(Received for publication, September 12, 1995; and in revised form, November 6, 1995)
From the
The intracellular Ca pump blocker,
thapsigargin, induces emptying of Ca
pools and entry
of DDT
MF-2 smooth muscle cells into a quiescent
G
-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 Ca
pools and growth in
thapsigargin-arrested DDT
MF-2 cells are exactly mimicked by
the three essential fatty acids, arachidonic, linoleic, and
-linolenic acids. The EC
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 Ca
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 Ca
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
Ca
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 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
stored within intracellular pools and released
through activation of intracellular Ca
channels,
provides a significant source of these Ca
signals(1) . Evidence suggests that intracellular pools
of Ca
exist within ER (
)or subfractions
thereof (1, 2, 3) . Accumulation of
Ca
within pools is mediated by intracellular
Ca
pumps of the sarcoplasmic/endoplasmic reticulum
calcium ATPase (SERCA) family which are widely distributed within the
ER of most cells(4, 5) . The Ca
accumulated within Ca
pools appears to control
a number of other functions in addition to serving as a source for
cytosolic Ca
signals. Thus, intraluminal
Ca
appears to be the trigger that activates
Ca
entry across the plasma membrane following
Ca
pool release(3, 6) . Intraluminal
Ca
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
-binding proteins, several of which
function as molecular chaperones(10, 11) .
Additionally, we have shown that the Ca
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 pump inhibitors thapsigargin (16) and 2,5-di-tert-butylhydroquinone (17) deplete intracellular Ca
pools and
concomitantly promote entry of DDT
MF-2 smooth muscle cells
into a stable growth-arrested G
-like
state(12, 13) . Although growth-arrested, the
Ca
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
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
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
pump protein within
1-3 h(14) , and agonist-releasable Ca
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 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 nonessential fatty acids. The
results provide not only further information on the link between
Ca
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.
In previous studies we have determined that the growth of
DDTMF-2 smooth muscle cells is profoundly altered by
intracellular Ca
pump blockers, including
thapsigargin, 2,5-di-tert-butylhydroquinone, and cyclopiazonic
acid(2, 11, 12) . In each case,
Ca
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
-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
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
pools that remain empty (that is,
unresponsive to Ca
mobilizing agonists or
Ca
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
MF-2 cells) in the absence of thapsigargin caused
appearance of new functional Ca
pump protein
(determined by measuring Ca
pump phosphorylated
intermediate) in 1-3 h, reappearance of functional Ca
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 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 ITS+ (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.
Figure 1:
Effects of serum, serum supplements,
and growth factors on growth recovery of thapsigargin-arrested
DDTMF-2 cells. Thapsigargin-treated cells were exposed to
the following growth conditions: standard conditions (2.5% serum); high serum (20% serum); high serum together with
8 µM thapsigargin (20% + 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 µg/ml insulin; a combination of 62.5
µg/ml insulin, 62.5 µg/ml transferrin, 62.5 ng/ml selenious
acid, 190 µM linoleic, and 1% BSA (ITS+).
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 ± S.D. of cell numbers
obtained from quadruplicate wells.
Experiments conclusively indicated that the active
component in this supplement was linoleic acid itself (Fig. 2).
Thus, 190 µM linoleic acid, the final concentration
present in ITS+ 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 ITS+, including 1% BSA,
either alone or in combination, had no recovery-inducing activity. The
EC of linoleic acid in the presence of 1% BSA was
approximately 5 µM (Fig. 3). Significant growth
recovery could be observed at linoleic acid concentrations lower than 1
µM and a maximal effect at approximately 100
µM. 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 µM linoleic acid (not shown); however, without BSA,
linoleic acid above 1 µM caused cell lysis and death as a
result of membrane perturbation.
Figure 2:
The active component in ITS+
responsible for recovery of thapsigargin-arrested DDTMF-2
cells is linoleic acid. Conditions for growth were: 2.5% serum
(standard conditions); ``ITS+'' (commercially obtained
62.5 µg/ml insulin, 62.5 µg/ml transferrin, 62.5 ng/ml
selenious acid, 190 µM linoleic acid, and 1% BSA);
``ITS'' (as for ITS+ but without linoleic acid or BSA);
insulin (62.5 µg/ml); transferrin (62.5 µg/ml); selenious acid
(62.5 ng/ml); BSA (1%); BSA + ITS; linoleic acid (LA)/BSA
(190 µM linoleic acid with 1% BSA); ITS LA/BSA (equivalent
to ITS+, but formulated entirely in the laboratory, cf.
commercially obtained ITS+). All solutions were in 2.5% serum.
After 72 h cell numbers were obtained in quadruplicate (±S.D.)
as described under ``Experimental
Procedures.''
Figure 3:
Dose dependence of linoleic acid-induced
growth recovery of thapsigargin-arrested DDTMF-2 cells.
Cells were thapsigargin-treated then exposed to linoleic acid at the
concentrations shown (together with 1% BSA) for 72 h as described under
``Experimental Procedures.'' Results are means ± S.D.
of cell numbers determined in quadruplicate
wells.
Key to determine was the
specificity of fatty acid-induced growth recovery of Ca 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
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 µM, 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) .
Figure 4:
Specificity of fatty acid-mediated growth
recovery of thapsigargin-arrested DDTMF-2 cells. Each of
the fatty acids shown was added at 100 µM together with 1%
fatty acid-free BSA in the presence of 2.5% serum. After 72 h cell
numbers (±S.D.) were obtained from quadruplicate wells as
described under ``Experimental
Procedures.''
Figure 5:
Dose dependence of arachidonic
acid-induced growth recovery of thapsigargin-arrested
DDTMF-2 cells. Thapsigargin-treated cells were exposed to
arachidonic acid at the concentrations shown (together with 1% BSA) for
72 h as described under ``Experimental Procedures.'' Results
are means ± S.D. of cell numbers obtained from quadruplicate
wells.
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 nonesterified fatty acid in the undiluted calf serum used was measured as approximately 530 µM; 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 µM, agreeing with the half-maximal effectiveness of linoleic or arachidonic acids given in Fig. 3and Fig. 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.
Figure 6:
Concentration dependence of serum-induced
growth recovery of thapsigargin-arrested DDTMF-2 cells.
Thapsigargin-treated cells were exposed to levels of serum shown or
DMEM without serum for 72 h as described under ``Experimental
Procedures.'' Results are means ± S.D. of cell numbers
obtained from quadruplicate wells.
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
thapsigargin-treated 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. 7where 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
µM 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 pump blockade and emptying of Ca
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).
Figure 7:
Assessment of the effects of serum or
linoleic acid on the sensitivity of DDTMF-2 cell growth to
thapsigargin. Normal cells were grown in the presence of the indicated
thapsigargin concentrations together with either standard 2.5% serum
(
), 20% serum (
), or 100 µM linoleic acid
together with 1% BSA and 2.5% serum (
). Cell numbers
(±S.D.) were obtained after 72 h from quadruplicate wells as
described under ``Experimental
Procedures.''
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 DDTMF-2
cells at up to 500 µM (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 µM; at 100
µM 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
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
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 pool depletion. Such recovery of
growth was the end result measured after 3 days. Previously we
demonstrated that one of the initial events following high serum
treatment of pool-depleted cells was induction of new functional SERCA
pump activity and inositol 1,4,5-trisphosphate-releasable
Ca
pools(13, 14) . Obviously it was
important to assess whether a similar early expression of
Ca
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
pools. Cells after treatment with thapsigargin have no measurable
inositol 1,4,5-trisphosphate-sensitive Ca
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
response to 10 µM bradykinin (Fig. 8A). 6 h after treatment of similarly arrested
cells with 100 µM 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 contained 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. 8reveal that this is the case and that regardless of
whether recovery is induced by serum or arachidonic acid, the
appearance of bradykinin-sensitive Ca
pools was
completely prevented with either cycloheximide or puromycin.
Figure 8:
Arachidonic acid and 20% serum both induce
recovery of agonist-sensitive Ca pools in
thapsigargin-arrested DDT
MF-2 cells via a process dependent
on protein synthesis. Cells were thapsigargin-treated under standard
conditions, then exposed for 6 h to either standard 2.5% serum (control), 20% serum, 20% serum with 16 µM puromycin, or 20% serum with 35 µM cycloheximide (CHX) (A) or 2.5% serum with 1% BSA (control), 100 µM arachidonic acid with 1% BSA (AA), 100 µM arachidonic acid with 1% BSA and 16
µM puromycin, or 100 µM arachidonic acid with
1% BSA and 35 µM cycloheximide (CHX) (B). Puromycin and cycloheximide were both added 30 min prior
to addition of high serum or arachidonic acid. Under each condition
Ca
release was measured in response to 10 µM bradykinin (BK) added at the arrow. Conditions
were otherwise as stated under ``Experimental Procedures,''
and results are typical of three similar
experiments.
The
specificity of fatty acid-induced appearance of functional
Ca pools was almost identical to that for growth
induction. Thus, as shown in Fig. 9, 6 h of treatment of
thapsigargin-arrested cells with 100 µM arachidonic or
linoleic acids caused complete induction of functional Ca
pools; 100 µM linolenic acid consistently induced
appearance of Ca
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
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 µM 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 µM arachidonic acid the
rate of onset of the bradykinin-activated Ca
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
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
but also a slower onset of Ca
release.
Figure 9:
Fatty acid specificity of
agonist-sensitive Ca pool recovery in
thapsigargin-arrested DDT
MF-2 cells. Thapsigargin-treated
cells were exposed for 6 h to either 100 µM arachidonic
acid (AA), 100 µM linoleic acid (LA),
100 µM
-linolenic acid (LnA), 100
µM palmitic acid (PA), or 100 µM
stearic acid (SA), in each case added with 1% BSA in the
presence of the standard growth medium containing 2.5% serum.
Ca
release was measured in response to 10 µM bradykinin (BK) added at the arrow. Results are
typical of three similar experiments, and conditions were otherwise as
stated under ``Experimental
Procedures.''
Figure 10:
Concentration dependence of arachidonic
acid-induced recovery of agonist-sensitive Ca pools
in thapsigargin-arrested DDT
MF-2 cells.
Thapsigargin-treated cells were exposed to arachidonic acid (AA) at the concentrations shown with 1% BSA for 1 h. Cells
were washed and recovery continued for a further 18 h in standard
growth medium containing 2.5% serum. Control cells received 1% BSA
without arachidonic acid. Ca
release was measured in
response to 10 µM bradykinin (BK) added at the arrow. Conditions were otherwise as stated under
``Experimental Procedures.'' Results are typical of three
similar experiments.
An important question is whether arachidonic acid
acts directly to stimulate Ca 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 µM 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, 29, 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 µM.
Figure 11:
Ca pool recovery in
thapsigargin-arrested DDT
MF-2 cells is activated by
arachidonic acid (AA) but not by 5,8,11,14-eicosatetraynoic
acid (ETYA). Thapsigargin-treated cells were exposed to either
100 µM arachidonic acid with 1% BSA, 100 µM ETYA with 1% BSA, or 1% BSA alone (control) for 6 h.
Ca
release in response to 10 µM bradykinin was measured as described under ``Experimental
Procedures.'' Results are typical of three similar
experiments.
The results presented
here provide compelling evidence that essential fatty acids can induce
recovery of cells that have been growth arrested by Ca pool depletion. This recovery involves both the return of
Ca
pumping pools and the transition of cells from a
G
-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
activity. The latter
appears unlikely since preliminary experiments have shown no effect of
phospholipase A
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. (
)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
pool depletion.