(Received for publication, August 7, 1995; and in revised form, December 20, 1995)
From the
The effects of extracellular Na on
store-dependent Ca
influx were compared for
transfected Chinese hamster ovary cells expressing the bovine cardiac
Na
-Ca
exchanger (CK1.4 cells) and
vector-transfected control cells. Store-dependent Ca
influx was elicited by depletion of intracellular Ca
stores with ionomycin, thapsigargin, or extracellular ATP, a
purinergic agonist. In each case, the rise in
[Ca
]
upon the addition
of extracellular Ca
was reduced in CK1.4 cells
compared with control cells at physiological
[Na
]
. When
Li
or NMDG was substituted for Na
,
the CK1.4 cells showed a greater rise in
[Ca
]
than control
cells over the subsequent 3 min after the addition of
Ca
. Under
Na
-free conditions, SK& 96365 (50
µM), a blocker of store-operated Ca
channels, nearly abolished the thapsigargin-induced rise in
[Ca
]
in the control
cells but only partially inhibited this response in the CK1.4 cells. We
conclude that in the CK1.4 cells, Ca
entry through
store-operated channels was counteracted by
Na
-dependent Ca
efflux at physiological
[Na
]
, whereas
Ca
entry was enhanced through
Na
-dependent Ca
influx in the Na
-free medium. We examined the
effects of thapsigargin on Ba
entry in the CK1.4
cells because Ba
is transported by the
Na
-Ca
exchanger, but it enters these
cells only poorly through store-operated channels, and it is not
sequestered by intracellular organelles. Thapsigargin treatment
stimulated Ba
influx in a Na
-free
medium, consistent with an acceleration of Ba
entry
through the Na
-Ca
exchanger. We
conclude that organellar Ca
release induces a
regulatory activation of Na
-Ca
exchange activity.
Agents that promote the production of 1,4,5-inositol
trisphosphate (InsP) (
)give rise to a biphasic
increase in cytosolic Ca
. The initial, transient
phase is primarily due to release of Ca
from
intracellular stores, whereas the more prolonged plateau phase involves
an accelerated influx of extracellular Ca
. The
Ca
influx pathway involves low conductance
Ca
channels and is activated, through a poorly
understood mechanism, by the loss of Ca
from the
InsP
-sensitive
stores(1, 2, 3, 4) . This process is
designated as capacitative Ca
entry (3) or
store-dependent Ca
influx (SDCI)(4) . When
cells are exposed to a Ca
-mobilizing agent in the
absence of extracellular Ca
, the SDCI pathway remains
activated (even after removal of the Ca
-mobilizing
agent) until Ca
is restored and the
InsP
-sensitive stores refill with Ca
.
Inhibitors of sarco(endo)plasmic reticulum Ca
ATPase
such as thapsigargin (Tg) prevent Ca
reaccumulation
by the InsP
-sensitive stores, resulting in prolonged
activation of SDCI(5) .
The
Na-Ca
exchanger is a
carrier-mediated transport process that couples the transmembrane
movement of 3 Na
ions to the movement of a single
Ca
in the opposite direction. In cardiac cells, it is
widely accepted that the exchanger transports a portion of the
Ca
released from the sarcoplasmic reticulum out of
the cells by Na
-dependent
Ca
efflux and in this way regulates the amount of
stored Ca
available for release during subsequent
beats (reviewed in (6) ). However, in many other types of cells
the downstream response to Ca
-mobilizing agents
depends more on the sustained influx of Ca
following
agonist addition than on the magnitude of the Ca
transient itself. For noncardiac cells, there is a wealth of
evidence supporting the generalized activity of
Na
-Ca
exchange in mediating
Ca
efflux and in modulating the Ca
content of intracellular stores(7) . However, the precise
physiological role of the exchanger and its interactions with other
Ca
hemeostatic mechanisms is not as clearly defined
in noncardiac cells as in myocardial cells.
A major difficulty in
investigations of exchanger function in intact cells is the absence of
a selective inhibitor for exchange activity. In this report, we utilize
transfected CHO cells to bypass this limitation. CHO cells do not
normally express Na-Ca
exchange
activity, but after transfection with an expression vector coding for
the bovine cardiac Na
-Ca
exchanger,
high levels of activity are observed(8, 9) . Comparing
the effects of Na
on Ca
mobilization
between transfected and vector-transfected control cells provides a
means of identifying functional roles of exchange activity in relation
to other cellular mechanisms for intracellular Ca
handling. The results of this study indicate that Ca
efflux via the Na
-Ca
exchanger
limits the rise in [Ca
]
during sustained Ca
entry, suggesting a
potential modulatory function for Na
-Ca
exchange in the Ca
signaling process.
Additional evidence suggests that the exchanger itself undergoes a
regulatory activation during Ca
release from
intracellular stores.
Figure 1:
Rate of store refilling
in CK1.4 cells (left panel) and vector-transfected controls (right panel). Intracellular Ca stores were
depleted by treatment with ionomycin and placed in nominally
Ca
-free Na-PSS containing 0.3% bovine serum albumin (cf. ``Experimental Procedures''). CaCl
(1 mM) was added at 30 s (arrows), and after
the desired interval, 3 mM EGTA was added (
) followed
by 0.3 mM ATP 30 s later (
). Individual traces are
displaced vertically by one 340/380 ratio unit for clarity; note the
different ordinate scales for the left and right
panels. For the control cells, the
[Ca
]
values at the
peak of the Ca
transients were 95, 110, 220, and 190
nM after exposure to 1 mM CaCl
for 0.5,
1, 2.5, and 5 min, respectively. For the CK1.4 cells, the peak
[Ca
]
values were 40,
113, and 160 after 0.5, 2.5, and 5 min of exposure,
respectively.
For the CK1.4
cells, the rate of store refilling was markedly increased in a
Na-free medium (Li substitution). As shown in Fig. 2(left panels), the increase in
[Ca
]
upon the addition of
CaCl
in Li-PSS was larger than in Na-PSS, and the
[Ca
]
transients elicited by ATP
were markedly increased; in contrast, there was no significant
difference between the sodium- and lithium-based media for the control
cells (right panels, Fig. 2). The peaks of the
Ca
transients in the CK1.4 cells after 30 and 150 s
of exposure to Ca
were 48 ± 2 nM (n = 5) and 140 ± 13 nM (n = 3) in Na-PSS as compared with 140 ± 22 nM (n = 3) and 427 ± 83 nM (n = 5) in Li-PSS, respectively. The differences between the
sodium- and lithium-based media were highly significant (p < 0.01). The comparable values for the control cells were 112
± 31 nM (n = 5) and 327 ± 46
nM (n = 8) in Na-PSS versus 109
± 25 nM (n = 3) and 312 ± 64
nM (n = 5) in Li-PSS. The difference between
the CK1.4 cells and the control cells in Na-PSS (150 s) was significant (p < 0.01); other comparisons showed no significant
differences. The larger size of the
[Ca
]
transient for the CK1.4
cells in the Na
-free medium was primarily a reflection
of the increased amount of Ca
in the
InsP
-sensitive stores. As shown below (see Fig. 8),
the ATP-evoked [Ca
]
transient
in cells containing equivalent amounts of stored Ca
was smaller in Na-PSS than in Li-PSS, but the effect of
Na
was small compared with that shown in Fig. 2. The results in Fig. 1and Fig. 2indicate
that extracellular Na
reduces the rate of store
refilling in CHO cells expressing the
Na
-Ca
exchanger.
Figure 2:
Effect of Na-PSS and Li-PSS on store
refilling in CK1.4 cells (A and B) and
vector-transfected controls (C and D). Fura-2-loaded
cells were treated with ionomycin and placed in nominally
Ca-free Na-PSS (bold traces; error bars
down) or Li-PSS (light traces; error bars up).
CaCl
(1 mM) was added at 30 s (arrows),
and after either 30 (A and C) or 150 s (B and D), 3 mM EGTA was added (
) followed
by 0.3 mM ATP 10 s later (
). The results are the mean
values for three to five determinations in two (control) and three
(CK1.4) independent experiments.
Figure 8:
Effect of Na on
[Ca
]
transients
elicited by ATP or ionomycin. CK1.4 cells were loaded with fura-2,
preincubated in 100 µl of Na-PSS + 1 mM CaCl
, and added to a cuvette containing 3 ml of Na- or
Li-PSS containing 0.3 mM EGTA. At the times indicated, 0.3
mM ATP (left panel) or 2 µM ionomycin (right panel) were added to the cuvette. The data in each
panel are the mean values from six to eight determinations in three
experiments.
Figure 3:
Effect of Tg on Mn entry
in CK1.4 cells (left panel) and control cells (right
panel). Fura-2-loaded cells were pretreated with Tg and placed in
Na-PSS + 1 mM CaCl
. MnCl
(0.2
mM) was added at 30 s, and the fluorescence (emission, 510 nm;
excitation, 360 nm) was monitored. The results are the mean values of
four to six determinations from three independent experiments with each
cell type.
The right panel of Fig. 4shows that the addition of 1 mM CaCl to Tg-treated control cells produced a marked
increase in [Ca
]
, which was
essentially identical in Na-PSS or Li-PSS. For CK1.4 cells, however (left panel, Fig. 4), the rise in
[Ca
]
was greatly reduced in
Na-PSS (trace c) compared with Li-PSS (trace a). Traces b and d depict the rise in
[Ca
]
in Li-PSS and Na-PSS,
respectively, for CK1.4 cells that had not been treated with Tg. For
these cells, the rise in [Ca
]
in Li-PSS (trace b) was significantly greater than in
Na-PSS (trace d) but was still much less than that shown by
Tg-treated cells in Li-PSS (trace a). Similar results were
obtained when NMDG was used as the Na
substitute
instead of Li
(data not shown). The average values of
[Ca
]
observed between 90 and
120 s after the addition of CaCl
for the data shown in Fig. 4are summarized in Table 1. Compared with the
vector-transfected control cells, Tg-treated CK1.4 cells exhibited a
reduced [Ca
]
in Na-PSS but an
elevated [Ca
]
in Li-PSS (p < 0.01 in each case). The initial portions of the traces from
the CK1.4 cells and control cells in Na-PSS are directly compared in
the inset to the right panel of Fig. 4. The
increases in [Ca
]
are identical
initially but as [Ca
]
approaches 100 nM, the rise in
[Ca
]
slows dramatically in the
CK1.4 cells; the difference between the two types of cells becomes
statistically significant (p < 0.05) at all times after the asterisk shown in the inset.
Figure 4:
Effect of Na on Ca
entry in Tg-treated CHO cells.
Fura-2-loaded CK1.4 cells (left panel) or control cells (right panel) were pretreated with or without 200 nM Tg and placed in nominally Ca
-free Na- or
Li-PSS. CaCl
(1 mM) and EGTA (3 mM) were
added as indicated. For the CK1.4 cells, the traces labeled a and c correspond to Tg-treated cells in Li-PSS and
Na-PSS, respectively; the traces labeled b and d correspond to untreated cells in Li-PSS and Na-PSS. For the
control cells (right panel) the data are presented with error bars down for Li-PSS and error bars up for
Na-PSS. The results are the mean values of five determinations from
four independent experiments except for trace d, which
represents nine determinations from five experiments. Inset,
direct comparison of data for Tg-treated CK1.4 cells and control cells.
The differences between the two types of cells are significant (p < 0.05 or less) for all points following the asterisk.
A similar pattern of
results was obtained in Ca
flux
experiments. The data in Fig. 5depict
Ca
uptake by vector-transfected control
and CK1.4 cells in Na-PSS and in Na-free PSS (NMDG substitution).
Ca
accumulation under these conditions
is markedly enhanced at alkaline pH, and so these experiments were
conducted at an external pH of 8.0. As shown in the right panel of Fig. 5, Tg treatment (filled symbols)
stimulated
Ca
uptake in the control
cells in both Na-PSS and in NMDG-PSS. For the CK1.4 cells, however (left panel, Fig. 5), Tg had practically no effect on
Ca
uptake in Na-PSS, although in
NMDG-PSS it stimulated
Ca
uptake as well
as in the control cells. The results in NMDG-PSS were similar to those
obtained with Li-PSS (data not shown). Thus, the conclusions of the
Ca
experiments are consistent with those
obtained in the fura-2 measurements: Tg-induced Ca
entry was markedly reduced in the CK1.4 cells by the presence of
extracellular Na
.
Figure 5:
Effect of Tg on Ca
uptake in Na
-free
media. CK1.4 cells (left panel) or control cells (right
panel) were treated with Tg (filled symbols) in
Ca
-free Na-PSS and assayed for
Ca
uptake in either Na-PSS (squares) or NMDG-PSS (circles). The unfilled
symbols represent data for cells not treated with Tg. The
transport assays were conducted at pH 8.0. The results are the means of
two separate experiments for each cell
type.
In many types of cells,
Ca entry via the SDCI pathway is blocked by SK&
96365(15, 16) . As shown in the right panel of Fig. 6, 50 µM SK& 96365 sharply
reduced the rise in [Ca
]
in
vector-transfected control cells that had been treated with Tg prior to
adding 1 mM CaCl
; SK& was equally effective
in Na- or in Li-PSS. In contrast, SK& only partially inhibited the
rise in [Ca
]
for Tg-treated
CK1.4 cells in Li-PSS (left panel). Thus, in the CK1.4 cells a
substantial fraction of Ca
enters the cell via a
pathway that is insensitive to inhibition by SK& 96365; because
this pathway is absent in the control cells, it probably reflects
Ca
influx via reverse
Na
-Ca
exchange. Incubation of the
Tg-treated cells under Na-free conditions for 5 min prior to adding
CaCl
reduced the increase in
[Ca
]
and increased its
sensitivity to SK& 96365, consistent with a reduced
[Na
]
and decreased contribution
of Ca
influx via the exchanger (data not shown). Note
also that the decline in [Ca
]
after addition of EGTA was slowed by the presence of SK&
96365 (left panel, Fig. 6), consistent with previous
reports of an inhibitory effect of this agent on Ca
efflux(16, 17) .
Figure 6:
Effect
of SK& 96365 on Ca entry in Tg-treated CK1.4
cells and vector-transfected controls. Control cells (right
panel) were pretreated with 200 nM Tg and placed in
nominally Ca
-free Na-PSS or Li-PSS with or without 50
µM SK& 96365. CaCl
(1 mM) and
EGTA (3 mM) were added as indicated. For the CK1.4 cells (left panel), only results with Li-PSS are shown, and the data
with SK& 96365 are depicted with downward error bars;
note the slower decline in [Ca
]
after the addition of EGTA in the presence of SK&
96365. For the control cells, the data in Li-PSS are shown with downward error bars. The results are the means of three to six
determinations in three experiments for the control cells and seven or
eight determinations for five or six experiments for the CK1.4
cells.
Figure 7:
Effect of Na on
Ca
efflux in CK1.4 cells. CK1.4 cells were loaded
with fura-2 and preincubated for 1 min in 100 µl of Li-PSS
containing 1 mM CaCl
plus 200 nM Tg. The
cells were then diluted into a cuvette containing 3 ml of Na-PSS or
Li-PSS, and [Ca
]
was
monitored continuously thereafter. The inset depicts the
initial declines in [Ca
]
in Li-PSS (filled symbols) and Na-PSS (open
symbols) as first order plots; the data points were fit to third
order polynomials in each case. The data are the mean values from six
to eight determinations in four
experiments.
The data in the inset of Fig. 7depict the decline in
[Ca]
during the first 40 s of
the experiment, expressed as a first order plot. Nonlinear first order
plots were observed in both Na- and Li-PSS. At equivalent
concentrations of cytosolic Ca
, the rate of decline
in [Ca
]
was approximately
2.5-fold greater in Na-PSS than in Li-PSS throughout the concentration
range examined.
Figure 9:
Effect of ATP in presence of extracellular
Ca in CK1.4 cells (left panel) and control
cells (right panel). For these experiments, cells were
preincubated in Na-PSS containing 1 mM CaCl
prior
to adding them to the cuvette. The cuvette contained nominally
Ca
-free PSS plus 0.3 mM EGTA with either 140
mM NaCl (traces labeled c), 140 mM LiCl (a), or 40 mM NaCl + 100 mM KCl (b) as the principal cations. After 30 s of incubation, the
following additions were made: 0.3 mM ATP plus 1 mM CaCl
(top panel), 0.3 mM ATP alone (center panel), or 1 mM CaCl
alone (bottom panel). The stock mixture of 15 mM ATP +
50 mM CaCl
added to the cuvette formed a
precipitate that dissolved immediately upon dilution in the cuvette.
The results with error bars are the mean values from three or
four determinations from three experiments; where error bars
are not shown, mean values from two separate experiments are
presented.
The effects of ATP in the presence extracellular
Ca are compared for vector-transfected control cells
and the CK1.4 cells in Fig. 9. The cuvette solutions in these
experiments initially contained 0.3 mM EGTA and PSS with 140
mM NaCl, 140 mM LiCl, or 40 mM NaCl +
100 mM KCl as the principal salts. The effects of adding 0.3
mM ATP plus 1 mM CaCl
(upper
traces, Fig. 9), ATP alone (center traces, Fig. 9), or CaCl
alone (lower traces, Fig. 9) were examined for each cell type. For the control cells (right panels, Fig. 9), the addition of ATP alone
produced a transient rise in
[Ca
]
, whereas the addition of
ATP + 1 mM CaCl
elicited a sustained increase
in [Ca
]
, which was higher than
that evoked by the addition of Ca
alone. There were
no major differences in the behavior of the control cells among the
various media, although there was a tendency for the sustained phase of
Ca
entry to be reduced in the 40/100 sodium/potassium
medium (trace b, upper right panel, Fig. 9);
the latter effect is probably due to the reduced driving force for
Ca
entry in cells depolarized by the high potassium
concentration.
For the CK1.4 cells (left panel, Fig. 9), dramatic differences were observed among the different
media when ATP and Ca were added together. In 140
mM Na
(trace c; upper left
panel, Fig. 9), the initial
[Ca
]
transient was followed by
a sustained phase that was slightly but significantly reduced (p < 0.05) compared with that observed in the control cells. The
levels of [Ca
]
attained in the
Na-free or 40 mM Na
media (traces a and b; upper left panel, Fig. 9) were
much higher than those produced by the addition of Ca
alone (lower left panel, Fig. 9) and were
markedly elevated compared with the sustained
[Ca
]
levels in the
vector-transfected control cells, particularly in the case of Li-PSS.
Ca
entry was similarly enhanced when NMDG was used as
the Na
substitute (data not shown). As in the case of
Tg-treated cells, the sustained increases in
[Ca
]
in the low
[Na
] media were only partially inhibited by
SK& 96365 (data not shown), suggesting that a portion of the
Ca
entry under these conditions was conducted by the
Na
-Ca
exchange system. Intracellular
Ca
stores refilled rapidly after ATP addition
(assessed by ionomycin-induced Ca
release; data not
shown) indicating that Ca
sequestration was not
blocked under these experimental conditions. The pronounced increase in
[Ca
]
in the low
[Na
] media suggests that exchange activity
might have been accelerated during the release of Ca
from InsP
-sensitive stores. This possibility is
explored further in the experiments described below.
The data in Fig. 10(right panel) show the effects of adding 1
mM BaCl to control cells with or without prior
treatment with Tg. Ba
entry produces an initial
abrupt rise in the 350/390 ratio in these experiments, which is
probably due to small amounts of extracellular fura-2. The subsequent
gradual rise in the 350/390 ratio reflects Ba
entry
into the cells and is only slightly enhanced in Tg-treated cells (trace a, Fig. 10) compared with untreated cells (trace b, Fig. 10). No difference between Li-PSS and
Na-PSS was observed, and so the results with both media have been
combined in each trace. Ba
influx therefore occurs
only weakly through the SDCI pathway in these cells. Note that after
the addition of EGTA at 180 s, only a small decline in the fura-2
signal was observed, indicating that Ba
is not
readily transported out of the cell under these conditions.
Figure 10:
Effect of Tg on Ba influx in CHO cells. Control (right panel) and CK1.4 (left panel) cells were loaded with fura-2, treated with or
without 200 nM Tg for 1 min in Ca
-free
Na-PSS + 0.3 mM EGTA, and placed in a cuvette containing
Ca
-free Na- or Li-PSS with 0.3 mM EGTA.
BaCl
(1 mM) was added at 30 s, and EGTA (10
mM) was added at 180 s. No difference was observed between Na-
or Li-PSS for the control cells, and the combined results are shown.
For the CK1.4 cells, no effect of Tg was observed in Na-PSS, and the
combined results with and without Tg are shown. Control cells, trace a, Tg-treated cells in Na- or Li-PSS; trace b,
nontreated cells in Na- or Li-PSS. CK1.4 cells: trace a, Tg-treated cells in Li-PSS; trace b, nontreated cells in
Li-PSS; trace c, Tg-treated and nontreated cells in Na-PSS.
The results are the mean values of four determinations from three
experiments for the CK1.4 cells and six or seven determinations from
three experiments for the control cells.
In CK1.4
cells (left panel, Fig. 10), Ba entry
in Li-PSS is substantially increased by Tg (trace a, Fig. 10) compared with untreated cells (trace b, Fig. 10); the fura-2 ratios attained in trace a (Fig. 10) are significantly higher than observed in the
vector-transfected control cells. SK& 96365 (50 µM)
had no effect on Ba
entry in the CK1.4 cells (data
not shown), consistent with the absence of significant barium entry via
store-operated channels. In Na-PSS, Ba
influx (trace c, Fig. 10) was only slightly less than in the
nontreated cells in Li-PSS (trace b, Fig. 10); no
difference was observed between Tg-treated and untreated cells in
Na-PSS, and both conditions have been combined in trace c (Fig. 10). Because a significant acceleration of
Ba
influx is observed only in Tg-treated CK1.4 cells
under Na
-free conditions, we conclude that Tg
treatment accelerates Na
-Ca
exchange
activity.
In the experiments described above, the cells had been
pretreated with Tg in Ca-free Na-PSS, and it is
conceivable that increased Na
entry might be
responsible for the subsequent acceleration of Ba
entry by the Na
-Ca
exchanger.
Therefore, we determined whether Tg could accelerate Ba
entry when added to the cells in a Na
-free
medium. CK1.4 cells were loaded with fura-2 and placed in a cuvette
containing either Ca
-free Na-PSS or Li-PSS and 0.3
mM EGTA. After 30 s, either 200 nM Tg or vehicle
(dimethyl sulfoxide) was added to the cuvette, and 1 mM BaCl
was added 2 min later. As shown in Fig. 11, in the absence of Tg treatment, there was little or no
difference between Na- or Li-PSS for barium entry in the CK1.4 cells (A) or the control cells (C). For both types of cells (Fig. 11, B and D), the addition of Tg
resulted in a slowly developing transient rise in the 350/390
fluorescence ratio, which undoubtedly reflects the gradual release of
Ca
from internal stores. (The choice of 350/390
excitation wavelengths optimizes the Ba
signal but
still allows detection of increases in
[Ca
]
.) For the Tg-treated
control cells (Fig. 11D), no difference was observed in
Ba
entry between the Na-PSS and the Li-PSS; the
ratios were not significantly higher than those observed in the absence
of Tg, again indicating that Ba
does not enter these
cells efficiently by the SDCI pathway. However, the Tg-treated CK1.4
cells (Fig. 11B) exhibited an accelerated
Ba
entry in Li-PSS (trace a) compared with
Na-PSS (trace b). We conclude that the Tg-induced acceleration
of Ba
entry via Na
-Ca
exchange is independent of a possible effect of Tg on
Na
entry. When the CK1.4 cells were treated with 200
nM ionomycin (which does not transport Ba
; (24) ) in otherwise identical experiments, similar results were
obtained, i.e. ionomycin stimulated Ba
uptake in Li-PSS but not in Na-PSS (data not shown).
Figure 11:
Effect of Tg on Ba entry in the presence and the absence of extracellular
Na
. CK1.4 cells (left panel) and control
cells (right panel) were loaded with fura-2, washed once in
Ca
-free Na-PSS containing 0.3 mM EGTA, and
resuspended in cuvettes containing 3 ml of either Na-PSS or Li-PSS
(nominally Ca
-free plus 0.3 mM EGTA). Tg
(200 nM; B and D) or dimethyl sulfoxide (6
µl; A and C) was added at 30 s, and 1 mM BaCl
was added at 150 s. For B, trace
a, Tg-treated CK1.4 cells in Li-PSS; trace b, Tg-treated
CK1.4 cells in Na-PSS. In all experiments, data obtained in Li-PSS are
shown with error bars up. The results are the mean values of
ten or eleven determinations from six experiments for the CK1.4 cells
and four to eight determinations from four experiments for the control
cells.
The expression of the cardiac
Na-Ca
exchanger in a cell type that
does not normally exhibit this activity confers several new attributes
to cellular Ca
homeostasis. In the CK1.4 cells,
physiological concentrations of extracellular Na
retard store refilling and attenuate Tg-induced Ca
entry compared with control cells. Upon the addition of
Ca
to Tg-treated cells, the initial rise
in [Ca
]
is identical in CK1.4
cells and in control cells until [Ca
]
reaches 100 nM, when the increase slackens markedly in
the CK1.4 cells (inset, Fig. 4).
Na
-dependent Ca
efflux
provides the simplest explanation for these results. We suggest that a
portion of the Ca
entering the cell through
store-operated Ca
channels is transported back out of
the cell by the exchanger, thereby reducing net Ca
entry and attenuating the rise in
[Ca
]
during SDCI. In this view,
the exchanger generates circulatory movements of Ca
across the plasma membrane during Ca
channel
activity; this circulation could play an important role in
Ca
signaling processes, as suggested by Alkon and
Rasmussen(27) .
The effects of Na in stimulating Ca
efflux (Fig. 7) and
reducing the [Ca
]
transients
elicited by ATP or ionomycin (Fig. 8) lend support to this
interpretation. However, these data were obtained in
Ca
-free media and do not necessarily reflect the
exchanger's activity in the presence of physiological
[Ca
]
. On one hand, the
efficiency of the exchanger as a Ca
pump could be
substantially reduced by Ca
entry via
Na
-Ca
or
Ca
-Ca
exchanges. On the other hand, local gradients due to
Ca
channel activity might elevate
[Ca
]
beneath the plasma
membrane compared with that of the bulk cytosol. The exchanger's
rapid turnover (>2,000 s
; (25) and (26) ) and high K
for Ca
(4 µM; ref. 26) would ensure a high level of
efficiency in transporting Ca
from a region with
locally elevated [Ca
]
.
In
the absence of Na, store refilling and
Tg-induced Ca
influx are accelerated in CK1.4 cells
compared with control cells (Fig. 1, Fig. 2, Fig. 4, and Fig. 5and Table 1). Under these
conditions, Ca
efflux via the exchanger is blocked
and Ca
enters the cell both by SK&-sensitive
channels (Fig. 6) and by ``reverse mode''
Na
-Ca
exchange
(Na
-dependent Ca
influx), a process that is insensitive to the SK& compound.
An unexpected feature of our results is that exchange activity is
accelerated during Ca
release from internal stores.
In Na-free or low [Na
] media, the plateau
levels of [Ca
]
following the
simultaneous addition of ATP and Ca
to
CK1.4 cells were greatly enhanced compared with either the addition of
Ca
alone or the responses of the control cells (Fig. 9). Although these observations are consistent with an
increase in exchange activity, alterations in other Ca
handling pathways could also contribute to the results.
The
Ba experiments provide firm support for activation of
the exchanger during Ca
release. The fura-2 signal
for Ba
provides a better index of divalent cation
influx than that for Ca
because Ba
is not sequestered by intracellular organelles, and its cytosolic
concentration should therefore be unaffected by blockade of
sarco(endo)plasmic reticulum Ca
ATPase activity.
Moreover, Ba
entry via store operated Ca
channels appears to be minimal in CHO cells ( Fig. 10and Fig. 11), allowing a better assessment of alterations in
exchange activity. With CK1.4 cells, Tg differentially stimulates
Ba
entry in Li-PSS compared with Na-PSS but has no
such effect with the control cells (Fig. 10, 11). Similar
results were obtained using ionomycin to release Ca
from internal stores (data not shown). Recent experiments
indicate that ATP also evokes a transient increase in exchange-mediated
Ba
influx in the CK1.4 cells. (
)
It is
important to distinguish between regulatory activation of exchange
activity and increased activity that is simply due to a rise in the
concentration of Na or Ca
as a
transport substrate. Although increased Ca
efflux via
the exchanger following organellar Ca
release has
been reported on numerous occasions, to our knowledge a linkage between
Ca
release and regulatory activation of exchange
activity has not been shown previously in any cell type. In our
experiments, enhanced exchange activity was measured as
Na
-dependent Ba
influx
and could be observed in a Na
-free medium (Fig. 11). We therefore conclude that exchange activity is
activated by a regulatory process during Ca
release
from internal stores.
The mechanism of activation is uncertain. Two
modes of regulation of the cardiac exchanger have been described:
ATP-dependent regulation and secondary Ca activation(28, 29, 30, 31) . A
phosphorylation mechanism has been suggested for ATP-dependent
activation of exchange activity in squid giant axons(28) , but
experiments with sarcolemmal membrane patches (29, 30, 31) and our previously published
results with CK1.4 cells (9) raise doubts as to the relevance
of this mechanism for the cardiac exchanger. Moreover, preliminary
experiments indicate that the Tg-induced increase in Ba
entry is not inhibited by the nonspecific protein kinase
inhibitor staurosporine (1 µM; data not shown). Thus, it
seems unlikely that exchange activity is accelerated by a protein
kinase-dependent mechanism. Other suggested modalities of ATP-dependent
regulation, such as aminophospholipid translocase activity (30) or cytoskeletal alterations (9) , have not yet
been tested.
Secondary activation by Ca would seem
to be a plausible mechanism, because acceleration of exchange activity
is linked to Ca
release from internal stores. This
mode of regulation involves the interaction of Ca
with regulatory sites distinct from the transport sites for
Ca
that lie within the central hydrophilic domain of
the exchanger. The molecular identity of these sites has recently been
described by Philipson and his colleagues(32, 33) .
Secondary Ca
activation has been extensively studied
in squid giant axons(28, 34) , barnacle muscle (35) , myocardial cells(36) , and cardiac sarcolemmal
membrane patches (29, 31, 37) , but its
physiological importance is not well understood.
The data in Fig. 9(ATP) and Fig. 11(Tg) are consistent with
secondary Ca activation as the mechanism accelerating
exchange activity, because in both cases the increased Ca
or Ba
influx was associated with an elevation
in [Ca
]
. However, under
conditions where the cells were pretreated with Tg, there does not
appear to be an elevation of [Ca
]
compared with untreated cells prior to adding extracellular
Ca
( Fig. 4and Fig. 6) or
Ba
(Fig. 10). Thus, it appears that
accelerated exchange activity does not necessarily correlate with
increased cytosolic Ca
. Experiments currently under
way suggest that Ca
-dependent changes in
exchange activity involve complex interactions between cytosolic
Ca
and the Ca
content of internal
stores. Additional studies will be required to resolve these issues.
Regardless of the precise mechanism(s) involved, our findings
indicate that Ca release from intracellular stores is
coupled to regulatory activation of Na
-Ca
exchange activity. The exchanger is potentially a focal point for
a variety of regulatory influences, as suggested by reports that the
sensitivity of the exchanger to secondary activation by Ca
can itself be modulated by an ATP-dependent
mechanism(19) . Activation of exchange activity during
Ca
release could therefore provide an adjustable
negative feedback mechanism for controlling the amount of
Ca
that is resequestered by the sarco(endo)plasmic
reticulum and, as demonstrated in this report, for limiting net
Ca
entry into the cell during Ca
channel activity. Na
-Ca
exchange activity thus provides a potentially rich source of
regulatory control for cellular Ca
traffic.