A close correlation was observed between intracellular Ca2+ pool depletion and refiling and the onset of DNA synthesis and proliferation of DDT,MF-2 smooth muscle cells. The intracellular Ca2+ pump inhibitors 2,5-di-tert-butylhydroquinone (DBHQ) and thapsigargin (TG) specificafly emptied identical inositol 1,4,5-trisphosphate (InsP3)-sensitive Ca2+ pools and both arrested cell growth at concentrations corresponding to Ca2+ pump blockade. However, an important distinction was observed between the two inhibitors with respect to their reversibility of action. Upon removal of DBHQ from DBHQ-arrested cells, Ca2+ pools immediately refilled, and 14 hr later cells entered S phase followed by normal cell proliferation; the time for entry into S phase was identical to that for cells released from confluence arrest. Although TG irreversibly blocked Ca2+ pumping and emptied Ca2+ pools, high serum treatment of TG-arrested cells induced recovery of functional Ca2+ pools in 6 hr (via probable synthesis of new pump); thereafter cells proceeded to S phase and normal cell proliferation within the same time period (14 hr) as that foUlowing release of DBHQ-arrested cells. The precise relationship between Ca2+ pump blockade and growth arrest indicates that Ca2+ pool emptying maintains cells in a Go-like quiescent state; upon refilling of pools, normal progression into the cell cycle is resumed. It is possible that a specific cell cycle event necessary for Go to G, transition depends upon signals generated from the InsP3-sensitive Ca2+ pool.

Parathyroid hormone (PTH) 1 plays a central role in plasma Ca 2ϩ homeostasis, and many tissues that are not involved in Ca 2ϩ regulation also express PTH receptors (1). Two PTH receptor subtypes (types 1 and 2) have been identified; their cDNA sequences share 70% similarity (2-5) and identify them as members of a subfamily of G protein-coupled receptors to which the receptors for calcitonin, secretin, ACTH, and glucagon also belong.It has long been accepted that PTH stimulates an increase in both intracellular cyclic AMP and [Ca 2ϩ ] i in many cell types (6, 7). The ability of PTH to activate two signaling pathways was generally assumed to result from its interaction with the two receptor subtypes, an assumption that gained support from the observation that the two pathways could be differentially stimulated by truncated forms of PTH (8). More recently, expression of recombinant receptors has established that type 1 (3, 9, 10) and type 2 (11) PTH receptors are each alone capable of stimulating increases in both [Ca 2ϩ ] i and intracellular cyclic AMP. Other members of the family of receptors to which the PTH receptor belongs share this ability to independently stimulate cyclic AMP formation and an increase in [Ca 2ϩ ] i (3,(12)(13)(14).In some cells, the increase in [Ca 2ϩ ] i evoked by PTH is mediated largely by effects of cyclic AMP on Ca 2ϩ entry (7), but in osteoblasts and kidney cells (6) and cells expressing recombinant type 1 (3, 9, 10) or type 2 (11) PTH receptors, PTH also stimulates release of intracellular Ca 2ϩ stores. The means whereby PTH causes such Ca 2ϩ mobilization is unclear. In some cells, PTH stimulates formation of inositol 1,4,5-trisphosphate (IP 3 ) (3, 6), which presumably then causes Ca 2ϩ mobilization via IP 3 receptors. In other situations, PTH and agonists that stimulate IP 3 formation appear to release Ca 2ϩ from different intracellular stores (9, 15), and PTH-stimulated Ca 2ϩ mobilization occurs without detectable formation of IP 3 (10, 15) and in the presence of antagonists of IP 3 receptors (9, 10). Furthermore, PTH-evoked Ca 2ϩ release appears not to be mediated by ryanodine receptors (9) or by either cyclic ADP-ribose or NAADP ϩ (9), both of which have been shown to stimulate Ca 2ϩ release in other cells (16). In short, the ability of PTH to stimulate Ca 2ϩ mobilization cannot easily be explained by the properties of known intracellular signaling pathways.In the present study, we examine the mechanisms underlying the effects of PTH on intracellular Ca 2ϩ stores in human embryonic kidney 293 cells stably expressing type 1 PTH receptors (HEK/PTH-R1 cells). EXPERIMENTAL PROCEDURESMaterials-The full-length cDNA encoding the human type 1 PTH receptor in the vector, pcDNAI/neo, was a gift from Dr. K. Seuwen (Basel, Switzerland) (5). HEK 293 cells were from the European Collection of Animal Cell Cultures (Porton Down, UK). Human PTH (1-34), Rp-8-Br-cAMPS, ionomycin, Xestospongin A, wortmannin, and H89 were from Calbiochem (Nottingham, UK). Fura-2AM and Cascade Blue wer...

Exposure of cells to the intracellular Ca2+ pump blocker, thapsigargin (TG), results in emptying of Ca2+ pools and termination of cell proliferation (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). DC-3F Chinese hamster lung cells were made resistant to TG by long-term stepwise exposure to increasing TG concentrations in culture (Gutheil, J. C., Hart, S. R., Belani, C. P., Melera, P. W., and Hussain, A. (1994) J. Biol. Chem. 269, 7976-7981). Since these cells (DC-3F/TG2) grow in the presence of TG, it was important to ascertain what Ca2+ pool function they retain. TG-resistant DC-3F/TG2 cells cultured with 2 microM TG had a doubling time (24 h) not significantly different from the parent DC-3F cells without TG. Analysis of TG-induced inhibition of 45Ca2+ uptake into permeabilized parent DC-3F cells revealed two distinct Ca2+ pump activities with 20,000-fold different sensitivities to TG; the IC50 values for TG were 200 pM and 4 microM, representing 80% and 20% of total pumping activity, respectively. Total pump activity in parent DC-3F and resistant DC-3F/TG2 cells was similar (0.23 +/- 0.10 and 0.18 +/- 0.08 nmol of Ca2+/10(6) cells, respectively). In DC-3F/TG2 cells, up to 100 nM TG had no effect on Ca2+ pumping; however, almost all pumping was blocked at higher TG concentrations with an IC50 of 5 microM. In both cell types, each Ca2+ pump activity (regardless of TG sensitivity) had high Ca2+ affinity (Km values congruent to 0.1 microM) and similar ATP dependence and vanadate sensitivity. In DC-3F cells, the TG-sensitive Ca2+ pool was releasable with inositol 1,4,5-trisphosphate (InsP3) or GTP and was oxalate-permeable; the TG-insensitive pool in these cells was not InsP3-releasable. 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 InsP3; unlike the parent cells, this pool was not oxalate-permeable, and GTP induced no Ca2+ transfer between pools in the presence of oxalate. Thus, whereas InsP3 releases Ca2+ only from the high TG sensitivity Ca2+ pumping pool in parent DC-3F cells, in resistant DC-3F/TG2 cells the TG-resistant Ca2+ pumping pool now contains functional InsP3 receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

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A. 90, 4986 -4990). Here we reveal that induction of this quiescent growth state with the Ca 2؉ pump blocker, thapsigargin, is correlated with the appearance of a novel caffeine-activated Ca 2؉ influx mechanism. Ca 2؉ influx through this mechanism is clearly distinct from and additive with Ca 2؉ entry through store-operated channels (SOCs). Whereas SOCmediated entry is activated seconds after Ca 2؉ 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 Ca 2؉ channel blockers SKF96365 or verapamil and is independent of modifiers of cyclic nucleotide levels. Growth arrest by thapsigargin-induced Ca 2؉ pool depletion can be reversed by treatment with 20% serum (Waldron, R.