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Title: The differential regulation of cyclic AMP phosphodiesterases in T lymphocytes
Author: Michie, Alison Mary
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1995
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Cyclic AMP phosphodiesterases were studied in T lymphocytes from different maturation stages, tissues and species. Although cyclic AMP phosphodiesterases were characterised in lymphocytes isolated from murine spleens, human tonsils, a murine thymoma cell line and a human mature T cell line, the main focus of this study was cyclic AMP PDE activities from murine thymocytes. With the use of selective phosphodiesterase inhibitors, the relative contribution that particular phosphodiesterase isoforms made towards total phosphodiesterase activity in the cells was assessed. Thus, cyclic GMP stimulated, PDE2 activity and cyclic AMP specific PDE4 activity were shown to provide the major cyclic AMP hydrolysing activities in murine thymocytes. PDE4 activity predominated (~80% total) in the absence of cyclic GMP. However, on addition of low (10?M) concentrations of cyclic GMP, PDE2 activity constituted the major PDE activity in thymocytes (~80% total). The PDE4 selective inhibitor rolipram inhibited murine thymocyte PDE4 activity in a dose-dependent manner (IC50 ~65nM). PDE2 activity was stimulated in a dose-dependent fashion (EC50 ~l?M) by cyclic GMP and was selectively inhibited by the compound, EHNA (IC50 ~4?M). No calcium/calmodulin stimulated, PDEl activity or cyclic GMP-inhibited, PDE3 activity was found to be present in murine thymocytes. The non-selective PDE inhibitor IBMX elicited >98% inhibition of PDE activity in thymocytes indicating the absence of IBMX- insensitive, PDE7 activity in these cells. FPLC analysis confirmed these findings, revealing a rolipram inhibited PDE4 activity and a cyclic GMP stimulated, EHNA inhibited, PDE2 activity, but no PDEl, PDE3 or PDE7 activity. The selective inhibitors identified the profile of phosphodiesterase activities in T cells from other sources. Thus, PDE2 activity was also found to be present in murine splenic lymphocytes, proliferating T3.2 thymoma cell line derived from murine thymus and the human leukemic T cell line, Jurkat. In contrast to murine thymocytes, PDE3 appeared to be present in human tonsillar T lymphocytes, the human T cell line Jurkat and the thymoma cell line, T3.2. Similar to murine thymocytes, PDE4 activities were found to be present in the murine thymoma cell line, T3.2 and the human T cell line, Jurkat. Moreover, primary lymphocytes isolated from murine thymuses, spleens and human tonsils did not contain Ca2+/CaM stimulated, PDE1 activity. In contrast, the proliferating human Jurkat T cell line contained calcium/calmodulin-stimulated PDEl and an IBMX insensitive PDE activity. The differential distributions of phosphodiesterase activities within these lymphoid cells indicated species and possibly maturation-dependent differences of phosphodiesterase expression within these cells. Upon ligation of the TCR/CD3 complex with either the mitogenic lectin phytohaemagluttinin or anti-TCR/anti-CD3 monoclonal antibodies, phosphodiesterase activities and cyclic AMP levels were found to be rapidly and differentially regulated in murine thymocytes. Within 5 minutes of challenge with PHA, there was a transient decrease (~83%) in PDE4 activity and in PDE2 activity (~40%), returning almost to basal in 20 minutes. Both anti-CD3 and anti-TCR antibodies also caused an initial reduction in the PDE4 activity (~50%) which was then followed by a sustained increase in activity. In contrast to that observed with PHA, anti-TCR/CD3 antisera had little effect on PDE2 activity. Cyclic AMP levels were also differentially regulated upon stimulation of thymocytes with either PHA or anti-CD3 monoclonal antibodies. Hence, there was a transient 2-fold elevation in cyclic AMP levels upon stimulation of thymocytes with PHA, which reached basal levels again within 30 minutes. In contrast anti-CD3 antibody stimulation of thymocytes led to a slow sustained elevation in cyclic AMP levels over a 30 minute period. Study of the mechanism controlling the anti-CD3-stimulated elevation in PDE4 activity in murine thymocytes revealed that the increase in this phosphodiesterase activity was dependent on protein tyrosine kinase and protein kinase C-mediated signals. This interpretation was based on findings that the tyrosine kinase inhibitor, genistein partially inhibited the TCR-mediated elevation in PDE4 activity. Moreover, the protein kinase C selective inhibitor chelerythrine blocked the rise of PDE4 activity and the phorbol ester, phorbol-12-myristate, 13- acetate caused an increase in PDE4 activity which was of a similar magnitude to the elevation seen in the presence of anti-CD3 antibodies. Interestingly, the elevation in PDE4 activity was blocked upon stimulation of thymocytes with TPA or anti-CD3 antibody in the presence cyclohexamide or actinomycin D. This indicated that a rapid induction of PDE4 activity occurred in murine thymocytes upon crosslinking of the TCR/CD3 complex. These data identify 'crosstalk' between cyclic AMP and PKC mediated signalling pathways.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral