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    Intracellular calcium pump expression, calcium pool function and cell growth

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    Author
    Waldron, Richard Taliesin
    Advisor
    Gill, Donald L.
    Date
    1996
    Type
    dissertation
    
    Metadata
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    Abstract
    Expression of functional intracellular Ca2+{ pumps is essential for operation of Ca2+ signaling pools. The aim of these studies was to detect functional pumps within Ca2+ pumping pools and identify the Ca2+ transport properties and specific Ca2+ signaling functions of these pools. (1) Using DDT{dollar}\sb1{dollar}MF-2 smooth muscle cells growth-arrested by exposure to the intracellular Ca2+ pump blocker thapsigargin (TG) at 3 {dollar}\mu{dollar}M for 24 hr, treatment with 20% serum for 6 hr without TG induced expression of functional Ca2+ pump protein detected as a 110 kDa TG-sensitive phosphorylated intermediate; 2.5% serum treatment resulted in no functional pump expression. Western analysis revealed only a slight serum-induced increase in total Ca2+ pump protein. The timing of appearance of new functional Ca2+ pump protein (first detected within 1 hr of high serum treatment) and the required duration of the high serum treatment (approx. 35 min.) for subsequent full recovery of Ca2+ pools and growth indicate the necessity of functional endoplasmic reticulum Ca2+ pumps in serum-induced cell growth and reflect a precise signaling period during which quiescent cells commit to a progression of events including Ca2+ pump expression, Ca2+ pool function, reentry into the cell cycle, and cell division. (2) Further studies utilized TG-resistant DC-3F/TG2 cells cultured with 2 {dollar}\mu{dollar}M TG; these cells have a doubling time (24 hr) not significantly different from parent DC-3F Chinese hamster lung cells without TG. TG inhibited {dollar}\rm \sp{lcub}45{rcub}Ca\sp{lcub}2+{rcub}{dollar} uptake of two distinct Ca2+ pump activities with 20,000-fold different sensitivities to TG within permeabilized parent DC-3F cells; the IC{dollar}\sb{lcub}50{rcub}{dollar} values for TG were 200 pM and 4 {dollar}\mu{dollar}M, representing 80% and 20% of total pumping activity, respectively. Total pump activity in parent DC-3F and resistant DC-3F/TG2 cells were similar (0.23 {dollar}\pm{dollar} 0.10 and 0.18 {dollar}\pm{dollar} 0.08 nmol Ca{dollar}\sp{lcub}2+{rcub}{dollar}/10{dollar}\sp6{dollar} cells, respectively), yet in DC-3F/TG2 cells, only TG-insensitive pumps were functional. In both cell types, each Ca2+ pump activity (regardless of TG-sensitivity) had high Ca2+ affinity (K{dollar}\sb{lcub}\rm m{rcub}{dollar} values {dollar}\simeq{dollar} 0.1 {dollar}\mu{dollar}M), and similar ATP-dependence and vanadate-sensitivity, In DC-3F cells, sensitivity to release with InsP{dollar}\sb3{dollar} or GTP and oxalate-permeability were exclusive to the TG-sensitive Ca2+ pool. GTP-induced Ca2+ uptake in the presence of oxalate indicated Ca2+ transfer between distinct pools in the DC-3F cells. In resistant DC-3F/TG2 cells, almost 50% of total TG-insensitive Ca2+ accumulation was releasable with InsP{dollar}\sb3{dollar}; unlike the parent cells this pool was not oxalate-permeable. These findings reveal that the TG-insensitive Ca2+ pumping pool within resistant DC-3F/TG2 cells contains functional InsP{dollar}\sb3{dollar} receptors. Measurements of cytosolic Ca2+ concentrations ((Ca2+{rcub}\rbrack\sb{lcub}\rm i{rcub}){dollar} within intact, fura-2 loaded parent DC-3F cells showed typical influx of Ca{dollar}\sp{lcub}2+{rcub}{dollar} through store-operated channels (SOCs) after TG-induced pool depletion. Unlike the parent DC-3F cells, DC-3F/TG2 cells had constitutively activated basal Ca2+ entry through SOCs which could be rapidly increased by transient external Ca2+ removal; thus, the machinery for activating Ca2+ entry is continuously operational. The effect of oleoyl lysophosphatidic acid (LPA) to release Ca2+ pools within DC-3F cells by interaction with receptors linked to generation of InsP{dollar}\sb3{dollar} was abolished by prior TG-addition. (Abstract shortened by UMI.)
    Description
    University of Maryland, Baltimore. Biochemistry and Molecular Biology. Ph.D. 1996
    Keyword
    Biology, Molecular
    Biology, Cell
    Biology, Animal Physiology
    calcium pools
    intracellular calcium pump
    Cell Growth Processes
    Identifier to cite or link to this item
    http://hdl.handle.net/10713/1433
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    Theses and Dissertations All Schools
    Theses and Dissertations School of Medicine

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