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Articles in PresS. Am J Physiol Renal Physiol (January 14, 2003). 10.1152/ajprenal.00121.2002
F-121.2002.R3
1
IMPAIRED ABILITY OF THE Na+/Ca2+ EXCHANGER
FROM THE DAHL/RAPP SALT SENSITIVE RAT TO
REGULATE CYTOSOLIC CALCIUM
E.F. Hwang, I. Williams, G. Kovacs, J. Peti-Peterdi, B. Siroky, W.C. Rice, E. Bates, E.M.
Schwiebert, M.T. Unlap, and P.D. Bell
Nephrology Research and Training Center, Departments of Medicine and Physiology,
Division of Nephrology, University of Alabama at Birmingham, AL 35294
Address correspondence to:
P. Darwin Bell, Ph.D.
University of Alabama at Birmingham
UAB Station
865 Sparks Center
Birmingham, AL 35294
Phone: (205) 934-3806
Fax:
(205) 934-1147
E-mail: [email protected]
Copyright (c) 2003 by the American Physiological Society.
F-121.2002.R3
2
ABSTRACT
We previously cloned NCX1 from mesangial cells of salt sensitive
(SNCX=NCX1.7) and salt resistant (RNCX=NCX1.3) Dahl/Rapp rats. The abilities of
2+
these isoforms to regulate [Ca ]i was assessed in fura 2 loaded OK cells expressing the
2+
vector (VOK), RNCX (ROK) and SNCX (SOK). Baseline [Ca ]i was 98 ± 20 nM (n =
12) in VOK and was significantly lower in ROK (44 ± 5 nM; n = 12) and SOK (47 ± 13
2+
nM; n = 12) cells. ATP at 100 µM increased [Ca ]i by 189 ± 55 nM (n = 12), 21 ± 9 nM
(n = 12), and 69 ± 18 nM (n = 12) in VOK, ROK, and SOK cells, respectively. ATP (1
2+
mM) or bradykinin (0.1 mM) caused large increases in [Ca ]i and ROK but not SOK
2+
cells were much more efficient in reducing [Ca ]i back to baseline levels. Parental
Sprague Dawley rat mesangial cells express both RNCX (SDRNCX) and SNCX
(SDSNCX).
SDRNCX and RNCX are identical at every amino acid residue but
SDSNCX and SNCX differ at amino acid 218 where it is isoleucine in SDSNCX and not
phenylalanine. OK cells expressing SDSNCX (SDSOK) reduced ATP(1mM)-induced
[Ca2+]i increase back to baseline at a rate equivalent to that for ROK cells. PKC downregulation significantly attenuated the rate at which ROK and SDSOK cells reduced
2+
ATP-induced [Ca ]i increase, but had no effect in SOK cells. The reduced efficiency of
2+
SNCX to regulate [Ca ]i is attributed, in part, to the isoleucine to phenylalanine
mutation at amino acid 218.
Key Words:
calcium
Sodium/calcium exchanger, mesangial cells, hypertension, cytosolic
F-121.2002.R3
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INTRODUCTION
+
In recent studies, Na /Ca
2+
exchange (NCX) activity has been shown to exist in
renal afferent and efferent arterioles and cultured mesangial cells (2, 20, 23, 33). It most
likely plays an important role in the regulation of cytosolic calcium concentration
2+
([Ca ]i) by serving as a Ca
2+
efflux pathway. Studies in vascular smooth muscle (36),
have further suggested that the exchanger has an important role in lowering agonist2+
induced elevations in [Ca ]i and it is possible that it serves a similar function in the renal
microcirculation. We have extended this proposal by suggesting that this process may
also involve the activation of protein kinase C (PKC). Phorbol esters, which activate
PKC, enhance exchanger activity in afferent arterioles and mesangial cells and this may
be due to PKC-induced translocation of the exchanger to the plasma membrane (20). It is
2+
also well known that vasoconstrictive agonists, which elevate [Ca ]i, also activate PKC
2+
through the Ca -diacylglycerol pathway. Although PKC can have multiple cellular
+
effects, one effect may be to enhance Na /Ca
2+
exchange which then serves to return
2+
agonist-induced elevations in [Ca ]i back to baseline levels.
+
In other studies we have found that this Na /Ca
2+
exchanger pathway may be
defective in the Dahl/Rapp salt sensitive (S) rat (33), a genetic model of salt dependent
hypertension (3, 30). Both freshly dissected afferent arterioles from the Dahl/Rapp salt
resistant (R) rats and from rabbit kidneys as well as cultured mesangial cells from R rats,
+
2+
all responded to the phorbol ester, PMA, with enhanced Na /Ca exchange (2, 8, 20, 23,
F-121.2002.R3
4
33). Whereas in afferent arterioles and cultured mesangial cells from S rats, PMA failed
+
to increase Na /Ca
2+
exchanger activity.
In S mesangial cells, PMA also failed to
stimulate translocation of the exchanger to the plasma membrane whereas in R mesangial
cells, PMA promoted translocation (20).
To determine if this difference between R and S rats was due to intrinsic
differences in the exchanger protein, NCX was cloned from cultured mesangial cells
from R and S rats (33). The clone from S rats denoted SNCX and from R rats denoted
RNCX differ at the amino acid level. One difference occurs at amino acid residue 218
where it is isoleucine in RNCX but is phenylalanine in SNCX. The significance of this
single amino acid difference is currently unknown. However, it may affect the regulation
of SNCX since this difference occurs in a region which is 43% homologous to a region
+
+
(amino acids 308-330) of the Na -K ATPase that is responsible for binding Ca
2+
(25).
The other difference between RNCX and SNCX occurs within the cytosolic loop at the
alternative splice site. This site is encoded by 6 different exons denoted A-F (16, 29). In
the RNCX clone, the alternative splice site is encoded by exons B and D, and in SNCX it
is encoded by exons B, D, and F (33). Although there is some indication that the
alternative splice site may be important in NCX regulation (27), the exact consequence of
the differences in exons expressed by these two clones at this site remains unknown.
RNCX and SNCX were expressed in an opossum kidney cell line (OK-PTH)
which does not express endogenous exchanger activity. It was found that the exchanger
+
activity of RNCX but not SNCX was enhanced by PMA (33). Thus, the PKC-Na /Ca
2+
exchanger pathway in the S rat appears to be defective at the level of the exchanger
protein.
F-121.2002.R3
5
The Dahl/Rapp rat model of hypertension is characterized by a marked increase in
blood pressure, decreased renal blood flow, and a progressive fall in glomerular filtration
rate when S rats are placed on an 8% NaCl diet (3). It has been suggested that this
2+
progressive renal vasoconstriction is due to a dysregulation of [Ca ]i in contractile
elements of the renal microcirculation of the S rat. One possible explanation for this
2+
+
derangement in [Ca ]i could be defective PKC regulation of the Na /Ca
2+
exchanger.
However, whether expression of RNCX versus SNCX actually results in differences in
2+
the regulation of [Ca ]i has not yet been addressed.
Therefore the purpose of these
studies was to express RNCX and SNCX in identical cellular environments (OK-PTH
2+
cells) and to determine if these two isoforms differ in their ability to help regulate [Ca ]i
2+
in response to agonist-induced elevations in [Ca ]i. Because RNCX and SNCX are both
expressed in mesangial cells of Sprague Dawley rats, these results were also compared to
those obtained for OK-PTH cells expressing the SNCX which was cloned from
mesangial cells of Sprague Dawley rats (SDSNCX). For these studies we used ATP to
2+
elevate [Ca ]i via the activation of purinergic receptors; several isotypes of this receptor
family were confirmed to be present in OK-PTH cells. In addition, bradykinin was also
used to elevate [Ca2+]i to ensure that the findings obtained with ATP were not peculiar to
purinergic receptors pathways.
F-121.2002.R3
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METHODS
Cell Cultures
Opossum proximal tubule kidney cells (OK-PTH, ATCC) were grown in MEM
media (Gibco Laboratories, Grand Island, NY) supplemented with 10% Fetalclone III
(Cellgro), 240 µg/ml L-glutamine (Gibco), 82 units/ml penicillin, 82 µg/ml streptomycin
(Sigma), in a humidified atmosphere under 95% air/5% CO2 at 37°C.
Media was
changed twice a week and cells were routinely passaged 72 hrs after seeding. Each OKPTH cell lot was used for no more than 30 passages.
Transfection of OK-PTH Cells with RNCX, SNCX, and SDSNCX
OK-PTH cells were transfected with pCDNA3.1/V5-His-TOPO (Invitrogen)
containing RNCX (pCDNA3.1-RNCX), SNCX (pCDNA3.1-SNCX) or SDSNCX
(pCDNA3.1-SDSNCX) cDNA using Lipofectin (BRL) according to manufacturer's
instructions. Transfectants were selected for using Geneticin at 500 µg/ml for 3 weeks.
After three weeks, transfected cells were incubated in the presence of 500 µM Ca
2+
and
20 µM of ionomycin for 30 min, washed and resuspended in complete media. This
2+
maneuver stimulates a significant rise in [Ca ]i and only cells with functional
2+
2+
exchangers will be able to lower [Ca ]i sufficiently to survive (Ca
killing) (15). This
process was repeated every 3 days in order to enrich the population of OK-PTH cells that
+
express functional Na /Ca
2+
exchanger.
Immunoblotting with Na+/Ca2+ Exchanger -Specific Antibody
F-121.2002.R3
7
Cells were lysed in a buffer containing 10 mM Tris, 0.5 mM NaCl, 0.5% Triton
X-100, 50 µg/ml aprotinin (Sigma, St. Louis, MO), 100 µg/ml leupeptin (Sigma), and
100 µg/ml pepstatin A (Sigma) adjusted to pH 7.2-7.4. Fifty microgram of protein was
run per lane and separated on 8% SDS polyacrylamide gels and then transferred to PVDF
membranes (Osmonics, Westborough, MA). Immunoblotting was performed with a
mouse monoclonal Na+/Ca2+ exchanger antibody with a dilution of 1:5000 (SWant,
Bellizona, Swizerland). Reactivity was detected by horseradish peroxidase-labeled goat
anti-mouse secondary antibody.
ECL chemiluminescence was used to visualize the
secondary antibody.
Na+/Ca2+ Exchanger Activity Measurement
Vector-transfected OK-PTH cells (VOK), or transfected OK-PTH cells
expressing RNCX (ROK), SNCX (SOK) or SDSNCX (SDOK) were grown to 80%
confluency in 100 mm cell culture dishes with MEM media supplemented with 10%
Fetalclone III and 82 µg/ml penicillin/streptomycin. Cells were harvested with a cell
scraper and incubated in media containing 24 µM fura 2-AM (TEF Labs, Austin, TX) for
1 hour at 37°C to allow loading of the dye into cells. Fura 2 loaded cells were pelleted at
700xg for 3 min and resuspended in Ringer solution (150 mM NaCl, 5 mM KCl, 1 mM
MgSO4, 1.6 mM Na2HPO4, 0.4 mM NaH2PO4, 5 mM D-glucose, 1.5 mM CaCl2, 10 mM
HEPES).
A small quantity of cells was then transferred to a chamber that was mounted on
an inverted microscope. After several mins, OK cells would settle to the bottom of the
chamber and adhere to the coverslip that formed the chamber’s bottom. After a period of
F-121.2002.R3
8
5 to 10 mins, it was possible to perfuse the chamber even at high flow rates with the cells
remaining attached to the coverslip.
2+
Single cell [Ca ]i measurements were performed
using dual-excitation wavelength fluorescence microscopy (Photon Technologies,
Princeton, NJ) with a Leitz compact photometer that had been converted to perform
photon counting. An adjustable photometery window was placed over a single cell with
magnification of 400X using an Olympus X40 UVFL lens. Excitation wavelengths were
set at 340 nm and 380 nm and alternated at 25 Hz. Emission wavelength was set at 510
nm, with data collection at a rate of 5 points/sec using PTI software. Background
corrections were made prior to the experimental measurements.
Baseline fura 2 ratios were measured for at least 100 sec in cells that were bathed
in Ringer solution at a rate of 1.7 ml/min. Cells were discarded if the baseline drifted
either up or down. After obtaining a stable baseline reading, either 100 µM or 1 mM
ATP or 100 µM bradykinin was added to the chamber at a rate of 1.7 ml/min to elevate
2+
[Ca ]i. Fura 2 ratio was monitored continuously before, during, and after addition of
ATP or bradykinin until the ratio returned to a stable baseline. All solutions had pH of
7.4 with temperature maintained at 37°C. In addition, there was no evidence of dye
leakage throughout the experiment.
The Effect of PKC Down-Regulation on Na+/Ca2+ Exchanger Activity
Previous studies have shown that PKC plays an important role in the activation of
NCX. In order to examine the role that PKC might play in the regulation of RNCX,
SNCX or SDSNCX, cells expressing RNCX (ROK), SNCX (SOK) or SDSNCX
(SDSOK) were treated with 300 nM PMA for 24 hr to down-regulate PKC prior to single
F-121.2002.R3
9
2+
cell [Ca ]i measurements. PMA was added directly to the culture media and cells were
maintained in the incubator throughout the 24 hr period.
Calibration of [Ca2+]i
2+
2+
Calibrations were performed to convert fura 2 ratios into [Ca ]i values. [Ca ]i
was calculated using the equation described by Grynkiewicz et al. (10):
2+
[Ca ]i = Kd × (Sf2/Sb2) × (R-Rmin)/(Rmax-R)
where Kd is the effective dissociation constant of fura 2, and has a value of 224 nM, R is
the fluorescence ratio obtained at 340 nm/380 nm, Rmin and Rmax are the ratios in absence
and presence of Ca
2+
respectively, and Sf2 and Sb2 are the emissions at 380 nm in the
absence and presence of Ca
2+
respectively. VOK, ROK, SDSOK or SOK cells were
loaded with 24 µM fura 2 for 1 hr, followed by resuspension in Ringer solution.
Calibration was accomplished after permeabilizing the cells with 5 µM ionomycin and
2+
measuring fluorescence at both wavelengths, 340 and 380 nm, under Ca -free (in 2 mM
2+
EGTA) or Ca -saturated (in 0.25 M CaCl2) conditions to obtain Rmin, Rmax, Sf2 , and
Sb2.
Genomic DNA Sequencing
The cDNAs for RNCX and SNCX show differences at amino cid 218 where it is
isoleucine in RNCX but is phenylalanine in SNCX and at the alternative splice site where
it is encoded by exons B and D and B, D, and F in RNCX and SNCX, respectively. In
order to address the question of whether or not the mutation observed at the cDNA level
F-121.2002.R3 10
occurs at the genomic level, genomic DNA was isolated from kidneys of salt sensitive
and salt resistant Dahl/Rapp rats using the Easy-DNA kit (Invitrogen) according to
manufacturer’s instructions. A 100 ng aliquot of the genomic DNA was used to amplify
the N-terminus of the Na+/Ca2+ exchanger gene using a forward and a reverse primer (5’gagaggatccgacaggcggcttctctttt-3’/5’-gaggagaattctgcaacccaagcgaacaca-3’).
PCR was
carried out by using the High Fidelity Supermix kit (Invitrogen). Using a minicycler (MJ
Research), PCR was carried out at 94oC for 2 min, 40 cycles at 94oC for 30 sec, 45oC for
30 sec, and 68oC for 45 sec followed by a long extension time of 10 min at 68oC. Ten
microliters of each PCR product were fractionated on a 1% Tris acetate-EDTA gel, the
band was excised and gel purified using Gene Clean (Amersham). A 2 µl aliquot of the
purified PCR fragment was ligated into pcDNA3.1GFPTOPO using the TOPO TA
Cloning Kit (Invitrogen).
The entire ligation mix was used to transform Top 10
competent E. Coli (Invitrogen) that were subsequently screened (34) and plasmid was
isolated from six positive colonies containing the N-terminus for RNCX or SNCX. The
plasmids were sent to SEQWRIGHT (Houston, TX) for sequencing and the DNASIS
Max program (Hitachi Software Engineering Co) was used for sequence alignments and
manipulations.
F-121.2002.R3 11
RESULTS
SNCX and RNCX Exchanger Levels
Using immunoblot analysis, the level of protein expression in VOK, ROK and
SOK cells was evaluated and the results are shown in Fig. 1. Low level of NCX protein
was detected in VOK cells and this level was at least ten fold less than that seen in ROK
and SOK cells. We reported in an earlier study that OK-PTH cells did not functionally
express the exchanger as evaluated by reverse-mode (33).
The important point is that
the expression of exchanger protein did not differ between ROK and SOK cells.
2+
Baseline [Ca ]i
2+
[Ca ]i was measured under baseline conditions in VOK, ROK and SOK cells.
+
The results are presented in Fig. 2 and demonstrate that the expression of Na /Ca
2+
2+
2+
exchanger in OK cells resulted in a significant reduction in [Ca ]i. Baseline [Ca ]i was
98 ± 20 nM in VOK cells while it was 44 ± 5 nM and 47 ± 13 nM (n=12) in cells
expressing RNCX (ROK) and SNCX (SOK), respectively. Thus the presence of the
+
Na /Ca
2+
2+
exchanger lowered resting [Ca ]i but there was no difference in baseline
2+
[Ca ]i between these two NCX isoforms.
2+
[Ca ]i Buffering Capacity
+
The Na /Ca
2+
exchanger is a Ca
2+
2+
efflux pathway that contributes to the
maintenance of low levels of [Ca ]i. The rationale for this series of experiments was to
F-121.2002.R3 12
perfuse a relatively low concentration of ATP (100 µM; at least it is a low concentration
for OK-PTH cells), and to assess the ability of the exchanger to extrude this ATPmediated Ca
2+
influx and mobilization.
The concentration of ATP and the rate of
perfusion were selected based on preliminary experiments that provided the optimal
separation of Ca
2+
transients between VOK and ROK cells. As shown in the example in
Fig. 3(a) and in the summary in Fig. 3(b), the largest Ca
2+
transients in response to ATP
2+
administration were observed in VOK cells; i.e., [Ca ]i increased by 189 ± 55 nM
(n=12). Thus this group demonstrated the poorest Ca
2+
buffering capacity in response to
2+
ATP. Both SOK and ROK cells were capable of maintaining lower levels of [Ca ]i with
2+
ATP administration. However the increase in [Ca ]i of 69 ± 18 nM (n=12) in the SOK
group with ATP administration was significantly greater than the 21 ± 9 nM (n=12)
2+
elevation in [Ca ]i obtained in the ROK group. Thus OK cells expressing the RNCX
2+
clone had a greater ability to buffer changes in [Ca ]i compared to cells expressing the
SNCX clone. The expressions of RNCX and SNCX were comparable in the two cell
lines (Fig. 1) so that it is highly unlikely that the difference in their abilities to buffer
2+
ATP-induced changes in [Ca ]i was due to differences in the expressions of these
exchangers.
2+
[Ca ]i Recovery Rate And Time
2+
The ability of the exchanger to buffer changes in [Ca ]i is one measure of
+
Na /Ca
2+
exchanger activity.
Another means of assessing exchanger activity is to
F-121.2002.R3 13
measure the initial rate of return (by taking the slope of the line) and the total time that is
2+
2+
required for [Ca ]i to approach baseline levels after agonist-induced increases in [Ca ]i.
Obviously, there are several Ca
2+
extrusion/sequestering mechanisms but, by comparing
the responses of OK-PTH cells expressing RNCX versus SNCX, it is possible to
2+
determine if there are differences in the rate of Ca
extrusion by these two isoforms. For
these experiments, ATP and bradykinin were used at concentrations of 1 and 0.1 mM,
respectively. These concentrations were found in preliminary experiments to produce
2+
rapid and maximal increases in [Ca ]i. Presumably, at these concentrations of ATP and
bradykinin, the magnitude of Ca
2+
influx and/or mobilization was sufficient to
overwhelm the cellular extrusion/sequestering mechanisms. As shown in Figs. 4a and 5a
and summarized in Figs. 4c and 5b, the presence of RNCX resulted in a significantly
2+
greater rate of return of [Ca ]i after the administration of 1 or 0.1 mM ATP and
bradykinin, respectively, compared to cells expressing SNCX. This can be seen in Figs.
2+
4a and 5a but is also statistically significant, when plotted as the initial rate of Ca
2+
recovery (Figs. 4c and 5b) or as the time required for [Ca ]i to approach baseline levels
(Figs. 4d and 5c). RNCX and SNCX are both expressed in mesangial cells of the
parental strain, Sprague Dawley rat (see accompanying manuscript) and are designated
SDRNCX and SDSNCX, respectively. While RNCX and SDRNCX are identical at
every amino acid residue, SNCX and SDSNCX differ at amino acid 218 where it is
isoleucine in SDSNCX but is phenylalanine in SNCX. To assess the effect of this single
amino acid difference, OK cells were transfected with SDSNCX and the ability of OK
2+
cells expressing SDSNCX (SDSOK) to reduce ATP (1mM)-induced [Ca ]i increase was
F-121.2002.R3 14
assessed as previously described. We found that SDSOK cells reduced ATP-induced
2+
[Ca ]i increase back to baseline level at a rate which was significantly greater than that
for SOK but equivalent to that for ROK (Figs. 4b, c, and d).
PKC Regulation
Previous studies have shown that basal exchanger activity is similar between
RNCX and SNCX isoforms and that PKC activation leads to enhanced exchanger activity
in OK-PTH cells that expressed RNCX but not SNCX (33). Since, in the presence of
extracellular ATP, exchanger activity was higher in ROK and SDSOK cells as compared
to the SOK cells, this difference could be due to ATP activation of PKC. To test this
possibility, ROK, SDSOK and SOK cells were either left untreated or pretreated with
300 nM PMA for 24 hrs, a maneuver that is known to down-regulate PKC activity. As
shown in Figs. 6(a-c), there is a distinct difference in the effects of PKC down-regulation
in ROK and SDSOK versus SOK cells. As shown in Figs. 6(a,b) the rate of return of
2+
[Ca ]i in ROK and SDSOK cells, after administration of ATP, was dramatically reduced
2+
with PKC inhibition. However, the rate of return of [Ca ]i in SOK cells (Fig. 6(c)) was
not greatly affected by prior treatment with PMA. As shown in the summary in Fig. 6(d)
2+
there was a highly significant decrease in the rate of Ca
recovery with PKC down-
regulation in ROK and SDSOK cells but not in SOK cells.
Genomic DNA Sequencing
The amino acid difference between RNCX and SNCX at residue 218 occurred as
the result of an A to T transversion which changed the codon for isoleucine (ATT) to that
F-121.2002.R3 15
for phenylalanine (TTT). In order to determine if this difference occurs at the genomic
level, the coding region of the Na+/Ca2+ exchanger gene from nucleotide 1 to 747 was
sequenced in both directions using genomic DNAs isolated from kidneys of salt sensitive
and salt resistant Dahl/Rapp rats. The sequence data (Fig. 7) show that the difference
between RNCX and SNCX that we observed in the cDNAs does not occur at the DNA
level.
F-121.2002.R3 16
DISCUSSION
+
The Na /Ca
2+
exchanger (NCX) is a 120 kDa transmembrane protein which
consists of five N-terminus and four C-terminus membrane spanning domains and a
cytosolic loop which comprises over 50% of the protein (11). There are at least four
different NCX exchangers encoded by separate genes (18, 24, 26). To date only NCX1
(which is also the cardiac isoform) has been demonstrated to occur in the tubules (4, 5),
blood vessels (2, 23) and mesangial cells of the kidney (2, 20, 22, 33). RNCX (NCX1.3)
and SNCX (NCX1.7) were previously cloned from mesangial cells of the Dahl/Rapp salt
resistant and salt sensitive rat, respectively (33). They are 100% homologous at the Cterminus membrane spanning domains and nearly identical in the N-terminus membrane
spanning domains except for a single amino acid difference at residue 218 where it is
isoleucine in RNCX and phenylalanine in SNCX. It should be noted that all other NCX1
isoforms that have been cloned have isoleucine at this site. Both RNCX and SNCX are
also highly homologus in the large cytoplasmic loop except at the alternative splice site
where RNCX is encoded by exons B and D whereas SNCX is encoded by exons B, D and
F. Thus, these differences in amino acid sequence may cause the lack of PKC sensitivity
+
of the Na /Ca
2+
exchanger in afferent arterioles and cultured mesangial cells from the
Dahl/Rapp salt sensitive rat. This PKC sensitivity of RNCX but not SNCX could also be
clearly demonstrated when these two isoforms were cloned into Opossum kidney
proximal tubule cells (OK-PTH).
In previous studies, exchanger activity in OK-PTH cells expressing RNCX
(ROK) and SNCX (SOK) was examined using a
45
2+
Ca -influx assay which involves
sodium loading cells followed by extracellular sodium removal. This stimulates the
F-121.2002.R3 17
+
reverse-mode Na /Ca
2+
2+
exchange (Ca
entry and Na exiting the cell) which is opposite
to how the exchanger normally operates under physiological conditions. Nevertheless,
these studies indicated that: 1) non-transfected OK-PTH cells had virtually no exchanger
activity; 2) basal exchanger activity was similar in ROK and SOK cells; 3) RNCX
activity increased with acute PMA treatment; and 4) SOK exchanger activity was
insensitive to PKC activation or PKC down-regulation by 24 hr pretreatment with PMA.
However these studies did not provide insights into whether this difference in PKC
sensitivity would have any consequences regarding the role of these exchangers in the
2+
regulation of [Ca ]i.
+
2+
2+
It has been suggested that Na /Ca exchange is involved in the extrusion of Ca
after agonist-induced elevations in [Ca2+]i (6). This issue was addressed in a recent study
by Slodzinski and Blaustein in which knockdown of exchanger activity was achieved by
pretreatment with antisense oligodeoxynucleotides of NCX (31).
They found that
2+
recovery from elevations in [Ca ]i was substantially prolonged in cells that were treated
2+
with antisense compared to control cells. This exchanger-mediated reduction in [Ca ]i
represents the forward-mode of the exchanger and more closely resembles the
+
physiological role of Na /Ca
2+
2+
exchange in [Ca ]i homeostasis. In the present study we
2+
used a similar approach except that we compared the regulation of [Ca ]i in cells that
were either vector-transfected or transfected with the RNCX, SNCX, or SDSNCX clone.
2+
In this manner we could directly determine if there were differences in [Ca ]i regulation
between RNCX and SNCX and whether those differences could be attributed to the
differences at the amino acid level.
F-121.2002.R3 18
2+
Initial studies were performed to measure baseline [Ca ]i in these three cells
+
types. We found that cells expressing Na /Ca
2+
2+
exchanger isoforms had lower baseline
2+
[Ca ]i compared to non-transfected cells. [Ca ]i regulation is complex, and is the result
of an ensemble of receptors, channels and transporters located at plasma and intracellular
2+
membrane sites. Nevertheless, the notion has been that at the plasma membrane, Ca 2+
ATPase, a high affinity and low-capacity Ca -extrusion mechanism, was important for
2+
setting the resting [Ca ]i while NCX, a low affinity and high-capacity Ca
2+
mechanism, primarily extruded Ca
2+
extrusion
2+
when [Ca ]i was elevated above baseline levels (1,
6, 9). Our studies indicate that NCX does contribute to the non-stimulated level of
2+
[Ca ]i at least in this cell type and under these conditions.
2+
In most studies of agonist-induced alterations in [Ca ]i, the initial paradigm is to
invoke Ca
2+
transients by rapidly applying sufficient hormone or agent to achieve a
2+
maximum [Ca ]i spike. In the present studies we wanted to examine the ability of the
2+
exchanger clones to handle a submaximal stimulus that would cause Ca
entry and
mobilization. We found that OK-PTH cells express P2X4, P2X5, and P2Y2 (data not
shown) and that a concentration of 100 µM ATP produced optimal discrimination
between the results obtained in the vector-transfected group versus those obtained in
ROK cells. In other cell types 100 µM ATP may produce maximal effects, however, this
may depend on, among other things, the number of P2 receptors that are on the cell
membrane. The important point is that during this exposure to
M levels of ATP, cells
2+
expressing NCX were much better able to prevent or minimize the increase in [Ca ]i.
Also, a major finding of this study, is that ROK cells were much better at buffering
F-121.2002.R3 19
2+
changes in [Ca ]i compared to SOK cells. Because these cell lines express comparable
levels of NCX (Fig. 1), this is direct evidence supporting the notion that the SNCX
2+
isoform is not as efficient or as effective in regulating [Ca ]i.
Other studies were performed to assess the ability of RNCX and SNCX to extrude
Ca
2+
2+
after maximal agonist-induced increases in [Ca ]i. Similar to what was observed in
the preceding experiments, the rate at which Ca
2+
declined after administration of 1 or
0.1 mM ATP or bradykinin, respectively, was slower and took longer to occur in SOK
cells versus ROK cells. These results further support our conclusion that SNCX is much
2+
less efficient in regulating agonist induced alterations in [Ca ]i. RNCX and SNCX are
both expressed in the parental strain, Sprague Dawley rat, and are designated SDRNCX
and SDSNCX, respectively. RNCX and SDRNCX are identical at every amino acid
residue while SNCX and SDSNCX differ at amino acid 218 where it is isoleucine in
SDSNCX but is phenylalanine in SNCX.
To determine if the single amino acid
difference at 218 contributes, in part, to the inability of SNCX to regulate ATP-induced
[Ca2+]i increase efficiently, we next examined the ability of cells expressing SDSNCX
(SDSOK) to regulate ATP(1mM)-induced [Ca2+]i increase. SDSOK cells reduced ATPinduced [Ca2+]i at a rate which was comparable to that for cells expressing RNCX. Thus,
the isoleucine to phenylalanine change in SNCX appeared to contribute, in part, to the
inability of this isoform to regulate agonist induced elevations in [Ca2+]i levels.
Because of the profound effect of the isoleucine to phenylalanine difference at
amino acid 218 on the ability of SNCX to regulate agonist-induced [Ca2+]i increase, we
next determined if the mutation occurred at the genomic level. This was accomplished
by sequencing the coding region of the Na+/Ca2+ exchanger gene from genomic DNA of
F-121.2002.R3 20
salt sensitive and salt resistant Dahl/Rapp rats spanning nucleotides 1 to 747. Our data
showed that the A to T transversion in the SNCX cDNA did not occur at the genomic
level. However, since we took great care in eliminating PCR artifacts in our original
study where we sequenced these isoforms, we feel confident that this mutation is not
likely to be a PCR artifact. We suggest the possibility that this mutation might occur in
the salt sensitive mesangial cell as a result of mRNA editing. Though rare, studies have
shown that this does occur in the eukaryotic cell and affects a number of proteins
including Apo B (32), glutamate receptor (17), and oxytocin receptor (7). Apo B has two
isoforms, Apo B-100 and Apo B-48. Apo B-100 is a 512 KDa protein that is synthesized
in the liver while Apo B-48 is a 240 KDa protein that consists of the N-terminus of Apo
B-100. Apo B-48 is synthesized in the small intestine, is developmentally regulated, and
is the result of CAA to UAA mutation which generates this truncated form of Apo B.
The glutamate receptor has also been shown to undergo mRNA editing which results in
Q to R mutation which arises as the result of a CAG to CGG codon change. Studies
showed that differences in oxytocin receptor mRNA sequences for different oxytocin
receptor populations in the endometrium are due to mRNA editing. mRNA editing of the
oxytocin receptor transcripts leads to differences in the observed responses to an oxytocin
challenge. Therefore, although rare, the possibility exists that NCX mRNA editing may
occur in addition to alternative splicing in mesangial cells of the salt sensitive Dahl/Rapp
rat.
+
Currently, the role that PKC plays in the activation of the Na /Ca
2+
exchanger is
not clearly understood. Our laboratory has demonstrated that PKC activates NCX in the
renal microcirculation of rabbits and normotensive rats (8) and in the cloned exchanger
RNCX (33). Also, work by Vigne et al. found that phorbol esters stimulated the
F-121.2002.R3 21
exchanger in cultured aortic smooth muscles (35). However, other studies in human
+
mesangial cells by Mene et al. failed to demonstrate enhanced reverse-mode Na /Ca
2+
exchange (21). In studying the regulation of NCX1, 2, & 3 Linck et al. (19) found that
NCX1 & 3 showed modest stimulation by both PKA and PKC agonists. Exchanger
activity appeared to be much more sensitive to PMA down-regulation. Finally, Iwamoto
et al. demonstrated that activation of PKC by agonists enhanced exchanger activity (12)
and phosphorylation of NCX1 (12-14) which occurred exclusively on serine residues.
Mutational analysis, however, indicated that activation of the exchanger did not require
direct phosphorylation of serine residues by PKC but did require the presence of the
cytosolic loop (12). Thus, the bulk of the current evidence favors PKC activation of
NCX1 but the mechanism by which this occur remains unclear. Our laboratory has
proposed that PKC may induce translocation of the exchanger, but whether this is
responsible for enhanced exchanger activity has also not been established.
The role of PKC in enhancing exchanger activity may be important based on the
following scheme. The binding of a vasoactive hormone to its receptor triggers the
activation of phospholipase C (PLC) which hydrolyses phosphatidyl inositol 4,5bisphosphate (PIP2) and elevates diacylglycerol (DAG) and inositol 1,4,5-trisphosphate
(IP3). IP3 binds to its receptor on the endoplasmic reticulum (ER) and stimulates the
release of Ca
2+
2+
2+
which thereby initiates the rise in [Ca ]i. With an elevation of [Ca ]i
+
2+
and DAG, there is activation of PKC which in turn enhances Na /Ca exchange. Thus,
+
PKC increases the rate at which the Na /Ca
+
2+
2+
exchanger extrudes Ca
Na thereby reducing [Ca ]i back to baseline levels.
2+
in exchange for
F-121.2002.R3 22
In the present studies this scenario was, in part, tested by 24 hr pretreatment with
PMA, a maneuver that is known to down-regulate PKC activity. Studies in other cell
systems (28) have shown that extracellular ATP via P2 receptors does increase PKC
activity. We demonstrated that inhibition of PKC activity greatly affected the abilities of
2+
ROK and SDSOK cells to rapidly return [Ca ]i to control levels. In contrast, PKC
2+
inhibition did not affect the rate of return in [Ca ]i in SOK cells. It should be mentioned
2+
that the Ca transients obtained in the presence of PKC inhibition were much more labile
and variable compared to cells which were not treated with PMA. This may reflect the
2+
fact that PKC has multiple cellular effector sites including effects on other cellular Ca
regulatory processes.
Nevertheless, these results are in agreement with our previous
+
work examining reverse-mode Na /Ca
2+
exchange in ROK and SOK cells (33). They
extend this previous work by suggesting that this difference in PKC sensitivity may have
2+
important consequences in terms of the cellular regulation of [Ca ]i. Furthermore, these
results indicate that insensitivity of SNCX to PKC may be attributed, in part, to the single
amino acid difference at residue 218. However, future mutational analysis is needed to
determine the exact mechanism which underlies the basis of SNCX insensitivity to PKC.
+
In conclusion, we have found that an isoform of the Na /Ca
2+
exchanger that was
cloned from mesangial cells of the Dahl/Rapp salt sensitive rat has an impaired ability to
2+
regulate agonist-induced changes in [Ca ]i. The cause of this reduced efficiency in Ca
2+
extrusion appears to be due to a defect in PKC-activation of SNCX which may be
attributed, in part, to a single amino acid mutation at amino acid 218. We suggest that
+
this reduced ability of the Na /Ca
2+
2+
exchanger to regulate [Ca ]i in the renal
F-121.2002.R3 23
microcirculation may, in part, be responsible for the increased vascular resistance and
reduced GFR that is a hallmark of this form of hypertension.
F-121.2002.R3 24
ACKNOWLEDGMENTS
This work was supported by grants 2R01HL07457-20 (PDB), 1K01HL67718-01
(MTU), and T32HL07457-20 (EH) from the NHLBI.
Foundation postdoctoral fellow.
JPP is a National Kidney
F-121.2002.R3 25
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16.
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Kortenbruck G, Berger E, Speckmann EJ, and Musshoff U. RNA editing at
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Zhu Z, Tepel M, Neusser M, and Zidek W. Role of Na+-Ca2+ exchange in
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F-121.2002.R3 30
FIGURE LEGENDS
Fig. 1. Stably transfected OK-PTH cells express comparable levels of RNCX and SNCX
proteins. Cell lysate from OK-PTH cells expressing RNCX (ROK) or SNCX (SOK) was
isolated as described in Methods, fractionated on an 8% SDS polyacrylamide gel,
transferred to Immobylon and immunodetected with a monoclonal Na+/Ca2+ exchanger
antibody. (a) Representative immunoblot showing the 120 KDa band and (b) bar graph
showing relative densitometric units for the 120 KDa band in each sample. Means ±
S.E.M. (n = 3); *p < 0.05 compared with VOK.
2+
Fig. 2. Baseline [Ca ]i is higher in vector-transfected OK-PTH cells than in OK-PTH
2+
cells expressing RNCX or SNCX. Baseline [Ca ]i in vector-transfected (VOK) and
transfected OK-PTH cells expressing RNCX (ROK) and SNCX (SOK) was measured
2+
using fura 2. Baseline [Ca ]i was significantly higher in VOK cells than in ROK and
SOK cells. Data were analyzed for statistical significance using ANOVA. Means ±
*
S.E.M. (n = 12); p < 0.05 compared with VOK.
Fig. 3. OK-PTH cells expressing RNCX (ROK) showed greater ability to buffer ATP2+
induced [Ca ]i increase compared to vector-transfected (VOK) and transfected OKPTH cells expressing SNCX (SOK). The abilities of ROK and SOK to buffer ATP2+
induced [Ca ]i increase were assessed in VOK, ROK, and SOK cells following
2+
2+
treatment with 100 µM ATP to elevate [Ca ]i. (a) Representative [Ca ]i tracings of
2+
VOK, ROK, and SOK in response to 100 µM ATP, and (b) bar graph showing ∆[Ca ]i
2+
in VOK, ROK, and SOK cells. ROK and SOK had lower ∆[Ca ]i or greater capacity to
F-121.2002.R3 31
2+
buffer ATP-induced [Ca ]i increase compared to VOK, with ROK having the greatest
2+
[Ca ]i buffering ability. Data were analyzed for statistical significance using ANOVA.
*
Means ± S.E.M. (n=12); p < 0.05 compared with VOK.
Fig. 4. OK-PTH cells expressing RNCX (ROK) and SDSNCX (SDSOK) reduced ATP2+
induced [Ca ]i increase at a significantly greater rate than cells expressing SNCX
2+
(SOK). The abilities of ROK, SDSOK, and SOK cells to reduce ATP-induced [Ca ]i
increase back toward baseline were assessed in fura 2-loaded ROK, SOK, and SDSOK
2+
2+
cells following treatment with 1 mM ATP to elevate [Ca ]i. (a) Representative [Ca ]i
tracings of ROK and SOK and (b) SDSOK and SOK in response to 1mM ATP. (c,d) Bar
2+
graphs showing [Ca ]i recovery rates (c) and times (d) in ROK, SDSOK, and SOK cells.
Data were analyzed for statistical significance using ANOVA. Means ± S.E.M. (n =
*
12); p < 0.05 compared with VOK.
2+
Fig. 5. OK-PTH cells expressing RNCX (ROK) reduced bradykinin-induced [Ca ]i
increase at a significantly greater rate than cells expressing SNCX (SOK). The abilities
2+
of ROK and SOK cells to reduce bradykinin-induced [Ca ]i increase back toward
baseline were assessed in fura 2-loaded ROK and SOK cells following treatment with
100
2+
2+
M bradykinin to elevate [Ca ]i. (a) Representative [Ca ]i tracings of ROK and
2+
SOK and (b,c) bar graphs showing [Ca ]i recovery rates (b) and times (c) in ROK and
SOK cells. Data were analyzed for statistical significance using ANOVA. Means ±
*
S.E.M. (n = 9); p < 0.05 compared with SOK.
F-121.2002.R3 32
Fig. 6.
PKC down-regulation attenuates the ability of cells expressing RNCX and
2+
SDSNCX to reduce ATP-induced [Ca ]i back to baseline level. The effect of PKC
2+
down-regulation on [Ca ]i recovery rate in response to 1 mM ATP in ROK (a), SOK (b)
and SDSOK cells (c) was examined in fura 2 loaded cells following ATP treatment with
or without 24 hr PMA (300 nM) pretreatment to down-regulate PKC by single cell
2+
[Ca ]i measurements in comparison with non-PMA treated cells (CTL). (d) Bar graph
2+
showing the effect of PKC down-regulation on [Ca ]i recovery rate in ROK, SOK and
SDSOK cells. Data were analyzed for statistical significance using ANOVA. Means ±
*
S.E.M. (n = 12); p < 0.05 compared with non PMA treated cells.
Fig. 7. The genomic DNA sequence of SNCX shows ATT(I) but not TTT(F) at nucleotide
652-654. To determine if the isoleucine to phenylalanine mutation at amino acid 218
(shown in bold) in SNCX occurs at the genomic level, genomic DNA was isolated from
kidneys of salt sensitive and salt resistant rats and the nucleotide sequence corresponding
to the N-terminus of the Na+/Ca2+ exchanger was PCR amplified using Na+/Ca2+
exchanger specific primers and sequenced. The nucleotide sequence of SNCX and
RNCX spanning nucleotides 631-657 (amino acid 211-219) is shown for RNCX and
SNCX cDNA (c) and genomic DNA (g).
F-121.2002.R3 33
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