(Received for publication, November 2, 1995)
From the
Chronic dietary phosphate restriction is associated with
up-regulation of sodium-dependent phosphate (Na/P)
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.
Chronic dietary P 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.
Chronic P 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.
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 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?
To evaluate whether
P 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) .
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 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) .
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 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.
Figure 2:
Effect of calcium channel blockade on
Na/P 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.
Figure 4:
Effect of actinomycin on Na/P 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.
Figure 5:
Effect of calcium ionophores and calcium
chelators on NaPi-4 mRNA levels in P-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.
Using OK cells, an experimental model for renal epithelial
cells, we have demonstrated parallel increases in cytosolic calcium and
Na/P 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.
The observed increase in
cytosolic calcium in P-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.
Experimental maneuvers that impede entry of calcium into the cells
or that chelate calcium may prevent an increase in cytosolic calcium in
P-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.
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-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.
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-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.
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 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.
The parallel increases in NaPi-4
mRNA, NaPi-4 protein, and Na/P 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.
The observed
increase in NaPi-4 mRNA during P 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.