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


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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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. 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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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 (open circle ), 2 (), 6 (), or 10 (black-triangle) 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.

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).

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.

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).

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).

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).

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.


    DISCUSSION
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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.

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 right-arrow 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
<IT>J</IT> = <FR><NU><IT>V</IT><SUB>ox</SUB></NU><DE>1 + <FENCE><FR><NU><IT>K</IT><SUB>ox</SUB></NU><DE>[ox]</DE></FR></FENCE><SUP><IT>n</IT></SUP> <FENCE>1 + <FENCE><FR><NU>[P]</NU><DE><IT>K</IT><SUB>P − ox</SUB></DE></FR></FENCE><SUP><IT>n</IT></SUP></FENCE></DE></FR> + <FR><NU><IT>V</IT><SUB>P</SUB></NU><DE>1 + <FENCE><FR><NU><IT>K</IT><SUB>P</SUB></NU><DE>[P]</DE></FR></FENCE><SUP><IT>m</IT></SUP> <FENCE>1 + <FENCE><FR><NU>[ox]</NU><DE><IT>K</IT><SUB>ox − P</SUB></DE></FR></FENCE><SUP><IT>m</IT></SUP></FENCE></DE></FR>
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 (Delta µ) per cycle of the Ca2+ pump can be written
&Dgr;&mgr; = <IT>vk</IT>T ln <FENCE><FR><NU><IT>C</IT><SUB>i</SUB></NU><DE><IT>C</IT><SUB>o</SUB></DE></FR></FENCE>
where k is the Boltzmann's constant, T is the absolute temperature, upsilon  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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Cell Physiol 278(6):C1183-C1190
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