©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Novel Ca Entry Mechanism Is Turned On during Growth Arrest Induced by Ca Pool Depletion (*)

(Received for publication, August 14, 1995; and in revised form, September 13, 1995)

Carmen A. Ufret-Vincenty Alison D. Short (§) Amparo Alfonso (¶) Donald L. Gill (**)

From the Department of Biological Chemistry, University of Maryland School of Medicine, Baltimore, Maryland 21201

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Ca pool depletion with Ca pump blockers induces growth arrest of rapidly dividing DDT(1)MF-2 smooth muscle cells and causes cells to enter a stable, quiescent G(0)-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 Ca pump blocker, thapsigargin, is correlated with the appearance of a novel caffeine-activated Ca influx mechanism. Ca influx through this mechanism is clearly distinct from and additive with Ca entry through store-operated channels (SOCs). Whereas SOC-mediated entry is activated seconds after Ca 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 channel blockers SKF96365 or verapamil and is independent of modifiers of cyclic nucleotide levels. Growth arrest by thapsigargin-induced Ca 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 Ca pools and reentry of cells into the cell cycle correlates exactly with the disappearance of the caffeine-sensitive Ca influx mechanism. Therefore, appearance and function of this novel Ca entry mechanism are closely tied to Ca pool function and cell growth state and may provide an important means for modifying exit from or entry into the cell cycle.


INTRODUCTION

Ca inside intracellular pools not only provides a source for cytosolic Ca signals (1) but controls diverse cellular and lumenal functions including the activation of external Ca influx(2) , folding and processing of proteins in the endoplasmic reticulum(3) , and cell proliferation and cell cycle progression(4, 5, 6, 7) . Emptying of Ca pools in rapidly dividing DDT(1)MF-2 smooth muscle cells with the Ca pump blockers, thapsigargin or 2,5-di-tert-butylhydroquinone, induces entry of cells into a stable, quiescent G(0)-like growth state(5, 6) . Here we reveal that while in this Ca pool-depleted, growth-arrested state, cells turn on a novel methylxanthine-sensitive Ca influx mechanism. This influx process is clearly distinct from Ca entry through store-operated channels (SOCs), is independent of ryanodine receptor or InsP(3) receptor function, and is not related to changes in cyclic nucleotide levels. Induction of new pump synthesis and return of functional Ca pools by treatment of Ca pool-depleted cells with high serum or arachidonic acid causes the methylxanthine-sensitive Ca influx mechanism to be turned off, normal receptor-operated Ca signaling to resume, and re-entry of cells into the cell cycle. The function of this novel Ca influx mechanism and its precise turning on and off may be important events in the relationship between Ca signaling and the growth state of cells.


EXPERIMENTAL PROCEDURES

Growth of Cells and Thapsigargin Treatment

Culture of DDT(1)MF-2 smooth muscle cells was as described previously(8) . Thapsigargin-treated cells (attached to poly-L-lysine-coated glass coverslips in Dulbecco's modified Eagle's medium with 2.5% CalfPlus) had been exposed to 3 µM thapsigargin in culture for 18 h, followed by washing and culture in thapsigargin-free medium for 48 h as described previously(5, 6) . Untreated cells were exposed to the same conditions except without thapsigargin.

Measurement of Intracellular Ca

Intracellular free Ca measurements were as described previously (6, 9) . Briefly, coverslip-attached cells were transferred into HKM (107 mM NaCl, 6 mM KCl, 1.2 mM MgSO(4), 1.2 mM KH(2)PO(4), 1 mM CaCl(2), 11.5 mM glucose, 0.1% bovine serum albumin, 20 mM Hepes-KOH, pH 7.3) and loaded with 2 µM fura-2 acetoxymethyl ester for 10 min at 20 °C in the dark. After deesterification in fresh loading medium for 15 min at 20 °C, coverslips were inserted into a Dvorak-Stotler chamber. Groups of 5-10 fura-2-loaded cells were viewed through a Nikon 40times UV-fluor objective. Excitation at 340, 358, or 380 nm was generated using a PTI D103 light source, and fluorescence emission at 505 nm was monitored at 24 °C using a PTI D104 photometer. Free intracellular Ca concentrations were calculated from either 340/380 or 358/380 ratios of fluorescence intensities using the method of Grynkiewicz et al.(10) and a K(d) of 135 nM. Rates of Mn uptake were estimated by measuring Mn-dependent fluorescence quenching at 358 nm.

Materials and Miscellaneous Procedures

Thapsigargin was from LC Services Corp. Fura-2 acetoxymethyl ester was from Molecular Probes. Other compounds were from Sigma. The DDT(1)MF-2 cell line was originally obtained from Drs. James Norris and Lawrence Cornett, University of Arkansas.


RESULTS AND DISCUSSION

The Ca pump blocker, thapsigargin, empties intracellular Ca pools (11) and rapidly activates Ca entry through SOCs(2) . In many cells including DDT(1)MF-2 cells this entry becomes efficiently deactivated a few minutes after pool emptying(7) . SOCs can be transiently reactivated by brief removal of Ca(o)(12) . As shown in Fig. 1A, in DDT(1)MF-2 cells, even 24 h after thapsigargin-induced Ca pool depletion and the establishment of growth arrest, Ca(o) removal results in an immediate decrease in resting cytosolic Ca, presumably reflecting the contribution of some residual non-deactivated SOC activity. During the absence of Ca(o), SOC activity becomes reactivated as reflected by the large ``overshoot'' in cytosolic Ca observed upon readdition of Ca(o); the transient nature of the overshoot reflects a temporary activation of SOCs, which again undergo rapid deactivation. Normal DDT(1)MF-2 cells with filled Ca pools and presumably without any activated SOCs exhibit little change in cytosolic Ca as a result of transient Ca(o) removal. Interestingly, addition of 10 mM caffeine to the pool-depleted cells induces a further rapid and substantial increase in Ca(i), which also appears to undergo deactivation (Fig. 1A). This caffeine effect is not dependent on brief Ca(o) removal since, added directly to thapsigargin-arrested cells, caffeine induces the same large transient Ca(i) increase (Fig. 1B). Cells treated with the alternative Ca pump blocker, 2,5-di-tert-butylhydroquinone, which also induces pool emptying and growth arrest(6) , develop the same caffeine-induced Ca response (data not shown). Importantly, normal cells (that is untreated with Ca pump blockers) are devoid of any caffeine response (Fig. 1, A and B).


Figure 1: Caffeine-induced and SOC-induced Ca influx in normal and Ca pool-depleted DDT(1)MF-2 cells. [Ca] was measured in normal cells (Untreated) or cells treated with thapsigargin to empty Ca pools and induce growth arrest (TG-treated). A, untreated and thapsigargin-treated cells were treated with nominally Ca-free HKM for 6 min and then returned to normal HKM. 3 min later, 10 mM caffeine was added to both cell types. B, 10 mM caffeine was added to untreated and thapsigargin-treated cells at 30 s in normal HKM. C, thapsigargin-treated cells were treated at 30 s with Ca-free HKM and at 90 s with 10 mM caffeine; after 5 min in Ca-free HKM cells were returned to normal HKM.



The effect of caffeine is clearly on influx of Ca since it induces no change in the absence of Ca(o) (Fig. 1C). When Ca is added back in the presence of caffeine, a rapid and even larger transient of Ca is observed (Fig. 1C). The almost exact doubling in the size of the transient after readdition of Ca reflects clear additivity of SOC- and caffeine-mediated Ca entry, providing further evidence that the two mechanisms are independent. The Ca entry blocker SKF96365 at 50 µM completely blocks SOC-mediated Ca entry but even as high as 2 mM has no effect on caffeine-mediated Ca entry (data not shown). These data indicate that caffeine induces an influx of Ca distinct from that mediated by SOCs but only in Ca pool-depleted growth-arrested cells.

Even though both depend on Ca pool emptying, an interesting divergence between the two influx mechanisms is their time dependence of activation following pool depletion. SOC-mediated Ca influx becomes activated rapidly after thapsigargin-induced pool depletion but within 5 min has become almost completely deactivated (Fig. 2A). Indeed, the efficient turn-off of SOC-mediated entry prevents long-term increased cytosolic Ca levels following pool depletion and may be important in the survival of DDT(1)MF-2 cells, albeit in a growth-arrested state, following pool emptying(13, 14) ; cells in which SOC-mediated entry is not deactivated can enter an irreversible apoptotic cycle(13, 14) . Reactivation of SOCs can occur by transient Ca removal as early as a few minutes after pool emptying (Fig. 2A). At this time addition of caffeine has no effect (Fig. 2B), further supporting an SOC-independent action of caffeine. The time dependence of pool emptying and appearance of caffeine-induced influx is shown in Fig. 2, C-E. After 10 min of thapsigargin-induced pool emptying no caffeine effect can be detected. The shortest period of thapsigargin treatment after which a significant caffeine-induced Ca influx can occur is 30 min. However, immediately following this minimally effective period of thapsigargin treatment, the caffeine response is small; during a further period of 30-60 min after removal of thapsigargin the caffeine response becomes larger. In Fig. 2, C-E, after treatment with thapsigargin for the times shown, cells were incubated a further 60 min without thapsigargin. Also, with the shorter thapsigargin treatment times, onset of influx following caffeine addition is more variable in cells, some cells exhibiting a lag of 1-2 min prior to activation of influx (this is apparent in Fig. 2D in which the data are an average of 8 cells). With a longer time of thapsigargin treatment the caffeine effect becomes larger and more rapid in onset. The minimum time of thapsigargin treatment (30 min) required to induce appearance of the caffeine effect is significant since a 30-min treatment of DDT(1)MF-2 cells with thapsigargin is the time sufficient to induce not only sustained emptying of pools but also entry of cells into a stable quiescent growth state(5, 6, 7) .


Figure 2: Appearance of caffeine-induced Ca influx after thapsigargin-activated Ca pool depletion in DDT(1)MF-2 cells. A and B, normal cells were treated with 3 µM thapsigargin (TG) followed by exposure to Ca-free HKM (A) or addition of 10 mM caffeine (B) at the times shown. C-E, cells were treated with 3 µM thapsigargin for either 10 min (C), 30 min (D), or 180 min (E) and then washed with thapsigargin-free culture medium for 1 h. Intracellular Ca measurements were calculated from 340/380 ratios obtained from groups of 5-10 cells as described under ``Experimental Procedures.''



The specificity among methylxanthines in activating Ca entry indicates function of a novel influx mechanism. At 10 mM, 3,7-dimethyl-1-propargylxanthine (3,7-DMPX), a ryanodine receptor agonist 4-fold more effective than caffeine(15) , is completely ineffective in inducing Ca influx (Fig. 3A). In fact DDT(1)MF-2 cells have no measurable ryanodine receptor activity; caffeine or ryanodine have no effect on intracellular Ca release; nor is there any detectable ryanodine receptor protein in DDT(1)MF-2 cells as determined by Western analysis. Theophylline (1,3-dimethylxanthine) consistently induces a more rapid increase in cytosolic Ca than caffeine (1,3,7-trimethylxanthine) (Fig. 3B). The sensitivity to theophylline and caffeine is similar; 10 mM gives a maximal response with a sharp dose-response curve between 1 and 10 mM. This caffeine-sensitivity range is similar to that for ryanodine receptors(15, 16) . 1,7-Dimethylxanthine induces a slower but almost full effect (Fig. 3B) whereas 3,7-dimethylxanthine has no effect (not shown). This methylxanthine specificity is clearly distinct from that of ryanodine receptors on which the latter two dimethylxanthines are actually more effective than caffeine(16) .


Figure 3: Specificity of methylxanthine-induced Ca influx in Ca pool-depleted growth-arrested DDT(1)MF-2 cells. A, at the time shown additions were made of 10 mM caffeine or 10 mM 3,7-DMPX. B, additions of 10 mM theophylline or 10 mM 1,7-DMX were made at the time shown. For each trace, 358/380 ratios obtained as described under ``Experimental Procedures'' were normalized to base-line ratios in order to compare the relative change in intracellular Ca induced by each methylxanthine.



The methylxanthine-activated Ca entry is not related to changes in cyclic nucleotide levels. The potent phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX), has no effect on Ca entry at 10 mM. Addition of dibutyryl cyclic AMP, 8-bromo-cyclic AMP, forskolin (alone or in combination with IBMX), or dibutyryl cyclic GMP all have no effect. Methylxanthines are also effective adenosine receptor antagonists. However, the ineffectiveness of 3,7-DMPX, 3,7-DMX, and IBMX, which are potent antagonists of both A(1) and A(2) adenosine receptors(17) , militates strongly against Ca influx being adenosine receptor-mediated. DDT(1)MF-2 cells have no measurable voltage-operated Ca channel activity, and 50 µM verapamil has no effect on the influx induced by caffeine. InsP(3) receptors are reported to be inhibited by caffeine(18, 19) . Although there was no logical explanation of our results based on InsP(3) receptor alteration, examination of the effects of 10 mM caffeine on InsP(3)-mediated Ca release from permeabilized DDT(1)MF-2 cells revealed a negligible effect. Also, in contrast to the specificity described above, theophylline is reported to be much less potent on InsP(3) receptors than caffeine(15) .

The entry of Ca induced by caffeine is clearly transient and becomes deactivated within a few minutes. Readdition of caffeine without washing induces no further effect; however, a 5-min wash of caffeine-treated cells followed by caffeine reapplication results in return of the full Ca entry effect indicating that the entry mechanism becomes reactivated. More recent experiments (^3)have assessed cation selectivity and specificity of the caffeine-activated entry mechanism. These studies reveal that the entry mechanism is readily permeable to Mn. By assessing quench of cytosolic fura-2 in pool-depleted DDT(1)MF-2 cells, rates of Mn entry activated by different methylxanthines have been shown to correlate well with the effects on cytosolic Ca shown in Fig. 3. Also, we have observed that caffeine-activated Ca entry is blocked by other cations including Gd, La, and Co but is not blocked by Mn. In contrast, while SOC activation induces a modest influx of Mn, the entry of Ca through SOCs is substantially blocked by Mn, in keeping with the observations of others(20, 21) . These results provide evidence of yet another distinction between the methylxanthine- and SOC-mediated Ca entry mechanisms.

Further reinforcement of the correlation between Ca pool content, growth state, and operation of caffeine-induced Ca influx is derived from experiments in which thapsigargin-treated, growth-arrested cells are induced to reenter the growth cycle. We previously revealed that a 40-min treatment of thapsigargin-arrested cells with 20% serum induces synthesis of new pump protein, return of functional Ca pools, and resumption of normal growth(7) . Recent studies reveal that 10 µM arachidonic acid induces an identical recovery of Ca pools and cell growth. (^4)As shown in Fig. 4, the reappearance of a functional bradykinin-activated Ca pool is directly correlated with the disappearance of the caffeine-sensitive Ca entry mechanism. In thapsigargin-arrested cells, the absence of an InsP(3)-sensitive Ca pool is reflected by the lack of response to receptor agonists such as bradykinin (Fig. 4A); in these cells caffeine always activates a substantial influx of Ca (Fig. 4C). Thapsigargin-arrested cells treated with 20% serum recover from growth arrest and when examined 16 h later show normal bradykinin-induced Ca signals (Fig. 4B) and a complete absence of the caffeine-induced Ca response (Fig. 4D). Thus, cells have returned to the pregrowth-arrested state in which the caffeine-sensitive influx mechanism is turned off and normal agonist-sensitive Ca pools are functional. Significantly, further experiments have shown that the time of disappearance of caffeine-induced influx closely correlates with that for reappearance of Ca pools following high serum-induced recovery. 3 h after thapsigargin-arrested cells are induced to recover with high serum, a significant bradykinin-sensitive Ca signal is observable in groups of cells as well as a still measurable caffeine response (although as yet a single cell with both activities has not been observed). At 6 h after induction, the response to caffeine has completely disappeared and the bradykinin response is the same as a normal cell. We have shown that following serum induction of thapsigargin-arrested cells, new Ca pump protein appears as early as 1 h and pools become fully operational at 6 h(7) ; thereafter cells progress through G(1) and begin to enter G(s) 16 h later(6, 7) . This entire sequence of events, including cessation of the caffeine response, is identically activated by 10 µM arachidonic acid as opposed to 20% serum. From these experiments it is clear that function of the caffeine-sensitive Ca influx mechanism is restricted only to Ca pool-depleted, growth-arrested cells.


Figure 4: Serum-induced recovery of agonist-sensitive Ca pools in DDT(1)MF-2 cells is accompanied by a return to the caffeine-insensitive state. A and C, DDT(1)MF-2 cells were pool-depleted by treatment with 3 µM thapsigargin (TG) for 3 h followed by washing and continued culture in normal growth medium (containing 2.5% serum). After 16 h, cells were fura-2-loaded and the effects of 10 µM bradykinin (BK) (A) or 10 mM caffeine (C) measured on [Ca]. B and D, cells were thapsigargin-treated as above but were exposed to growth medium with 20% serum during the 16-h post-treatment time, followed by the same fura-2 loading and determination of the effects of 10 µM bradykinin (B) or 10 mM caffeine (D). Dye loading and 340/380 ratio fluorescence measurements on groups of 5-10 cells were as described under ``Experimental Procedures.''



The studies described here demonstrate specific activation of a novel and distinct Ca entry mechanism in pool-depleted growth-arrested DDT(1)MF-2 cells that permits a transient but substantial entry of Ca. Indeed, the levels of intracellular Ca achieved by activation of this mechanism are comparable with those attained by complete Ca pool emptying or activation of SOC-mediated entry. A Ca-conducting caffeine-sensitive influx channel was recently reported in adult gastric smooth muscle cells(22) ; however, in this case Ca influx was not rapidly deactivated and methylxanthine specificity was not examined. Whereas functional ryanodine receptors in the same cells precluded absolute proof that Ca influx was independent of ryanodine receptor function(23) , it is intriguing that methylxanthine-sensitive Ca entry channels might be expressed in nondividing smooth muscle cells. Our results are the first to provide evidence for a pharmacologically defined and apparently unique methylxanthine-sensitive Ca entry pathway. Since caffeine has been widely used as a means of probing the action of Ca release channels in many cell types(24) , the present results are significant in providing awareness of the existence of a distinct caffeine-activated Cainflux pathway. It is possible that this mechanism retains certain structural and/or functional similarities with ryanodine receptors. At this stage we do not know whether protein synthesis is required for turning on the entry mechanism. Another intriguing area of investigation will be to determine the means by which deactivation occurs. It is likely that operation of the influx pathway is transient within the cell cycle and/or restricted to discrete cell growth states.

Most significantly, although the physiological activation of the caffeine-sensitive Ca entry mechanism has yet to be characterized, the appearance of this clearly defined and substantial Ca entry mechanism under such specific conditions of pool emptying and growth arrest indicates a potentially important role in mediating Ca signals during transition into and out of the cell cycle or during cell division when Ca pools undergo substantial reorganization. As such, pharmacological modification of this channel may provide an important means for controlling cell growth.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants NS19304 and GM15407, by National Science Foundation Grant DCB 9307746, and by the award of a postdoctoral fellowship from the Maryland Heart Association, Maryland Affiliate (to A. D. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Dept. of Pharmacology, Cambridge University, Tennis Court Road, Cambridge CB2 1QJ, United Kingdom.

Recipient of a postdoctoral fellowship from Formación del Personal Investigador en el Extranjero, Ministerio de Educación y Ciencia, Spain.

**
To whom correspondence should be addressed: Dept. of Biological Chemistry, University of Maryland School of Medicine, 108 North Greene St., Baltimore, MD 21201. Office Tel.: 410-706-2593; Laboratory Tel.: 410-706-7247; Fax: 410-706-6676.

(^1)
The abbreviations used are: SOC, store-operated channel; InsP(3), inositol 1,4,5-trisphosphate; 3,7-DMPX, 3,7-dimethyl-1-propargylxanthine; IBMX, 3-isobutyl-1-methylxanthine; 1,7-DMX, 1,7-dimethylxanthine; HKM, Hepes-buffered Kreb's medium.

(^2)
Whereas discrete channel proteins have yet to be identified, the convention, SOC, was adopted in May 1995 at the International Conference on Receptor-regulated Calcium Influx, Pacific Grove, CA, to provide a general description of Ca store-activated influx channels variously described as capacitative Ca entry, depletion-activated channels, and I.

(^3)
C. A. Ufret-Vincenty, A. Alfonso, A. D. Short, and D. L. Gill, manuscript in preparation.

(^4)
M. N. Graber, A. Alfonso, and D. L. Gill, submitted for publication.


ACKNOWLEDGEMENTS

We thank Dr. Tarun K. Ghosh, 3M Pharmaceuticals, for helpful advice and discussions and Thuyly Nguyen for expert technical assistance.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.