Department of Pharmacology and Physiology, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Newark, New Jersey 07103
Submitted 28 April 2003 ; accepted in final form 27 May 2003
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ABSTRACT |
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fura 2; NCX1.1; myocyte
A major mechanism of exchanger regulation involves the binding of Ca2+ to sites within the hydrophilic domain (11). Many studies indicate that these sites must be filled with Ca2+ for the exchanger to work in any of its known modes of operationNa+/Ca2+, Na+/Na+, or Ca2+/Ca2+ exchange. Experiments with excised patches suggest that the KD for Ca2+ for the activation of exchange activity is 300-600 nM, whereas studies with intact cardiac myocytes or transfected cells have resulted in a lower range of values (25-125 nM) (reviewed in Refs. 6 and 18). The selectivity of the regulatory binding sites has not been systematically examined. Ba2+ was shown to activate exchange activity at higher concentrations than Ca2+ (24), but thus far no other Ca2+ surrogates have been tested.
La3+ has been widely used to block Ca2+-dependent transport activities including Ca2+ channels, ATP-dependent Ca2+ pumps, and Na+/Ca2+ exchange. Under the assumption that La3+ is impermeant, it has also been used in cardiac cells and tissues to displace Ca2+ from sarcolemmal binding sites (9) and to assess changes in plasma membrane permeability during ischemia (see, e.g., Refs. 19 and 23). Some reports questioned the assumption of La3+ impermeance in cardiac myocytes (4, 25). Indeed, Peeters et al. (15) presented convincing evidence that La3+ gains entry into ventricular cells by Na+/Ca2+ exchange; indirect evidence that La3+ is transported by the Na+/Ca2+ exchanger in chromaffin cells has also been presented (17). Here we provide definitive evidence that La3+ is transported by the Na+/Ca2+ exchanger and that it is also an extremely potent regulatory activator of exchange activity.
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EXPERIMENTAL PROCEDURES |
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Solutions. Na-physiological salt solution (PSS) contained (in mM) 140 NaCl, 5 KCl, 1 MgCl2, 10 glucose, and 20 3-(N-morpholino)propanesulfonic acid (MOPS), buffered to pH 7.4 with Tris. K-PSS contained (in mM) 140 KCl, 1 MgCl2, 10 glucose, and 20 MOPS, buffered to pH 7.4 with Tris. Mixtures of Na-PSS and K-PSS were prepared to contain 20 mM NaCl and 120 mM KCl (20/120 Na/K-PSS). Biochemicals were purchased from Sigma, unless indicated otherwise, and cell culture media, including fetal bovine serum, were from Life Technologies.
Fura 2 imaging. Cells were grown on 25-mm circular coverslips and
loaded with fura 2 by incubating the coverslips for 30 min at room temperature
in Na-PSS containing 1 mM CaCl2, 1% bovine serum albumin, 0.25 mM
sulfinpyrazone (to retard fura 2 transport from the cell), and 3 µMfura 2
AM (Molecular Probes). The coverslips were then washed in Na-PSS + 1 mM
CaCl2, placed in a stainless steel holder (bath volume 0.8 ml;
Molecular Probes), and viewed in a Zeiss Axiovert 100 microscope coupled to an
Attofluor digital imaging system. Alternating excitation at 334 and 380 nm was
obtained through the use of appropriate filters, and fluorescence was observed
at >510 nm. Forty to sixty individual cells were selected and monitored
simultaneously from each coverslip.
Experimental protocol. For all experiments, cells were washed in
Na-PSS + 0.3 mM EGTA and then treated in the same medium with 200 µM ATP (a
purinergic agonist) + 2 µM thapsigargin, an inhibitor of the
sarco(endo)plasmic reticulum Ca2+-ATPase, to release
Ca2+ from internal stores. In some experiments, the medium was
subsequently changed to 20/120 Na/K-PSS and 1 µg/ml gramicidin was applied
to permeabilize the plasma membrane to monovalent cations. Experiments were
begun 10 min after the application of ATP + thapsigargin. Changes in the
bath medium were carried out by manually applying
4 ml of the solutions
specified in individual experiments over a period of 15 s. All experiments
were carried out at room temperature. Each trace shown in Figs.
2,
3,
4,
5,
6 is representative of
experiments carried out with five or more coverslips. Statistical analyses
utilized Student's t-test for unpaired samples.
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Calibration and titration of fura 2 with La3+.
Fluorescence was measured at 510 nM with a cuvette-based Photon Technology
International RF-M 2001 fluorometer. A quartz cuvette contained 3.0 ml of 0.15
M KCl, 10 mM MOPS-Tris, 0.1 mM EGTA, and 0.1 µM fura 2, pH 7.1 (room
temperature). Excitation spectra (300-450 nm) were measured after each
successive addition of aliquots (0.5-2 µl) of 20 mM LaCl3.
La3+ concentrations ([La3+]) were computed with the MAXC
program (2) to correct for
effects of pH and ionic strength, assuming a pKD for the
La-EGTA complex of 15.79 (22).
All fluorescence values were corrected for background fluorescence (no fura
2). It should be noted that at La3+-to-EGTA ratios >0.8 (25
pM La3+), the MAXC program gave a cautionary message stating that
the computed concentrations might not be reliable.
Calibration of the fura 2 signal to obtain cytosolic [La3+]
([La3+]i) was not possible with the conventional
approach of Grynkiewicz et al.
(7). This was because fura 2 in
the cells could not be saturated with La3+ to obtain a value for
the maximal 334-to-380 ratio (Rmax) or for the ratio of
fluorescence at 380-nm excitation for unbound vs. La3+-saturated
fura 2 (Sf/Sb). La3+ uptake into the cells by
exchange activity attained a steady-state value that is far below
Rmax (cf. Fig. 2),
and ionomycin, a Ca2+ ionophore that is typically used to obtain
Rmax values for Ca2+, did not transport La3+
(0.1-20 mM) into the cell. Rmax and minimal 334-to-380 ratio
(Rmin) values for Ca2+ under the conditions of our
experiments are typically 6-7 and 0.4-0.5, respectively, with
Sf/Sb values of 5-6. As a rough estimate of
[La3+]i, based on the data in
Fig. 1B, we assume
that Rmax and Sf/Sb for La3+ are
2.4- and 1.8-fold greater, respectively, than the corresponding values for
Ca2+ and that the KD for the fura
2-La3+ complex is 30 pM. With these assumptions, we calculate that
[La3+]i 7-15 pM at a 334-to-380 ratio of 1.0.
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RESULTS |
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In Fig. 1C, the
fluorescence at 340-nm excitation in the presence of EGTA was subtracted from
the corresponding fluorescence values at each La3+ concentration
and the differences, F340 - F340(E), were plotted vs.
the free [La3+], computed as described in EXPERIMENTAL
PROCEDURES. The F340 - F340(E) values increased
nearly linearly with [La3+] before abruptly reaching a saturating
value; the [La3+] at which F340 - F340(E) was
half-maximal was 27 pM. Corresponding data obtained with Ca2+
instead of La3+ were well fit by a rectangular hyperbola and
yielded a half-maximal value at 257 nM (data not shown), a value close to the
previously determined KD for Ca2+ of 274 nM
under slightly different conditions at 22°C
(21). The reasons for the
unusual concentration profile for La3+ are not known with
certainty, but undoubtedly a major factor is the unreliability of the
estimates of free [La3+] at total La3+-to-EGTA ratios
exceeding 0.8 ([La3+] > 25 pM). It is clear, nevertheless, that
fura 2 has an affinity for La3+ that is 4 orders of magnitude
higher than for Ca2+, a finding that is consistent with the
exceedingly high affinity of La3+ for EGTA, which itself is nearly
5 orders of magnitude higher than that for Ca2+
(22).
La3+ transport by Na+/Ca2+ exchanger. CHO cells expressing the wild-type bovine cardiac Na+/Ca2+ exchanger (NCX1.1) were loaded with fura 2 to determine whether La3+ enters the cell by an NCX-mediated exchange for internal Na+. To eliminate possible interference by Ca2+, the cells were treated for 10 min with a combination of ATP, a purinergic agonist that elicits the rapid release of Ca2+ from internal stores, and thapsigargin, an inhibitor of the sarco(endo)plasmic reticulum Ca2+-ATPase. The cells were then placed in a medium containing 20 mM NaCl and 120 mM KCl (20/120 Na/K-PSS), and the plasma membrane was made permeable to monovalent cations by addition of 1 µg/ml gramicidin. As described elsewhere (5), gramicidin clamps cytosolic [Na+] at 20 mM and ensures a constant driving force for reverse exchange activity.
As shown in Fig.
2A, the addition of 0.1 mM La3+ to the
external medium resulted in an increase in the fura 2 ratio, reflecting
La3+ entry into the cell. La3+ uptake followed a
sigmoidal time course and did not attain a maximal rate of uptake until 40-50
s after La3+ addition; the early portion of the time course is
shown more clearly on the expanded time scale in
Fig. 2C. As discussed
below, we propose that the sigmoidal time course reflects a positive feedback
process due to the interaction of La3+ with the regulatory
Ca2+ binding sites of the exchanger. This interpretation is
supported by the data in Fig.
2B, which depicts an identical experiment using cells
expressing the (241-680) deletion mutant of NCX1.1. This mutant is
missing a large portion of the exchanger's regulatory domain and does not
require cytosolic Ca2+ to activate exchange activity. In contrast
to cells expressing the wild-type NCX1.1, the
(241-680) cells
accumulated La3+ immediately after its addition to the external
medium (Fig. 2C).
For both the wild-type and the mutant exchangers, the fura 2 signal approached an apparent steady state after several minutes of La3+ uptake. The addition of EGTA at this point (Fig. 2) resulted in a slow decline in the fura 2 ratio. This decline occurred at the same rate whether or not Na+ was present in medium (cf. Fig. 3B), indicating that it is not due to exchange-mediated La3+ efflux. Perhaps cytosolic La3+ is transported out of the cell by the plasma membrane ATPase or is slowly sequestered within an intracellular compartment. In any event, the results indicate that once La3+ enters the cell, it does not readily escape.
What is responsible for the apparent steady state of La3+ uptake? It seems likely that the accumulation of cytosolic La3+ inhibits the exchanger, resulting in a progressive inhibition of La3+ uptake. The results presented below in Fig. 5 support this interpretation. The subsequent addition of 1 mM CaCl2 produced a rapid increase in the fura 2 ratio (Fig. 2); most of the Ca2+ uptake under these conditions is due to Na+/Ca2+ exchange, indicating that the exchanger was not completely inactive at these [La3+]i. These results also indicate that the cytosolic fura 2 was not saturated with La3+ under these conditions.
Figure 3 displays the
results of several control experiments.
Figure 3A demonstrates
that La3+ accumulation was negligible when parental CHO cells,
devoid of Na+/Ca2+ exchange activity, were used in an
experiment identical to that shown in Fig.
2. Thus La3+ accumulation required the presence of the
exchanger. Figure 3B
shows that La3+ uptake by the wild-type exchanger required the
presence of cytosolic Na+. In this experiment, the cells were
treated with gramicidin in a Na+-free medium (K-PSS) to deplete the
cells of cytosolic Na+. Very little La3+ accumulation
was observed under these conditions (Fig.
3B). La3+ was removed, and the
[Na+] was increased to 20 mM. A second addition of La3+
then yielded results very similar to those depicted in
Fig. 2A. In this
experiment, EGTA was added in a Na+-free medium; the slow decline
in the fura 2 signal was similar to that seen in the presence of
Na+. Thus the apparent falloff in cytosolic La3+ under
these conditions is not due to exchange-mediated La3+ efflux. The
data in Fig. 3C address the
dependence of La3+ uptake on the external [Na+]. In this
experiment, the gramicidin treatment was omitted and cells expressing the
(241-680) mutant were exposed to 0.1 mM La3+ in the presence
of 140 mM Na+. A significant uptake of La3+ was
observed, but the rate of uptake was markedly accelerated when external
Na+ was removed (K+ replacement; see
Fig. 3C, bottom). Thus, as expected for Na+/Ca2+ exchange
activity, La3+ uptake is substantially reduced at high
extracellular [Na+] compared with Na+-free conditions.
The rates of La3+ uptake were similar at 0.01, 0.1, and 1 mM
La3+ (data not shown).
Regulatory activation of La3+ uptake by
Ca2+. For cells expressing the wild-type exchanger,
the low rate of La3+ uptake during the lag phase
(Fig. 2C) suggested
that the exchanger was in a deactivated state, reflecting the low
[Ca2+]i (20 nM) under these conditions. To test
this hypothesis, we conducted the experiment depicted in
Fig. 4, in which we used
Ca2+ influx through store-operated channels to elevate cytosolic
Ca2+ before initiating La3+ uptake. The store-operated
channels were activated by release of Ca2+ from the endoplasmic
reticulum as a consequence of pretreatment with ATP and thapsigargin.
Gramicidin was omitted in these experiments, because the resulting membrane
depolarization would inhibit Ca2+ influx through the store-operated
channels.
As shown in the control trace in Fig. 4A, when a Na+-free medium containing 0.1 mM La3+ was applied to the cells, La3+ uptake occurred slowly at first but then increased gradually over the ensuing several minutes. The sigmoidal time course of La3+ uptake was qualitatively similar to that shown in Fig. 2A, but the rate of La3+ uptake was much slower. The reduced exchange activity under these conditions probably reflects a lower initial [Na+]i than for the experiment in Fig. 2, as well as the progressive loss of cytosolic Na+ in the absence of external Na+. In the other traces in Fig. 4A, Na-PSS containing either 0.1 or 0.3 mM Ca2+ was applied for 30 s before initiation of La3+ uptake. Increases in cytosolic [Ca2+] were observed, reflecting Ca2+ entry through store-operated channels; as expected, the rise in [Ca2+]i was greater for 0.3 mM Ca2+ than for 0.1 mM Ca2+. The subsequent rates of La3+ accumulation were significantly higher after application of either solution than for the cells under control conditions (Fig. 4C; n = 5-6).
The initial lag in the time course of La3+ uptake appeared to be
eliminated or greatly reduced for the trace with 0.3 mM Ca2+. This
is shown in Fig. 4B,
which displays the fura 2 traces on an expanded time scale. For the trace
where 0.3 mM Ca2+ was applied, the data points after the initiation
of La3+ uptake represent a combination of a decline in
[Ca2+]i and a rise in [La3+]i and
the individual contributions of each ion cannot be determined. Assuming that
the linear portion of the trace reflects only La3+ accumulation,
extrapolating this portion of the trace back to the initial fura 2 ratio
suggests that La3+ uptake had begun without appreciable delay. In
contrast, a linear extrapolation of the trace for 0.1 mM Ca2+
suggests that there was at least a 30-s lag phase before the linear phase of
La3+ uptake began. Similar results were obtained in five other
experiments. The rise in cytosolic Ca2+ and the acceleration of
La3+ uptake were both abolished when SK&F-96365 (50 µM), a
blocker of store-operated Ca2+ channels
(13), was included in the
Ca2+-containing solution (data not shown). In similar experiments
with cells expressing the (241-680) mutant, the rate of La3+
uptake was not affected when [Ca2+]i was increased
before addition of La3+ (data not shown). We conclude that the
increased cytosolic Ca2+ in these experiments accelerated
La3+ uptake and reduced or abolished the initial lag phase because
of the activation of exchange activity through the regulatory Ca2+
binding sites.
Regulatory activation of Ca2+ uptake by La3+. In the experiment depicted in Fig. 5, we demonstrate the converse of the above relation, i.e., that cytosolic La3+ stimulates exchange-mediated Ca2+ uptake in cells expressing the wild-type exchanger. For the traces in Fig. 5A, the bath medium (Na-PSS + 0.3 mM EGTA) was replaced with Na+-free solution (K-PSS) containing either 0.1 mM EGTA (control) or 0.1 mM La3+. La3+ uptake occurred for 90 s, at which time the medium was replaced with Na-PSS + 0.1 mM EGTA (see Fig. 5A, bottom). The elevation in [La3+]i was maintained over the ensuing 90 s, and then Ca2+ uptake by Na+/Ca2+ exchange was initiated by replacing the medium with K-PSS + 0.1 mM Ca2+. Ca2+ uptake in the cells containing La3+ occurred rapidly, whereas for the control cells, there was a delay before rapid Ca2+ uptake was observed.
This behavior can be discerned more clearly in
Fig. 5B, which
displays the data on an expanded time scale. For the control trace,
Ca2+ entered the cells slowly at first and there was only a small,
gradual rise in [Ca2+]i. After 20 s, there was an
acceleration of Ca2+ uptake leading to a rapid rise in
[Ca2+]i (Fig. 5,
A and B). This behavior is analogous to what
happens during La3+ uptake and indicates that Ca2+
accelerates its own uptake through positive feedback mediated by the
regulatory Ca2+ binding sites. In the case of the cells that had
previously taken up a low level of La3+, rapid Ca2+
uptake occurred within the mixing time of the apparatus. The results of five
such experiments are summarized in Fig.
5C, which displays the rate of rise in the fura 2 signal
over an interval of 5-15 s after the solution change to K-PSS + 0.1 mM
Ca2+. The rates of Ca2+ uptake were 20-fold higher for
the cells with cytosolic La3+ compared with control cells.
Figure 5, D-F,
shows the results of similar experiments conducted with cells expressing the
(241-680) deletion mutant, in which activity is not regulated by
cytosolic Ca2+. It should be noted that Ca2+ uptake
began nearly immediately after the solution change, in sharp contrast to the
behavior of the wild-type cells. The presence of cytosolic La3+ did
not stimulate exchange activity in the mutant cells; indeed, La3+
inhibited the rate of Ca2+ uptake by 32%
(Fig. 5F). The
inhibition of exchange activity may reflect binding of La3+ to the
translocation sites of the exchanger.
We conclude that cytosolic La3+ activated the wild-type exchanger by binding to the regulatory Ca2+ binding sites, leading to a rapid Ca2+ influx at the time of the solution switch. Given the high affinity of fura 2 for La3+ (Fig. 1), the exchanger's regulatory Ca2+ binding sites appear to have an extremely high affinity for La3+.
Na+/La3+ exchange in neonatal cardiac myocytes. It is difficult to measure reverse-mode (Ca2+ influx) Na+/Ca2+ exchange activity unambiguously in cardiac myocytes because these cells contain voltage-activated Ca2+ channels whose activity would overwhelm the exchanger unless high concentrations of channel blockers were used. Moreover, Ca2+ entry into the cells initiates contraction, possibly leading to optical artifacts in connection with fura 2 measurements. Similar concerns would be applicable if Ba2+ were used instead of Ca2+. Because La3+ blocks cardiac Ca2+ channels and does not promote myocyte contraction (15), we felt that La3+ might be a useful Ca2+ surrogate for the measurement of exchange activity in these cells.
To explore this possibility, we treated neonatal rat cardiac myocytes with caffeine and thapsigargin to remove Ca2+ from the sarcoplasmic reticulum. The cells were then treated similarly to the transfected CHO cells, i.e., gramicidin was applied in Ca2+-free 20/120 Na/K-PSS and La3+ uptake was initiated by adding 0.1 mM La3+. As shown in Fig. 6, La3+ uptake by the myocytes occurred after a lag period and attained an apparent steady state after several minutes, just like the results obtained with the CHO cells in Fig. 2.
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DISCUSSION |
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In addition to being a substrate for transport, La3+ activates exchange activity through its interactions with the regulatory Ca2+ activation site. The evidence for this conclusion is as follows. 1) Cells expressing the wild-type exchanger displayed a lag period before maximal rates of La3+ uptake were attained (Fig. 2). 2) La3+ uptake was accelerated, and the lag period was abolished, when the exchanger was activated by an increase in [Ca2+]i before initiation of La3+ entry (Fig. 4). We conclude that the lag period reflects low exchange activity due to the absence of Ca2+ activation and that as La3+ accumulates within the cytosol it binds to the regulatory Ca2+ binding site and accelerates activity. A sigmoidal time course was also observed for exchange-mediated Ca2+ uptake (Fig. 5, A and B), for reasons that are entirely analogous to those discussed above, i.e., Ca2+ entry was initially slow but increased because of positive feedback as cytosolic Ca2+ progressively activated the exchanger.
Two additional observations support this interpretation. 1) With
the (241-680) mutant, which lacks the regulatory Ca2+
binding sites, La3+ uptake began without delay
(Fig. 2, B and
C) and the rate of La3+ uptake was not
increased by an increase in [Ca2+]i (data not shown).
Similarly, Ca2+ uptake by the
(241-680) mutant occurred
without delay (Fig.
5B). 2) Cytosolic La3+ accelerated
Ca2+ uptake and abolished the lag phase in cells expressing the
wild-type exchanger. As shown in Fig.
5C, the initial rate of Ca2+ uptake increased
20-fold after the uptake of a small quantity of La3+. Cytosolic
La3+ did not accelerate the activity of the mutant exchanger and in
fact inhibited activity (Fig.
5F). We conclude that La3+ binds to the
regulatory Ca2+ activation site of the wild-type exchanger and
accelerates exchange activity.
La3+ is effective at remarkably low concentrations. For the
cells expressing the wild-type exchanger, maximal rates of uptake for
La3+ occur at 334-to-380 ratios <0.8, as shown in
Fig. 2C. Moreover,
La3+ stimulates Ca2+ uptake by the wild-type exchanger
at similar 334-to-380 ratios, as shown in
Fig. 5B. Although we
cannot assign a precise value for [La3+]i in these
experiments, we can roughly estimate that a 334-to-380 ratio of 0.8
corresponds to [La3+]i 5-15 pM (see
EXPERIMENTAL PROCEDURES). We conclude that the regulatory
Ca2+ binding site in the wild-type exchanger has an affinity for
La3+ that is 3-4 orders of magnitude higher than for
Ca2+.
At similarly low concentrations, La3+ partially inhibits
Na+/Ca2+ exchange activity in (241-680) cells
(Fig. 5, E and
F), perhaps by binding to the exchanger's Ca2+
translocation sites. This inhibitory effect would also be expected to occur in
cells expressing the wild-type exchanger as well but cannot be detected
because the effects of regulatory activation by La3+ predominate.
Inhibition of exchange activity probably accounts for the time-dependent
decline in La3+ uptake shown in Figs.
2 and
6.
La3+ provides a useful means of assessing the properties of the exchange activity in cardiac myocytes. Measuring the reverse (Ca2+ influx) mode of exchanger operation has many advantages over the forward (Ca2+ efflux) mode in terms of assessing regulatory influences on exchange activity. In cardiac myocytes, however, Ca2+ or Ba2+ uptake through Ca2+ channels makes it difficult to develop an unambiguous assay for reverse exchange activity without adding high concentrations of channel blockers. Because La3+ is itself an effective blocker of Ca2+ channels, exchange activity can be easily assessed by using La3+ as a Ca2+ surrogate. As shown in Fig. 6, La3+ uptake displayed a sigmoidal time course in the myocytes, similar in all respects to that seen in the transfected CHO cells. Thus La3+ uptake measurements should provide a straightforward experimental approach for investigating the regulation of exchange activity in cardiac myocytes.
What are the physiological implications of the results presented here? The exceedingly low concentrations of La3+ that activate and partially inhibit the exchanger suggest that exchange activity might be similarly affected by endogenous heavy metal ions, which are present at low concentrations in most cells. Such an effect would have gone undetected by the common procedures used to measure exchange activity because these procedures use chelating agents such as EGTA or EGTA-based fluorescent probes that would be expected to chelate endogenous metals. We are currently developing approaches for examining the possible role of endogenous metal ions in regulating exchange activity.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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