From the
Although the Na
The Na
The
Na
The regulatory properties
of the cardiac Na
For the cardiac
Na
In the present study, we provide the first evidence
that the Na
SDS-PAGE and immunoblotting were performed
essentially as described
(25) . The immunoblots were visualized
using the ECL detection system (Amersham Corp.).
Phosphoamino
acid analysis was performed on the phosphorylated
Na
As shown in Fig. 6, treatment of cells with PDGF-BB or
In the present study, we demonstrated that the
Na
PDGF-BB
was the most potent stimulator of exchanger phosphorylation among the
agents tested. Tryptic phosphopeptide map analysis revealed that the
same four major phosphopeptides (P1, P2, P3, and P4) were generated in
unstimulated as well as PDGF-BB- or PMA-stimulated cells, suggesting
that the exchanger phosphorylation occurs at multiple sites
(Fig. 4). Phosphorylation of P1 and, to a lesser extent, of P2
was enhanced in PDGF-BB- and PMA-treated cells, and the phosphopeptide
maps were essentially the same for these cells. P1 contains major
site(s) for phosphorylation by PDGF-BB and phorbol ester, because
Regulation of the
Na
In
this study, we determined the effects of PDGF-BB and other agents on
Na
In contrast to other growth factors tested,
angiotensin II did not significantly increase both
Na
PDGF
and
Regulation of the Na
/Ca
exchanger
is one of the major Ca
extrusion systems in excitable
tissues, little is known about its regulation via protein
phosphorylation. We now present evidence that the
Na
/Ca
exchanger is phosphorylated in
quiescent and growth factor-stimulated cultured aortic smooth muscle
cells. The Na
/Ca
exchanger was
isolated from
P-labeled cells by immunoprecipitation with
a specific polyclonal antibody. Phosphorylation of the exchanger was
increased by up to 1.7-fold in response to platelet-derived growth
factor-BB (PDGF-BB),
-thrombin, or phorbol 12-myristate 13-acetate
(PMA). However, angiotensin II did not enhance the phosphorylation
significantly. The extent of phosphorylation appeared to correlate with
the growth factor-induced increase in cell 1,2-diacylglycerol. At least
four phosphopeptides (P1 to P4) were detected by tryptic phosphopeptide
map analysis of the phosphorylated exchanger, suggesting that
phosphorylation occurred at multiple sites. PDGF-BB and PMA increased
phosphorylation of the same phosphopeptides (in particular P1).
Phosphorylated amino acids were exclusively serine residues in both
quiescent and stimulated cells. We found that growth factors enhanced
Na
/Ca
exchange activity and that
there was a good correlation between the growth factor-induced
stimulations of phosphorylation and exchange activity. PDGF-BB-induced
activation of the exchanger was abolished by prior long treatment of
cells with PMA. These results suggest that the
Na
/Ca
exchanger is activated by
protein kinase C-dependent phosphorylation in response to growth
factors in vascular smooth muscle cells.
/Ca
exchanger of the
plasma membrane is an electrogenic transporter with a
3Na
/1Ca
stoichiometry that plays a
key role in regulating
[Ca
]
(
)
in many mammalian cells. The physiological importance of
Na
/Ca
exchange has been studied
extensively in excitable tissues such as cardiac muscle, vascular
smooth muscle, and nerve fiber (for review, see Ref. 1). The exchanger
is primarily responsible for the removal of excess Ca
from cells during agonist or electrical stimulation.
/Ca
exchanger (NCX1) has been
cloned from heart
(2) , aorta
(3) , kidney
(4) ,
and brain
(5, 6) . By hydropathy analysis, the cardiac
clone is modeled to have an amino-terminal cleaved signal sequence, 11
transmembrane segments, and a large hydrophilic cytoplasmic domain
between the fifth and sixth transmembrane segments
(2) . The
clones isolated from different tissues are highly homologous to the
cardiac clone, except for the diversity found in a small region near
the carboxyl end of the intracellular domain, which is generated by
alternative splicing
(3, 6) . Recently, a new
Na
/Ca
exchanger isoform (NCX2) has
been cloned that is a product of a different gene and abundantly
expressed only in brain and skeletal muscle
(7) . NCX1 and NCX2
have amino acid sequence identity of 65%, but their functional
properties are rather similar
(7) .
/Ca
exchanger have
most extensively been characterized using giant excised membrane
patches from cardiomyocytes or Xenopus laevis oocytes
expressing the cardiac exchanger. The cardiac exchanger activity is
modulated positively by cytoplasmic Ca
and ATP
(8) and negatively by cytoplasmic Na
(9) and exchanger inhibitory peptide
(10) .
Interestingly, most of these regulatory properties can be abolished by
treating the exchanger with chymotrypsin from the cytoplasmic side
(8) or deletion mutagenesis of the cytoplasmic domain
(11) . Therefore, the cytoplasmic domain is thought to be
involved in the regulation of the exchanger.
/Ca
exchanger, there is currently
no strong evidence that protein phosphorylation is involved in the
regulation of its activity. In squid giant axons, however, there is
much evidence suggesting that protein kinase-dependent phosphorylation
is responsible for stimulation of the exchanger activity induced by
MgATP
(12, 13, 14) . On the other hand, the
Na
/Ca
exchanger in vascular smooth
muscle cells is inhibited by depleting cellular ATP
(15) .
Furthermore, phorbol esters
(16) , 8-bromo-cGMP
(17) ,
norepinephrine and high K
(18) , and PDGF-BB
(19) were reported to stimulate the
Na
/Ca
exchange activity in smooth
muscle cells and tissues. These findings suggest that phosphorylation
of the exchanger itself or associated ancillary proteins may be
involved in the exchanger activation in nerve and vascular smooth
muscle cells.
/Ca
exchanger in rat
aortic smooth muscle cells is phosphorylated and concomitantly
activated in response to PDGF-BB,
-thrombin, or phorbol ester. The
regulation of this exchanger by growth factors is very interesting,
since elevation of [Ca
]
has been suggested to be important for cell proliferation
(20, 21) . Some of these results have been presented in
abstract form
(22) .
Cell Culture
Vascular smooth muscle cells were
isolated from the thoracic aorta of male Wistar rats (200-300 g)
by enzymatic dispersion as described by Chamley et al. (23) . The resulting cells were grown for 4-5 days in
Dulbecco's modified Eagle's medium supplemented with 10%
heat-inactivated fetal calf serum, 100 units/ml penicillin, and 100
µg/ml streptomycin at 37 °C in a humidified atmosphere with 5%
CO. After reaching confluence, cells were cultured in
serum-free medium for an additional 24-48 h to enhance
redifferentiation.
Antibody Production
A polyclonal
anti-Na/Ca
exchanger antibody was
raised by immunizing rabbits with a maltose-binding protein fusion
protein containing a portion of the cytoplasmic domain (amino acids
273-769) of the dog cardiac Na
/Ca
exchanger
(2) . Preparation of the fusion protein and
affinity purification of the antibody were described previously
(24) .
Immunoprecipitation and Immunoblotting
Confluent
smooth muscle cells in 100-mm dishes were labeled for 5 h at 37 °C
in a phosphate-free, serum-free medium containing
[P]orthophosphate (10 MBq/ml). The cells were
stimulated with growth factors for 2-20 min and washed twice with
ice-cold phosphate-buffered saline. Cell monolayers were scraped,
suspended in 800 µl of ice-cold buffer A (50 m
M Hepes/Tris
(pH 7.4), 150 m
M NaCl, 3 m
M KCl, 25 m
M sodium pyrophosphate, 10 m
M ATP, 5 m
M EDTA, 1
m
M phenylmethylsulfonyl fluoride, 1 m
M benzamidine
hydrochloride, 10 units/ml aprotinin, 0.5 µg/ml leupeptin, and 0.5
µg/ml pepstatin A), and then centrifuged for 15 min at 100,000
g. The pellet was solubilized in 800 µl of
ice-cold buffer A containing 1% C
E
for 15 min,
and the lysate was centrifuged for 30 min at 100,000
g. The solubilized sample was pretreated with protein
A-Sepharose beads at 4 °C for 1 h and then incubated with an
anti-Na
/Ca
exchanger antibody at 4
°C for 1 h. To this mixture, 50 µl of a 50% slurry of protein
A-Sepharose beads previously blocked for at least 1 h with a
C
E
extract of Escherichia coli was
added and the resultant mixture was incubated for 1 h at 4 °C in a
rotating shaker. The beads were then washed eight times with buffer A
containing 1% C
E
. Labeled proteins solubilized
from beads by boiling in Laemmli's buffer were subjected to
SDS-PAGE on a 7.5% gel and visualized by Bioimage analyzer (BAS2000,
Fuji Film Co.).
Phosphopeptide Mapping
Phosphopeptide mapping
analysis was carried out as described previously
(26) , except
that P-labeled Na
/Ca
exchanger protein, which was recovered from SDS-PAGE gel slices
and resuspended in 500 µl of 50 m
M NH
HCO
(pH 8.0), was digested with 20 µg of TPCK-treated trypsin for
16 h, instead of 24 h, at 37 °C.
Phosphoamino Acid Analysis
Phosphoamino acid
analysis was performed by the method of Cooper et al. (27) with modification. Briefly, the P-labeled
Na
/Ca
exchanger protein was first
digested with TPCK-treated trypsin as described above and then
hydrolyzed with 7
M HCl at 105 °C for 1 h. The hydrolysate
was dried, washed four times with 500 µl of water, and then
suspended in 10 µl of water containing internal standards (10
µg each of phosphoserine, phosphothreonine, and phosphotyrosine).
Aliquots of the samples were applied onto thin-layer cellulose plates
and amino acids were separated by electrophoresis for 1 h at 800 V in
pyridine:acetic acid:water (5:45:950, v/v/v) (pH 3.6). Amino acids were
reacted with ninhydrin and visualized using Bioimage analyzer.
Measurement of
Na
For
Na -dependent
Ca
Uptake
loading, confluent cells in 24-well dishes were
incubated in 1 ml of BSS (10 m
M Hepes/Tris (pH 7.4), 146
m
M NaCl, 4 m
M KCl, 2 m
M MgCl
, 1
m
M CaCl
, 10 m
M glucose, and 0.1% BSA)
containing 1 m
M ouabain, 10 µ
M monensin, 10
m
M NaHCO
, and 3 m
M NaH
PO
at 37 °C for 20 min. Cells were
then incubated for 3 min with a Ca
-free Na
loading solution to reduce elevated
[Ca
]
to a basal level.
Ca
uptake was initiated by switching the
medium to a Na
-free BSS (replacing NaCl with equimolar
KCl) or normal BSS, both of which contained 370 KBq/ml
Ca
, 1 m
M ouabain, and 10
µ
M verapamil. After a 30-s incubation,
Ca
uptake was stopped by washing cells
five times with an ice-cold solution containing 10 m
M Hepes/Tris (pH 7.4), 120 m
M choline chloride, and 10
m
M LaCl
. For the growth factor treatment, the
solutions contained growth factors from 10 min before the start of
Ca
uptake measurement. Cells were
solubilized in 1% SDS and 10 m
M Na
B
O
, and aliquots were taken
for determination of radioactivity and protein.
Na
/Ca
exchange activity was
estimated by subtracting
Ca
uptake
activity in the normal BSS from that in Na
-free BSS.
Protein was measured by a modified Lowry method
(28) with BSA
as a standard. Measurement of
[Ca
]
-[Ca
]
in monolayered cultured cells was measured using fura-2 as a
fluorescent Ca
indicator as described previously
(29) . Measurement of
[Na
]
-Cell
Na
content was measured by equilibrating cells with
BSS containing
Na
(740 KBq/ml), and
[Na
]
was calculated by
adopting 4.5 µl/mg protein as the total intracellular water space
(30) .
Measurement of 1,2-Diacylglycerol
Confluent cells
in 12-well dishes were stimulated with PDGF-BB in 1 ml of BSS. After
appropriate intervals, incubation was terminated by adding 1 ml of
ice-cold methanol. After extraction of lipids, 1,2-diacylglycerol was
measured with the sn-1,2-diacylglycerol assay reagent system
(Amersham) using a thin layer chromatography to separate phosphatidic
acid converted from 1,2-diacylglycerol.
Statistical Analysis
Experimental results are
expressed as means ± S.D. Significant differences were assessed
with a one-way analysis of variance followed by Dunnett's test. A
p value of <0.05 was considered significant.
Materials
CaCl
and
NaCl were obtained from DuPont NEN, and
[
P]orthophosphate and
sn-1,2-diacylglycerol assay reagent system were obtained from
Amersham. BSA (fatty acid-free),
-thrombin, cyclopiazonic acid,
monensin, ouabain, PMA, and verapamil were from Sigma. PDGF-BB was
purchased from Becton Dickson Laboratories. Ionomycin was from
Calbiochem. Fura-2/acetoxymetylester was from Dojindo Laboratories.
Angiotensin II was from the Peptide Institute. C
E
was purchased from Nikkol Chemical Co. Ltd. (Tokyo, Japan). All
other chemicals were of analytical grade.
Phosphorylation of the
Na
We investigated growth factor-induced
phosphorylation of the Na/Ca
Exchanger by PDGF-BB
and Other Agents
/Ca
exchanger by immunoprecipitation from serum-depleted rat aortic
smooth muscle cells metabolically labeled with
[
P]orthophosphate. The antibody used, which was
raised against a portion of the cytoplasmic domain of the dog cardiac
Na
/Ca
exchanger and
affinity-purified, recognized a broad band covering the expected
molecular mass for the Na
/Ca
exchanger (125 kDa), when the immunoprecipitated material was
analyzed by SDS-PAGE and protein staining (Fig. 1 A). This
broad band may be caused by glycosylation of the exchanger protein
(1) or the presence of alternatively spliced exchanger isoforms
(3, 6) . We found that this protein band was
phosphorylated in serum-depleted, unstimulated cells
(Fig. 1 B). When these cells were treated with 10 or 20
ng/ml PDGF-BB for 10 min, the extent of phosphorylation increased to
146 ± 9% or 166 ± 15% ( n = 3) of that for
the untreated cells (Fig. 1 B). Treatment of cells with
10 n
M
-thrombin or 100 n
M PMA for 10 min also
increased the phosphorylation to 140 ± 9% or 145 ± 11%
( n = 3), respectively (Fig. 1, B and
C). However, 100 n
M angiotensin II did not increase
the phosphorylation significantly (118 ± 6%, n =
3) under the equivalent experimental conditions
(Fig. 1 C). Thus PDGF-BB,
-thrombin, and PMA induced
Na
/Ca
exchanger phosphorylation in
serum-depleted aortic smooth muscle cells. We found that removal of
extracellular Ca
decreased the basal and
PDGF-BB-induced exchanger phosphorylation slightly; in 4 m
M EGTA, the basal phosphorylation was 83% ( n = 2) of
the control value, whereas PDGF-BB (10 ng/ml, 10 min) increased the
exchanger phosphorylation to 132% ( n = 2) under the
same conditions.
Figure 1:
Immunological detection of
phosphorylated Na/Ca
exchanger in
unstimulated and growth factor-stimulated smooth muscle cells. Rat
aortic smooth muscle cells labeled with ( panels B and
C) or without ( panel A)
[
P]orthophosphate (10 MBq/ml) were treated with
growth factors for 10 min and then solubilized with 1%
C
E
lysis buffer. Cell lysates were incubated
with a polyclonal antibody against the dog cardiac
Na
/Ca
exchanger (1/100 dilution),
and the resultant immunoprecipitates were subjected to SDS-PAGE. In
panel A, a nitrocellulose transfer of gel was probed
with the polyclonal antibody and visualized by using ECL detection
system. The upper band is the Na
/Ca
exchanger, whereas the lower band is immunoglobulin derived from
the immunoprecipitate. In panels B and C,
phosphorylated proteins in the gels were visualized by Bioimage
analyzer. In each panel, C, unstimulated cells; PDGF 10 and 20, cells stimulated with 10 or 20 ng/ml
PDGF-BB; T, cells stimulated with 10 n
M
-thrombin; AII, cells stimulated with 100 n
M angiotensin II; PMA, cells stimulated with 100 n
M PMA. Molecular mass markers (in kDa) are shown on the
left.
A time course of PDGF-BB-induced phosphorylation of
the Na/Ca
exchanger is shown in Fig.
2, and the results of three similar experiments analyzed
densitometrically are summarized in Fig. 3. In the same latter figure,
we also show the time courses of PDGF-BB-induced changes in
[Ca
]
and a
1,2-diacylglycerol level. The PDGF-BB-induced phosphorylation increased
with time, reaching 169 ± 17% ( n = 3) of the
control level at 20 min. Under the same conditions,
[Ca
]
reached a peak
(450 ± 40 n
M, n = 4) within 2 min,
followed by a slow decline. The formation of 1,2-diacylglycerol, on the
other hand, increased from a basal level of 91 ± 8 pmol/10
cells ( n = 4) to a high level of 191 ± 20
pmol/10
cells at 20 min. In contrast to PDGF-BB, 100 n
M angiotensin II, which did not enhance the exchanger
phosphorylation significantly (Fig. 1 C), increased the
formation of 1,2-diacylglycerol to a lower level (126 ± 16
pmol/10
cells ( n = 4) at 10 min), as
compared with PDGF-BB (189 ± 30 pmol/10
cells ( n = 4) at 10 min).
Phosphopeptide Mapping and Phosphoamino Acid Analysis of
the Na
To characterize sites for the basal and
stimuli-enhanced phosphorylation in the
Na/Ca
Exchanger
/Ca
exchanger, we performed
two-dimensional tryptic phosphopeptide mapping as described under
``Experimental Procedures'' (Fig. 4). Tryptic digestion of
the
P-labeled Na
/Ca
exchanger in unstimulated cells generated four major
phosphopeptides (P1, P2, P3, and P4). Occasionally one minor spot (P5),
which may represent a partially digested phosphopeptide, was observed.
Stimulation of cells with PDGF-BB (20 ng/ml) and PMA (100 n
M)
for 20 min resulted in a significant increase in the amounts of
P label incorporated into P1 and, to a lesser extent, into
P2. In unstimulated cells, P1 was weakly phosphorylated, representing
less than 15% of the total
P label incorporated. In
contrast, it accounted for 40-50% of the total
P
label in PDGF-BB- and PMA-stimulated cells. These results indicate that
phosphorylation of the Na
/Ca
exchanger occurs at multiple sites and that PDGF-BB and PMA
enhance phosphorylation of the same phosphopeptides.
/Ca
exchanger (Fig. 5). The
Na
/Ca
exchanger phosphorylation in
the basal, and PDGF-BB- and PMA-stimulated cells occurred exclusively
on serine residues, not on threonine or tyrosine residues. The result
indicates the involvement of serine kinase(s) in the
Na
/Ca
exchanger phosphorylation.
Effect of PDGF-BB and Other Agents on
Na
We examined the effect of treatment with PDGF-BB
(10 or 20 ng/ml), /Ca
Exchanger
Activity
-thrombin (10 n
M), angiotensin II (100
n
M), or PMA (100 n
M) for 10 min on
Na
-dependent
Ca
uptake (reverse mode of the
Na
/Ca
exchanger) in
Na
-loaded rat aortic smooth muscle cells, as described
under ``Experimental Procedures'' (Fig. 6). Because these
agents except for PMA increased
[Ca
]
from basal levels
of 100-150 n
M to peak levels of 400-550 n
M and because Na
/Ca
exchange
depends on [Ca
]
, cells
had been exposed to a Ca
-free solution containing
these agents during the last 3 min of the 10-min treatment in order to
reduce [Ca
]
to a basal
level.
-thrombin caused a significant increase (120-130% of the
control value) in Na
-dependent
Ca
uptake. The extent of such
PDGF-BB-induced activation was similar, when cells had been incubated
with 30 to 146 m
M Na
and loaded with
different levels of Na
(data not shown). It should be
mentioned that cell Na
loading itself was not affected
by prior treatment with PDGF-BB; when cells were equilibrated with 146
m
M
Na
in the presence and
absence of 20 ng/ml PDGF-BB,
[
Na
]
immediately after the transfer of cells to the
Ca
uptake medium were 97 ± 4 and
99 ± 5 m
M ( n = 4) in control and
PDGF-BB-treated cells, respectively. On the other hand, angiotensin II
did not affect Na
-dependent
Ca
uptake (Fig. 6). However, PMA
enhanced the
Ca
uptake significantly. It
is important to note that there is apparent parallelism between these
growth factor-induced increases in Na
/Ca
exchange activity and the phosphorylation of the exchanger
(Fig. 6).
Figure 6:
Correlation between phosphorylation and
activity of Na/Ca
exchanger. Cells
cultured in 24-well dish were stimulated with no agent ( C), 10
or 20 ng/ml PDGF-BB ( PDGF 10 or 20), 10
n
M
-thrombin ( T), 100 n
M angiotensin II
( AII), or 100 n
M PMA for 10 min.
Na
-dependent
Ca
uptake
was determined as described under ``Experimental
Procedures.''
P incorporation into the
immunoprecipitated Na
/Ca
exchanger
(see Fig. 1) was quantified by densitometry using Bioimage analyzer.
Results are presented as the mean ± S.D. ( n = 3
or 4). *, significant difference from unstimulated
cells.
We also examined whether PDGF-BB was able to enhance
Na-dependent decline in
[Ca
]
via the forward
mode of Na
/Ca
exchanger in aortic
smooth muscle cells (Fig. 7). We treated cells with 50 µ
M cyclopiazonic acid in Ca
- and
Na
-free, high pH (pH 8.8) BSS containing 20 m
M Mg
, which inhibits Ca
extrusion via the Na
/Ca
exchanger and the ATP-dependent Ca
pump
(31) . After [Ca
]
reached a plateau level of about 300 n
M (about 2 min
later), Na
was added extracellularly to a final
concentration of 50 m
M. In unstimulated cells,
Na
evoked a decline in
[Ca
]
(initial rate of
decline, 33 ± 6 n
M/10 s ( n = 3)) (Fig.
7 A), although addition of choline chloride, in place of NaCl,
did not affect [Ca
]
(data not shown). When cells were stimulated with 10 ng/ml
PDGF-BB for 20 min, the initial rate of
Na
-induced
[Ca
]
decline
significantly increased to 89 ± 12 n
M/10 s ( n = 3), and [Ca
]
reached a lower steady state level (Fig. 7 A).
Interestingly, the PDGF-BB-induced stimulation of
Na
-dependent decline in
[Ca
]
was abolished
(initial rate of decline, 36 ± 8 n
M/10 s ( n = 3)) by pretreatment of cells with 100 n
M PMA for
24 h, suggesting that protein kinase C is involved in such activation
of Na
/Ca
exchanger
(Fig. 7 B).
Figure 7:
Effect
of PDGF-BB on Na-dependent
[Ca
] decline measured in PMA-pretreated
cells in Na
- and Ca
-free high
pH/high Mg
medium. Cells were treated with or without
10 ng/ml PDGF-BB for 20 min in BSS after they had been incubated with
( panel B) or without ( panel A) 100
n
M PMA for 24 h. Then they were transferred to a
Ca
- and Na
-free, high pH medium (pH
8.8) containing 20 m
M Mg
and 50 µ
M cyclopiazonic acid. After [Ca
] reached
a plateau level (about 300 n
M), NaCl was applied to the medium
to a final concentration of 50 m
M to induce
Na
-dependent [Ca
]
decline.
/Ca
exchanger was phosphorylated
in quiescent smooth muscle cells and that the phosphorylation was
enhanced by up to 1.7-fold in response to PDGF-BB,
-thrombin, or
PMA (Figs. 1, 3, and 6). This is the first demonstration of
phosphorylation of the Na
/Ca
exchanger in response to the physiological ligands.
P label incorporation into P1, which accounted for less
than 15% of the total exchanger phosphorylation, increased to
40-50% of the total phosphorylation after PDGF-BB and PMA
treatment. Furthermore, phosphorylated amino acids in the exchanger
were exclusively serine residues in both quiescent and PDGF-BB- or
PMA-stimulated cells (Fig. 5), indicating the involvement of
serine protein kinase(s). Taken together, these results suggest that
the Na
/Ca
exchanger in vascular
smooth muscle cells is phosphorylated in response to PDGF-BB or
-thrombin via a protein kinase C-dependent pathway. We do not
know, however, whether protein kinase C directly phosphorylates the
exchanger or whether it phosphorylates and activates a secondary
serine/threonine kinase. The involvement of protein kinase C is
consistent with the well known facts that signaling by PDGF-BB and
-thrombin occurs through activation of phospholipase C-
1 and
phospholipase C-
1, respectively, that produce inositol
1,4,5-triphosphate and 1,2-diacylglycerol from phosphatidylinositol
4,5-bisphosphate
(32) . Indeed, we observed elevation of
1,2-diacylglycerol in PDGF-BB-stimulated cells (Fig. 3). At
present, however, we cannot rule out the possibility that other protein
kinases such as Ca
/calmodulin kinase II were also
involved in the exchanger phosphorylation, because PDGF-BB induced a
prolonged increase in [Ca
]
(Fig. 3) and because increased
[Ca
]
seemed to be able
to enhance the exchanger phosphorylation (see ``Results'').
Figure 4:
Two-dimensional tryptic phosphopeptide
maps of Na/Ca
exchanger. Cells
labeled with [
P]orthophosphate were stimulated
with no agent ( C), PDGF-BB (20 ng/ml), or PMA (100
n
M) for 20 min. The phosphorylated
Na
/Ca
exchanger was isolated by
immunoprecipitation and subsequent SDS-PAGE. The
Na
/Ca
exchanger protein eluted from
gels was digested with TPCK-treated trypsin and subjected to
two-dimensional phosphopeptide mapping as described under
``Experimental Procedures.'' Phosphopeptides were visualized
by Bioimage analyzer. The right bottom panel schematically represents the location of phosphopeptides and
direction of peptide migration
( arrow).
Figure 5:
Phosphoamino acid analysis of
phosphorylated Na/Ca
exchanger.
Cells labeled with [
P]orthophosphate were
stimulated with no agents ( C), PDGF-BB (20 ng/ml), or PMA (100
n
M) for 20 min. The phosphorylated
Na
/Ca
exchanger was isolated by
immunoprecipitation and subsequent SDS-PAGE. After tryptic digestion
and acid hydrolysis, phosphoamino acids were analyzed by thin-layer
electrophoresis and then visualized by Bioimage analyzer.
P-Ser, P-Thy, and P-Tyr represent the positions of phosphoserine, phosphothreonine, and
phosphotyrosine, respectively.
Figure 3:
Time courses of PDGF-BB-induced changes in
Na/Ca
exchanger phosphorylation,
[Ca
], and 1,2-diacylglycerol level. In
cells stimulated with 10 ng/ml PDGF-BB for indicated periods of time,
changes in the Na
/Ca
exchanger
phosphorylation, [Ca
], and
the1,2-diacylglycerol content were determined as described under
``Experimental Procedures.''
P incorporation
into the Na
/Ca
exchanger
( cf. Fig. 2) was quantified by densitometry using Bioimage
analyzer. Results are presented as the mean ± S.D. ( n = 3 or 4). *, significant difference from cells at 0
min.
Primary structure of the Na/Ca
exchanger in rat aortic smooth muscle cells is highly homologous
to the canine cardiac exchanger except for the NH
-terminal
portion, which is presumably a cleaved signal sequence, and part (amino
acid residues 570-621) of the large cytoplasmic domain
(3) . The cytoplasmic domain is considered to be involved in the
regulation of the exchanger as briefly discussed in the introduction.
The cytoplasmic domain of the smooth muscle
Na
/Ca
exchanger contains several
candidate phosphorylation site sequences for serine/threonine kinases
such as protein kinase C, Ca
/calmodulin kinase II,
and cAMP-dependent protein kinase
(33) . One of such sequences
Arg-Lys-Ala-Val-Ser
, which is also conserved in the
cardiac Na
/Ca
exchanger
(2) ,
is a good candidate for the phosphorylation site.
/Ca
exchanger in vascular smooth
muscle cells is important to maintain cellular Ca
homeostasis. Recently, phorbol esters
(16) , cGMP
(17) , norepinephrine and high K
(18) ,
and PDGF-BB
(19) have been reported to stimulate
Na
/Ca
exchange in A7r5 cells, rat
aortic smooth muscle cells, and rabbit aortic rings. However, there has
been no information about the mechanism by which these agents enhance
the exchange activity, except that norepinephrine and high K
were later shown to cause cytoplasmic Na
retention that may partially explain the observed increase in
Na
/Ca
exchange
(34) .
-dependent
Ca
uptake (the reverse mode of
Na
/Ca
exchange) in rat aortic smooth
muscle cells. We found that PDGF-BB,
-thrombin, and PMA
significantly enhanced the
Na
-dependent
Ca
uptake (Fig. 6). Furthermore,
PDGF-BB stimulated Na
-dependent
[Ca
]
decline under
conditions in which the plasma membrane and sarcoplasmic reticulum
Ca
pumps were inhibited (Fig. 7). It should be
noted that PDGF-BB stimulated
Na
-dependent
Ca
uptake only by about 1.3-fold,
whereas it stimulated
Na
-dependent
[Ca
]
decline to a
greater extent (about 3-fold when the initial rate of
[Ca
]
decline was
measured) ( cf. Figs. 6 and 7). For this apparent discrepancy,
we are currently unable to provide an explanation. It should also be
noted that stimulation of
Na
-dependent
[Ca
]
decline by
PDGF-BB was abolished by prolonged pretreatment of cells with PMA, a
procedure that down-regulates the protein kinase C content in the
plasma membrane
(35) . All these results suggest that activity
of the Na
/Ca
exchanger is positively
regulated by growth factors and that the PDGF-BB-induced increase in
Na
/Ca
exchange activity, like the
phosphorylation of the exchanger, is mediated via a protein kinase
C-dependent pathway.
/Ca
exchange and phosphorylation
of the exchanger (Figs. 1 C and 6).
-Thrombin and PMA
induced intermediate levels of activation of these parameters
(Fig. 6). Thus, there is apparently a good correlation between
the extents of stimulation of Na
/Ca
exchange and phosphorylation of the exchanger induced by
different growth factors. We found that angiotensin II increased
1,2-diacylglycerol content to a low level under our experimental
conditions when compared with PDGF-BB (see ``Results'').
Therefore, the inability of angiotensin II to enhance
Na
/Ca
exchange and the exchanger
phosphorylation seems to be due to a low level of 1,2-diacylglycerol
produced. Production of different levels of 1,2-diacylglycerol in
response to PDGF-BB and angiotensin II seems to indicate that these two
growth factors differ in their capacity to activate protein kinase C in
the primary cultured smooth muscle cells used in this study.
-thrombin are powerful mitogens for cells of mesenchymal
origin including vascular smooth muscle cells
(36) . They
increase [Ca
]
transiently and induce cascades of events such as activation of
ion transporters, phosphorylation of proteins, and expression of a
number of genes that are associated with cell proliferation. The
increase in [Ca
]
may
be required for mitogenic signaling at least in certain cell types,
since Ca
blockers and chelators are able to inhibit
growth factor-induced mitogenesis in those cells
(20, 21) . On the other hand, transient exposure of
growth-arrested vascular smooth muscle or other types of cells to PDGF,
-thrombin, or phorbol esters result in activation of the
Na
/H
exchanger and
Na
/K
/Cl
cotransporter
(37, 38, 39) . In addition,
phorbol esters are able to stimulate the plasma membrane Ca
pump in vascular smooth muscle cells and other cell types
(29, 40) . We presented evidence suggesting that protein
kinase C directly phosphorylates and activates the plasma membrane
Ca
-ATPase
(41, 42) . Our finding that
PDGF-BB,
-thrombin, and PMA stimulate the
Na
/Ca
exchanger in aortic smooth
muscle cells indicate that this exchanger is also one of the targets
involved in the growth factor-induced ionic changes that may be
required for the cellular proliferative cycle to proceed properly.
/Ca
exchanger
has been studied most extensively in canine cardiomyocytes and squid
giant axons
(1) . In squid giant axons, ATP and its slowly
hydrolyzable analog ATP
S have been shown to activate the
Na
/Ca
exchanger in the presence of
Mg
and micromolar cytoplasmic Ca
(13) , and inorganic phosphate and vanadate have been
shown to markedly enhance the ATP-dependent stimulation
(12, 14) . These findings strongly support the view that
the Na
/Ca
exchanger is regulated by
phosphorylation in squid axons. In contrast, ATP-dependent stimulation
of Na
/Ca
exchange in cardiomyocytes
under patch clamp conditions does not require intracellular
Ca
, cannot be mimicked by ATP
S, and is not
enhanced by phosphatase inhibitors
(43) . These results suggest
no involvement of kinases or phosphatases in regulation of the cardiac
exchanger, although it was once reported that the exchange activity of
cardiac sarcolemma vesicles was modulated by a calmodulin-dependent
kinase and phosphatase
(44) . The stimulation of the cardiac
exchanger by ATP under patch conditions is thus thought to be caused
indirectly by phospholipid asymmetry in the sarcolemma generated by a
lipid flipase
(45) . Therefore regulation of the
Na
/Ca
exchanger by phosphorylation
currently seems to be a property shared only by squid axon and smooth
muscle exchangers, although we have no information regarding how
similar are these mammalian and non-mammalian exchanger isoforms with
respect to their structure and regulation. Regulation of the cardiac
isoform by protein phosphorylation obviously requires further
investigation.
], intracellular Ca
concentration; PDGF, platelet-derived growth factor;
C
E
, octaethylene glycol
mono- n-dodecyl ether; PAGE, polyacrylamide gel
electrophoresis; ECL, enhanced chemiluminescence; TPCK,
tosylamide-2-phenylethylchloromethyl ketone; Na
,
intracellular Na
; Na
, extracellular
Na
; BSS, balanced salt solution; BSA, bovine serum
albumin; PMA, phorbol 12-myristate 13-acetate; ATP
S, adenosine
5`- O-(thiotriphosphate).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.