Nephrology Research and Training Center, Departments of Medicine, Physiology, and Psychiatry, University of Alabama at Birmingham, Birmingham, Alabama 35294
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ABSTRACT |
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The purpose of these studies was to determine whether there is a
defect in protein kinase C (PKC) regulation of the
Na+/Ca2+
exchanger in cultured mesangial cells (MC) from Dahl/Rapp
salt-sensitive (S) and salt-resistant (R) rats. R and S MCs were
cultured, grown on coverslips, and loaded with fura 2 for measurement
of single cell cytosolic calcium concentration
([Ca2+]i)
in a microscope-based photometry system. Studies were performed in
cells that were exposed to serum (serum fed) and in cells that were
serum deprived for 24 h. Baseline
[Ca2+]i
values measured in a Ringer solution containing 150 mM NaCl were
similar between R and S MCs in both serum-fed and serum-deprived groups, although baseline
[Ca2+]i
values were uniformly higher in the serum-deprived groups. Exchanger
activity was assessed by reducing extracellular Na
(Nae) from 150 to 2 mM, which
resulted in movement of Na+ out of
and Ca2+ into these cells
(reverse-mode
Na+/Ca2+
exchange). PKC was activated in these cells with 15-min exposure to 100 nM phorbol 12-myristate 13-acetate (PMA). In the absence of PMA, the
change in
[Ca2+]i
([Ca2+]i)
with reduction in Nae was similar
between R and S MCs in both serum-fed and serum-deprived groups,
although the magnitude of
[Ca2+]i
was enhanced by serum deprivation. In both serum-fed and serum-deprived groups, PMA significantly increased
[Ca2+]i
in R but not S MCs. Upregulation of exchanger activity in R MCs could
be abolished by prior 24-h exposure to PMA, a maneuver that
downregulates PKC activity. Other studies were performed to evaluate
exchanger protein expression using monoclonal and polyclonal
antibodies. Immunoblots of PMA-treated cells revealed an increase in
the levels of 70- and 120-kDa proteins in the crude membrane fraction
of R but not S MCs, an increase which was abrogated by prior 24-h PMA
pretreatment and corresponded to reduction in the 70-kDa protein in the
crude cytosolic fraction. These data demonstrate that PKC enhances
Na+/Ca2+
exchange activity in MCs from R but not from S rats, suggesting that
there may be a defect in the
PKC-Na+/Ca2+
exchange regulation pathway in MCs of S rats.
sodium/calcium exchanger; Dahl rat; immunoblots; translocation
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INTRODUCTION |
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THE SODIUM/CALCIUM exchanger, which is expressed in a variety of tissues including kidney, is involved in the regulation of intracellular calcium concentration ([Ca2+]i). It functions as a bidirectional plasma membrane antiporter transporting three Na+ ions for one Ca2+ ion (18) and is, therefore, influenced by the electrochemical gradients for both Na+ and Ca2+. Since there is normally a large inwardly directed electrochemical gradient for Na+, exchange activity results in the electrogenic movement of Na+ into and Ca2+ out of the cell. This is referred to as the forward-mode operation of the exchanger. Experimentally, the exchanger can function in the reverse mode by decreasing extracellular Na (Nae) resulting in Ca2+ entry that is associated with a decrease in intracellular Na+ concentration ([Na+]i) (3, 20). Previous work has established the existence and some of the functional characteristics of the renal Na+/Ca2+ exchanger via reverse-mode exchange in renal mesangial cells (MC), afferent and efferent arterioles, and epithelial tubular segments (5, 6, 20, 23, 38).
At the molecular level, cardiac and rod photoreceptor forms of the Na+/Ca2+ exchanger have been cloned and found to be encoded by different genes (25, 29). The cardiac form of the exchanger is 970 amino acids in size with 11 transmembrane spanning regions. Philipson et al. (27) have developed both polyclonal and monoclonal antibodies to the Na+/Ca2+ exchanger found in canine cardiac sarcolemma. These antibodies are immunoreactive with proteins that are 70, 120, and 160 kDa in size. The 160-kDa protein is seen under nonreducing conditions. The 120-kDa protein is thought to be the fully functional protein, whereas the 70-kDa protein may be a proteolytic fragment (27). Recent work has suggested that the canine cardiac exchanger has a high homology with exchangers derived from rat renal tissue (5, 11, 30).
Currently, there is a lack of information concerning regulation of the Na+/Ca2+ exchanger and potential alterations in exchange regulation that might occur in certain disease processes. In this regard, we have been particularly interested in the role of protein kinase C (PKC) as a regulator of the exchanger. Previous work by other laboratories (19, 37) has resulted in conflicting data, with PKC activation resulting in no change, stimulation, or inhibition of the exchanger. In our studies, we have found that phorbol 12-myristate 13-acetate (PMA) stimulation of PKC results in enhanced Na+/Ca2+ exchange activity in afferent arterioles from rabbit and rat kidney (6, 24). These results are supported by recent studies that demonstrated a direct PKC phosphorylation of the exchanger protein (10). Therefore, phosphorylation by PKC may play a role in the regulation of Na+/Ca2+ exchange activity (37).
As shown in the accompanying study (24), PKC regulation of Na+/Ca2+ exchange appears to be different in isolated afferent arterioles from Dahl/Rapp salt-sensitive (S) vs. salt-resistant (R) rats. Specifically, we have found that PMA stimulation of exchange activity is present in arterioles from R rats, whereas in S arterioles, PKC activation does not alter Na+/Ca2+ exchange. These results suggest that there may be a defect in the PKC-Na+/Ca2+ exchange regulatory pathway in afferent arterioles from S rats.
The current studies were conducted to determine whether differences in PKC activation of the exchanger would likewise occur in MCs cultured from S and R rats. Use of cultured cells would eliminate residual hemodynamic and humoral influences that might be present in the isolated arteriolar studies. Likewise, cultured MCs allowed us to use quantitative immunoblots to detect Na+/Ca2+ exchanger protein and to determine the effects of PKC activation on the expression of the Na+/Ca2+ exchanger in MCs from S and R rats.
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METHODS |
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Animals. Animals used in this study were male Dahl/Rapp salt-sensitive (S) and salt-resistant (R) rats (28) (Harlan Sprague Dawley, Indianapolis, IN) weighing ~50 g and fed standard rat chow (Prolab RMH 1000, Agway; ~170-180 meq/kg of both Na and K) and water ad libitum. As outlined in recent reports (12, 33), S rats from Harlan Sprague Dawley were genetically contaminated as early as March 1993. It was noted that some of the S animals failed to develop hypertension when placed on a high-salt diet. Animals used in our studies were obtained after the S colony had been regenerated. In concurrent studies, we reported that S rats became hypertensive on an 8% NaCl diet and that they were devoid of the previous contamination using PCR and DNA analysis (17, 24).
Isolation and culture of glomerular MCs. Kidneys from both S and R rats were aseptically removed, and glomerular MC were isolated and cultured as previously described with minor modifications (21). In brief, renal cortical tissue was minced with a razor blade and passed through a 70-mesh copper sieve (Fisher Scientific, Pittsburgh, PA). Tissue was then passed through progressively smaller nylon sieves (Tetko, Briar Cliff Manor, NY) ranging in size from 315 to 75 µm to separate glomeruli from the remaining kidney tissue. Glomeruli were then treated with collagenase (Sigma Chemical, St. Louis, MO) and plated onto 60 × 15-mm petri dishes (Costar, Cambridge, MA). Cells were grown in RPMI 1640 media (Life Technologies, Gaithersburg, MD) supplemented with 20% fetal bovine serum (Intergen, Purchase, NY), 240 µg/ml L-glutamine (GIBCO), 82 U/ml penicillin and 82 µg/ml streptomycin (Sigma), and 2 µg/µl amphotericin B for 21 days in humidified 95% air-5% CO2 at 37°C. Media was changed twice a week. Cells were then subcultured and plated onto two 40 × 12-mm glass coverslips in petri dishes and were grown to confluence for an additional 21 days before being used in calcium measurement experiments. Previous studies identified these cells as MC. Electron microscopy demonstrated prominent microfilaments, dense bodies, well-developed rough endoplasmic reticulum, gap junctions, and attachment plaques. Vimentin, a cytoskeletal filament, was also found in these cells (14, 21, 34, 39).
Protein preparation. Cultured MC were either untreated or treated with 100 nM PMA (Sigma) for 15 min in RPMI 1640 with or without 24-h PMA pretreatment to downregulate PKC. MC were washed three times in PBS, harvested in 5 ml of PBS, and centrifuged at 400 g for 5 min at room temperature. The supernatant was discarded, and the pellet was resuspended in 1 ml of homogenizing buffer (20 mM Tris and 1 mM EGTA, pH 7.0). Cells were then homogenized using a Teflon-on-glass homogenizer and centrifuged for 20 min at 16,000 g to separate the crude membrane and cytosolic fractions. The supernatant was transferred to new tubes, centrifuged at 40,000 g, and the supernatant was saved and classified as the crude cytosolic extract. The 16,000 g pellet, containing membranes, was resuspended in 1 ml of SDS lysis buffer (1% SDS, 1 mM sodium vanadate, and 10 mM Tris · HCl, pH 7.4), boiled for 5 min, and centrifuged at 16,000 g for 20 min. The supernatant was saved and classified as the crude membrane fraction. Whole cell homogenates of dog heart (supplied by Dr. Gilbert Hageman) or rat heart were used as positive controls for immunoblotting. Protein concentrations were determined using the Lowry method (15).
Antibodies. The monoclonal antibody C-2C12 is an IgM that reacts with three proteins, 160, 120, and 70 kDa, in canine sarcolemma. This antibody was obtained as previously described (7) from a mouse immunized with isolated reconstituted canine cardiac Na+/Ca2+ exchanger. The epitope for the antibody is on the intracellular side of the plasma membrane in the region of amino acids 371-525. C-2C12 was the generous gift of Dr. Kenneth D. Philipson.
The polyclonal antibody 11-13 is an IgG that also reacts with
three proteins, 160, 120, and 70 kDa, in canine sarcolemma. The
antibody was formed in rabbit against a reconstituted, purified Na+/Ca2+
exchanger from canine cardiac sarcolemma (27). This antibody was
obtained from SWant, Bellinzona, Switzerland.
Immunoblot. Fifty micrograms of S and R MC protein samples to be immunoblotted were separated by 7.5% SDS-PAGE and transferred for 1.25 h onto nitrocellulose electrophoretically. The blots were blocked in 5% nonfat dry milk in PBS at room temperature for 1 h. After blocking, blots were incubated in 5% nonfat dry milk in PBS with a 1:1,000 dilution of either the monoclonal (IgM) or the polyclonal (IgG) antibody at 4°C overnight. After three washes in PBS, blots were incubated in 5% nonfat dry milk in PBS with a 1:1,500 dilution of either horseradish peroxidase-conjugated goat anti-mouse IgM or horseradish peroxidase-conjugated goat anti-rabbit IgG (Hyclone, Logan, UT) at room temperature for 4 h. Blots were then washed five times with PBS (10 min per wash) and developed using enhanced chemiluminescence (Amersham, Arlington Heights, IL).
Measurement of intracellular calcium
concentration.
Na+/Ca2+
exchange activity experiments were performed on cells that had either been incubated in RPMI 1640 media with 20% fetal bovine serum until
the time of the experiment (serum fed) or RPMI 1640 media alone for 24 h prior to measurements of
[Ca2+]i
(serum deprived).
[Ca2+]i
was measured using the fluorescent dye fura 2-AM (Molecular Probes,
Eugene, OR). Cells were incubated in media containing 5 µM fura 2-AM
dissolved in DMSO for 1 h at 37°C to allow loading of the dye into
cells.
[Ca2+]i
measurements were performed using dual-excitation wavelength fluorescence microscopy (Photon Technologies International, South Brunswick, NJ). Coverslips containing either S or R MC were positioned in the microscope (Leitz) stage chamber and bathed in a 150 mM Ringer
solution. Single cell
[Ca2+]i
measurements were performed using a compact Leitz photometer (6) with a
variable diaphragm. Excitation wavelengths were set at 340 and 380 nm,
and the emission wavelength was 510 nm. The Ringer solution contained
(in mM) 4.2 KCl, 0.8 K2HPO4,
0.5 MgCl2, 0.4 MgSO4, 20 HEPES, 5.5 D-glucose, 5.0 L-alanine, 1.1 CaCl2, and 140 NaCl.
N-methyl-D-glucamine
was used to isosmotically replace
Na+ (all reagents from Sigma, St.
Louis, MO). No differences in functional responses were noted between
these two Na+ replacements. All
solutions were bubbled with O2, pH
was 7.4, and temperature was maintained at 37°C. Baseline
measurements were made after 15 min in the 150 mM Ringer solution.
Reducing the 150 mM NaCl solution to 2 mM resulted in a transient
increase in cytosolic Ca2+. The
fura 2 ratio was monitored continuously before, during, and after the
exchange to a lower Nae. In
addition, there was no evidence for dye leakage throughout the
experiment. Activity of the reverse-mode exchanger, expressed as
[Ca2+]i,
was assessed by measurement of the difference between baseline [Ca2+]i
and the maximum increase in
[Ca2+]i.
Results and conclusions are the same whether expressing the data as the
[Ca2+]i
or as the initial rate of increase in
[Ca2+]i
to the removal of bath Na+.
To assess PMA regulation of the Na+/Ca2+ exchanger, cells were incubated for 15 min in 150 mM NaCl containing 100 nM PMA prior to and during the reductions in bath Na+ (Nae). In some experiments, cells were incubated for 24 h in RPMI 1640 containing 100 nM PMA. In these experiments, 100 nM PMA was also present during the reduction in Nae. Calibrations were performed to convert fura 2 ratios into [Ca2+]i values. Composition of the calibration solution was chosen to approximate the intracellular milieu and consisted of 115 mM KCl, 20 mM NaCl, 10 mM MOPS, 1.1 mM MgCl2, 1 µM fura 2 pentapotassium salt (Molecular Probes, Eugene, OR), 3 mM Ca2+ or no Ca2+, and 3 mM EGTA. [Ca2+]i was calculated by the following equation (from Ref. 8)
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Values are means ± SE. Analysis was done using Student's unpaired t-test or ANOVA single-factor test. Significance was taken as P < 0.05.
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RESULTS |
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Reports by Mené et al. (19) indicate that
Na+/Ca2+
exchange activity in MC is enhanced by serum depriving the cells for 24 h. We confirmed this observation in serum-fed vs. serum-deprived (24 h
prior to Ca2+ measurements) S and
R MC by evaluating
[Ca2+]i
upon removal of Nae. Table
1 lists the baseline
[Ca2+]i
values for each treatment group used in this study. It can be seen that
serum deprivation increases baseline
[Ca2+]i
measurements in both R and S MC compared with serum-fed MC. However, no
differences in baseline
[Ca2+]i
were seen between S and R within the serum-fed or serum-deprived groups. In response to a reduction in
Nae, increases in
[Ca2+]i
in both S and R MC were greater in the serum-deprived vs. serum-fed groups (compare control responses in Figs.
1 and 2).
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PMA was used to determine whether PKC affects
Na+/Ca2+
exchange activity. MC were incubated in 100 nM PMA for 15 min with (to downregulate PKC) or without (to activate PKC) 24-h PMA pretreatment (26).
Na+/Ca2+
exchange activity was measured as the change in
[Ca2+]i
([Ca2+]i)
in response to the reduction of
Nae. Figure 1 shows the
[Ca2+]i
upon removal of Nae in control and
15-min PMA-treated serum-fed S and R MC.
[Ca2+]i
for S control MC was 167 ± 43 nM
(n = 21) and was unaltered with 15-min
PMA treatment (202 ± 30 nM, n = 24).
[Ca2+]i
for R controls was 92 ± 20 nM (n = 22) and increased significantly to 242 ± 62 nM
(n = 30) in response to 15-min PMA
treatment. Figure 2 shows the results of
15-min PMA treatment on exchange activity in S and R MC that have been
serum deprived for 24 h. Control
[Ca2+]i
values were 604 ± 118 nM (n = 12)
and 566 ± 118 nM (n = 11) for S
and R MC, respectively. After 15-min PMA treatment, S
[Ca2+]i
was not statistically different compared with the control response (840 ± 143 nM, n = 15), whereas R
[Ca2+]i
increased significantly to 1,187 ± 178 nM
(n = 19). Thus, after 15-min
incubation in 100 nM PMA, exchange activity was significantly increased
in serum-fed and serum-deprived R but not S MC compared with untreated
cells. Treatment of MC with 100 nM PMA for 24 h downregulates PKC
activity, so that this maneuver should abolish PKC stimulation in
response to 15-min PMA incubation. As shown in Fig.
3, 24-h PMA pretreatment abrogated the
increase in exchange activity in R MC obtained with addition of PMA for
15 min.
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As a result of the above findings, we sought to determine whether PMA
affects the amount of immunodectable
Na+/Ca2+
exchange protein in MC incubated with 100 nM PMA for 15 min with or
without 24-h PMA pretreatment. The levels of the
Na+/Ca2+
exchanger in the crude membrane and cytosolic fractions did not change
significantly in response to either PMA treatment in S MC (Figs.
4 and 5). In R cells,
however, 15-min PMA treatment increased the amount of both 120-kDa and
70-kDa proteins in the crude membrane fraction by 2.1- and 4.1-fold,
respectively, whereas 24-h PMA pretreatment, to downregulate PKC,
abrogated these increases (Fig. 4). The two PMA treatments also caused
significant decreases in the 70-kDa protein in the crude cytosolic
fraction of R MC. Therefore, both functional and antibody localization
studies indicate differences in PKC regulation of the
Na+/Ca2+
exchanger in cultured MC between S and R rats.
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DISCUSSION |
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The purpose of the present studies was to examine, using functional and
immunologic techniques, expression and regulation of the
Na+/Ca2+
exchanger in cultured MCs. The development of monoclonal and polyclonal
antibodies to the exchanger has provided a means of directly
demonstrating the existence of the exchanger in a number of tissues
including cardiac sarcolemma (36), proximal tubule, glomeruli (5), and
connecting tubule (31). To our knowledge, the exchanger has not been
directly immunolocalized in MC. Dominguez et al. (5) reported no
immunolocalization of the exchanger to whole glomeruli. In the present
study, protein bands of 120 and 70 kDa in size were detected in S and R
MC with both a monoclonal and a polyclonal antibody directed against
the canine cardiac sarcolemma
Na+/Ca2+
exchanger. The same size bands were found in studies (data not shown)
where canine and rat heart homogenates were used as positive control.
Although the amount of exchanger protein was considerably less in MC
compared with heart tissue, the clear presence of these bands serves as
a positive identification of the
Na+/Ca2+
exchanger in MC. Our ability to detect the 120- and 70-kDa bands in MC
can be attributed to two things; the use of serum free media prior to
treatments and the use of a polyclonal antibody, 11-13, which
is much more sensitive than the monoclonal antibody, C-2C12, in
immunoblot studies. In addition, there was no difference in the amount
of exchanger protein between untreated S and R MC. This finding is
consistent with functional studies
(left of Figs. 1 and 2) that indicate
no major differences in basal
Na+/Ca2+
exchange activity between S and R MC.
Previous functional studies have demonstrated
Na+/Ca2+
exchange activity in several cell types including vascular smooth
muscle cells and MC (1-3, 6, 16, 19, 20, 22, 23, 32). Several
different functional assays have been developed to study Na+/Ca2+
exchange including the measurement of tension in isolated smooth muscle
rings in response to reduction of
Nae (1). A more direct approach
has been to measure changes in
[Ca2+]i
using Ca2+-sensitive fluorescent
probes (3, 6, 20, 38). Other work involved the use of
45Ca efflux studies as a way of
assessing
Na+/Ca2+
exchange activity (16). In the present studies, we measured [Ca2+]i
in MC during reductions in Nae.
Reducing Nae results in an increase in
[Ca2+]i,
which reflects the operation of the exchanger in what has been termed
the reverse mode. That is, when
Nae is reduced,
Na+ exits while
Ca2+ enters the cell. In previous
work (23), we have demonstrated that this increase in
[Ca2+]i
is associated with changes in
[Na+]i,
suggesting that the increase in
[Ca2+]i
is the direct result of a decrease in
[Na+]i
that occurs, at least in part, through the
Na+/Ca2+
exchanger. In addition, recent studies have demonstrated that the
increase in
[Ca2+]i
with reduction of Nae can be
blocked by pretreatment of the cell with an antisense oligonucleotide
directed against the
Na+/Ca2+
exchanger (38). These studies and others (3, 6, 19, 23) clearly
indicate that the increase in
[Ca2+]i
during reduction in Nae occurs
through the
Na+/Ca2+ exchanger.
Presently, the regulation of the
Na+/Ca2+
exchanger in MC or in any other tissues is not well understood. To
study possible modes of regulation, we first needed to determine
appropriate conditions for studying
Na+/Ca2+
exchange activity. Mené et al. (19) reported that serum
deprivation of cells prior to measuring
[Ca2+]i
enhanced
Na+/Ca2+
exchange activity, resulting in a larger influx in
Ca2+ after
Nae reduction. Our results are
consistent with those of Mené et al. (19). Serum-deprived cells
had significantly greater baseline
Ca2+ values and larger
Ca2+ influx responses compared
with serum-fed cells. Interestingly, using both serum-fed and
serum-deprived cells allowed us to examine exchanger regulation at two
different levels of exchanger activity. The fact that pattern of
responses between S and R cells were the same in both serum-fed and
serum-deprived groups strengthens the conclusions of this study.
Although we have no direct knowledge regarding the mechanism for
enhanced activity after serum deprivation, one explanation concerns the
elevated basal
[Ca2+]i
in the serum-deprived group. Previous studies (4) have shown that
elevated
[Ca2+]i
directly enhances
Na+/Ca2+
exchange activity. Although the mechanism whereby cytosolic
Ca2+ regulates exchange activity
is not known, the elevated
[Ca2+]i
levels in the serum-deprived group may help explain the enhanced exchange activity found in this group.
Several studies from other laboratories (13, 37) have shown that activation of PKC results in enhanced Na+/Ca2+ exchange activity in vascular smooth muscle cells and in cells transfected with the exchanger. Although other workers have not found this same stimulatory effect of PKC (19) in MCs, these studies, nevertheless, prompted us to determine whether PKC can alter exchange activity in cultured MC. We found that a 15-min PMA treatment, which activates PKC in MC (9), resulted in enhanced Na+/Ca2+ exchange activity in R but not S MC in both serum-fed and serum-deprived cells. Our studies also showed that 24-h PMA pretreatment, a maneuver that downregulates PKC, prior to the 15-min treatment, abrogated the increase in Na+/Ca2+ exchange activity in R MC. Thus PKC appears to upregulate Na+/Ca2+ exchanger at least in R MC. PMA stimulation of the exchanger has also been found in MC derived from the Sprague-Dawley rat (data not shown). A similar upregulation has been shown in afferent arterioles from rabbit and R rat kidney (6, 24). Taken together, these results suggest that the lack of a significant stimulation of exchange activity in S MC represents a defect in the PKC-Na+/Ca2+ exchange regulatory pathway.
Although the site of defect in the PKC-Na+/Ca2+ exchange regulatory pathway remains to be determined, recent work by our laboratory (17) showed no differences in PKC isoform expression between R and S MCs, suggesting that the site of defect may be downstream of PKC. One possibility was that the enhanced Na+/Ca2+ exchange activity seen in R MC might be due to a recruitment of exchangers to the plasma membrane, a phenomenon which may be missing in S MCs. Therefore, the next goal of this study was to determine whether Na+/Ca2+ exchange protein levels at the plasma membrane were affected by PMA stimulation. MC from S and R rats were either untreated or treated with PMA for 15 min with or without 24-h PMA pretreatment. Immunoblots of crude membrane fractions of these cells showed an increase in the amount of immunoreactive proteins after 15-min PMA treatment in R but not S MC. This increase in immunoreactive proteins in crude membrane extracts was abrogated by 24-h PMA pretreatment. Although we do not know the specific mechanism for this increase in membrane protein in the R cells, it is possible that PKC activation causes a recruitment of exchangers from a cytosolic pool to the plasma membrane, an idea which is supported, in part, by our results which indicated that the 70-kDa band was significantly reduced in the cytosol of R MC after the 15-min PMA treatment. It is unlikely that the increased membrane Na+/Ca2+ exchanger levels in R MC are due to an activation of protein synthesis, since the time period of PMA stimulation was only 15 min, much too short for protein synthesis to occur. In addition, since PKC can directly phosphorylate the exchanger (10), the relationship between Na+/Ca2+ exchanger phosphorylation and possible exchanger recruitment to the plasma membrane is unclear. Further work in this area is needed to determine the exact mechanism of PMA-stimulated increases in Na+/Ca2+ exchange protein levels.
Taken together, the functional and immunologic data suggest that there is a difference in the Na+/Ca2+ exchanger response to PKC activation in R and S MC. PMA treatment resulted in increases of Na+/Ca2+ exchange activity and membrane levels of the exchanger in R MC. In contrast, the S MC exhibited neither enhanced exchange activity nor increased membrane protein levels in response to PMA treatment. These studies should lead to further work to identify which step in the PKC-Na+/Ca2+ exchange regulatory pathway is defective in S MC and the consequences that this defect may have on the development of salt-dependent hypertension.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge Dr. Kenneth Philipson for the gift of the monoclonal antibodies used in this study. We also thank Clay Isbell and Cameron Nixon for technical assistance and Martha Yeager for secretarial support.
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FOOTNOTES |
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This work was funded by the National Heart, Lung, and Blood Institute Grant HL-50163 (to P. D. Bell).
Address for reprint requests and other correspondence: P. D. Bell, Univ. of Alabama at Birmingham, UAB Station, 865 Sparks Center, Birmingham, AL 35294 (E-mail: dbell{at}nrtc.dom.uab.edu).
Received 18 September 1997; accepted in final form 10 December 1998.
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REFERENCES |
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---|
1.
Ashida, T.,
Y. Kawano,
H. Yoshimi,
M. Kuramochi,
and
T. Omae.
Effects of dietary salt on sodium-calcium exchange and ATP-driven calcium pump in arterial smooth muscle of Dahl rats.
J. Hypertens.
10:
1335-1341,
1992[Medline].
2.
Ashida, T.,
M. Kuramochi,
and
T. Omae.
Increased sodium-calcium exchange in arterial smooth muscle of spontaneously hypertensive rats.
Hypertension
13:
890-895,
1989[Abstract].
3.
Batlle, D. C.,
M. Godinich,
M. S. LaPointe,
E. Munoz,
F. Carone,
and
N. Mehring.
Extracellular Na+ dependency of free cytosolic Ca2+ regulation in aortic vascular smooth muscle cells.
Am. J. Physiol.
261 (Cell Physiol. 30):
C845-C856,
1991
4.
Dipolo, R.,
and
L. Beauge.
Regulation of Na+-Ca2+ exchange. An overview.
Ann. NY Acad. Sci.
639:
101-111,
1991.
5.
Dominguez, J. H.,
M. Juhaszova,
S. B. Kleiboeker,
C. C. Hale,
and
H. A. Feister.
Na+-Ca2+ exchanger of rat proximal tubule: gene expression and subcellular localization.
Am. J. Physiol.
263 (Renal Fluid Electrolyte Physiol. 32):
F945-F950,
1992
6.
Fowler, B. C.,
P. K. Carmines,
L. D. Nelson,
and
P. D. Bell.
Characterization of sodium-calcium exchange in rabbit renal arterioles.
Kidney Int.
50:
1856-1862,
1996[Medline].
7.
Frank, J. S.,
G. Mottino,
D. Reid,
R. S. Molday,
and
K. D. Philipson.
Distribution of the Na+-Ca2+ exchange protein in mammalian cardiac myocytes: an immunofluorescence and immunocolloidal Gold-labeling study.
J. Cell Biol.
117:
337-345,
1990[Abstract].
8.
Grynkiewicz, G.,
M. Poenie,
and
R. Y. Tsien.
A new generation of Ca2+ indicators with greatly improved fluorescence properties.
J. Biol. Chem.
260:
3440-3450,
1985[Abstract].
9.
Isbell, J. C.,
S. T. Christian,
N. A. Mashburn,
and
P. D. Bell.
A non-radioactive fluorescent method for measuring protein kinase C activity.
Life Sci.
57:
1701-1707,
1995[Medline].
10.
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
11.
Kofuji, P.,
W. J. Lederer,
and
D. H. Schulze.
Na/Ca exchanger isoforms expressed in kidney.
Am. J. Physiol.
265 (Renal Fluid Electrolyte Physiol. 34):
F598-F603,
1993
12.
Lewis, J. L.,
R. J. Russell,
and
D. G. Warnock.
Analysis of the genetic contamination of salt-sensitive Dahl/Rapp rats.
Hypertension
24:
255-259,
1994[Abstract].
13.
Linck, B.,
Z. Qiu,
Z. He,
Q. Tong,
D. W. Hilgemann,
and
K. D. Philipson.
Functional comparison of the three isoforms of the Na+/Ca2+ exchanger (NCX1, NCX2, NCX3).
Am. J. Physiol.
274 (Cell Physiol. 43):
C415-C423,
1998
14.
Lovett, D. H.,
J. L. Ryan,
and
R. B. Sterzel.
A thymocyte-activating factor derived from glomerular mesangial cells.
J. Immunol.
130:
1796-1801,
1983
15.
Lowry, O. H.,
N. F. Rosebrough,
A. L. Farr,
and
R. J. Randall.
Protein measurement with the Folin phenol reagent.
J. Biol. Chem.
193:
265-275,
1951
16.
Lyu, R.-M.,
L. Smith,
and
J. B. Smith.
Ca2+ influx via Na+-Ca2+ exchange in immortalized aortic myocytes. I. Dependence on [Na+]i and inhibition by external Na+.
Am. J. Physiol.
263 (Cell Physiol. 32):
C628-C634,
1992
17.
Mashburn, N. A.,
T. Wilborn,
G. V. W. Johnson,
J. Schafer,
and
P. D. Bell.
Protein kinase C isozymes in cultured mesangial cells from salt sensitive and salt-resistant rats (Abstract).
J. Am. Soc. Nephrol.
7:
1440,
1996.
18.
McNaughton, P. A.
Fundamental properties of the Na+-Ca2+ exchanger. An overview.
Ann. NY Acad. Sci.
639:
2-9,
1991[Medline].
19.
Mené, P.,
F. Pugliese,
and
G. A. Cinotti.
Regulation of Na+-Ca2+ exchange in cultured human mesangial cells.
Am. J. Physiol.
261 (Renal Fluid Electrolyte Physiol. 30):
F466-F473,
1991
20.
Mené, P.,
F. Pugliese,
T. Faraggiana,
and
G. A. Cinotti.
Identification and characteristics of a Na+/Ca2+ exchanger in cultured human mesangial cells.
Kidney Int.
38:
1199-1205,
1990[Medline].
21.
Misra, R. P.
Isolation of glomeruli from mammalian kidneys by graded sieving.
Am. J. Clin. Pathol.
58:
135-139,
1972[Medline].
22.
Nabel, E. G.,
B. C. Berk,
T. A. Brock,
and
T. W. Smith.
Na+-Ca2+ exchange in cultured vascular smooth muscle cells.
Circ. Res.
62:
486-493,
1988[Abstract].
23.
Nelson, L. D.,
N. A. Mashburn,
and
P. D. Bell.
Altered sodium-calcium exchange in afferent arterioles of the spontaneously hypertensive rat.
Kidney Int.
50:
1889-1896,
1996[Medline].
24.
Nelson, L. D.,
M. T. Unlap,
J. L. Lewis,
and
P. D. Bell.
Renal arteriolar Na+/Ca2+ exchange in salt-sensitive hypertension.
Am. J. Physiol.
276 (Renal Physiol. 45):
F567-F573,
1999
25.
Nicoll, D. A.,
S. Longoni,
and
K. D. Philipson.
Molecular cloning and functional expression of the cardiac sarcolemmal Na+-Ca2+ exchanger.
Science
250:
562-565,
1990[Medline].
26.
Nishizuka, Y.
The Albert Lasker awards. The family of protein kinase C for signal transduction.
JAMA
262:
1826-1833,
1989[Abstract].
27.
Philipson, K. D.,
S. Longoni,
and
R. Ward.
Purification of the cardiac Na+-Ca2+ exchange protein.
Biochim. Biophys. Acta
945:
298-306,
1988[Medline].
28.
Rapp, J. P.,
and
H. Dene.
Development and characteristics of inbred strains of Dahl salt-sensitive and salt-resistant rats.
Hypertension
7:
340-349,
1985[Abstract].
29.
Reilander, H.,
A. Achilles,
U. Friedel,
G. Maul,
F. Lottspeich,
and
N. J. Cook.
Primary structure and functional expression of the Na/Ca,K-exchanger from bovine rod photoreceptors.
EMBO J.
11:
1689-1695,
1992[Abstract].
30.
Reilly, R. F.,
and
C. A. Shugrue.
cDNA cloning of the renal Na+-Ca2+ exchanger.
Am. J. Physiol.
262 (Renal Fluid Electrolyte Physiol. 31):
F1105-F1109,
1992
31.
Reilly, R. F.,
C. A. Shugrue,
D. Lattanzi,
and
D. Biemesderfer.
Immunolocalization of the Na+/Ca2+ exchanger in rabbit kidney.
Am. J. Physiol.
265 (Renal Fluid Electrolyte Physiol. 34):
F327-F332,
1993
32.
Smith, J. B.,
T. Zheng,
and
L. Smith.
Relationship between cytosolic free Ca2+ and Na+-Ca2+ exchange in aortic muscle cells.
Am. J. Physiol.
256 (Cell Physiol. 25):
C147-C154,
1989
33.
St. Lezin, E. M.,
M. Pravenec,
A. Wong,
J.-M. Wang,
T. Merriouns,
S. Newton,
D. E. Stec,
R. J. Roman,
D. Lau,
R. C. Morris,
and
T. W. Kurtz.
Genetic contamination of Dahl SS/Jr rats: impact on studies of salt-sensitive hypertension.
Hypertension
23:
786-790,
1994[Abstract].
34.
Tagouri, Y. M.,
P. W. Sanders,
M. M. Picken,
G. P. Siegal,
J. D. Kerby,
and
G. A. Herrera.
In vitro AL-amyloid formation by rat and human mesangial cells.
Lab. Invest.
74:
290-302,
1996[Medline].
35.
Tsien, R. Y.
Fluorescent probes of cell signaling.
Annu. Rev. Neurosci.
12:
227-253,
1989[Medline].
36.
Vemuri, R.,
M. E. Haberland,
D. Fong,
and
K. D. Philipson.
Identification of the cardiac sarcolemmal Na+-Ca2+ exchanger using monoclonal antibodies.
J. Membr. Biol.
118:
279-283,
1990[Medline].
37.
Vigne, P.,
J.-P. Breittmayers,
D. Duval,
C. Frelin,
and
M. Lazdunski.
The Na+/Ca2+ antiporter in aortic smooth muscle cells. Characterization and demonstration of an activation by phorbol esters.
J. Biol. Chem.
263:
8078-8083,
1988
38.
White, K. E.,
F. A. Gesek,
and
P. A. Friedman.
Identification and antisense inhibition of Na+/Ca2+ exchange in renal epithelial cells.
Ann. NY Acad. Sci.
779:
115-118,
1996[Abstract].
39.
Zhu, L.,
G. A. Herrera,
J. E. Murphy-Ullrich,
Z. Q. Huang,
and
P. W. Sanders.
Pathogenesis of glomerulosclerosis in light chain deposition disease: role for transforming growth factor-beta.
Am. J. Pathol.
147:
375-385,
1995[Abstract].