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(Received for publication, November 2, 1995) From the
Chronic dietary phosphate restriction is associated with
up-regulation of sodium-dependent phosphate (Na/P Chronic dietary P Chronic P Opossum
kidney (OK) cells, a continuous line of renal epithelial cells, are a
useful experimental model for investigation of the cellular mechanisms
involved in the adaptation to P
To evaluate whether
P To provide a reference for total RNA per
lane, the Northern blots were stripped and rehybridized with a probe
for the glyceraldehyde-3-phosphate dehydrogenase ``housekeeping
gene.'' Degenerate forward
(5`-AA(A/G)-TGG-GGT-GAT-GCT-GGT-GC(C/T)-G-3`) and reverse
(5`-CAT-GCC-AGT-GAG-(C/T)TT-CCC-GTT-C-3`) primer pairs for the rat,
rabbit, and human glyceraldehyde-3-phosphate dehydrogenase cDNAs (20) were a kind gift of James Schafer (Department of
Physiology, University of Alabama at Birmingham). After reverse
transcription-PCR of OK cell RNA, these primers were used to synthesize
a
Figure 1:
Effect of phosphate restriction and
verapamil on cytosolic calcium in OK cells. OK cells were preincubated
for 24 h in serum-free medium containing P
Figure 2:
Effect of calcium channel blockade on
Na/P
Figure 4:
Effect of actinomycin on Na/P
Figure 5:
Effect of calcium ionophores and calcium
chelators on NaPi-4 mRNA levels in P
Using OK cells, an experimental model for renal epithelial
cells, we have demonstrated parallel increases in cytosolic calcium and
Na/P The observed increase in
cytosolic calcium in P Experimental maneuvers that impede entry of calcium into the cells
or that chelate calcium may prevent an increase in cytosolic calcium in
P Even in the presence of a net influx of calcium into
cells, a calcium chelator prevents the anticipated increase in
cytosolic calcium. We found that the calcium chelator quin-2/AM
prevented the increase in NaPi-4 mRNA in P Calcium ionophores promote entry of calcium into the
cell, also favoring an increase in cytosolic calcium. If the increase
in cytosolic calcium mediates the increase in NaPi-4 mRNA in
P It is possible that an
increase in cytosolic calcium produces multiple nonspecific cellular
changes, rather than a specific increase in certain cellular functions.
The lack of change in sodium-dependent glucose transporter mRNA during
P The parallel increases in NaPi-4
mRNA, NaPi-4 protein, and Na/P The observed
increase in NaPi-4 mRNA during P
Volume 271,
Number 7,
Issue of February 16, 1996 pp. 3902-3906
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)
cotransport by renal proximal tubular epithelial cells in association
with increases in Na/P cotransporter mRNA and protein. We
investigated whether changes in cytosolic calcium mediate this adaptive
response in opossum kidney cells, a continuous line of renal epithelial
cells. After 24 h of phosphate depletion, steady-state cytosolic
calcium levels were increased; this increase was observed at
physiologic levels of phosphate restriction and was prevented by the
calcium channel blocker verapamil. Chronic phosphate depletion was also
associated with parallel increases in Na/P cotransport
activity, Na/P cotransporter mRNA, and Na/P cotransporter protein, all of which were blocked in
verapamil-treated cells. Actinomycin D, at a dose that prevented the
increase in NaPi-4 mRNA during phosphate depletion, also prevented the
increase in Na/P cotransport activity. Incubation with the
calcium ionophore ionomycin or A23187 reproduced the increase in
Na/P cotransporter mRNA in phosphate-replete cells.
Conversely, chelation of cytosolic calcium by quin-2/AM prevented the
increase in Na/P cotransporter mRNA in phosphate-depleted
cells. The effect of an increase in cytosolic calcium was specific for
the Na/P cotransporter as mRNA levels for the
sodium-dependent glucose transporter were not affected. Our
observations suggest that chronic phosphate restriction increases
steady-state cytosolic calcium, which, in turn, increases transcription
of Na/P cotransporter mRNA, thereby stimulating Na/P cotransport activity.
restriction results in widespread
changes in the physiologic function of multiple cell types. These
include impaired insulin secretion in pancreatic islet cells, impaired
norepinephrine metabolism in synaptosomes, and impaired phagocytosis in
polymorphonuclear cells(1, 2, 3) .
P-depleted cells also have elevated cytosolic calcium
levels. In vivo administration of the calcium channel blocker
verapamil to rats during chronic dietary P restriction
prevents both the increase in cytosolic calcium as well as the
characteristic physiologic changes(1, 2, 3) .
These observations suggest that an increase in cytosolic calcium is an
important cellular mediator of many physiologic changes associated with
phosphate restriction. deprivation is also
associated with an increase in P reabsorption by the
kidney, which is mediated by a specific, membrane-bound
sodium-dependent phosphate (Na/P) (
)cotransporter in the proximal tubule (4, 5) This adaptive increase in Na/P cotransport activity is associated with parallel increases in the
Na/P cotransporter mRNA and
protein(6, 7) , suggesting a transcriptional cellular
mechanism. Moreover, recent studies suggest that changes in cytosolic
calcium can affect mRNA levels by modulating gene
transcription(8, 9, 10) , providing a
potential cellular mechanism for some of the observed effects of
P depletion. On the basis of these previous investigations,
we hypothesized that chronic P restriction increases
cytosolic calcium in renal tubular epithelial cells and that this
change, in turn, produces the observed increases in Na/P cotransporter mRNA, protein, and transport activity. restriction, in isolation
from the multiple systemic changes associated with in vivo P deprivation (4, 5) The recent
cloning of the OK cell Na/P cotransporter NaPi-4 cDNA (11) permits quantification of the corresponding mRNA levels.
In addition, we have raised a rabbit polyclonal antibody against NaPi-4
protein (12) to enable direct measurements of NaPi-4 protein
levels. We have recently demonstrated in OK cells parallel increases in
Na/P cotransport activity, Na/P cotransporter
NaPi-4 mRNA, and NaPi-4 protein during chronic P restriction(12) , similar to the changes observed in
P-deprived rats(6, 7) . Moreover, our
preliminary studies detected an increase in cytosolic calcium in
P-depleted OK cells. Using the newly available molecular
tools and the NaPi-4 antibody, we investigated whether an increase in
cytosolic calcium might mediate the increases in NaPi-4 mRNA, NaPi-4
protein, and Na/P cotransport activity associated with
chronic P restriction of OK cells. Specifically, we
addressed three experimental questions. 1) Do maneuvers that prevent
the increase in cytosolic calcium during P depletion
(calcium channel blockers or calcium chelators) prevent the increase in
NaPi-4 mRNA? 2) Do other experimental maneuvers that increase cytosolic
calcium in P-replete cells (calcium ionophores) also
produce an increase in NaPi-4 mRNA? 3) Are the effects of cytosolic
calcium specific for NaPi-4 mRNA?
Materials
OK cells were a gift of Judith Cole
(University of Missouri, Columbia, MO). Culture media and dishes were
from Life Technologies, Inc. Fura-2/AM was from Teflabs (Austin, TX).
The verapamil enantiomers S(-)-verapamil and R(+)-verapamil were from Research Biochemicals Inc.
(Natick, MA). Enhanced chemiluminescent (ECL) kits for developing the
Western blots were from Amersham (Buckinghamshire, United Kingdom).
Peptide N-glycosidase F was from Boehringer Mannheim.
Multi-antigen peptides for antibody production were synthesized by
Research Genetics (Huntsville, AL). PCR primers were synthesized by
Operon Technologies, Inc. (Alameda, CA). All isotopes were from DuPont
NEN. All other reagents were from Sigma.Cell Culture and Measurement of Na/P
OK cells were grown on 35-mm culture dishes in
minimum essential medium with the addition of 10% fetal calf serum, 2
mM glutamine, 50 IU/ml penicillin, and 50 µg/ml
streptomycin. They were kept in an incubator at 37 °C with 95% air
and 5% CO Cotransport
. The medium was changed every other day. At
weekly intervals, the cells were detached from the plates with 0.05%
trypsin, 0.02% EDTA and subcultured at a 1:10 dilution. P transport was measured as previously reported by us(13) .
After reaching confluence, the cells were rendered quiescent by a 16-h
incubation with serum-free minimum essential medium containing 1 mM P and 1% bovine serum albumin, followed by a 24-h
incubation in serum-free medium containing normal or low P concentrations. After aspirating the medium and washing, the
cells were incubated at 37 °C for 5 min with transport solution
(137 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl, 1.2 mM MgSO
, 14.0 mM HEPES, and 0.1 mM KHPO
, pH 7.4). The transport
solution was aspirated; the cells were washed three times with ice-cold
137 mM NaCl and 14 mM HEPES, pH 7.4, to stop
phosphate uptake; and P uptake was quantified by
scintillation counting. Sodium-independent P
uptake was
determined by substituting 137 mMN-methylglucamine
chloride for NaCl in the transport solution. Sodium-dependent
cotransport was calculated as the difference in P uptake
with and without sodium.Measurement of Cytosolic Calcium
After the
appropriate preincubation, the cells were detached with EDTA,
resuspended in serum-free medium, and loaded with Fura-2/AM for 40 min
at room temperature. They were washed and resuspended in balanced salt
solution (127 mM NaCl, 3.8 mM KCl, 1.2 mM KHPO, 1.2 mM CaCl,
0.8 mM MgCl, 5 mM glucose, and 10 mM HEPES, pH 7.4). The cells were transferred to 2-ml plastic
cuvettes, continuously stirred at room temperature, and excited
alternatively at wavelengths of 340 and 380 nm, with fluorescence
emission measured at 510 nm, using a Photon Technology International
Delta scanner. Cytosolic calcium was calculated from the ratio of
emissions at the two excitation wavelengths, as previously validated in
OK cells(14) . The calcium channel blocker verapamil was used
to prevent the increase in cytosolic calcium during P depletion. In addition, we used two biologically active
enantiomers of verapamil: S(-)-verapamil, which also
blocks calcium channels, and R(+)-verapamil, which does
not(15) . In some studies, the calcium ionophores ionomycin and
A23187 were used to increase cytosolic
calcium(16, 17) . In other studies, the calcium
chelator quin-2/AM was used to prevent an increase in cytosolic calcium (17) . The medium was supplemented with 10 mM sodium
pyruvate to prevent intracellular ATP depletion in the presence of
quin-2/AM.Measurement of Na/P
After reverse transcription-PCR of OK cell RNA, forward
(5`-TCC AGC ACA TCT ACC TCC ATC ATC-3`) and reverse (5`-AGT GGG GTA ATG
GCT GAA GTG AAC-3`) primers were used to synthesize a probe for NaPi-4
mRNA. The 0.8-kilobase PCR product was sequenced by a dye-labeled
dideoxynucleotide sequencing kit (Applied Biosystems Inc.), and
homology to the published NaPi-4 cDNA sequence (11) was
confirmed. The NaPi-4 PCR fragment was radiolabeled with
[ Cotransporter mRNA
LevelsP]dCTP by random priming and hybridized with OK
cell RNA using Northern analysis. Total RNA was isolated from OK cells
by the guanidium isothiocyanate method(18) , purified by phenol
and chloroform extraction, precipitated with isopropyl alcohol, and
washed with 70% ethanol. Total RNA was quantified by spectrophotometry,
denatured with 37% formaldehyde, separated on 1% agarose gel, and
transferred to a nylon membrane. After baking the membranes at 80
°C for 2 h, the blots were prehybridized at 42 °C with 5
SSPE (sodium chloride/sodium phosphate/EDTA buffer), 2% SDS,
and 50% formamide. After hybridization with the NaPi-4 probe, the
membrane was washed twice at room temperature with 2 SSPE and
0.1% SDS, followed by one wash at room temperature with 0.5
SSPE and 0.2% SDS and one wash at 42 °C with 0.4 SSPE and
0.2% SDS. The blots were exposed to Kodak X-AR film, and the bands were
quantified by densitometry. NaPi-4 cDNA hybridized with Northern blots
from OK cell RNA detected a single band at 2.5 kilobases,
comparable in size to NaPi-4 mRNA (11) .
depletion nonspecifically induces the transcription of
genes for other cell membrane-bound transporters, we also measured mRNA
levels for the renal sodium-dependent glucose transporter (SGT).
Forward (5`-AGC-TCA-TTC-GCA-ATG-CAG-CC-3`) and reverse
(5`-TGT-CCG-TGT-AAA-TCA-CCG-CC-3`) primers to the published sequence of
the human SGT cDNA (19) (at sites of homology to the rabbit and
rat SGT cDNAs) were selected with the MacVector software program and
synthesized commercially. Following reverse transcription-PCR of OK
cell RNA, this primer pair was used to make a 600-base pair probe.
Sequencing of this probe established >90% homology to the human SGT
sequence. When this probe was hybridized with OK cell RNA Northern
blots, it detected a single
2.8-kilobase signal, representing OK
cell SGT mRNA(12) .
400-base pair probe. Sequencing of the PCR fragment revealed a
90% homology to the published sequence for human
glyceraldehyde-3-phosphate dehydrogenase cDNA. When this probe was
hybridized with OK cell RNA Northern blots, it detected a single
1.4-kilobase signal, representing the OK cell
glyceraldehyde-3-phosphate dehydrogenase mRNA(12) .
Raising and Purification of Rabbit Polyclonal Anti-NaPi-4
Antibody
The 8-amino acid peptide RSPARLPK (amino acids
566-573) from the published amino acid sequence of NaPi-4 (11) was found to have both high hydrophilic and antigenic
indices, making it a promising epitope for the generation of
antibodies. Multi-antigen peptides were synthesized, with the peptide
coupled to polylysine to increase its immunogenicity. Rabbits were
immunized with 1 mg of multi-antigen peptides suspended in
Freund's complete adjuvant, followed by booster multi-antigen
peptide injections suspended in Freund's incomplete adjuvant at 4
and 6 weeks. Blood samples obtained 10 days after the second booster
revealed high antibody titers by enzyme-linked immunosorbent assay in
the antisera of three of the four rabbits. The antibodies were
precipitated from the antiserum with 50% ammonium sulfate saturation
and centrifuged at 10,000 g for 15 min at 4 °C.
The precipitate was dialyzed against 10 mM Tris, pH 7.6. The
dialysate was passed three times through an affinity column coupled to
goat anti-rabbit IgG. Further purification of the antibody was
performed by passing the eluate through a second affinity column to
which the NaPi-4 peptide was bound. Antibody bound to the column was
then eluted and dialyzed against Tris-buffered saline, pH 7.6. The
final purified antibody was used to probe the Western blots from OK
cell crude membrane protein.Preparation of Western Blots
OK cells were scraped
into 5 mM HEPES/KOH, pH 7.2, and homogenized with a syringe
through an 18-gauge needle. The resulting suspension was centrifuged (5
min at 1000 rpm). The supernatant was centrifuged for 30 min at 40,000
g at 4 °C. The pellet was resuspended in 100
mM mannitol/Tris-HCl, pH 7.2, with addition of the following
protease inhibitors: 1 µM leupeptin, 1 µM soybean trypsin inhibitor, 0.1 µM pepstatin, and 1
µM aprotinin. The crude membrane preparation was frozen in
liquid nitrogen until future use. It was then thawed, boiled with SDS
sample buffer, run on SDS-polyacrylamide gel, and transferred to a
nitrocellulose membrane. Nonspecific binding was blocked with 5% nonfat
dry milk for 2 h. The membrane was then incubated with rabbit
anti-NaPi-4 antibody. Finally, the membrane was incubated with goat
anti-rabbit IgG (1:20,000) conjugated to horseradish peroxidase. The
Western blots were developed using the ECL kit. The purified rabbit
polyclonal anti-NaPi-4 antibody detected an intense band at 70
kDa, corresponding to the predicted size of NaPi-4
protein(11) . This band was eliminated when the antibody was
preincubated with the NaPi-4 peptide (data not shown). A second band at
84 kDa was eliminated when the crude membrane preparation was
deglycosylated by an 18-h incubation at 37 °C with peptide N-glycosidase F (data not shown), suggesting that it
represents the glycosylated form of NaPi-4, analogous to the
glycosylated form of the rat Na/P
cotransporter (NaPi-2) (21) . Because the phosphate transport characteristics are
similar for glycosylated and deglycosylated NaPi-2
protein(21) , we deglycosylated the crude membrane preparation
prior to loading subsequent gels so as to quantify total NaPi-4
protein. An additional (weaker) band detected at 51 kDa probably
represents a degradation product of NaPi-4 protein.
Changes in Cytosolic Calcium during Phosphate
Depletion
Incubation of OK cells in a P-free medium
for 24 h was associated with an approximate doubling of steady-state
cytosolic calcium levels, as compared with P-replete cells (Fig. 1). This change was not limited to total P depletion; rather, progressive P restriction within
the physiologic range was associated with progressive increases in
cytosolic calcium (Fig. 1A). The calcium channel
blocker verapamil prevented the increase in cytosolic calcium in
P-depleted cells, without affecting steady-state cytosolic
calcium levels in cells incubated with 1 mM P (Fig. 1B). S(-)-Verapamil
reproduced the effect of verapamil on cytosolic calcium, whereas R(+)-verapamil did not, confirming the specificity of the
former enantiomer for the calcium channel.
concentrations
between 0 and 1 mM. The cells were then loaded with Fura-2/AM
for 40 min, washed and resuspended in balanced salt solution, and
excited alternatively at wavelengths of 340 and 380 nm, with
fluorescence emission measured at 510 nm, using a Photon Technology
International Delta scanner. Cytosolic calcium was calculated from the
ratio of emissions at the two excitation wavelengths. A, the
cells were incubated with medium containing 0, 0.2, 0.4, 0.6, 0.8, or
1.0 mM P (Phos) prior to measurement of
cytosolic calcium. B, the cells were incubated with the
calcium channel blocker verapamil (Ver; 100 µM)
to prevent the increase in cytosolic calcium during P depletion. In addition, we used two biologically active
enantiomers of verapamil: S(-)-verapamil, which also
blocks calcium channels, and R(+)-verapamil, which does
not. Values are means ± S.E. of three experiments. *, p < 0.05; , p < 0.01; , p < 0.0001 versus cells incubated with 1 mM P, as
calculated by analysis of variance. Con,
control.
Effect of Calcium Channel Blockade on Na/P
Incubation of OK cells in a 0 mM P Cotransport Activity, NaPi-4 mRNA, and NaPi-4 Protein during
Phosphate Depletion medium for 24 h was associated with a significant
increase in Na/P cotransport activity, as compared with
cells incubated with 1 mM P (Fig. 2A). Chronic P depletion was
also associated with parallel increases in NaPi-4 mRNA and protein, as
compared with P-replete cells (Fig. 2B and
3). Verapamil completely prevented the increases in NaPi-4 mRNA, NaPi-4
protein, and Na/P cotransport activity in
P-depleted cells, without affecting any of these parameters
in P-replete cells. S(-)-Verapamil
reproduced the inhibitory effects of verapamil, whereas R(+)-verapamil did not.
cotransport activity and NaPi-4 mRNA levels during
phosphate depletion. OK cells were preincubated in serum-free medium
containing 1 or 0 mM phosphate ± 100 µM verapamil (Ver), R(+)-verapamil, or S(-)-verapamil for 24 h. A, the medium was
aspirated, the cells were washed and then incubated for 5 min at 37
°C with transport solution, and P uptake was
quantified by scintillation counting. Values are means ± S.E. of
three experiments. , p < 0.001 versus cells
incubated with 1 mM P
, as calculated by analysis
of variance. B, total RNA was extracted, separated on 1%
agarose (10 µg of RNA/lane), transferred to nylon membranes, and
hybridized sequentially with an NaPi-4 cDNA probe and a probe for
glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Con, control.
Dependence of Adaptation to Phosphate Depletion on Gene
Transcription
Further experiments were performed to determine
whether gene transcription was required for the chronic adaptation to
phosphate depletion. Actinomycin D (0.25 µg/ml), an inhibitor of
DNA transcription, completely abolished the increase in Na/P cotransport during chronic (24 h) P depletion (Fig. 4A). Parallel Northern analysis confirmed that
this dose of actinomycin prevented an increase in NaPi-4 mRNA levels
during P depletion (Fig. 4B).
cotransport and NaPi-4 mRNA during chronic phosphate depletion of
OK cells. OK cells were incubated with 1 or 0 mM P for 24 h ± actinomycin (Actino, Act;
0.25 µg/ml). A, Na/P cotransport was measured
after the incubation. Values are means ± S.E. of four
experiments. , p < 0.001 versus 1 mM P, by Student's t test. B,
total RNA was extracted, separated on 1% agarose (10 µg of
RNA/lane), transferred to nylon membranes, and hybridized with the
NaPi-4 cDNA probe. Veh, vehicle.
Effect of Calcium Ionophores on NaPi-4
mRNA
Additional studies were performed to evaluate whether other
experimental maneuvers that increase cytosolic calcium reproduce the
effect of P depletion on the Na/P cotransporter
mRNA. The calcium ionophores ionomycin and A23187 both increased NaPi-4
mRNA levels in OK cells incubated with 1 mM P,
without affecting mRNA levels for the glyceraldehyde-3-phosphate
dehydrogenase housekeeping gene (Fig. 5). Moreover, neither
ionophore changed mRNA levels for an unrelated membrane-bound
transporter, the sodium-dependent glucose transporter. Interestingly,
there was no additive effect between P depletion and the
calcium ionophores on NaPi-4 mRNA levels in OK cells.
-depleted and
P-replete OK cells. The cells were incubated for 24 h in
serum-free medium containing 1 or 0 mM P ±
vehicle, ionomycin (Iono; 1 µM), A23187 (1
µM), or quin-2/AM (20 µM). Total RNA was
extracted, separated on 1% agarose (10 µg of RNA/lane), transferred
to nylon membranes, and hybridized sequentially with an NaPi-4 cDNA
probe, a probe for SGT, and a probe for glyceraldehyde-3-phosphate
dehydrogenase (GAPDH). Con,
control.
Effect of Calcium Chelators on NaPi-4
mRNA
Additional studies demonstrated that the calcium chelator
quin-2/AM could prevent the increase in NaPi-4 mRNA during P depletion of OK cells (Fig. 5). In contrast, the calcium
chelator did not affect mRNA levels for the sodium-dependent glucose
transporter.
cotransporter NaPi-4 mRNA during chronic (24 h)
phosphate depletion. These observations suggest that an increase in
cytosolic calcium mediates the increase in NaPi-4 mRNA in
P-restricted OK cells. Alternatively, P depletion may increase cytosolic calcium and NaPi-4 mRNA by two
independent cellular mechanisms. To establish causality between the
changes in cytosolic calcium and NaPi-4 mRNA, several conditions must
be satisfied. First, prevention of the increase in cytosolic calcium
should prevent the increase in NaPi-4 mRNA in P-depleted
cells. Second, other experimental maneuvers that increase cytosolic
calcium should also increase NaPi-4 mRNA. Finally, the effect of
cytosolic calcium should be specific for NaPi-4 mRNA, without affecting
other membrane-bound transporters.-restricted OK cells is in agreement
with similar findings in pancreatic islet cells, synaptosomes, and
polymorphonuclear cells from rats(1, 2, 3) .
Chronic in vivo P deprivation is associated with
multiple systemic changes (e.g. decreased parathyroid hormone
and increased calcitriol and insulin), which may themselves affect cell
function(4, 5) . Because the increase in cytosolic
calcium in this study occurred in cultured cells, in isolation from
systemic factors, it must reflect an intrinsic effect of P restriction. Moreover, the finding of increased cytosolic calcium
even during partial P restriction (Fig. 1A)
establishes the physiologic significance of this effect. The cellular
mechanism whereby P restriction increases cytosolic calcium
in OK cells remains to be established. Levi et al.(2) have reported a decrease in the activity of the
Ca-ATPase in pancreatic islet cell membranes from
P
-restricted rats. This physiologic defect, by impairing
cellular extrusion of calcium, would favor an increase in cytosolic
calcium due to unopposed entry of calcium from the extracellular space. -restricted cells. Thus, we have observed that incubation
with the calcium channel blocker verapamil prevented the increase in
NaPi-4 mRNA. Verapamil has multiple effects on cellular function that
are independent of calcium channel blockade. Thus, for example,
multiple immune functions of lymphocytes are inhibited by both S(-)-verapamil and R(+)-verapamil, even
though only the former enantiomer blocks the calcium
channel(15) . Therefore, demonstrating inhibition of a
particular cellular function by verapamil does not necessarily
establish that cytosolic calcium mediates that function. In this study,
however, the adaptations to P restriction were blocked only
by S(-)-verapamil, and not by R(+)-verapamil. This discrepancy suggests that the
increase in cytosolic calcium is in fact mediating the corresponding
increases in Na/P cotransport activity, NaPi-4 mRNA, and
NaPi-4 protein.-restricted OK
cells (Fig. 5). This observation adds further weight to the
hypothesis that the increase in cytosolic calcium mediates the increase
in NaPi-4 mRNA.-depleted cells, then calcium ionophores should increase
NaPi-4 mRNA levels even in P-replete cells. Indeed, we
found that incubation with two different calcium ionophores, ionomycin
and A23187, reproduced the increase in NaPi-4 mRNA in
P-replete OK cells (Fig. 5). Moreover, the effects
of P restriction and the calcium ionophores on NaPi-4 mRNA
were not additive, suggesting that both experimental maneuvers are
mediated by a common biochemical pathway. restriction and calcium ionophore incubation (two
experimental maneuvers that increase steady-state cytosolic calcium)
suggests, however, that the effects are specific for the membrane-bound
Na/P cotransporter. cotransport activity in
P-depleted OK cells are in agreement with our previous
report(12) . These observations suggest that the increase in
NaPi-4 mRNA mediates the increase in Na/P cotransport
activity during chronic P depletion. The prevention by
actinomycin D of the increase in both NaPi-4 mRNA and Na/P cotransport activity during P depletion (Fig. 4) lends weight to this conclusion. depletion may reflect
either an increase in transcription rate or an increase in mRNA
stability. Our finding that actinomycin blocks the increase in NaPi-4
mRNA in P-restricted cells (Fig. 4) tends to support
a transcriptional effect of cytosolic calcium. The recent demonstration
of a direct modulation of transcription factors by calcium (8, 9) suggests that increased cytosolic calcium may
interact with a transcription factor for NaPi-4, thereby increasing
NaPi-4 mRNA levels. On the basis of our experimental findings, we
propose that phosphate depletion increases cytosolic calcium in renal
tubular epithelial cells. The increase in cytosolic calcium modulates a
transcription factor for the Na/P cotransporter gene. This
effect, in turn, increases Na/P cotransporter mRNA and
protein levels, ultimately up-regulating Na/P cotransport
activity. Confirmation of this novel cellular mechanism will require a
more detailed knowledge of the promoter and enhancer regions associated
with the Na/P cotransporter DNA.
),
sodium-dependent phosphate; OK, opossum kidney; PCR, polymerase chain
reaction; SGT, sodium-dependent glucose transporter.
We thank David G. Warnock for ongoing advice, support,
and encouragement and Angela Myracle and Joyce Watkins for maintaining
the OK cells.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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