Intracellular calcium pump expression, calcium pool function and cell growth

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.)