ATP stimulation of
Na+/Ca2+
exchange in cardiac sarcolemmal vesicles
Graciela
Berberián1,
Cecilia
Hidalgo2,
Reinaldo
Dipolo3, and
Luis
Beaugé1
1 Instituto de
Investigación Médica Mercedes y Martín Ferreyra,
5000 Córdoba, Argentina;
2 Centro de Estudios
Científicos de Santiago, Santiago 9; and Departamento de
Fisiología y Biofísica, Facultad de Medicina,
Universidad de Chile, Santiago 7, Chile; and
3 Laboratorio de
Permeabilidad Iónica, Instituto Venezolano de
Investigaciones Científicas, Caracas 1020-A,
Venezuela
 |
ABSTRACT |
In cardiac
sarcolemmal vesicles, MgATP stimulates
Na+/Ca2+
exchange with the following characteristics:
1) increases 10-fold the apparent
affinity for cytosolic Ca2+;
2) a Michaelis constant for ATP of
~500 µM; 3) requires micromolar vanadate while millimolar concentrations are inhibitory;
4) not observed in the presence of
20 µM eosin alone but reinstated when vanadate is added;
5) mimicked by adenosine
5'-O-(3-thiotriphosphate), without the need for vanadate, but not by
,
-methyleneadenosine 5'-triphosphate; and 6) not
affected by unspecific protein alkaline phosphatase but abolished by a
phosphatidylinositol-specific phospholipase C (PI-PLC). The PI-PLC
effect is counteracted by phosphatidylinositol. In addition, in the
absence of ATP,
L-
-phosphatidylinositol
4,5-bisphosphate (PIP2) was able
to stimulate the exchanger activity in vesicles pretreated with PI-PLC.
This MgATP stimulation is not related to phosphorylation of the
carrier, whereas phosphorylation appeared in the phosphoinositides,
mainly PIP2, that
coimmunoprecipitate with the exchanger. Vesicles incubated with MgATP
and no Ca2+ show a marked
synthesis of
L-
-phosphatidylinositol
4-monophosphate (PIP) with little production of
PIP2; in the presence of 1 µM Ca2+, the net synthesis of PIP is
smaller, whereas that of PIP2
increases ninefold. These results indicate that
PIP2 is involved in the MgATP
stimulation of the cardiac
Na+/Ca2+
exchanger through a fast phosphorylation chain: a
Ca2+-independent PIP formation
followed by a Ca2+-dependent
synthesis of PIP2.
phosphorylation; phosphoinositides; membrane transport
 |
INTRODUCTION |
IN INTACT CELLS, the
Na+/Ca2+
exchanger is modulated by MgATP (2, 6, 13, 24). The nucleotide
stimulates all partial reactions of the carrier
(Na+o/Ca2+i, Na+i/Ca2+o,
Ca2+o/Ca2+i, and
Na+o/Na+i
exchanges, where subscripts o and i refer to extracellular and
intracellular, respectively). Among the kinetics effects, there is an
increase in the apparent affinity of the transporting sites for
Na+o and Ca2+i (6) as well as that of the
regulatory site for intracellular
Ca2+ (14). Most of the kinetic
data on the MgATP stimulation of Na+/Ca2+
exchange by ATP comes from work in injected and dialyzed squid axons.
In fact, until recently, this effect was difficult to
demonstrate in isolated membrane vesicles. Thus, in
cardiac sarcolemmal vesicles, an early reported MgATP activation
(9) was not seen by others (see Refs. 35, 37), perhaps because some
component that regulates the exchanger is modified or lost during
membrane isolation (35). However, recent work (11) has shown that in
giant membrane patches excised from cardiac myocytes MgATP does
activate the
Na+/Ca2+
exchange current. That finding encouraged us to reinvestigate the MgATP
actions in cardiac sarcolemmal vesicles. In addition, and with the
consideration of all preparations being used, the mechanism(s) of this
MgATP regulation is still under debate. Enzyme-catalyzed reactions at
phosphoryl centers are numerous, in particular those involving ATP (active transport, muscle contraction, oxidative phosphorylation, and photosynthesis). Phosphorylation reactions are
also involved in important modulations by protein kinases and
phosphatases. Experiments in dialyzed axons using metal(III)ATP complexes (17), nonhydrolyzable ATP analogs (13), and vanadate (19)
favor a phosphorylation-dephosphorylation mechanism (such as a
kinase-phosphatase system; see Ref. 15). On the other hand, initial
experiments on giant excised patches from cardiac myocytes suggested
that changes in membrane phospholipid distribution, caused by a
MgATP-dependent amino phospholipid translocase (flippase), could be
responsible for the MgATP effect (11). A basic unresolved question is
whether the exchanger is phosphorylated. Several attempts to find
phosphorylation of the carrier by MgATP have been unsuccessful (1),
including the dog cardiac antiporter expressed in COS cells (12). On
the other hand, while this work was in preparation, a series of
experiments indicated at least two possible ways to account for the
MgATP stimulation. On the one hand, a direct phosphorylation of the
exchanger, through a direct or indirect action of the protein kinase C
(PKC), that correlates with the transport rate was shown in cultured
smooth muscle (27). On the other hand, the observed stimulation seen in
giant excised patches from cardiac myocytes disappeared after
pretreatment with a phospholipase C specific for phosphatidylinositol
(PI-PLC), while
L-
-phosphatidylinositol 4,5-bisphosphate (PIP2) mimicked
the MgATP stimulation (25). The results presented here indicate that,
in cardiac sarcolemmal vesicles, MgATP stimulation of
Na+/Ca2+
exchange involves the synthesis of
PIP2 from
L-
-phosphatidylinositol (PI)
through a fast phosphorylation chain: a
Ca2+-independent formation of
L-
-phosphatidylinositol
4-monophosphate (PIP) followed by a
Ca2+-dependent synthesis of
PIP2. Conversely, phosphorylation
of the carrier itself was not detected. An additional important
conclusion is that all structures and enzymes responsible for MgATP
stimulation are membrane bound and copurify with the exchange activity.
Finally, although the actual mechanism is not known,
PIP2 stimulation seems to require
an intimate association with the carrier, since the newly synthesized
phosphoinositides and the exchanger coimmunoprecipitate. Parts of this
work have already been presented in abstract form (3, 5).
 |
METHODS |
Preparation of cardiac membrane vesicles.
Cardiac membrane vesicles were prepared by differential centrifugation
from beef heart obtained immediately after killing the animal (36). The
vesicles were loaded in a solution of 160 mM NaCl, 20 mM
3-(N-morpholino)propanesulfonic acid
(MOPS)-tris(hydroxymethyl)aminomethane (Tris) (pH 7.4), and 0.1 mM
EDTA; aliquots were stored in the same solution (2-3 mg
protein/ml) at
80°C. The orientation of the vesicles was
estimated using the asymmetric properties of the
Na+-K+-ATPase.
Na+-K+-ATPase
activity was measured after 30-min preincubation of membranes (0.5 mg/ml) at 20°C in 20 mM MOPS-Tris, pH 7.1, with or without sodium
dodecyl sulfate (SDS) (0.3 mg/ml). The basic composition of the assay
medium was (in mM) 130 NaCl, 20 KCl, 30 MOPS-Tris (pH 7.4), 3 MgCl2, and 3 ATP
([
-32P]ATP). The
vesicles (data not shown) were usually ~40% inside out, 35%
right-side out, and 25% leaky (see also Ref. 9). Total protein was
measured by the methods of Lowry et al. (32) with modifications (34)
using bovine serum albumin as standard.
Na+
gradient-dependent
Ca2+ uptake.
The Ca2+ uptake was estimated at
37°C by using
45CaCl2.
In routine assays, 2 µl of membranes were diluted in 100 µl of the
uptake solutions. These solutions had either low (10 mM) or high (160 mM) Na+. Low
Na+ had the equivalent to 260 mosmol N-methylglucamine chloride
(NMG-Cl) or bis-tris-propane-Cl (BTP). The concentration of external
ionized Mg2+ was kept constant at
5 mM. In addition, the usual extravesicular solutions contained 0.5 mM
vanadate and a final Ca2+ concentration
([Ca2+]) of
0.8-1.0 µM. In some cases, vanadate was absent, and in others, 20 µM eosin, a Ca2+ pump
inhibitor (22), was added. The composition of other solutions used is
given in the table and figure legends. The reaction was terminated
after 10 s (except in the time-dependent experiments) by adding 1 ml of
ice-cold quenching medium [200 mM KCl, 1 mM ethylene
glycol-bis(
-aminoethyl
ether)-N,N,N',N'-tetraacetic acid (EGTA), and 5 mM MOPS-Tris, pH 7.4]. The vesicles were then harvested by filtration on GF/F (Whatman) glass-fiber filters. To
correct for unspecific
45Ca2+
bound to vesicles and filters, blanks were run in which vesicles were
diluted into 160 mM NaCl-20 mM MOPS-Tris, pH 7.4, containing the same
concentration of Ca2+ as the assay
medium. Experimental points are the means of duplicate or triplicate
determinations, and each experiment was repeated at least twice.
Because most of the experiments consumed a large amount of vesicles, in
many cases different designs correspond to different vesicle
preparations; this may well explain the observed variability in the
Na+/Ca2+
exchange flux values (see Ref. 36). More details are given in the
corresponding figure and table legends.
Phosphorylation of cardiac membrane vesicles.
Vesicles (80-100 µg) were incubated for 10 s at 37°C in a
media with composition similar to the uptake solutions [20 mM
MOPS-Tris (pH 7.4), 20 mM NaCl (osmolarity was matched with NMG-Cl),
0.1 mM EGTA, 0.3 mM vanadate, 0.25 or 0.5 mM
[
-32P]ATP (500 counts · min
1 · pmol
1),
3 mM Mg2+, and 0.8 µM
Ca2+]. The methods of
stopping the reaction depended on the final assay that was programmed:
1) for immunoprecipitation studies, it was done by dilution (1:100, vol/vol) in an ice-cold solution containing 160 mM NaCl, 20 mM MOPS-Tris (pH 7.4), 10 mM EDTA, 1 mM
EGTA, 0.5 mM vanadate plus 0.25% Nonident P-40, 0.5% Triton X-100,
and antiprotease cocktail [1 µg/ml leupeptin, pepstatin, and
aprotinin and 0.2 mM phenylmethylsulfonyl fluoride (PMSF)]; and
2) for lipids, extraction by adding
1 ml ice-cold HCl and then 2 ml of an ice-cold mixture of 1:1 (vol/vol)
chloroform-methanol.
Immunoprecipitation, SDS-polyacrylamide gel electrophoresis, and
immunobloting.
Samples of cardiac membrane vesicles (80-100 µg protein) were
diluted 1:100 (vol/vol) in an ice-cold solution containing 160 mM NaCl,
20 mM MOPS-Tris (pH 7.4), 10 mM EDTA, 1 mM EGTA, 0.5 mM vanadate plus
0.25% Nonident P-40, 0.5% Triton X-100, and antiprotease cocktail (1 µg/ml leupeptin, pepstatin, and aprotinin and 0.2 mM PMSF). After 10 min at 0°C, the suspension was sonicated for 40 s and centrifuged
in an Airfuge centrifuge for 5 min. After centrifugation, the
supernatant was incubated for 3 h at room temperature with a 300-fold
dilution of the guinea pig serum (negative controls were run in
parallel with nonimmune guinea pig serum). Protein A-agarose (50 µl/ml, from ICN Biomedicals) was then added, and after an additional
1-h incubation at room temperature, the suspension was centrifuged and
the pellet was rinsed three times by repeated centrifugation and
resuspension. The immunoprecipitated proteins were eluted by adding 5 µl of five times concentrated SDS sample buffer, were heated for 30 min at 37°C, and were subjected to SDS-polyacrylamide gel
electrophoresis (PAGE) or to organic extraction for thin-layer
chromatography (TLC) separation of the lipids that coprecipitated.
Discontinuous SDS-PAGE was carried out according to the method
described by Laemmli (29). The proteins in the gel were visualized by
Coomassie blue R-250 staining and/or autoradiograph
(radiolabeled samples) or electrotransferred to polyvinylidene
difluoride (PVDF) transfer membranes for Western blots.
Guinea pig antibodies against the
NH2-terminal portion of the bovine
cardiac
Na+/Ca2+
exchanger (anti-NCX antiserum) (1) and anti-guinea pig immunoglobulin G-alkaline phosphatase conjugate (Sigma A.7686) were used. For Western
blots, the antibody was first adsorbed with nonimmnune guinea pig serum
immobilized in PVDF membranes. The molecular mass standards were GIBCO
prestained high molecular weight range.
Extraction and TLC separation of phospholipids.
Phospholipids were extracted from cardiac membrane vesicles that had
been incubated with
[
-32P]ATP and the
uptake solution without or with
Ca2+. Once the reaction was
stopped with 1 ml of ice-cold 1 N HCl, the lipids were extracted by
adding 2 ml of an ice-cold chloroform-methanol mixture (1:1, vol/vol).
The suspension was vortexed for 30 s, and the tubes were placed in ice
for ~30 min. The organic phase containing the lipids was carefully
removed, poured into conical tubes, and dried under a stream of
N2 gas while the tubes remained in
water at room temperature. A second extraction was not used because
initial results showed that it added minimal amount of additional
material. The dried lipids were redissolved in 10 µl of
chloroform-methanol (1:1, vol/vol) that were then applied in spots of
~3 mm diameter on high-efficiency TLC plates (Analtech, Newark, DE).
This procedure was repeated with another 10 µl of the same solution.
The plates (20 cm × 10 cm) were preactivated at 140°C for 1 h. The composition of solvent system was
methanol-chloroform-water-concentrated NH4OH (48:40:10:5, vol/vol) (21).
The 32P-labeled phospholipids were
visualized in an autoradiograph of the plates. The identification of
phosphatidylinositol mono- and bisphosphate was done by comigrating
commercial standards and submitting the plates to an atmosphere of
saturated iodine vapor. The places where the labeled spots were
localized were removed and counted in a liquid scintillation counter.
Solutions.
All solutions were made with deionized ultrapure water (18
water,
Milli-Q, Millipore). NaCl and KCl were from Baker Ultrex; all other
chemicals were reagent grade. ATP, adenosine
5'-O-(3-thiotriphosphate) (ATP
S), and
,
-methyleneadenosine 5'-triphosphate
(AMP-PCP), from Boehringer Mannheim, were transformed into Tris salts
by passing them through an Amberlite IR-120-P column. Ouabain,
digitoxigenin, Tris-OH, NMG, MOPS, BTP, alkaline phosphatase (type
VII-NLA from bovine intestinal mucous), PI-PLC (from
Bacillus cereus), PI, and
PIP2 were obtained from Sigma
Chemical. 45Ca, as chloride salt,
and [
-32P]ATP were
purchased from New England Nuclear. Calculations of free
[Mg2+] were done
taking a dissociation constant
(Kd) of
0.091 mM for MgATP (7) and 0.159 mM for MgAMP-PCP (38). The
Kd for MgATP
S was assumed to be equal to that of ATP. Free
[Ca2+] were estimated
by means of the MaxChelator program from Chris Patton taking the same
Kd values for ATP
and ATP
S.
 |
RESULTS |
Time dependence,
Ca2+ ionophore,
and exchange inhibitory peptide effects on the
Na+
gradient-dependent
Ca2+ uptake in
the absence and presence of ATP.
Two points had to be first established without ambiguity:
1) that the method for measuring
Ca2+ uptake indeed estimated the
45Ca2+
that went into the vesicles, and 2)
that our definition of Ca2+ fluxes
through the
Na+/Ca2+
exchanger, as the Na+
gradient-dependent Ca2+ uptake,
was correct. The first point is answered in the experiments illustrated
in Fig. 1, where the
Na+ gradient-dependent influx of
Ca2+ was measured as a function of
time in the absence (A) and presence (B) of 1 mM ATP. In both cases, the
other components of the incubation solutions were identical, among them
1 µM free Ca2+ and 5 mM free
Mg2+. The uptake of
Ca2+ can be considered linear at
10 s in both conditions; therefore, that time was used in most assays.
In addition, it is clear that the uptake in the presence of ATP doubles
that observed in its absence, i.e., there is an ATP stimulation of
Ca2+ influx in these vesicles.
That the observed fluxes are indeed such and not merely
Ca2+ absorbed to some
extravesicular matrix (9, 36) is shown by the fact that the addition of
30 µM (final concentration) of the
Ca2+ ionophore A-23187 produces a
release of all radioactivity present in the vesicles, and that is seen
without and with ATP. Notice that to inhibit the cardiac
Ca2+ pump vanadate was always
present (300-500 µM) in the extravesicular solution. We also
performed similar experiments but adding 20 µg oligomycin/mg total
protein to the incubation solutions. This oligomycin concentration was
more than enough to completely inhibit mitochondrial
Ca2+-ATPase (8). The results (not
shown) were identical to those of Fig. 1,
A and
B, thus ruling out artifacts due to
contamination with mitochondrial fragments.

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 1.
Time course of a Na+
gradient-dependent uptake of Ca2+
in bovine sarcolemmal vesicles. Vesicles were loaded with 160 mM NaCl,
20 mM MOPS-Tris (pH 7.4 at 37°C), and 0.1 mM EDTA. To measure
45Ca2+
influx, vesicles were incubated at 37°C in solutions containing
high (160 mM) or low (10 mM) NaCl, 5 mM free
Mg2+, 20 mM MOPS-Tris (pH 7.4 at
37°C), 0.1 mM EGTA, 0.5 mM vanadate, 0.1 mM digitoxigenin, and 1 µM Ca2+ without
(A) or with
(B) 1 mM ATP. Low
Na+ solutions contained equivalent
to 260 mosmol N-methylglucamine
chloride (NMG-Cl). At time indicated by arrows, 1 mM EGTA without ( )
or with ( ) 30 µM (all final concentrations) ionophore A-23187 was
added. We defined Na+ gradient
dependent to difference between
Ca2+ uptake in low minus that in
high extravesicular Na+. Data
points are means of duplicate determinations. Note the following:
1) in both
A and
B, uptake of
Ca2+ can be considered linear up
to 10 s; 2) ATP increases
Na+ gradient-dependent
Ca2+ uptake (compare
A with
B) by ~2-fold; and
3)
45Ca2+
accumulates into intravesicular compartment, and it is not
superficially bound, since upon addition of excess EGTA, radioactivity
from vesicles was lost only in presence of
Ca2+ ionophore. , Control
uptakes.
|
|
To test for the specificity of these fluxes as expression of
Na+/Ca2+
exchange activity, we used the exchange inhibitory peptide (XIP). This
peptide inhibits the exchanger by interacting with a small domain in
the inner large "regulatory" loop of the protein (31). In Table
1, it can be seen that at 20 µM
concentration XIP inhibits the Na+
gradient-dependent Ca2+ uptake.
There are some characteristics of that inhibition that are interesting:
the inhibition is not total, it increases when the vesicles are
preincubated with XIP, and it is more pronounced in the absence of ATP.
A partial inhibition of the
Na+/Ca2+
exchanger by XIP has already been reported for cardiac vesicles (28)
and for dialyzed squid axons (18); as a difference with our
preparation, in the squid axons the inhibition was the same without and
with ATP (18). In that preparation, XIP had to be injected; therefore,
the actual concentration inside the axon was not known. Given the fact
that in cardiac vesicles there was an increment in inhibition after
preincubation, it is possible that the dose used here was submaximal.
At any rate, and taken together, the results of this section indicate
that the Na+ gradient-dependent
Ca2+ uptake indeed represents
Ca2+ flux through the
Na+-Ca2+
countertransport system.
View this table:
[in this window]
[in a new window]
|
Table 1.
Effect of XIP on a Na+ gradient-dependent
Ca2+ uptake in bovine heart sarcolemmal vesicles in
the absence and presence of ATP
|
|
Extravesicular
Ca2+
concentration and ATP effect on the
Na+
gradient-dependent
Ca2+ uptake.
In intact cells, MgATP increases the affinity of the transporting and
regulatory sites for intracellular
Ca2+ (6). To investigate if this
is the case in our preparation, we studied the variation of
Na+ gradient-dependent
45Ca2+
uptake as a function of the extravesicular
[Ca2+] in the absence
and presence of ATP. Figure 2 summarizes
the results of three different experiments showing that all ATP
stimulation can be explained by an increase in the apparent affinity of
intracellular sites for Ca2+.
Actually, with the Michaelis constant for
Ca2+ values curve fitted, they
changed from 1.03 ± 0.08 µM in the absence of ATP to 0.12 ± 0.01 µM in its presence, i.e., ~10-fold decrease. In the
experiments described above, we used NMG as the Na+ replacement. If BTP was used
instead, the same results were obtained (data not shown), ruling out
artifacts due to salt-dependent MgATP stimulation. On the other hand,
the results shown in Fig. 2 may account for the variable results found
by different authors in the sense that if
[Ca2+] were
nonlimiting, stimulation by MgATP would not be detected. In fact,
lithium salts, commonly used as
Na+ replacement, usually have
Ca2+ as contaminant (for
references, see Ref. 36).

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 2.
Stimulation by extravesicular Ca2+
of a Na+ gradient-dependent uptake
of Ca2+ in bovine heart
sarcolemmal vesicles. Vesicles loaded with 160 mM NaCl, 20 mM MOPS-Tris
(pH 7.4 at 37°C), and 0.1 mM EDTA were incubated for 10 s in
solutions containing high (160 mM) or low (10 mM) NaCl, 20 mM MOPS-Tris
(pH 7.4 at 37°C), 5 mM free
Mg2+, 0.5 mM vanadate, 0.1 mM
digitoxigenin, 0.1 mM EGTA, and variable
Ca2+ concentrations without (open
symbols) or with (solid symbols) 1 mM ATP. Low
Na+ solutions contained equivalent
to 260 mosmol NMG-Cl. All data points are means ± SE of triplicate
determinations. Note the following:
1) both
Ca2+ activation curves can be
adequately fitted to a Michaelian function, and
2) addition of 1 mM ATP produced a
shift in the Michaelis constant for
Ca2+ from 1.03 ± 0.08 to 0.12 ± 0.01 µM.
|
|
Apparent affinity for ATP, reversibility, and nucleotide specificity
for stimulation of
Na+/Ca2+
exchange.
Figure 3 summarizes three experiments
showing that when ATP is added at the beginning of the uptake period
(10 s), together with 1 µM Ca2+,
it stimulates the exchanger following a hyperbolic curve with a
Michaelis constant for ATP of 490 ± 34 µM
(n = 3). However, in one case, the
curve was not Michaelian, reaching a plateau at 1-2 mM ATP. In
following up with the matter, we decided to investigate what was the
response when the vesicles were preincubated for 10 s with ATP, in the
absence and presence of Ca2+,
before the uptake period. The results of these experiments are presented in Table 2. When the vesicles are
preincubated with 1 mM ATP in the absence of
Ca2+ and the nucleotide
concentration is drastically reduced (to 26 µM) by dilution during
the Ca2+ uptake period, there is
no stimulation of the exchanger, i.e., the
Na+ gradient-dependent influx of
Ca2+ is not different from that
observed without or with 26 µM ATP. However, a very interesting
result is that preincubation with ATP and no
Ca2+ increases the apparent
affinity for the nucleotide; notice that the stimulation produced by
0.1 mM is identical to that observed with 1 mM ATP when the nucleotide
concentration in the preincubation is maintained during the uptake
period. On the other hand, when preincubation with ATP (0.1 and 0.5 mM)
was done in the presence of 1 µM
Ca2+, the stimulation of the
exchanger was identical without and with a large dilution of the ATP
concentration during the uptake period. These results indicate three
important points: 1) a
Ca2+-dependent reaction is
required for the MgATP stimulation of
Na+/Ca2+
exchange, 2) a
Ca2+-independent process seems
also associated with that effect, and 3) these reactions must be very fast
since stimulation with 1 mM ATP is the same with or without
preincubation.

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 3.
ATP stimulation curve of a Na+
gradient-dependent uptake of Ca2+
in bovine heart sarcolemmal vesicles. Vesicles loaded with 160 mM NaCl,
20 mM MOPS-Tris (pH 7.4 at 37°C), and 0.1 mM EDTA were incubated
for 10 s in solutions containing high (160 mM) or low (10 mM) NaCl, 20 mM MOPS-Tris (pH 7.4), 5 mM free
Mg2+, 0.5 mM vanadate, 0.1 mM
digitoxigenin, 0.1 mM EGTA, 1 µM
Ca2+, and variable concentrations
of ATP. Low Na+ solutions
contained equivalent to 260 mosmol NMG-Cl. All data points are means ± SE of triplicate determinations and were fitted to a Michaelian
equation. Michaelis constant for ATP was 0.480 ± 0.034 mM. See text
for more details.
|
|
View this table:
[in this window]
[in a new window]
|
Table 2.
Effects of preincubation with MgATP, in the absence and presence of
1 µM Ca2+, on ATP stimulation of a
Na+ gradient-dependent Ca2+ uptake
in bovine heart sarcolemmal vesicles
|
|
The ATP stimulation described above could have more than one
explanation. For instance, it could adhere to a "regulatory
nonphosphorylating" role of the nucleotide or, alternatively,
require the phosphorylation of a key structure, not necessarily the
carrier itself. To distinguish between these possibilities, we followed
the actions of two ATP analogs. One was the nonphosphorylating AMP-PCP,
which in other transport systems can sustain the nonphosphorylating
regulatory role of ATP (39); the other was ATP
S, which can act as
substrate for kinases but not for ATPases (10, 23) and in squid axons stimulates
Na+/Ca2+
exchange. The results are illustrated in Table
3. AMP-PCP, at 2 mM concentration, failed
to stimulate the exchange. On the other hand, ATP
S behaved exactly
as ATP; this similarity included the inability to sustain stimulation
if it is removed after a preincubation period in the absence of
Ca2+.
View this table:
[in this window]
[in a new window]
|
Table 3.
Comparative effects of ATP, AMP-PCP, and ATP S on a
Na+ gradient-dependent Ca2+ uptake in
bovine heart sarcolemmal vesicles
|
|
Need for and effects of vanadate.
To inhibit the Ca2+ pump, the
extravesicular solutions contained 200-500 µM vanadate. However,
vanadium, depending on the conditions (pH, ionic strength, etc.), can
acquire different states (vanadate, metavanadate, vanadyl, etc.); in
addition, we cannot be sure that a mixture of forms is present in our
case. This raises the possibility that some artifacts, including some
derived from complexing Ca2+,
could be introduced in the estimation of the fluxes. To remove vanadate, we took advantage of a recent observation showing that eosin,
at 20 µM concentration, can completely block the
Ca2+ pump in this preparation
(22). Accordingly, we explored the effects of ATP on
Na+/Ca2+
exchange in the presence of 20 µM eosin. Table
4 summarizes these experiments. The first
row shows experiments done in the presence of high extravesicular
Na+, where
Ca2+ uptake through the
Na+/Ca2+
exchanger is inhibited, so the main route for
Ca2+ entry is expected to be the
Ca2+ pump. That this is actually
the case is indicated by the entries in the second row, left column, of
Table 4 where addition of 20 µM eosin inhibits most of this
Ca2+ influx; however, to our
surprise, under these conditions, ATP also failed to stimulate
Na+/Ca2+
exchange (second row, right column with low extravesicular
[Na+]). As before, in
the presence of 200-500 µM vanadate, an ATP stimulation of the
exchanger was observed (third row). Comparison of the entries in
rows 3 and
4 of Table 4 indicate that eosin by
itself does not inhibit the ATP stimulation because that stimulation in
the presence of vanadate is roughly the same with and without eosin. At
this stage, we must point out that in the absence of ATP the
Na+ gradient-dependent
Ca2+ fluxes were the same in eosin
alone, vanadate alone, and eosin plus vanadate (not shown). Finally,
the last row describes extremely important results: ATP
S, which as
expected, is not a substrate for the
Ca2+ pump (left column),
stimulates
Na+/Ca2+
exchange even in the absence of vanadate. Therefore, whatever the
reason for the need of vanadate to see ATP stimulation of the exchanger
(see DISCUSSION), it has nothing to
do with artifacts in the estimation of the
Ca2+ uptake.
View this table:
[in this window]
[in a new window]
|
Table 4.
Effects of eosin and vanadate, separate or in combination, on
Ca2+ uptake of sarcolemmal vesicles in the presence of
high (gradient independent) and low (gradient dependent) extravesicular
[Na+]
|
|
There is another important aspect regarding vanadate effects on this
countertransport system. In squid axons, and in the presence of ATP,
the response of
Na+/Ca2+
exchange to vanadate is biphasic: a marked stimulation at low concentrations (mean affinity constant of ~5-10 µM) followed
by inhibition at higher concentrations (inhibition constant of ~1.5 mM) (16). Therefore, we explored whether a similar response was seen in
cardiac sarcolemmal vesicles. The results of one of these experiments,
described in Table 5, fully reproduce those observed in squid axons: 1) in the
absence of ATP, vanadate does not affect
Na+/Ca2+
exchange, and 2) in the presence of
1 mM ATP, there is a marked stimulation of the exchanger at 300 µM
vanadate, which is considerably reduced when the vanadate concentration
is 3 mM.
View this table:
[in this window]
[in a new window]
|
Table 5.
Effect of low (0.3 mM) and high (3 mM) vanadate concentrations on a
Na+ gradient-dependent Ca2+ uptake in
bovine heart sarcolemmal vesicles in absence and presence of ATP
|
|
Absence of carrier phosphorylation and lack of effect of alkaline
phosphatase.
So far we found that in cardiac sarcolemmal vesicles MgATP stimulation
of the
Na+/Ca2+
exchanger can be mimicked by MgATP
S, which is substrate for kinases,
whereas AMP-PCP, which is a nonhydrolyzable nucleotide, does not. The
kinase phosphorylation hypothesis is strong; the point is what
structures become phosphorylated. In searching for the target
molecules, we initially investigated the exchange carrier. To that end,
vesicles were incubated under identical conditions where
Ca2+ uptake was measured but in
the presence of 0.5 mM of
[
-32P]ATP. The
exchanger was immunoprecipitated with a specific
anti-Na+/Ca2+
exchanger antibody, subjected to SDS-PAGE, and blotted. Despite the
fact that the
Na+/Ca2+
exchange carrier was detected in the transferred membrane by using the
same specific antibody, a 10-day exposure to an X-ray film showed no
32P labeling. These results (not
shown) indicated that, under our experimental conditions, the carrier
does not become phosphorylated.
In dialyzed squid giant axons, the microinjection of an unspecific
alkaline phosphatase reverts all MgATP stimulation of
Na+/Ca2+
exchange (DiPolo and Beaugé, unpublished data). We therefore decided to explore that possibility in our preparation. To that aim,
the vesicles were preincubated for 2 min at 37°C with a
concentration of alkaline phosphatase similar to that used in axons
(200 U/ml), and then the system was assayed for MgATP stimulation of a
Na+ gradient-dependent
Ca2+ uptake. As indicated in Table
6, both basal and MgATP-stimulated Na+/Ca2+
exchange were insensitive to the presence of alkaline phosphatase. In
control experiments, with the same solutions used in the transport assays, we confirmed the effectiveness of the phosphatase using p-nitrophenyl phosphate as substrate
(data not shown). Note that the vanadate concentrations were 0.2 mM in
these cases. Although the inhibition constant for vanadate inhibition
of this particular phosphatase is >1 mM (data not shown), we decided
to reduce vanadate concentration a little.
View this table:
[in this window]
[in a new window]
|
Table 6.
Effect of an unspecific alkaline phosphatase on the
Na+ gradient-dependent Ca2+ uptake
in bovine heart sarcolemmal vesicles in the absence and presence
of ATP
|
|
Involvement of phosphoinositides in the MgATP stimulation of the
Na+/Ca2+
exchanger.
At the time these studies were done, a report indicating that in
cardiac sarcolemmal vesicles enriched with phosphoinositides there was
an increase in
Na+/Ca2+
exchange was published (33). The authors suggested that
phosphoinositides, particularly
PIP2, could mediate the MgATP
effects. If that hypothesis was correct, it could be that newly
synthesized (32P labeled)
phosphoinositides coprecipitated with the exchanger. This
possibility was explored by subjecting to TLC analysis a chloroform-methanol extraction of the immunoprecipitated exchanger previously incubated with radioactive ATP. The actual experimental conditions were 10 s at 37°C in the presence of 500 µM
[
-32P]ATP, 5 mM
Mg2+, 0.3 mM vanadate, 1 µM
Ca2+, and low
Na+. Figure
4 illustrates one of the autoradiographs
and indicates that our expectations were borne out. In Fig. 4,
lane 3 (organic extraction of the
immunoprecipitated exchanger), we can clearly see a spot corresponding
to PIP2 and another small spot
below which could represent
lyso-PIP2 (30).

View larger version (36K):
[in this window]
[in a new window]
|
Fig. 4.
One-dimensional thin-layer chromatography (TLC) separation of lipid
extracts from immunoprecipitated protein of
Na+-Ca2+
cardiac sarcolemmal membranes. Shown is an autoradiograph of
32P-labeled phospholipids from the
following samples: lane 1, heated (5 min at 80°C) denatured vesicles reacted with phosphorylating media
and exposed to NH2-terminal
portion of the bovine cardiac
Na+/Ca2+
exchanger (anti-NCX antibody); lane 2,
native vesicles exposed to phosphorylating media and then to nonimmune
guinea pig serum; lane 3, native
vesicles exposed to phosphorylating media and then to anti-NCX
antibody. Cardiac sarcolemmal vesicles (100 µg) were incubated for 10 s at 37°C in 20 mM MOPS-Tris (pH 7.4), 150 mM NMG-Cl, 20 mM NaCl,
0.1 mM EGTA, 0.3 mM vanadate, 0.5 mM
[ -32P]ATP, 3 mM
Mg2+, equivalent of 260 mosmol
NMG-Cl, and 0.8 µM Ca2+. Samples
were subjected to immunoprecipitation and then to chloroform extraction
and TLC separation as indicated in
METHODS. Note the following:
1) on right are indicated
positions of respective standards for phosphatidylinositol
4-monophosphate (PIP) and phosphatidylinositol 4,5-bisphosphate
(PIP2);
2) TLC were run in a mobile phase of
chloroform-methanol-H2O-concentrated
NH4OH (40:48:10:5, vol/vol) using
20 cm × 10 cm plates (high-efficiency TLC); and
3) only in lane
3 is phosphorylated
PIP2 detected.
|
|
The immediate next step, and following a recent publication from
Hilgemann and Ball (25), was to deplete and reinsert these compounds in
the membrane vesicles. The initial experiment was to follow the MgATP
effects in vesicles without and with previous exposure to PI-PLC. The
vesicles were preincubated for 4 min at 37°C in the absence of
Na+ gradient and of extravesicular
Ca2+ (160 mM NaCl, 20 mM
MOPS-Tris, pH 7.4) without or with 2 U/ml PI-PLC. After that time, the
Na+ gradient-dependent
Ca2+ uptake was measured in the
absence and presence of 1 mM ATP or 1 mM ATP
S under the usual
incubation conditions. Three experiments, each one done in triplicate,
are summarized in Table 7. They unmistakably show that pretreatment with PI-PLC completely blocks the
stimulation of
Na+/Ca2+
exchange by 1 mM of both ATP and ATP
S. The values for
Ca2+ uptake, which were the same
in the treated vesicles under the three conditions, are higher than
those observed in control vesicles in the absence of nucleotides;
although not statistically significant, this was consistently found. In
complementary experiments, vesicles initially subjected to the action
of PI-PLC (also 4 min at 37°C) were incubated, after PLC removal,
for 5 min at 0°C and for an additional minute at 37°C, with no
ligands, 200 µM PI, or 50 µM PIP2. Immediately thereafter,
45Ca2+
uptake was assayed in the same incubation solutions with and without 1 mM ATP. One of these experiments, listed in Table
8, shows additional important points: PI by
itself does not induce stimulation of
Na+/Ca2+
exchange, but it is able to restore the MgATP stimulation that had been
lost by treatment of the vesicles with PI-PLC. In addition, in the
absence of ATP, PIP2 produces a
stimulation of the exchanger similar to that obtained with 1 mM of the
nucleotide plus 200 µM PI.
View this table:
[in this window]
[in a new window]
|
Table 7.
Effect of preincubation with PI-PLC on Na+
gradient-dependent Ca2+ uptake in bovine heart
sarcolemmal vesicles in the absence and presence of ATP and
ATP S
|
|
View this table:
[in this window]
[in a new window]
|
Table 8.
Effects of PI, without and with ATP, and of PIP2 on
Na+ gradient-dependent Ca2+ uptake
in bovine heart sarcolemmal vesicles pretreated with PI-PLC
|
|
Relevant to these last findings are the following observations
described above: 1) if the vesicles
are preincubated with MgATP, stimulation of the exchanger persists upon
its removal only if there is Ca2+
in the preincubation solution, and
2) preincubation with MgATP and
without Ca2+ increases the
apparent affinity for the nucleotide effect. All together, these
results suggest that MgATP stimulation occurs via phosphatidylinol
phosphorylation that is Ca2+
dependent and very fast. On that basis, we decided to investigate the
32P labeling from
[
-32P]ATP of
phosphoinositides in membrane vesicles incubated for 10 s at
37°C with 0.5 mM
[
-32P]ATP in the
usual Ca2+ uptake solutions in the
absence and presence of 1 µM
Ca2+. A typical result of one of
the several experiments performed is illustrated in Fig.
5. In the absence of
Ca2+, there is a marked synthesis
of PIP with a minute production of
PIP2; in the presence of
Ca2+, the net synthesis of PIP is
reduced and that of PIP2 increases ninefold. These results are quite satisfying for they can explain what
happened with the experiment on fluxes. When there was ATP without
Ca2+ during the preincubation, a
lot of PIP was formed, but, because this compound is not effective in
stimulating the exchanger, there was no stimulation upon removal of the
nucleotide during the Ca2+ uptake
period. On the other hand, it produced enough PIP to serve as substrate
for PIP2 production during the
uptake period with ATP; that can account for the increase in the
apparent affinity for the nucleotide under those circumstances. In
addition, to concur with the notion that
PIP2 is responsible for the MgATP stimulation of
Na+/Ca2+
exchange, these results show that the production of
PIP2 is a very fast reaction.
Actually, it is so fast that with only a 5-s incubation we obtained
~80% of the levels of synthesis found at 10 s (6.4 ± 0.1 pmol
PIP2/mg protein at 5 s compared
with 8.3 ± 0.7 pmol PIP2/mg
protein at 10 s).

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 5.
32P labeling from
[ -32P]ATP of
phosphoinositides in membrane vesicles incubated with 0.5 mM
[ -32P]ATP in usual
Ca2+ uptake solutions in absence
and presence of 1 µM Ca2+.
A: autoradiograph of a 1-dimensional
TLC separation of 32P-labeled
phosphoinositides. Aliquots of 50-µg membrane vesicles were
incubated at 37°C in 20 mM MOPS-Tris (pH 7.4), 150 mM NMG-Cl, 20 mM
NaCl, 0.1 mM EGTA, 0.3 mM vanadate, 0.25 mM
[ -32P]ATP, and 5 mM
Mg2+ in absence
(lanes 1 and
2) and presence
(lanes 3 and
4) of 0.8 µM
Ca2+. After 10 s, reaction
was stopped and lipids were extracted as indicated in
METHODS. TLC plate was developed
in a mobile-phase of
chloroform-methanol-H2O-concentrated
NH4OH (40:48:10:5, vol/vol) using
20 cm × 10 cm high-efficiency TLC. Positions of PIP and
PIP2 standards are indicated.
B: absolute values of
32P incorporation in PIP and
PIP2 spots by comigrating
commercial standards and submitting plates to an atmosphere of
saturated iodine vapor. Data are expressed as pmol/mg protein present
in initial solution extracted with chloroform-methanol. Note the
following: 1) in absence of
Ca2+, a substantial net synthesis
of PIP coexists with a small production of
PIP2;
2) in presence of
Ca2+, net synthesis of PIP is
markedly reduced, and it is accompanied by ~9-fold increase in net
synthesis of PIP2. See text for
details.
|
|
 |
DISCUSSION |
There are three pieces of evidence indicating that the
45Ca2+
fluxes considered in this work in fact go through the
Na+/Ca2+
exchanger: 1) the radioactivity
measured is indeed inside the vesicles for it is rapidly lost when the
A-23187 Ca2+ ionophore is added to
the medium; 2) the uptake, both in
the absence and presence of ATP, is inhibited on the cytosolic
(extravesicular) side by XIP, a specific inhibitor of the
Na+/Ca2+
exchanger; and 3) we are dealing
with a Na+ gradient-dependent (or
inhibitable by Na+ on the
cis-side) flux. With these
considerations, there are two main questions to answer, Why was MgATP
stimulation not always detected in this preparation (see Ref. 37) or
was seen with different characteristics (9)? What are the mechanisms
underlying this regulation?
With regard to the first question, it must also be noted that our
results do not coincide completely with others who found an ATP effect
in heart preparations. For instance, the
Ca2+ concentration needed to see
the effect is much smaller than the 50 µM reported before for these
vesicles (9); actually, had we used that concentration, our system
would have been saturated and the stimulation by the nucleotide missed.
In addition, in our hands, stimulation by MgATP is insensitive to
nonspecific alkaline phosphatases. Also, compared with initial excised
patch data (11), we observed 1) a
much lower Michaelis constant for ATP,
2) ATP
S stimulation, and
3) an unexpected requirement for vanadate. Regarding later patch-clamp data (25), our results are in
much closer agreement (see below). Most likely, the disagreements are
related to the possibility that preparations like vesicles and membrane
patches are structurally and biochemically incomplete, lacking some
soluble, or loosely membrane attached, component required for the
metabolic regulation of the exchanger. Relevant to this point is the
observation that in squid optic nerve, even in the presence of
vanadate, MgATP stimulates the exchanger only when a soluble nerve
cytosolic factor is added (4, 20). Even more, if the axons are
subjected to prolonged dialysis, the ATP stimulation is lost (4) and
can be recovered when the cytosolic factor is injected into the axons
(20). This line of reasoning can also apply to exchangers expressed in
alien cells that may lack the biochemical machinery present in the
natural host cells.
With regard to the mechanism of stimulation, the first thing to notice
is that MgATP produces a 10-fold increase in the apparent affinity for
cytosolic Ca2+, i.e., stimulation
does not happen at saturating Ca2+
concentrations. An increment in the apparent affinity for cytosolic Ca2+, both for regulatory and
transport sites, due to MgATP has also been seen in dialyzed squid
axons (16). Needless to say, the molecular mechanisms underlying those
affinity changes remain unknown; however, in our case, they seem
related to the production of PIP2.
In this regard, our results concur with recent work of Hilgemann and
Ball (25) with the additional information that the effect of MgATP
involves a cascade of at least two reactions: 1) synthesis of PIP from PI that
does not require Ca2+ and
2) synthesis of
PIP2 from PIP for which the
presence of Ca2+ is essential.
Notice that the need of a
Ca2+-dependent phosphorylation
mechanism to account for MgATP stimulation of the
Na+/Ca2+
exchanger was proposed as early as in 1986 for squid giant axons (14).
The fact that PIP2
coimmunoprecipitates with the exchanger may speak of a close
association between these two molecules; however, and this must also be
stressed, the way(s) PIP2
interacts with the countertransport system remains unknown. However,
this may not necessarily be the only way for MgATP stimulation. On the
one hand, phosphorylation of the carrier molecule, induced by growth
factor (27) and PKC (26) and accompanied by stimulation of the
exchanger, has been reported in whole cells. On the other, when rat
heart exchanger is expressed in COS cells, the inhibition of
Na+/Ca2+
exchange after ATP depletion is similar to that observed when there is
a disruption of the cytoskeleton (12). All these results open the
possibility to multiple regulatory paths in this system.
Finally, an extremely interesting aspect of this work, and for which we
have no explanation, has to do with vanadate. This compound is needed
for MgATP and not for MgATP
S stimulation and produces a biphasic
response, stimulation followed by a decline. The original idea put
forward to account for similar results in squid axons was a protein
kinase-protein phosphatase system (2, 24). That idea was sustained by
some properties of vanadate: 1) it
stimulates tyrosine kinases (39), 2)
it is a powerful inhibitor of phosphotyrosil and less potent inhibitor
of serine/threonine phosphatases (40), and
3) it inhibits, with an inhibition
constant in the millimolar range, other protein kinases (unpublished
data). In our vesicle preparation, we did not detect phosphorylation of
the carrier but other protein bands did incorporate
32P; the same happens in squid
axon membranes, and that phosphorylation pattern is altered by
vanadate. Therefore, can we ensure that none of these proteins is
related to the metabolic regulation of the exchanger? No doubt in
isolated cardiac vesicles the PIP2 formation is essential MgATP stimulation of the exchanger, but is it
the only molecule involved in that process? Micromolar vanadate concentrations also interact with the metabolism of
phosphoinositides, stimulating PI phosphorylation (42), but nothing
is known about the effects of millimolar vanadate concentration on
phosphoinositides metabolism. Similar lack of information is found when
considering the interactions of ATP
S in these metabolic paths.
Actually, the efforts of our laboratories are presently oriented in
that direction.
 |
ACKNOWLEDGEMENTS |
We thank Myriam Siravegna for skillful technical assistance. We
also thank the generosity of Dr. Kenneth Philipson who provided us with
XIP and of Dr. John P. Reeves who gave us the antibody against the
NH2-terminal portion of the bovine
cardiac
Na+/Ca2+
exchanger. We are thankful to Frigorífico Bustos and
Beltrán (Córdoba, Argentina) for providing beef hearts.
 |
FOOTNOTES |
This work was supported by Consejo Nacional de Investigaciones
Científicas y Técnicas (Argentina) (CONICET) Grant
4904/97, CONICOR (Argentina) Grant 3511/95, Volkswagen-Stiftung Grant
I/72 122, Fundación Andes Grant C-12777/9, FONDECYT (Chile) Grant 1940369, and Consejo Nacional de Investigaciones Científicas y
Tecnológicas (Venezuela) Grant S1-2651. G. Berberián
and L. Beaugé are established investigators from
CONICET.
Address for reprint requests: L. Beaugé, Instituto de
Investigación Médica Mercedes y Martín Ferreyra,
Casilla de Correo 389, 5000 Córdoba, Argentina.
Received 5 August 1997; accepted in final form 21 November 1997.
 |
REFERENCES |
1.
Aceto, J. F.,
M. Condrescu,
C. Kroupis,
H. Nelson,
N. Nelson,
D. Nicoll,
K. D. Philipson,
and
J. P. Reeves.
Cloning and expression of the bovine cardiac Na+/Ca2+ exchanger.
Arch. Biochem. Biophys.
298:
553-560,
1992[Medline].
2.
Baker, P. F.,
and
H. G. Glitsch.
Voltage-dependent changes in the permeability of nerve membranes to calcium and other divalent.
Philos. Trans. R. Soc. Lond. B Biol. Sci.
270:
389-409,
1975[Medline].
3.
Beaugé, L., and G. Berberián. Membrane
components are involved in the ATP modulation of the
Na+/Ca2+
exchange in the heart (Abstract). Proc. Pan-Am. Assoc.
Biochem. Mol. Biol. 8th Pucón Chile 1996, p.
48.
4.
Beaugé, L.,
D. Delgado,
H. Rojas,
G. Berberián,
and
R. DiPolo.
A nerve cytosolic factor is required for Mg.ATP stimulation of a Na+ gradient-dependent Ca2+ uptake in plasma membrane vesicles from squid optic nerve.
Ann. NY Acad. Sci.
776:
208-216,
1996.
5.
Berberián, G.,
and
L. Beaugé.
ATP stimulation of a Na+ gradient-dependent Ca2+ uptake in cardiac sarcolemmmal vesicles.
Ann. NY Acad. Sci.
776:
282-283,
1996.
6.
Blaustein, M. P.
Effects of internal and external cations and ATP on sodium-calcium and calcium-calcium exchange in squid axons.
Biophys. J.
20:
79-110,
1977[Abstract].
7.
Campos, M.,
and
L. Beaugé.
Effects of magnesium and ATP on pre-steady-state phosphorylation kinetics of the Na+,K+-ATPase.
Biochim. Biophys. Acta
1105:
51-60,
1992[Medline].
8.
Caroni, P.,
and
E. Carafoli.
An ATP-dependent Ca2+-pumping system in dog heart sarcolemma.
Nature
283:
765-767,
1980[Medline].
9.
Caroni, P.,
and
E. Carafoli.
The regulation of Na+/Ca2+ exchanger of heart sarcolemma.
Eur. J. Biochem.
132:
451-460,
1983[Abstract].
10.
Cassidy, P.,
P. Hoar,
and
G. Kerrick.
Irreversible thiophosphorylation and activation of tension in functionally skinned rabbit ileum strip by [35S]ATP
S.
J. Biol. Chem.
21:
11148-11153,
1979.
11.
Collins, A.,
A. V. Somlyo,
and
D. W. Hilgemann.
The giant cardiac membrane patch method: stimulation of outward Na+/Ca2+ exchange current by MgATP.
J. Physiol. (Lond.)
454:
27-57,
1992[Abstract].
12.
Condrescu, M.,
J. P. Gardner,
G. Chernaya,
J. F. Aceto,
C. Kroupis,
and
J. C. Reeves.
ATP-dependent regulation of sodium-calcium exchange in Chinese hamster ovary.
J. Biol. Chem.
270:
9137-9146,
1995[Abstract/Free Full Text].
13.
DiPolo, R.
The effect of ATP on Ca efflux in dialyzed squid giant axons.
J. Gen. Physiol.
64:
503-517,
1974[Abstract/Free Full Text].
14.
DiPolo, R.,
and
L. Beaugé.
In squid axons reverse Na+/Ca2+ exchange requires internal Ca2+ and/or ATP.
Biochim. Biophys. Acta
854:
298-306,
1986.
15.
DiPolo, R.,
and
L. Beaugé.
In squid axons ATP modulates Na+/Ca2+ exchange by a Ca2+-dependent phosphorylation.
Biochim. Biophys. Acta
897:
347-353,
1987[Medline].
16.
DiPolo, R.,
and
L. Beaugé.
Regulation of the Na+/Ca2+ exchange. An overview.
Ann. NY Acad. Sci.
639:
100-111,
1991[Medline].
17.
DiPolo, R.,
and
L. Beaugé.
Effects of some metal-ATP complexes on Na+/Ca2+ exchange internally dialized squid axons.
J. Physiol. (Lond.)
462:
71-86,
1993[Abstract].
18.
DiPolo, R.,
and
L. Beaugé.
Cardiac sarcolemmal Na+-Ca2+-inhibiting peptides XIP and FMRF-amide also inhibit Na+/Ca2+ exchange in squid axons.
Am. J. Physiol.
267 (Cell Physiol. 36):
C307-C311,
1994[Abstract/Free Full Text].
19.
DiPolo, R.,
and
L. Beaugé.
Effects of vanadate on Mg-ATP stimulation of Na+/Ca2+ exchange support kinase-phosphatase modulation in squid axon.
Am. J. Physiol.
266 (Cell Physiol. 35):
C1382-C1391,
1994[Abstract/Free Full Text].
20.
DiPolo, R.,
G. Berberián,
D. Delgado,
H. Rojas,
and
L. Beaugé.
A novel cytoplasmic soluble protein is required for nucleotide (MgATP) modulation of Na+/Ca2+ exchange in squid nerve fibers.
FEBS Lett.
401:
6-10,
1997[Medline].
21.
Ferrell, J. E.,
and
W. H. Huestis.
Phosphoinositide metabolism and the morphology of human erythrocytes.
J. Cell Biol.
98:
1992-1998,
1984[Abstract].
22.
Gatto, C.,
C. C. Hale,
and
M. A. Milanick.
Eosin, a potent inhibitor of the plasma membrane Ca2+ pump does not inhibit the cardiac Na+/Ca2+ exchanger (Abstract).
Biophys. J.
66:
331,
1994.
23.
Gratecos, D.,
and
E. Fisher.
Adenosine 5'-O-(3-thiotriphosphate) in the control of phosphorylase activity.
Biochem. Biophys. Res. Commun.
58:
960-967,
1974[Medline].
24.
Haworth, R. A.,
A. B. Goknur,
D. R. Hunter,
J. O. Hegge,
and
H. A. Berkoff.
Inhibition of Ca2+ influx in isolated adult rat heart cells by ATP depletion.
Circ. Res.
60:
586-594,
1987[Abstract].
25.
Hilgemann, D. W.,
and
R. Ball.
Regulation of cardiac Na+,Ca2+ exchange and KATP potassium channels by PIP2.
Science
273:
956-960,
1996[Abstract].
26.
Iwamoto, T.,
Y. Pan,
S. Wakabayashi,
T. Imagawa,
H. I. Yamanaka,
and
M. Shigekawa.
Phosphorylation-dependent regulation of cardiac Na+-Ca2+ via protein kinase C.
J. Biol. Chem.
271:
13609-13615,
1996[Abstract/Free Full Text].
27.
Iwamoto, T.,
S. Wakabayashi,
and
M. Shigekawa.
Growth factor-induced phosphorylation and activation of aortic smooth muscle Na+/Ca2+ exchanger.
J. Biol. Chem.
270:
8996-9001,
1995[Abstract/Free Full Text].
28.
Kleiboeker, S. B.,
M. A. Milanick,
and
C. C. Hale.
Interactions of the exchange inhibitory peptide with Na+/Ca2+ exchange in bovine cardiac sarcolemmal vesicles and ferret red cells.
J. Biol. Chem.
267:
17836-17841,
1992[Abstract/Free Full Text].
29.
Laemmli, U. K.
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227:
680-685,
1970[Medline].
30.
Lagos, L.,
and
J. Vergara.
Phosphoinositides in frog skeletal muscle: a quantitative analysis.
Biochim. Biophys. Acta
1043:
235-244,
1990[Medline].
31.
Li, Z.,
D. Nicoll,
A. Collins,
D. W. Hilgemann,
A. G. Filoteo,
J. T. Penniston,
J. N. West,
J. M. Tomich,
and
K. D. Philipson.
Identification of a peptide inhibitor of cardiac sarcolemmal Na+-Ca2+ exchanger.
J. Biol. Chem.
266:
1014-1020,
1991[Abstract/Free Full Text].
32.
Lowry, O. H.,
N. J. Rosebrough,
A. L. Farr,
and
R. J. Randall.
Protein measurements with the Folin phenol reagent.
J. Biol. Chem.
193:
265-275,
1951[Free Full Text].
33.
Luciani, S.,
M. Antolini,
S. Bova,
G. Cargnelli,
F. Cusinato,
P. Debetto,
L. Trevisi,
and
R. Varotto.
Inhibition of cardiac sarcolemmal sodium-calcium exchanger by glycerophosphoinositol 4-phosphate and glycerophosphoinositol 4,5-bisphosphate.
Biochem. Biophys. Res. Commun.
206:
674-680,
1995[Medline].
34.
Markwell, M. A. K.,
S. M. Hass,
L. L. Bieber,
and
N. E. Tolbert.
A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples.
Anal. Biochem.
87:
206-210,
1978[Medline].
35.
Reeves, J. P.
The sarcolemma sodium-calcium exchange system.
Curr. Top. Membr. Transp.
25:
77-119,
1985.
36.
Reeves, J. P.
Sodium-calcium exchange activity in plasma membrane vesicles.
In: Cellular Calcium, edited by J. G. McCormack,
and P. H. Cobbold. Oxford, UK: Oxford Univ. Press, 1991, p. 283-297.
37.
Reeves, J. P.,
and
K. D. Philipson.
Sodium-calcium exchange activity in plasma membrane vesicles
In: Na+/Ca2+ Exchange, edited by T. Allen,
D. Noble,
and H. Reuter. Oxford, UK: Oxford Univ. Press, 1989, p. 27-53.
38.
Rossi, R.,
and
J. P. Garrahan.
Steady-state kinetic analysis of the Na+,K+-ATPase. The effect of adenosine 5'[
,
-methylene]triphosphate on substrate kinetics.
Biochim. Biophys. Acta
981:
85-94,
1989.
39.
Simons, T. J. B.
The interaction of ATP analogues possessing a blocked
-phosphate group with the sodium pump in human red cells.
J. Physiol. (Lond.)
244:
731-739,
1975[Abstract].
40.
Swarup, G.,
S. Cohen,
and
D. L. Garbers.
Inhibition of membrane phosphotyrosyl-protein phosphatase activity by vanadate.
Biochem. Biophys. Res. Commun.
107:
1104-1109,
1982[Medline].
41.
Tracey, A. S.,
and
M. J. Gresser.
Interaction of vanadate with phenol and tyrosine: implications for the effects of vanadate on system regulated by tyrosine phosphorylation.
Biochemistry
83:
609-613,
1986.
42.
Yang, D. C.,
A. B. Brown,
and
T. M. Chan.
Stimulation of tyrosine-specific protein phosphorylation and phosphatidylinositol phosphorylation by orthovanadate in rat liver plasma membrane.
Arch. Biochem. Biophys.
274:
659-662,
1989[Medline].
AJP Cell Physiol 274(3):C724-C733
0363-6143/98 $5.00
Copyright © 1998 the American Physiological Society