial cells from human donor material were incorporated into this study. AtTymetrix gene-arrays were used to compare native eye tissue with its respective cell-line. A TP levels were determined using a luciferinlluciferase Bioluminescence kit, specifically designed for A TP quantification. This assay was used to determine levels in ocular fluids and in culture medium of cells maintained under various experimental conditions. The etTects of ATP and its breakdown products on growth of FHL-124 and hTERT RPE were investigated using the patch assay technique. Expression of intracellular signalling proteins, SAPK and ERK were detennined using western blot methods. Immunocytochemistry was employed to detennine the presence of cell surface ecto-nucleotidases and cytokeratin. Results: AtTymetrix gene microarrays revealed that FHL-124 and hTERT RPE cell-lines showed strong agreement in gene expression with respect to their native tissues. Moreover, cell specific markers were also expressed in the cell lines. Analysis of the A TP content of aqueous humour from cataract patients and sub-retinal fluid showed mean concentrations of 37.8 ± 7.7nM and 0.362 ± 0.172nM respectively. Challenging both FHL 124 and hTERT RPE cells with increased levels of NaCI, to induce an osmotic stress, induced a release of A TP into the culture medium. The detectable levels were to stress levels related. The addition of low concentrations of ATP (as low as lOOpM) induced a significant inhibition of growth in both cell types. The A TP metabolite adenosine at 100pM also gave a significant inhibition of growth in the two cell lines. The specific A 1 receptor agonist CPA added at l00pM reproduced the adenosine effects. Immunocytochemical staining in addition to the gene array analyses, identified the presence of cell-surface 5'ectonucleotidases in both cell types. 100pM A TP, adenosine and CPA increased the expression of pSAPK in both cell types within I hr. In the case of hTERT RPE cells, all agonists led to decreased levels of pERK within Ihr, while in FHL 124 cells this response was only observed with ATP and CPA. Conclusions: A TP is present in human aqueous humour and sub-retinal fluid. Moreover, FHL 124 and hTERT cells can release A TP in response to environmental stresses. Cell surface ecto-nucleotidases are present in these cells and therefore have the potential to generate A TP metabolites, such as ADP and adenosine. Physiological levels of A TP and its metabolites can inhibit cell growth and it is adenosine that is most likely to play the greater role.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available