Cyclic AMP, ATPases and growth control of mastocytoma cells

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dc.contributor.advisor Ralph, R.K. en
dc.contributor.author Twigden, D. G. en
dc.date.accessioned 2007-08-28T09:40:54Z en
dc.date.available 2007-08-28T09:40:54Z en
dc.date.issued 1980 en
dc.identifier THESIS 80-109 en
dc.identifier.citation Thesis (PhD)--University of Auckland, 1980 en
dc.identifier.uri http://hdl.handle.net/2292/1569 en
dc.description Full text is available to authenticated members of The University of Auckland only. en
dc.description.abstract Depriving P815 mouse mastocytoma cells of ca2+ ions by growing them with EGTA inhibited their growth. This effect was fully reversed by Ca2+ or partially reversed by Mg2+. Growth of mastocytoma cells was also inhibited by 10-4M DBcAMP (N6, 02'-dibutyryladenosine 3',5'-cyclic monophosphate) plus 10-3M theophylline, and the drug-treated cells were significantly more sensitive to fluctuations in extracellular [Ca2+] than untreated cells. Early to mid-log phase cells had low Mg2+-ecto-ATPase activity, and ca2+-ecto-ATPase activity was not detected with these cells. However, both Mg2+-stimulated and Ca2+-stimulated ecto-ATPase activities increased in parallel during late-log and approaching stationary phase growth. The results were consistent with increased Mg2+- and Ca2+-stimulated ecto-ATPases being associated with inhibition of mastocytoma cell growth and they suggested that the activities might be the expression of one enzyme with different Vmax's when stimulated by Mg2+ or Ca2+. Magnesium was the more effective stimulatory cation at all stages of growth. A KME for Mg2+ of 0.5mM was determined for Mg2+-ATPase and a KME for Ca2+ of 0.65mM for Ca2+-ATPase of cells in late-log phase growth. A Km for ATP of 0.87mM was determined for both Mg2+- and Ca2+-stimulated ATP hydrolysis. DBcAMP plus theophylline treatment of P815 mastocytoma cell cultures caused Mg2+-ecto-ATPase activity to increase 4.8 to 6.3-fold and Ca2+-ecto-ATPase activity to increase 6.8 to 9.1-fold. The Km for ATP of Mg2+- and Ca2+-stimulated ATP hydrolysis remained unchanged compared to untreated cells. However, the Ca2+-binding affinity of the ecto-ATPase increased significantly compared to untreated cells, and the Mg2+-binding affinity of the ecto-ATPase decreased simultaneously, giving a KME for Ca2+ of 0.20m.M and a KME for Mg2+ of 1.80mM for the Ca2+- or Mg2+-stimulatede ecto-ATPases. These results are consistent with several reports that there is an increase in Ca2+ bound to the plasma membrane of growth-arrested (eg. after serum deprivation, or DBcAMP treatment) or quiescent cells. The effect of DBcAMP plus theophylline on cell growth and ecto-ATPase activity was reversed upon reseeding drug-treated cells in fresh medium without drug. The approximately 2-fold decrease in ecto-ATPase activity in a 7.5h period at the time of reinitiation of cell growth was consistent with an involvement of Ca2+- and Mg2+-stimulated ecto-ATPases in growth regulation of P815 mouse mastocytoma cells. Phosphorylation of membranes by cAMP-dependent protein kinases is known to increase Ca2+ binding, and to decrease the permeability of membranes to Ca2+. DBcAMP plus theophylline-treated P815 mastocytoma cells develop increased amounts of cell surface-associated acidic polysaccharides (Davis and Ralph, 1975) and these complexes are known to be good Ca2+ ligands. Thus, a consequence of treating mastocytoma cells with DBcAMP plus theophylline may be to increase phosphorylation of their plasma membranes and the amount of associated acidic polysaccharide, thereby increasing the Ca2+ affinity of plasma membrane constituents (eg. the ecto-ATPase), reducing the ca2+ permeability of the plasma membrane, and decreasing growth. That some metabolic event such as cAMP-dependent phosphorylation was required to activate ecto-ATPase activity was shown by there being no increase in ecto-ATPase activity for ≥ 2h after drug addition to cultures. en
dc.language.iso en en
dc.publisher ResearchSpace@Auckland en
dc.relation.ispartof PhD Thesis - University of Auckland en
dc.relation.isreferencedby UoA9921872014002091 en
dc.rights Restricted Item. Available to authenticated members of The University of Auckland en
dc.rights Items in ResearchSpace are protected by copyright, with all rights reserved, unless otherwise indicated. en
dc.rights.uri https://researchspace.auckland.ac.nz/docs/uoa-docs/rights.htm en
dc.title Cyclic AMP, ATPases and growth control of mastocytoma cells en
dc.type Thesis en
thesis.degree.grantor The University of Auckland en
thesis.degree.level Doctoral en
thesis.degree.name PhD en
dc.rights.holder Copyright: The author en


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