Separate entry pathways for phosphate and oxalate in rat brain
microsomes
X.-J.
Meng,
R. T.
Timmer,
R. B.
Gunn, and
R. F.
Abercrombie
Department of Physiology, Emory University School of Medicine,
Atlanta, Georgia 30322
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ABSTRACT |
ATP-dependent 45Ca uptake in rat brain
microsomes was measured in intracellular-like media containing
different concentrations of PO4 and oxalate. In the absence
of divalent anions, there was a transient 45Ca
accumulation, lasting only a few minutes. Addition of PO4
did not change the initial accumulation but added a second stage that increased with PO4 concentration. Accumulation during the
second stage was inhibited by the following anion transport inhibitors: niflumic acid (50 µM),
4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS; 250 µM),
and DIDS (3-5 µM); accumulation during the initial stage was
unaffected. Higher concentrations of DIDS (100 µM), however,
inhibited the initial stage as well. Uptake was unaffected by 20 mM Na,
an activator, or 1 mM arsenate, an inhibitor of Na-PO4 cotransport. An oxalate-supported 45Ca uptake was larger,
less sensitive to DIDS, and enhanced by the catalytic subunit of
protein kinase A (40 U/ml). Combinations of PO4 and oxalate
had activating and inhibitory effects that could be explained by
PO4 inhibition of an oxalate-dependent pathway, but not
vice versa. These results support the existence of separate transport
pathways for oxalate and PO4 in brain endoplasmic reticulum.
4,4'-diisothiocyanostilbene-2,2'-disulfonic acid; 4,4'-dinitrostilbene-2,2'-disulfonic acid; niflumic acid; endoplasmic reticulum; adenosine 3',5'-cyclic
monophosphate; active calcium transport; calcium homeostasis
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INTRODUCTION |
THE SARCOPLASMIC RETICULUM (SR) of muscle and
endoplasmic reticulum (ER) of nonmuscle tissue accumulate and release
Ca2+ and help control and coordinate intracellular
Ca2+ concentration
([Ca2+])-sensitive processes within the
cytosol. Microsomes from brain (12) and muscle (1) stoichiometrically
take up Ca2+ and divalent anions, such as PO4
or oxalate, which allows more Ca2+ to be sequestered and
stored in the lumen.
Oxalate, a dicarboxylic anion, traditionally has been used to
experimentally extend the linear time course of Ca2+ uptake
in microsomes, thus making 45Ca fluxes easier to measure.
Because the solubility product of Ca2+ oxalate (~2 × 10
8 M2) is significantly below
that of calcium phosphate (~5 × 10
6
M2), the upper limit of allowable intraluminal free
Ca2+ ([Ca2+]er) is
significantly lower with oxalate compared with PO4.
Ca2+ accumulation should be greater with oxalate than with
PO4 (1), both because of oxalate's greater ability to trap
Ca2+ and prevent efflux and because active Ca2+
uptake is enhanced by low [Ca2+]er
(6, 7, 26).
Because physiological concentrations of divalent anions may influence
intracellular Ca2+ distribution (11), we set out to
describe better the divalent anion pathways that have a measurable
influence on active Ca2+ uptake into ER. For this purpose,
we measured the ATP-dependent 45Ca flux in microsomes with
different concentrations of oxalate and/or PO4 and with
inhibitors of anion transport. To test if the anion transport pathways
could be regulated by cAMP-dependent processes, we added the catalytic
subunit of protein kinase A (PKA) to the transport assay.
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MATERIALS AND METHODS |
Materials.
Ca2+ ionophore (ETH-129) and polyvinylchloride were
from Fluka Chemie (Buchs, Switzerland); DIDS (disodium salt) was from
Molecular Probes (Eugene, OR); 5-nitro-2-(3-phenylpropylamino)benzoic
acid (NPPB) was from Research Biochemicals International (Natick, MA); 4-4'-dinitrostilbene-2-2'-disulfonic acid (DNDS)
was from Pfaltz and Bauer (Waterbury, CT); niflumic acid, MgATP,
phosphocreatine, creatine phosphokinase (from rabbit muscle), PKA,
catalytic subunit (from beef heart), and Brilliant blue G were from
Sigma Chemical (St. Louis, MO). Potassium chloride, Ca2+
chloride, magnesium chloride, potassium phosphate, potassium hydroxide,
hydrochloric acid, sucrose, ethyl alcohol, o-phosphoric acid
85%, tetrahydrofuran (aldehyde free), and HEPES were from Fisher
Scientific (Fair Lawn, NJ); Chelex-100 was from Bio-Rad (Hercules, CA).
45CaCl2 in aqueous solution (~40 µCi/µg
Ca2+) was from Amersham (Arlington Heights, IL).
Isolation of rat brain microsomes.
Whole rat brain microsomes were isolated by methods similar to those
previously described (19). Male Sprague-Dawley rats, 150-200 g
each, were decapitated after brief anesthesia with CO2. The
brains were removed, minced, and homogenized on ice, using a Wheaton
40-ml Dounce-type homogenizer (type B pestle), in ~10 ml of ice-cold
homogenization solution for each brain. To make the homogenization
solution (0.30 M sucrose and 2 mM HEPES, pH 7.4 at room temperature)
Ca2+ free, it was filtered through a Chelex-100
ion-exchanger column. The brain homogenate was kept at 4°C and spun
at 1,500 g for 10 min; the pellet was discarded. The
supernatant was collected and spun at 17,000 g for 40 min; this
pellet was also discarded, and the supernatant was spun at 100,000 g for 1 h. The final supernatant was discarded. The microsomes
were collected by resuspending the final pellet in
Ca2+-free KCl buffer solution containing (in mM) 150 KCl,
1.4 MgCl2, and 20 HEPES, (pH 7.4 at room temperature) and
were kept on ice for ~2 h until used. We previously (27) showed that
our preparation of microsomes had an ATP-dependent Ca2+
uptake that was sensitive to thapsigargin and insensitive to the
mitochondrial uncoupler FCCP and was reduced very little (~10%) by a
concentration of digitonin (10 µM) known to permeabilize plasma
membranes; thus these preparations have little plasma or mitochondrial
membrane contamination. The protein concentration in the microsome
preparation (usually 0.3-0.7 µg/µl) was measured by the
Bradford (2) method using BSA as the standard.
Solutions.
Ca2+ was first removed by passing solutions through a
Chelex-100 ion-exchange column, and then magnesium, Ca2+,
and other components were added. The uptake solutions contained tracer
45Ca, and 150 mM KCl, 1.4 mM MgCl2, 20 mM HEPES
(pH 7.25 at 37°C), with or without 3 mM MgATP, 10 mM
phosphocreatine, and 10 U/ml creatine phosphokinase;
KH2PO4, potassium oxalate, and
CaCl2 were added at the concentrations indicated in the
figure legends.
Free Ca2+ measurement.
Ca2+-sensitive minielectrodes constructed from plastic
Eppendorf pipettes (Brinkman Instruments, Westbury, NY) were used to measure the free (ionized) Ca2+ of solutions. The
Ca2+-sensitive tip was formed from the Ca2+
ionophore ETH-129, polyvinylchloride, and tetrahydrofuran, combined in
the ratio of 33:7:60 (ml/g/ml). After construction, electrodes were
allowed to equilibrate in a buffer of pCa 6.75 overnight. Electrodes
were calibrated using pCa buffers of 6.75 and 5.0, and electrode
voltages were detected by an FD 223 electrometer (World Precision
Instruments, Sarasota, FL). The [Ca2+] was
measured in the following uptake solutions containing 5 µM total
CaCl2: 2.8 µM [Ca2+] in media
with no PO4 or oxalate; 2.4 µM
[Ca2+] in 10 mM PO4 media; 1.3 µM
[Ca2+] in 10 mM oxalate; and 1.2 µM
[Ca2+] in 10 mM PO4 plus 10 mM
oxalate. In 3 and 2 µM total CaCl2, the free
[Ca2+] were proportionately lower. The 5 µM
Ca2+ solutions with PO4 were 100-fold lower
than the solubility limit for Ca2+-HPO4,
whereas those with oxylate were 3-fold below the solubility limit for
Ca2+-oxylate monohydrate, the least soluble form, because
about one-half of the total oxylate is in soluble ion pairs with K and
Mg (24).
45Ca uptake measurement.
A 300-µl aliquot of microsome suspension was preincubated at 37°C
for 10 min. Uptake of 45Ca was initiated by the addition of
1 ml warmed buffer solution (37°C) containing ~0.2 µCi/ml of
the isotope. Microsomes were allowed to accumulate Ca2+ for
known time intervals, and the 45Ca uptake was then
terminated by the addition of 3 ml ice-cold wash solution containing
(in mM) 150 KCl, 1.4 MgCl2, 20 HEPES, and 2 KH2PO4, (pH 7.4 at room temperature). Each
cooled sample was drawn into a 5-ml syringe and was filtered through a
glass fiber filter (Whatman GF/B) under vacuum. Each filter was washed three times with 5 ml of the ice-cold wash solution before being placed
in a vial with 5 ml Ecoscint A scintillation fluid (National Diagnostics, Atlanta, GA). The radioactivity was measured in a Beckman
LS 7500 liquid scintillation counter. ATP-dependent Ca2+
uptake (expressed as µmol/g protein) was determined by subtracting 45Ca accumulation in the absence of MgATP, phosphocreatine,
and creatine phosphokinase from 45Ca accumulation in the
presence of MgATP, phosphocreatine, and creatine phosphokinase.
Statistics.
Results are presented as means ± SE. Comparisons were made by paired
Student's t-test. The number (n) refers to the
total number of replicates. For example, the number of replicates
was two or three for each experiment, and each experiment was repeated at least one time with a different preparation of microsomes. Each
experiment, i.e., microsome preparation, required the killing of two or
three rats.
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RESULTS |
Effect of PO4.
Results shown in Fig. 1 were qualitatively
similar to those of Fulceri et al. (12), although our experiments were
performed at much lower but physiological
[Ca2+]. Microsomes were preincubated without
PO4 (Fig. 1A) or with PO4 (Fig.
1B). After time 0, PO4 was present at the
concentrations indicated in the legend of Fig. 1. Two periods of
Ca2+ uptake were evident: a rapid initial stage (1-2
min) that was independent of the PO4 concentration and a
second, slower, PO4-dependent stage that lasted for 30 min
or more. The amount of Ca2+ accumulated in the
PO4-independent initial stage was ~1 µmol/g protein. In
terms of microsomal water [0.003 l water/g protein (20)],
this would be ~0.3 mM or a Ca2+ accumulation of 60-fold
over the 5 µM external [Ca2+]. In the absence
of PO4, microsomes rapidly lost the Ca2+ that
had accumulated during the initial stage, a result different from that
found by Fulceri et al. (12) at higher (~50 µM) Ca2+.
We interpret the rapid initial stage as resulting from intrinsic activity of the Ca2+ pump and the slow
PO4-dependent stage as requiring, in addition, luminal
PO4 accumulation. Because the initial stage of
45Ca uptake was unaffected by 0-10 mM PO4,
we conclude that the activity of the Ca2+ pump was
unaffected by this range of PO4 concentrations. The transient Ca2+ accumulation in the absence of
PO4 suggests that a delayed change in the properties of the
microsome system permits the accumulated Ca2+ to be
released, although sufficient MgATP is always present to provide energy
to the Ca2+ pump.

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Fig. 1.
Time course of PO4-dependent 45Ca uptake in rat
brain microsomes. ATP-dependent 45Ca uptake was measured
after microsomes were incubated 1-30 min at 37°C in 5 µM
Ca2+ and 150 mM KCl buffer containing 0 ( ), 2 ( ), 6 ( ), or 10 ( ) mM PO4 (pH 7.25). Microsomes were
preincubated for 10 min at 37°C before starting 45Ca
uptake (n = 6 replicate experiments). A: preincubation
media contained 0 PO4. B: preincubation media
contained the same concentration of PO4 as that in the
incubation solution. The zero PO4 data are the same as in
A.
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Na independence.
The PO4-dependent portion of Ca2+ uptake was
unaffected by the Na concentration (0-20 mM; Fig.
2A). We conclude that Na had no
direct effect on the Ca2+ pump, nor did it have an indirect
effect, such as enhancing PO4 transport, either of which
would be expected to change 45Ca accumulation. In 10 mM
PO4, the second stage of Ca2+ uptake was
unaffected by 1 mM arsenate (Fig. 2B), a congener of
PO4 and an inhibitor of Na-PO4 cotransport in
the kidney and in erythrocytes. Arsenate (1 mM) causes 99% inhibition
of Na-PO4 cotransport in the kidney (15) and 15%
inhibition of the erythrocyte Na-PO4 cotransporter (22).
Thus neither Na nor a Na-PO4 cotransport inhibitor
measurably changed the PO4-dependent portion of
45Ca uptake, suggesting that Na-PO4 cotransport
is an unimportant pathway for PO4 flux in brain microsomes.

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Fig. 2.
Effect of Na and arsenate on ATP-dependent 45Ca uptake with
10 mM PO4 and 5 µM Ca2+ (n = 6).
A: ATP-dependent 45Ca uptake at 15 min with 0, 10, and 20 mM NaCl added to the bath constituents. B: time course
of ATP-dependent 45Ca uptake with 10 mM PO4 at
1, 5, 15, and 20 min, with or without arsenate (1 mM).
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Anion transport inhibitors.
Niflumic acid (50 µM; Fig. 3A),
DNDS (250 µM; Fig. 3B), and DIDS (5 µM; Fig. 3C)
did not alter the initial stage but blocked a substantial part of the
second, PO4-dependent stage of 45Ca
accumulation. At a higher concentration of 100 µM DIDS, the initial
stage was blocked as well (Fig. 3C). Each of these substances inhibits anion transport in other systems. Niflumic acid binds to the
external aspect of the erythrocyte anion exchanger system with a
potency that depends on the internal chloride concentration (16).
Niflumic acid (50 µM) also prevents long openings of
Ca2+-activated Cl
channels in inside-out
membrane patches from ventricular myocytes (5). DNDS is a potent
inhibitor of erythrocyte anion exchanger-1 (band 3)-mediated
anion exchange (10) but is without effect on erythrocyte (21) or renal
(23) Na-PO4 cotransport. DIDS is a
bifunctional reagent that can irreversibly react with lysine and
histidine residues and inhibit anion transport (3). DIDS also binds to,
and inhibits, the SR Ca2+ pump (4); in addition, it has
been shown to inhibit the Na-K-ATPase of renal tissue by reacting at
the cytoplasmic surface with specific lysine residues (14). In our
experiments, lower concentrations of DIDS (3-5 µM) only blocked
the PO4-dependent Ca2+ uptake, probably by
inhibiting PO4 transport. The complete inhibition of all
Ca2+ transport at higher concentrations of DIDS (100 µM)
may be a result of it inhibiting the Ca2+-ATPase.

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Fig. 3.
Effect of anion transport inhibitors on ATP-dependent 45Ca
uptake with 10 mM PO4 and 5 µM Ca2+. ATP
dependence of 45Ca uptake was measured at different
incubation times (1-30 min), with or without inhibitors (n = 6). A: 50 µM niflumic acid. B: 250 µM
4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS).
C: 5 or 100 µM DIDS.
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Oxalate.
Oxalate (10 mM) increased the ATP-dependent 45Ca
accumulation rate >50% over that with 10 mM PO4 (Fig.
4 compared with Fig. 1). The data of Fig. 4
were fit to straight lines through the origin; the initial stage is not
apparent here, as the first data point is at 5 min. [The expected
45Ca uptake at 1 min (1 µmol/g protein) falls near the
line of continuous linear uptake in 10 or 15 mM oxalate.] In our
experiments (pH 7.25 at 37°C), ~70% of total PO4
would be present as divalent PO4
(HPO2
4), since PO4 has a
pK of 6.9 in these solutions. However, the difference in uptake between
PO4 and oxalate solutions is not likely attributable to the
differences in the divalent anion concentrations (7 vs. 10 mM). When the total PO4 concentration was increased to 12 mM so that the divalent PO4 was 8.4 mM, Ca2+
accumulation was 5.3 ± 0.3 µmol/g at 20 min, still much less than
the accumulation of 11.9 ± 1.2 µmol/g at 20 min with 10 mM oxalate
in paired experiments (data not shown).

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Fig. 4.
Time course of ATP-dependent 45Ca uptake in the absence of
PO4 at different concentrations of oxalate.
45Ca was measured from 0 to 20 min at 37°C and with 5 µM Ca2+ and 5, 10, and 15 mM oxalate (n = 6).
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The higher Ca2+ accumulation rate in oxalate compared with
that in PO4 is possibly related to differences in their
respective solubility products with Ca2+. Because the
Ca2+-PO4 solubility product is higher, the
limiting free Ca2+ would be higher in microsomes containing
PO4 compared with those containing an equal amount of
oxalate. Because high intraluminal Ca2+ reduces the
Ca2+ uptake in brain microsomes (7, 26), lower uptake in
PO4 is expected. This is the usual, and our preferred,
explanation for the PO4-oxalate difference. However, other
explanations are possible. For example, intraluminal free oxalate and
PO4 concentrations could be below their equilibrium levels,
with oxalate transport intrinsically faster than PO4
transport, or oxalate may enhance Ca2+ transport by
facilitating Ca2+ delivery to the pump more effectively
than PO4, i.e., an external chelator effect (19).
Oxalate-PO4 pathways.
The effects of the anion channel inhibitor NPPB (25) or niflumic acid,
in the presence of 10 mM PO4 or 10 mM oxalate, are compared
in Fig. 5A. Oxalate-supported,
ATP-dependent 45Ca uptake was approximately threefold
greater than PO4-supported uptake in these experiments.
This approximately threefold difference was maintained when the
accumulation was inhibited by NPPB (5 µM) or by niflumic acid (50 µM); that is, the greater activation in oxalate was independent of
the absolute value of the accumulation. This suggests that these
inhibitors reduce the number of common (shared) anion transporters,
that they have proportional effects on two independent anion
transporters, or that they directly act on the ATP-dependent
Ca2+ pump. Because the initial stage of 45Ca
uptake was unaffected by niflumic acid (Fig. 3A), it seems unlikely that the ATP-dependent Ca2+ pump is influenced by
this inhibitor. Observations given below support the view that there
are different anion transporters for oxalate and PO4.
Therefore, we propose that niflumic acid has similar effects on two
anion transport systems.

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Fig. 5.
Comparison of inhibitors on PO4 or oxalate-stimulated
45Ca uptake. A: effect of chloride channel blockers
5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB; 5 µM) and niflumic
acid (50 µM) on 20 min 45Ca uptake with 10 mM
PO4 or 10 mM oxalate. Total Ca2+ concentration
was 3 µM (n = 12). B: time course of ATP-dependent
45Ca uptake with 10 mM PO4 or 10 mM oxalate,
with or without anion transport inhibitor DIDS (3 µM). Total
Ca2+ concentration was 2 µM (n = 6).
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Uptake in both PO4 and oxalate media was reduced by 3 µM
DIDS (Fig. 5B). The effect of DIDS at 20 min was, however, more
potent in PO4 (60 ± 12% reduction, n = 9)
compared with oxalate (24 ± 5% reduction, n = 9), suggesting
differences between DIDS inhibition of PO4 vs.
oxalate transport mechanisms or differences in DIDS action in these
media. This can also be seen in the experiment of Fig.
6C where 3 µM DIDS reduced uptake
by 57 ± 7% in PO4 and by 24 ± 7% in oxalate
(n = 9). Data in Fig. 5B (total
Ca2+ = 2 µM) were taken from different experimental
preparations than that in Fig. 5A (total Ca2+ = 3 µM). It may be noted that, in experiments with lower total Ca2+, the absolute values of uptake in PO4 and
oxalate were lower. The PO4-oxalate differences between
Fig. 5, A and B, are in the range of variability we
found with different microsome preparations; uptake in oxalate was
always greater.

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Fig. 6.
Effect of protein kinase catalytic subunit on ATP-dependent
45Ca uptake with 10 mM PO4 or 10 mM oxalate and
3 µM Ca2+. Error bars show SE; ** significance at
P = 0.01 for protein kinase A (PKA) addition; all samples taken
at 20 min. A: ATP-dependent 45Ca uptake control or
with PKA (40 U/ml; n = 11-12). B: ATP-dependent
45Ca uptake control or with NPPB (10 µM) or NPPB (10 µM) plus PKA (40 U/ml; n = 11-12). C:
ATP-dependent 45Ca uptake control or with DIDS (3 µM) or
DIDS (3 µM) plus PKA (40 U/ml; n = 9).
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One explanation for the weaker DIDS inhibition in oxalate could be that
oxalate partially protects the DIDS inhibitory site. If this were so,
then lowering the oxalate concentration might allow DIDS to inhibit
more effectively. In a separate set of experiments, the DIDS inhibition
was compared at 5 and 10 mM oxalate for 20 min uptake. At 10 mM
oxalate, 5 µM DIDS reduced uptake from 11.9 ± 1.2 to 9.4 ± 0.8 µmol/g protein (21 ± 13% reduction, n = 12). At 5 mM
oxalate, DIDS reduced uptake from 3.5 ± 0.3 to 2.4 ± 0.3 µmol/g
protein (31 ± 16% reduction, n = 12). Thus the percentage of
DIDS inhibition in 5 mM oxalate was not significantly greater than in
10 mM oxalate and was less than the DIDS inhibition in experiments
performed at the same time in 12 mM PO4. In PO4
media, DIDS reduced uptake from 5.3 ± 0.3 to 2.5 ± 0.1 (54 ± 12%
reduction, n = 9).
PKA catalytic subunit.
Several anion transport systems are regulated by cAMP-dependent
phosphorylation through PKA (8, 13, 18). To test for regulation of
oxalate- or PO4-supported uptake, we added the catalytic subunit of PKA to microsomes, as shown in Fig. 6. Under only one condition did Ca2+ uptake show a statistically significant
(P < 0.01) increase with PKA addition: with 10 mM oxalate and
without inhibitors (Fig. 6A). This was a specific effect of the
enzyme because a heat-denatured solution of PKA had no effect on
Ca2+ uptake in 10 mM oxalate (data not shown). The increase
of Ca2+ uptake with intact PKA in oxalate disappeared when
NPPB was present (Fig. 6B). When DIDS was present (Fig.
6C), the activation by PKA fell to slightly below the
statistically significant level (P = 0.06). Ca2+
uptake was unaffected by PKA in the presence of PO4 under
all conditions tested. These results suggest that oxalate and
PO4 support Ca2+ uptake through pathways with
different sensitivities to PKA.
Phosphate-oxalate interactions.
The interactions between PO4- and oxalate-supported
Ca2+ uptake are shown in Figs.
7 and 8. In the
experiments of Fig. 7, the added PO4 replaced molar
equivalents of oxalate. In the experiments of Fig. 8, the oxalate
concentration was held constant at 10 mM, with 5 mM (Fig. 8B)
or 10 mM (Fig. 8A) added PO4. If PO4
and oxalate were present in equal concentrations [5 mM (Fig. 7)
or 10 mM (Fig. 8A)], uptake was below that with oxalate
alone. However, if a small amount (5 mM) of PO4 was added
to 10 mM oxalate, uptake increased beyond that with oxalate alone (Fig.
8B). These results suggest that PO4 can either
increase or inhibit uptake in the presence of oxalate, depending on the
relative concentrations of the two divalent anions. Two transport
pathways, one for PO4 and one for oxalate, can explain the
biphasic effects of PO4; this is shown in Fig.
9 using a phenomenological model that is presented in the DISCUSSION.

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Fig. 7.
Interaction between PO4 and oxalate. ATP-dependent
45Ca uptake, at 3 µM total Ca2+, was measured
at 20 min. Phosphate was increased from 0 to 10 mM by replacing equal
moles of oxalate (n = 5-6).
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Fig. 8.
Competition between PO4 and oxalate during 45Ca
uptake (20 min, 3 µM Ca2+). A: ATP-dependent
45Ca uptake with PO4 alone (10 mM), oxalate
alone (10 mM), or with both PO4 and oxalate together at 10 mM (n = 9). B: ATP-dependent 45Ca uptake
with PO4 alone (5 mM), PO4 (5 mM)
plus oxalate (10 mM), or oxalate alone (10 mM; n = 6).
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Fig. 9.
Model of oxalate and PO4 competition. Data points are
average ATP-dependent 45Ca uptake at 20 min. Smooth curves
were drawn according to the equation given in the
DISCUSSION with the maximal flux of oxalate and
PO4 = 16 and 8 µmol/g (20 min), respectively; the
apparent dissociation constants for transport of oxalate and
PO4 = 8 and 7 mM, respectively; the apparent
inhibitor constants for oxalate to PO4 transport and
PO4 to oxalate transport = 100 and 5 mM, respectively; the
no. of identical (cooperative) sites for oxalate and phosphate = 4 and
2, respectively. A: PO4 replaces oxalate.
B: PO4 is added to 10 mM oxalate.
[PO4] in mM.
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DISCUSSION |
Several features of Ca2+ uptake by ER microsomes are
underlined by this study: 1) the ability of divalent anions to
alter intraluminal Ca2+, 2) the existence of
specific divalent anion transport pathways in ER, 3)
modification of an oxalate pathway by the PO4 concentration or PKA, and 4) the requirement for physiological
PO4 (or other precipitating anion) to maintain an ER
Ca2+ load in brain microsomes. These features are shown
schematically in Fig. 10.

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Fig. 10.
Hypothesized model for anion effects on endoplasmic reticulum (ER)
Ca2+ accumulation. Phosphate (HPO4) and oxalate
(OX) enter the lumen of the ER by different pathways with different
sensitivities to PKA and DIDS. Within the lumen, these anions
([A2 ]) complex Ca2+ and lower
[Ca2+], relieving inhibition of the
Ca2+ pump. Brackets denote concentration.
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We assume that, for oxalate or PO4 to support
Ca2+ uptake, it must be present within the ER lumen,
trapping intraluminal Ca2+, lowering
[Ca2+]er, and relieving inhibition
of the Ca2+ pump. Transport of these ions is
required if they are to have their effects from within the ER lumen.
Therefore, inhibitors such as niflumic acid, NPPB, DIDS, or the
activator PKA likely change the divalent anion-dependent portion of
45Ca uptake by modifying divalent anion transport in the ER.
Separate pathways.
Our data are consistent with a hypothesis of separate, but similar,
anion transport pathways for oxalate and PO4. Both are partially inhibited by NPPB (5 µM), niflumic acid (50 µM), and DIDS
(3 µM). However, DIDS inhibited a greater percentage of the flux in
10 mM PO4 than in 10 mM oxalate. If oxalate protects a DIDS
inhibitory site, protection of the site should be affected by changing
the oxalate concentration. We found, however, that lowering the oxalate
concentration from 10 to 5 mM did not significantly change the
proportion of DIDS inhibition.
The catalytic subunit of PKA enhanced oxalate-dependent, but not
PO4-dependent, Ca2+ uptake. In the presence of
NPPB or DIDS, the PO4-dependent uptake remained unaffected
by PKA. In the presence of NPPB, the oxalate-dependent uptake was
unaffected by PKA. In the presence of DIDS, however, the
oxalate-dependent uptake may have increased ~20% with PKA, although
this was not quite significant (P = 0.06). These results are
consistent with there being separate pathways for oxalate and
PO4; the oxalate pathway is less sensitive to DIDS and
enhanced by PKA. Both pathways were inhibited by NPPB and niflumic
acid, as is observed with some other anion channels (and carriers).
The sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) 2 is the
isoform of the ER Ca2+ pump that is expressed in heart
muscle and in nerve. Phospholamban, which interacts with the SERCA2 of
heart, slow-twitch skeletal, and smooth muscle, has not been found to
be expressed in neurons, to our knowledge. Therefore, phosphorylation
of phospholamban by PKA, which would result in relief of inhibition of
the Ca2+ pump, cannot (with the present knowledge) explain
the observed effect of PKA on Ca2+ uptake in a neuronal
preparation. Additionally, there is evidence against an effect of PKA
on the Ca2+ pump per se (either via phospholamban or via
any other means), since PKA had its effect only in the presence of
oxalate (not PO4), whereas the Ca2+ pump must
be driving Ca2+ uptake under both of these conditions.
Phosphate and oxalate competition.
As had been observed many years ago for skeletal muscle microsomes (1),
there is a striking interference among divalent anions during active
Ca2+ uptake. We found that increasing the PO4
concentration above ~5 mM markedly inhibited the ability of 10 mM
oxalate to support 45Ca uptake. Inhibition occurred over a
narrow PO4 concentration range. The reciprocal inhibition
of the PO4 pathway by oxalate was less pronounced. This
suggests that multiple anion sites are involved in PO4 inhibition.
An alternate explanation for elevated PO4 inhibition of the
45Ca uptake could be that the pump rate is reduced via mass
action in the reaction: Ca2+ in the cytosol + ATP
Ca2+ in the ER + ADP + PO4. However, our data
in Fig. 1 do not demonstrate a direct inhibition of the
Ca2+ pump by PO4. We interpret the rapid
initial Ca2+ uptake as resulting from activity of the pump
alone. Figure 1 shows little, if any, difference in this initial stage
of Ca2+ uptake as the PO4 concentration is
increased from 0 to 10 mM.
As is shown in Fig. 9, A and B, the behavior of
45Ca uptake in oxalate and PO4 combinations can
be phenomenologically described by assuming two pathways of anion flux,
supporting Ca2+ accumulation: an oxalate pathway that is
inhibited by multiple occupancy of PO4 sites and a
PO4 pathway that is poorly inhibited by oxalate. The
equation used for the smooth curves in Fig. 9 is
where
J equals the net Ca2+ influx that accompanies the
two components of divalent anion fluxes; Vox
and VP represent the maximal flux of oxalate
and PO4, respectively;
K
1ox and
K
1P are the respective
apparent affinities for transport of oxalate and PO4;
K
1P-ox and
K
1ox-P are the apparent
inhibitory affinities of the first anion in the subscript on the
transport of the second anion; and n and m represent
the number of identical (cooperative) sites for substrate and inhibitor
on the oxalate ([ox]) and PO4 ([P])
pathways, respectively. The number of substrate and inhibitor sites
acting for each transport pathway has been arbitrarily chosen (for
simplicity) to be the same.
In the model, the difference in Ca2+ uptake in solutions of
oxalate or PO4 was phenomenologically accounted for by
adjusting the constants VP and
Vox. Fitting of the data obscures the likely reason
that uptake is greater in oxalate: that luminal Ca2+ is
lower and therefore inhibition of Ca2+ uptake is relieved
in oxalate. As the bath PO4 concentration rises in the
presence of oxalate, the luminal composition of divalent anions should
also change. The simplest assumption would be that the total
Ca2+ buffer present in the microsome lumen would increase
and, therefore, free intraluminal Ca2+ would fall,
resulting in relief of inhibition and greater Ca2+ uptake.
The result that increasing PO4 in the presence of oxalate reduced Ca2+ uptake is explained in the model by the
postulate that bath PO4 interferes with the oxalate
transport pathway, thus allowing less total Ca2+ to enter
the microsomes.
Parameters used for the smooth curves are given in the legend of Fig.
9. The rationale for choosing these parameters is as follows.
Cooperativity in activation kinetics of PO4 (m = 2)
and oxalate (n = 4) was chosen because of the weak effect of
low concentrations (2-5 mM) of divalent anions and steep
activation by higher concentrations. Cooperativity in inhibitory
kinetics of PO4 on the oxalate-supported uptake was chosen
for the same reason: weak (or no) inhibition by low PO4
concentrations and strong inhibition by higher concentrations. The
inhibitory constant Kox-P is large because 10 mM
oxalate did not prevent activation by PO4. The inhibitory
constant KP-ox is small, ~5 mM, because, beyond
this concentration, PO4 began to reduce uptake in the
presence of 10 mM oxalate.
Transient ATP-dependent Ca2+
uptake in absence of divalent anions.
At micromolar Ca2+, millimolar ATP, and in the complete
absence of divalent anions, brain microsomes transiently accumulated Ca2+. The accumulated Ca2+ remained within the
microsome for only a few minutes without divalent anions present. This
phenomenon was not reported at higher (nonphysiological)
[Ca2+] (12). Under more normal
[Ca2+], the ability of brain microsomes to
maintain their Ca2+ store appears to be strongly dependent
on the presence of divalent anions.
The [Ca2+] gradient generated in the absence of
divalent anions was much less than the maximum gradient allowable from
the available energy from ATP hydrolysis. Assuming no ER membrane
potential, the energy requirement (
µ) per cycle of the
Ca2+ pump can be written
where
k is the Boltzmann's constant, T is the absolute temperature,
is the number of Ca2+ transported per cycle, and
Ci and Co are the
[Ca2+] inside and outside the microsomes,
respectively. Assuming 100% efficiency of the pump, hydrolysis of ATP,
yielding ~600 meV of energy (17), could move two Ca2+
against a concentration gradient of >104 (kT = 26.7 meV at 37°C). Without divalent anions present, our microsomes
generated a gradient of only ~102. Thus accumulation was
far below the thermodynamic limit.
DIDS stoichiometry and irreversibility.
Assuming that DIDS reacts irreversibly with the Ca2+ pump
of brain ER, as it reacts with the muscle SR pump (4), the reaction with the pump at 5 µM DIDS must be much slower than the reaction with
the transport pathway for divalent ions. We measured the DIDS
concentration spectrophotometrically (9) in a microsome-free filtrate
to determine the amount of DIDS removed from solution by nonspecific
binding to microsomes (20 min, 37°C). Microsomes (75 µg/ml) bound
and removed 10, 33, and 43% of the DIDS (5 µM) in media containing
10 mM PO4, 10 mM oxalate, or 0 PO4/0 oxalate, respectively (data not shown). We attribute this reduction of free DIDS
concentration to nonspecific binding to microsomal proteins. Because
less DIDS was bound in PO4 than in oxalate media and yet DIDS inhibited a greater part of the uptake in PO4 than in
oxalate, DIDS binding to the divalent anion pathway does not dominate
the total DIDS binding. The lack of inhibition of the Ca2+
pump by low concentrations of DIDS cannot be attributed to its absorption and elimination from the media since at least 50% of DIDS
remained free in solution.
Ca2+ and anion flux.
Our measurements of 45Ca uptake in ER microsomes were made
with a total bath Ca2+ of 2-5 µM and a free
Ca2+ of ~1 µM. The Ca2+ (and anion) uptakes
in our experiments were 100 times lower (normalized to protein) than
that found in skeletal muscle microsomes (1) and 10 times lower than
that seen by Fulceri et al. (12) in brain microsomes at a higher free
Ca2+. With the greater absolute flux values, these authors
were able to measure PO4 (or oxalate) fluxes and determine
a stoichiometric (1:1) relationship between Ca2+ and
PO4 (or Ca2+ and oxalate) uptake. At a lower
physiological [Ca2+] and at the lower
Ca2+ (and anion) flux of our experiments, a
Ca2+-dependent PO4 flux was not detectable by
us above the background.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Otto Froehlich for critically reading the manuscript.
 |
FOOTNOTES |
This work was partially supported by National Institutes of Health
Grants NS-19194 (R. F. Abercrombie) and HL-28674 (R. B. Gunn).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: R. F. Abercrombie, Dept. of Physiology, Emory Univ., Atlanta, GA 30322 (E-mail: ron{at}physio.emory.edu).
Received 24 July 1998; accepted in final form 7 January 2000.
 |
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