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A novel Ca2+ entry mechanism is turned on during growth arrest induced by Ca2+ pool depletion
C A Ufret-Vincenty1, A D Short, A Alfonso
1Department of Biological Chemistry, University of Maryland School of Medicine, Baltimore 21201, USA.
Abstract:
Ca2+ pool depletion with Ca2+ pump blockers induces growth arrest of rapidly dividing DDT1MF-2 smooth muscle cells and causes cells to enter a stable, quiescent G0-like growth state (Short, A.D., Bian, J., Ghosh, T.K., Waldron, R.T., Rybak, S.L., and Gill, D.L. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 4986-4990). Here we reveal that induction of this quiescent growth state with the Ca2+ pump blocker, thapsigargin, is correlated with the appearance of a novel caffeine-activated Ca2+ influx mechanism. Ca2+ influx through this mechanism is clearly distinct from and additive with Ca2+ entry through store-operated channels (SOCs). Whereas SOC-mediated entry is activated seconds after Ca2+ pool release, caffeine-sensitive influx requires at least 30 min of pool emptying. Although activated in the 1-10 mM caffeine range, this mechanism has clearly distinct methylxanthine specificity from ryanodine receptors and is not modified by ryanodine. It is also unaffected by the Ca2+ channel blockers SKF96365 or verapamil and is independent of modifiers of cyclic nucleotide levels. Growth arrest by thapsigargin-induced Ca2+ pool depletion can be reversed by treatment with 20% serum (Waldron, R.T., Short, A.D., Meadows, J.J., Ghosh, T.K., and Gill, D.L. (1994) J. Biol. Chem. 269, 11927-11933). The serum-induced return of functional Ca2+ pools and reentry of cells into the cell cycle correlates exactly with the disappearance of the caffeine-sensitive Ca2+ influx mechanism. Therefore, appearance and function of this novel Ca2+ entry mechanism are closely tied to Ca2+ pool function and cell growth state and may provide an important means for modifying exit from or entry into the cell cycle.
Insights
Ca2+ pump blockers halt smooth muscle cell division, inducing a quiescent state. This state is linked to a new caffeine-activated Ca2+ influx, distinct from store-operated channels, and disappears upon cell cycle reentry.
Area of Science:
- Cell Biology
- Physiology
- Biochemistry
Background:
- Ca2+ pool depletion using Ca2+ pump blockers, like thapsigargin, arrests cell growth in DDT1MF-2 smooth muscle cells, inducing a quiescent state.
- This growth arrest is reversible by serum treatment, which restores Ca2+ pools and cell cycle reentry.
Purpose of the Study:
- To investigate the mechanisms underlying Ca2+ pool depletion-induced cell cycle arrest.
- To identify novel Ca2+ influx pathways associated with the quiescent state.
Main Methods:
- Induction of Ca2+ pool depletion using the Ca2+ pump blocker thapsigargin.
- Characterization of Ca2+ influx mechanisms using caffeine and various channel blockers (SKF96365, verapamil, ryanodine).
- Assessment of cell cycle status and Ca2+ pool function.
Main Results:
- Thapsigargin-induced growth arrest correlates with a novel caffeine-activated Ca2+ influx mechanism.
- This caffeine-sensitive Ca2+ influx is distinct from and additive to store-operated channels (SOCs), requiring prolonged pool emptying.
- The novel Ca2+ influx mechanism's activity is closely linked to Ca2+ pool status and cell growth state, disappearing upon serum-induced cell cycle reentry.
Conclusions:
- A novel caffeine-activated Ca2+ influx pathway is identified and closely associated with Ca2+ pool depletion and cell cycle arrest.
- This mechanism's appearance and disappearance correlate with cell cycle exit and reentry, suggesting a role in regulating cell growth.
- The findings offer potential targets for modulating cell cycle progression.