(Received for publication, August 14, 1995; and in revised form, September 13, 1995)
From the
Ca pool depletion with Ca
pump blockers induces growth arrest of rapidly dividing
DDT
MF-2 smooth muscle cells and causes cells to enter a
stable, quiescent G
-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.
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
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
-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
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.
The Ca pump blocker, thapsigargin, empties
intracellular Ca
pools (11) and rapidly
activates Ca
entry through SOCs(2) . In many
cells including DDT
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
(12) . As shown in Fig. 1A, in DDT
MF-2 cells, even 24 h after
thapsigargin-induced Ca
pool depletion and the
establishment of growth arrest, Ca
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
, SOC activity becomes reactivated as
reflected by the large ``overshoot'' in cytosolic
Ca
observed upon readdition of
Ca
; the transient nature of the overshoot
reflects a temporary activation of SOCs, which again undergo rapid
deactivation. Normal DDT
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
removal. Interestingly,
addition of 10 mM caffeine to the pool-depleted cells induces
a further rapid and substantial increase in
Ca
, which also appears to undergo
deactivation (Fig. 1A). This caffeine effect is not
dependent on brief Ca
removal since,
added directly to thapsigargin-arrested cells, caffeine induces the
same large transient Ca
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
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
(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
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
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
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
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
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
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
and A
adenosine
receptors(17) , militates strongly against Ca
influx being adenosine receptor-mediated. DDT
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
receptors are reported to be
inhibited by caffeine(18, 19) . Although there was no
logical explanation of our results based on InsP
receptor
alteration, examination of the effects of 10 mM caffeine on
InsP
-mediated Ca
release from
permeabilized DDT
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
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 (
)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
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. (
)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
-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
and
begin to enter G
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
MF-2
cells is accompanied by a return to the caffeine-insensitive state. A and C, DDT
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
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 Ca
influx 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.