Ca2+ pools and cell growth: arachidonic acid induces recovery of cells growth-arrested by Ca2+ pool depletion.

The intracellular Ca2+ pump blocker, thapsigargin, induces emptying of Ca2+ pools and entry of DDT1MF-2 smooth muscle cells into a quiescent G0-like growth state. Although thapsigargin blocks pumps essentially irreversibly, high serum (20%) induces appearance of new pump protein, return of functional pools, and reentry of cells into the cell cycle (Waldron, R. T., Short, A. D., Meadows, J. J., Ghosh, T. K., and Gill, D. L.(1994) J. Biol. Chem. 269, 11927-11933). Through analysis of the effects of defined serum components and growth supplements, we reveal here that the factors in serum responsible for inducing recovery of Ca2+ pools and growth in thapsigargin-arrested DDT1MF-2 cells are exactly mimicked by the three essential fatty acids, arachidonic, linoleic, and α-linolenic acids. The EC50 values for arachidonic and linoleic acids on growth induction of thapsigargin-arrested cells were the same, approximately 5 μM. Nonessential fatty acids, including myristic, palmitic, stearic, oleic, and arachidic acids, were without any effect. Although not proven to be the active component of serum, levels of arachidonic and linoleic acids in serum were sufficient to explain serum-induced growth recovery. Significantly, arachidonic or linoleic acids induced complete recovery of bradykinin-sensitive Ca2+ pools within 6 h of treatment of thapsigargin-arrested cells. Protein synthesis inhibitors (cycloheximide or puromycin) completely blocked the appearance of serum-induced or arachidonic acid-induced agonist-sensitive pools. The sensitivity and fatty acid specificity of Ca2+ pool recovery in thapsigargin-arrested cells were almost identical to that for growth recovery. No pool or growth recovery was observed with 5,8,11,14-eicosatetraynoic acid, the nonmetabolizable analogue of arachidonic acid, suggesting that conversion to eicosanoids underlies the pool and growth recovery induced by essential fatty acids. The results provide not only further information on the link between Ca2+ pools and cell growth but also evidence for a potentially important signaling pathway involved in inducing transition from a stationary to a proliferative growth state.

Cytosolic Ca 2ϩ signals control diverse cellular functions ranging from short term responses, including contraction and secretion, to longer term responses such as cell division and growth. Ca 2ϩ stored within intracellular pools and released through activation of intracellular Ca 2ϩ channels, provides a significant source of these Ca 2ϩ signals (1). Evidence suggests that intracellular pools of Ca 2ϩ exist within ER 1 or subfractions thereof (1)(2)(3). Accumulation of Ca 2ϩ within pools is mediated by intracellular Ca 2ϩ pumps of the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) family which are widely distributed within the ER of most cells (4,5). The Ca 2ϩ accumulated within Ca 2ϩ pools appears to control a number of other functions in addition to serving as a source for cytosolic Ca 2ϩ signals. Thus, intraluminal Ca 2ϩ appears to be the trigger that activates Ca 2ϩ entry across the plasma membrane following Ca 2ϩ pool release (3,6). Intraluminal Ca 2ϩ also influences certain essential ER functions, including the folding, processing, and assembly of proteins (7)(8)(9); such effects may be mediated by intraluminal Ca 2ϩ -binding proteins, several of which function as molecular chaperones (10,11). Additionally, we have shown that the Ca 2ϩ content of intracellular pools exerts a profound influence upon cell growth and the ability of cells to continue through the cell cycle (12)(13)(14)(15).
Studies reveal that the Ca 2ϩ pump inhibitors thapsigargin (16) and 2,5-di-tert-butylhydroquinone (17) deplete intracellular Ca 2ϩ pools and concomitantly promote entry of DDT 1 MF-2 smooth muscle cells into a stable growth-arrested G 0 -like state (12,13). Although growth-arrested, the Ca 2ϩ pool-depleted cells remain intact and viable and maintain normal cellular and subcellular morphology and mitochondrial function for up to 1 week (12,13). Whereas the inhibition of Ca 2ϩ pumps by thapsigargin is essentially irreversible (12,18,19), we recently revealed that high (20%) serum-treatment of thapsigargin-arrested cells induces reappearance of Ca 2ϩ pools and an orderly transition of quiescent cells back into the cell cycle (13,14). The high serum treatment induces expression of new functional Ca 2ϩ pump protein within 1-3 h (14), and agonist-releasable Ca 2ϩ pools reappear within 6 h. Cells begin to enter S-phase 16 h later and thereafter continue to proliferate normally (13,14).
An important question that remained to be answered was the nature of any active component within serum that was responsible for the recovery of pools and the growth of cells following growth arrest induced by Ca 2ϩ pump blockade. Presented here are studies examining the actions of different components from serum as well as the effects of a number of supplemental growth-promoting factors. The results reveal that essential fatty acids closely mimic the actions of high serum on pool recovery and resumption of growth. The effect is specific to the essential fatty acids arachidonic, linoleic, and linolenic acids and is not observed with a range of other non-essential fatty acids. The results provide not only further information on the link between Ca 2ϩ pools and cell growth but also evidence for a potentially important signaling pathway involved in inducing transition from a stationary to a proliferative growth state.

EXPERIMENTAL PROCEDURES
Cell Culture-DDT 1 MF-2 smooth muscle cells derived from hamster vas deferens were cultured in Dulbecco's modified Eagle's medium supplemented with 2.5% serum (Calf-Plus, Inovar, Gaithersburg, MD) as described previously (20). Calf-Plus is newborn calf serum supplemented with additional growth factors and is referred to as "serum" in this report. Fetal calf serum yields essentially identical results on growth and reversal of thapsigargin-induced growth arrest (13,14).
Growth Conditions and Measurement of Cell Proliferation-DDT 1 MF-2 smooth muscle cells were grown in 24-well dishes (1 ϫ 10 5 cells/well). Thapsigargin-treated cells were prepared by adding 3-8 M thapsigargin in DMEM with 2.5% serum for 3 h. Thapsigargin treatment was followed by three washes in thapsigargin-free DMEM with 2.5% serum followed by recovery under conditions specified. All recovery media contained DMEM with 2.5% serum together with additions as specified. For all cell proliferation experiments, the total time from the end of thapsigargin treatment until determination of cell number was 72 h. At the end of 72 h, cells were resuspended by pipette, transferred to cuvettes, and counted spectrophotometrically. For each experiment, standard curves were obtained for cell number (by direct counting) and light scattering measured by absorbance at 600 nm. Absorbance values obtained at different dilutions of cells were compared with the linear portion of the standard curve and values for cell number obtained. All measurements for cell number were obtained in quadruplicate, and results presented are typical of at least three different experiments.
Measurement of Cytosolic Free Ca 2ϩ -Cells were allowed to attach to poly-L-lysine-coated 25-mm glass coverslips in culture for at least 4 h prior to use. As described above, attached cells were treated with 3 M thapsigargin for 3 h then, after washing in thapsigargin-free DMEM with 2.5% serum, transferred to recovery media as indicated in figures. Cells were incubated for 6 h (unless otherwise noted) under appropriate recovery conditions in DMEM with 2.5% serum, at the end of which Ca 2ϩ levels were measured. Measurements of free cytosolic Ca 2ϩ were similar to those described previously (21). Attached cells were transferred to Hepes-buffered Kreb's medium (107 mM NaCl, 6 mM KCl, 1.2 mM MgSO 4 , 1 mM CaCl 2 , 1.2 mM KH 2 PO 4 , 11.5 mM glucose, 0.1% bovine serum albumin, 20 mM Hepes-KOH, pH 7.4) and loaded with fura-2/AM (2 M) for 10 min at 20°C in the dark. Cells were then washed in the same medium and loaded dye allowed to deesterify for 15 min at 20°C in the dark. Under these conditions, approximately 95% of the dye was confined to the cytoplasm as determined by the signal remaining after saponin permeabilization (21). Coverslips were inserted into a Dvorak- Materials and Miscellaneous Procedures-All fatty acids used were added to cells in DMEM with 2.5% serum containing 1% (w/v) fatty acid-free BSA. Thapsigargin was from LC Services, Corp., Woburn, MA. fura-2/acetoxymethyl ester was from Molecular Probes, Eugene, OR. ITS (insulin, transferrin, and selenious acid), ITSϩ (ITS with linoleic acid and BSA), insulin, transferrin, and selenious acid were from Collaborative Biomedical, Bedford, MA. All unsaturated fatty acids were from Cayman Chemical, Ann Arbor, MI. All other compounds were from Sigma. Heat inactivation of serum was for 30 min at the temperature shown. The DDT 1 MF-2 cell line was originally obtained from Drs. James Norris and Lawrence Cornett, University of Arkansas.

RESULTS AND DISCUSSION
In previous studies we have determined that the growth of DDT 1 MF-2 smooth muscle cells is profoundly altered by intracellular Ca 2ϩ pump blockers, including thapsigargin, 2,5-ditert-butylhydroquinone, and cyclopiazonic acid (2,11,12). In each case, Ca 2ϩ pool emptying is correlated with entry of cells into a growth-arrested state. DNA synthesis is not inhibited per se, and cells appear to progress through S-phase before entry into a stable G 0 -like quiescent state (12). Cells remain stable in this state for 7 days, maintaining viability, normal morphology and mitochondrial function, and approximately 20% of the protein synthesis observed in normal dividing cells (11). The blocking action of thapsigargin on intracellular Ca 2ϩ pumps is essentially irreversible (19,23), and even brief (30 min) treatment of cells with thapsigargin followed by extensive washing and culture in thapsigargin-free medium for up to 7 days results in Ca 2ϩ pools that remain empty (that is, unresponsive to Ca 2ϩ mobilizing agonists or Ca 2ϩ pump blockers) and in cells that remain in a quiescent nondividing state (11,12). However, we recently revealed that a brief treatment of thapsigargin-arrested cells with high serum (20% as opposed to the normal level of 2.5% used to grow DDT 1 MF-2 cells) in the absence of thapsigargin caused appearance of new functional Ca 2ϩ pump protein (determined by measuring Ca 2ϩ pump phosphorylated intermediate) in 1-3 h, reappearance of functional Ca 2ϩ pools in 3-6 h, followed by reentry of cells into the cell cycle (13,14).
Although intriguing, the basis of action of high serum in inducing growth recovery was unknown. Therefore, we sought to determine the active component(s) within serum responsible for pool and growth recovery. Initial studies involved the fractionation and modification of serum by heat inactivation, charcoal-stripping, and dialysis. As shown in Fig. 1, as compared with the effect of 20% serum on growth recovery of thapsigargin-arrested cells, 20% serum heat-treated at either 56 or 78°C gave lower recovery. However, in both cases cells did recover Thapsigargin-treated cells were exposed to the following growth conditions: standard conditions (2.5% serum); high serum (20% serum); high serum together with 8 M thapsigargin (20% ϩ Tg); 20% serum heat-inactivated either at 56°C (HI-56°) or 78°C (HI-78°); 10 ng/ml platelet-activating factor (PAF); 10 ng/ml platelet-derived growth factor (PDGF); 62.5 g/ml insulin; a combination of 62.5 g/ml insulin, 62.5 g/ml transferrin, 62.5 ng/ml selenious acid, 190 M linoleic, and 1% BSA (ITSϩ). All solutions contained 2.5% serum. Cell numbers were determined after 72 h under these conditions and compared with those immediately following thapsigargin treatment. Thapsigargin treatment and other procedures were as described under "Experimental Procedures." Results are means Ϯ S.D. of cell numbers obtained from quadruplicate wells. and the difference appeared due merely to a decreased rate of growth sustained by the heat-inactivated serum (as determined on normal, untreated cells). Charcoal-stripping of serum gave similar results. Serum dialysis proved inconsistent in its ability to remove agent(s) responsible for growth recovery. A number of growth agents, including platelet-activating factor, platelet-derived growth factor, insulin, and pituitary extract (not shown), did not induce growth recovery. Interestingly, the growth supplement known as ITSϩ (insulin, transferrin, selenious acid, and linoleic acid bound to BSA) was able to recover growth in thapsigargin-treated cells, almost as effectively as 20% serum.
Experiments conclusively indicated that the active component in this supplement was linoleic acid itself (Fig. 2). Thus, 190 M linoleic acid, the final concentration present in ITSϩ used in Fig. 1, added together with 1% (w/v) fatty acid-free BSA as carrier, induced cell recovery. In contrast, each of the other components of ITSϩ, including 1% BSA, either alone or in combination, had no recovery-inducing activity. The EC 50 of linoleic acid in the presence of 1% BSA was approximately 5 M (Fig. 3). Significant growth recovery could be observed at linoleic acid concentrations lower than 1 M and a maximal effect at approximately 100 M. In the presence of BSA a substantial fraction of the fatty acid is bound, particularly at the higher fatty acid levels. In the absence of carrier BSA, growth recovery could be attained with less than 1 M linoleic acid (not shown); however, without BSA, linoleic acid above 1 M caused cell lysis and death as a result of membrane perturbation.
Key to determine was the specificity of fatty acid-induced growth recovery of Ca 2ϩ pool-depleted cells. As shown in Fig. 4, an important pattern of specificity was observed among fatty acids tested. Consistently, all the nonessential fatty acids tested between 14 and 20 carbons, including the saturated fatty acids, myristic, palmitic, stearic, and arachidic acids, and the unsaturated fatty acid, oleic acid, did not give any significant recovery of growth. In contrast, all three of the essential fatty acids, linoleic, ␣-linolenic, and arachidonic acids, induced growth recovery of cells arrested by Ca 2ϩ pool depletion (Fig.  4). Arachidonic acid was consistently more effective (that is, induced a greater rate of recovery) than linoleic acid, which itself was more effective than linolenic acid. The arachidonic acid dose-response curve for inducing growth recovery (Fig. 5) was similar to that for linoleic acid; the half-maximal effectiveness was between 3 and 5 M, and significant recovery was usually seen with 100 nM arachidonic acid. The similar effectiveness of these closely related fatty acids is significant, indicating either a narrow structural requirement for their action or, as discussed below, that their effects on growth recovery are likely mediated by regulatory eicosanoids which can be specifically derived from each of the essential fatty acids (24).
An important question is whether essential fatty acids constitute the active component within serum-inducing growth recovery of pool-depleted cells. Whereas our results do not definitively prove that they are, evidence is consistent with this being the case. Heat inactivation, charcoal stripping, and dialysis are generally ineffective in removing fatty acids from serum, a large proportion of which is tightly bound to albumin. Analysis of the dose effectiveness of serum in inducing growth recovery in thapsigargin-arrested cells reveals a half-maximal effectiveness of approximately 7% serum (Fig. 6). Total nones- terified fatty acid in the undiluted calf serum used was measured as approximately 530 M; the essential fatty acids, linoleic, arachidonic, and linolenic acids, in combination, represent approximately 12% of total nonesterified fatty acid in serum and are in the ratio of approximately 90:10:1, respectively (25). Therefore, 7% serum contains a combined essential fatty acid concentration of approximately 5 M, agreeing with the halfmaximal effectiveness of linoleic or arachidonic acids given in Figs. 3 and 5. The somewhat broad concentration dependence of arachidonic and linoleic acids likely reflects dissociation from albumin which has a number of different binding sites for fatty acids over the sub-and low micromolar range (26). Albumin both protects cells from the detergent effects of fatty acids and provides a means of delivering fatty acids to cells; the actual free concentrations of fatty acids present in experiments are obviously considerably lower than the total added.
Although extremely unlikely based on the remarkable affinity and slow dissociation rate of thapsigargin from SERCA pump protein (19,23), a trivial explanation for the actions of essential fatty acids or serum on recovery of thapsigargintreated cells was possible removal or stripping of thapsigargin from cells. To determine any such effect, experiments were conducted to measure the effectiveness of thapsigargin on cells in the presence of fatty acids and serum. One such experiment is shown in Fig. 7 where the concentration dependence of thapsigargin in preventing growth of cells under standard conditions (2.5% serum) is compared with its effects either in the presence of 20% serum or 100 M linoleic acid (together with 1% BSA and 2.5% serum). It is clear that the effectiveness of thapsigargin is virtually identical under each condition, indicating that these agents do not bind, sequester, or otherwise prevent the inhibitory action of thapsigargin. The concentration dependence of thapsigargin on Ca 2ϩ pump blockade and emptying of Ca 2ϩ pools in intact cells is similar to that for growth inhibition (12,13), and similarly, serum, BSA, linoleic acid, or arachidonic acid had no measurable effects on the ability of thapsigargin to empty pools (data not shown).
In other control experiments, we examined the effects of essential and nonessential fatty acids on growth of normal cells, that is, cells not treated with pump blockers. Linoleic acid added to DDT 1 MF-2 cells at up to 500 M (with 1% BSA) under otherwise standard culture conditions had no significant effect on cell proliferation. Arachidonic acid actually had a significant growth inhibitory effect when added above 10 M; at 100 M the rate of cell growth was reduced by approximately 40%. Other nonessential fatty acids had no effect on normal cell growth. The effects of linoleic and arachidonic acids on normal cell growth concur well with those described by others on the effects of essential fatty acids on smooth muscle proliferation (27). These results indicate that reversal of growth arrest and induction of entry of quiescent Ca 2ϩ pool-depleted cells into the cell cycle is a specific action of essential fatty acids which is distinct from any general effects on cell proliferation. Indeed, taking into account its inhibitory action on cell growth rate, the action of arachidonic acid on recovery of quiescent cells in some of the above experiments is actually underestimated. In further control experiments, neither arachidonic nor linoleic acids had any measurable effect upon the size or function of Ca 2ϩ pools in normal cells.
From the above experiments, it is clear that arachidonic acid and the other essential fatty acids can mimic the action of high serum in promoting growth recovery of cells that have entered a stable quiescent state following Ca 2ϩ pool depletion. Such recovery of growth was the end result measured after 3 days. Previously we demonstrated that one of the initial events fol- lowing high serum treatment of pool-depleted cells was induction of new functional SERCA pump activity and inositol 1,4,5trisphosphate-releasable Ca 2ϩ pools (13,14). Obviously it was important to assess whether a similar early expression of Ca 2ϩ pools resulted from treatment with essential fatty acids or whether their effect on growth recovery was manifested after a different series of events. As shown in Fig. 8, there is clearly a rapid fatty acid-mediated induction of new Ca 2ϩ pools. Cells after treatment with thapsigargin have no measurable inositol 1,4,5-trisphosphate-sensitive Ca 2ϩ pools, and they remain without these pools for many days in a quiescent but otherwise viable state (12,13). After 6-h treatment of thapsigargin-arrested cells with 20% serum, the cells had regained fully operational pools as judged by a maximal Ca 2ϩ response to 10 M bradykinin (Fig. 8A). 6 h after treatment of similarly arrested cells with 100 M arachidonic acid and 1% BSA, an identical bradykinin-releasable pool was observed (Fig. 8B). This provides further evidence that the effects of high serum and arachidonic acid are equivalent and therefore that the action of high serum could be attributed to essential fatty acids con-tained within it. Although our recent studies revealed that high serum treatment induces the appearance of new pump protein (14), we had not previously determined whether protein synthesis was required for return of this activity. The results in Fig. 8 reveal that this is the case and that regardless of whether recovery is induced by serum or arachidonic acid, the appearance of bradykinin-sensitive Ca 2ϩ pools was completely prevented with either cycloheximide or puromycin.
The specificity of fatty acid-induced appearance of functional Ca 2ϩ pools was almost identical to that for growth induction. Thus, as shown in Fig. 9, 6 h of treatment of thapsigarginarrested cells with 100 M arachidonic or linoleic acids caused complete induction of functional Ca 2ϩ pools; 100 M linolenic acid consistently induced appearance of Ca 2ϩ pools that were less than maximal in this time period. The nonessential fatty acids, stearic and palmitic acid, did not result in any measurable bradykinin-sensitive Ca 2ϩ pools. The sensitivity of arachidonic acid-mediated pool recovery was also similar to that for arachidonic acid-induced growth recovery. As shown in Fig. 10, the response to 100 M arachidonic acid was maximal, lower concentrations giving a smaller response. Although the maximum peak height appeared to be approximately correlated with arachidonic acid level, at concentrations below 1 M arachidonic acid the rate of onset of the bradykinin-activated Ca 2ϩ signal was attenuated (data not shown). Thus, cells treated with 100 nM arachidonic acid showed a consistent recovery of pools, but the rate at which emptying occurred in response to bradykinin was slower. It is possible that functional Ca 2ϩ pools may be less extensively formed at this lower level of stimulation by arachidonic acid, resulting in not only a smaller amount of releasable Ca 2ϩ but also a slower onset of Ca 2ϩ release.
An important question is whether arachidonic acid acts directly to stimulate Ca 2ϩ pool regeneration and growth recovery or whether it becomes metabolized to an active derivative. From the results shown in Fig. 11, it is unlikely that arachidonic acid itself is the activating species. Thus, the structural analogue, eicosatetraynoic acid (ETYA), at 100 M did not induce any recovery of pools. This analogue is unable to undergo metabolism to active products via the lipoxygenase, cyclooxygenase, or monooxygenase pathways (28). ETYA has been shown to mimic the actions of arachidonic acid in cases where metabolism of the fatty acid is not required (28 -30). Based on this, it appears likely that arachidonic acid requires metabolism in order to induce cell recovery. ETYA also did not induce any growth of thapsigargin-arrested cells; and like arachidonic acid, ETYA induced some inhibition of the rate of growth of normal cells, reducing the rate by almost 50% at 100 M.
The results presented here provide compelling evidence that essential fatty acids can induce recovery of cells that have been growth arrested by Ca 2ϩ pool depletion. This recovery involves both the return of Ca 2ϩ pumping pools and the transition of cells from a G 0 -like growth state back into the cell cycle. The action of essential fatty acids is very similar to that of addition of high serum to cells (13,14). At present, we have not proven whether essential fatty acids are the active recovery-inducing component within serum or whether serum induces the formation of arachidonic acid or other fatty acids by, for example, stimulation of receptor-linked phospholipase A 2 activity. The latter appears unlikely since preliminary experiments have shown no effect of phospholipase A 2 inhibitors on serum-induced recovery. Moreover, the actual levels of arachidonic and linoleic acids present within serum appear sufficient to explain the actions of serum. The other important question is whether arachidonic acid requires conversion to eicosanoids in order to have its growth inducing effects. Obviously, the essential fatty acids, including arachidonic, linoleic, and linolenic acids, are each major substrates for conversion to prostaglandins, prostacyclins, thromboxanes, leukotrienes, and other eicosanoids (24). In view of the noneffectiveness of ETYA, it is likely that conversion is required. It is also clear that ETYA is an effective blocker of the entry of arachidonic acid and other essential fatty acids into each of the pathways through which eicosanoids are formed, including the cyclooxygenase, lipoxygenase, and monooxygenase pathways (24,28). Recent experiments indicate that ETYA also blocks arachidonic acid-induced pool and growth recovery. 2 However, further dissection of the effective metabolites is required before information on exactly which eicosanoid products are effective in mediating recovery of cells from growth arrest induced by Ca 2ϩ pool depletion.