Comparative data of Ca2+ transport in brain and skeletal muscle microsomes.

Abstract A vesicular membrane system (microsomes) was prepared from rabbit brain. Ca2+ uptake was observed in this preparation upon incubation with ATP and Mg2+. Tris-oxalate did not increase Ca2+ uptake. A decrease in adsorbed Ca2+, coupled to an increase in Ca2+ transport, was observed upon addition of 4 mm Pi to the incubating media. Ca2+ adsorption was higher in presence of 120 mm KCl than in a medium with 120 mm NaCl. Ca2+ transport was lower in a medium without supporting concentrations of monovalent cations. Acetyl phosphate as substrate would not support Ca2+ uptake in brain microsomes. Brain microsomal ATPase was activated by Ca2+ and Mg2+ similarly to that in skeletal muscle microsomes. Ca2+-dependent ATPase activity was higher in media containing 120 mm KCl than 120 mm NaCl. Ouabain did not inhibit this activity. The amount of Ca2+ bound by skeletal muscle microsomes in an oxalate-free medium was 2- to 4-fold higher than in brain microsomes. In these experiments, the Ca2+ Pi ratio was 0.15 to 0.30 both in brain and muscle microsomes. Ca2+ Pi ratios between 1.0 and 1.2 were obtained in skeletal muscle microsomes incubated in media containing 4 mm Tris-oxalate.

their uptake of Ca* is not increased by oxalate; however, ATPdependent Caz+ transport activated by oxalate was reported in crustacean peripheral nerve microsomes by Lieberman et al. (9). They also reported a lower total amount of Caz+ bound when KC1 (115 mM) was replaced by NaCl in the assay medium. Alonso and Walser (lo), with rat brain microsomes and a rapid perfusion incubation procedure, did not observe ATPdependent Ca2+ uptake. If KC1 was substituted by NaCl in the perfusion medium, there was some Ca2+ release from microsomes.
In this paper, the effect of monovalent cations in Ca2f uptake and ATPase activity of rabbit brain microsomes has been studied. Most of these experiments were designed to compare Ca2f transport and ATPase activity in brain and muscle microsomal fractions.

METHODS
Preparation of Microsomes-All operations were performed at 4O. Brains of two rabbits were homogenized with 3 volumes of ice-cold 120 mM KC1 in a Potter-Elvehjem tissue grinder. The homogenate was centrifuged at 1,500 x g for 10 min and the pellet was discarded. The supernatant fraction was centrifuged in a Sorvall RCB-B centrifuge at 10,000 X g for 10 min to remove mitochondria. The supernatant containing microsomes and soluble protein was then centrifuged at 41,000 x g for 1 hour. The supernatant was discarded and the pellet was washed once with 20 ml of either 120 mM KC1 or 240 mM sucrose. The material was dispersed in a Potter-Elvehjem tissue grinder and centrifuged at 41,000 X g for 45 min. The pellet was suspended at a protein concentration of 10 mg per ml in the solution used for washing. Muscle microsomes were prepared by essentially the same procedure. Protein was estimated by the biuret method. All preparations used were less than 2 hours old.
Standard Assay-Unless otherwise stated, the incubation medium consisted of 8 mM Tris-maleate buffer, pH 6.8; 4 mM MgSO4; 2 mM ATP; and the specified amounts of NaCI, KCl, 45CaC12. The total volume was usually 1.0 ml. The reaction was started by the addition of microsomal protein, and carried out at 37". The incubation time was usually 5 min. When ATPase activity was measured, the reaction was stopped by the addition of 0.1 ml of a 10% solution of trichloracetic acid. When Ca2+ uptake was measured, the reaction was stopped by removal of particles with Millipore filters, type HA, with 0.45 ~1 average pore size, as previously described (11). In all experiments, controls both with and without microsomes and without ATP were performed. The pH of the filtrates was measured   ATPase Activity-This enzyme was assayed by measuring the Pi content of the sample by the method of Fiske and SubbaRow (12) f Electron Microscopy-After incubation, microsomes were centrifuged at 80,000 X g for 30 min. The pellet obtained was fixed overnight at 5" in a 2% solution of OSOC in 120 mM KCl. After washing in distilled water, it was dehydrated in ethanol, stained with uranyl acetate, carefully removed from the centrifuge tube, and embedded in Epon.
The pellets were cut to have  Figs. 1 to 3 show electron micrographs of skeletal muscle and brain microsomes.
Vesicular structures with varying diameters were observed in the microsomal fractions of both tissues, although larger vesicles were predominant in the brain preparation.
Disrupted vesicles were also frequently found in brain microsomes.

Statistical AnalysisThe
Student t test was performed on _ E 1.0 paired observations of the same preparation, adopting absence of variation as a null hypothesis (13).
ChemicalsATP, ouabain, and acetyl phosphate were purchased from Sigma, and 46CaCl~ from Abbott Radio-Pharmaceuticals.
All other reagents were analytical grade. The dilithium salt of acetyl phosphate was converted to the Tris salt by passing it over a cooled Dowex 50 W-X8 column in the Tris form.

RESULTS
Ca2+ Transport in Brain MicrosomesFig. 4 and Table I show that Ca2+ binds to brain microsomes in the absence of ATP. This fraction will be referred to as "adsorbed Cati." Upon addition of ATP and Mg2+, larger amounts of Ca2f are bound. This will be referred to as "total Ca2+." With the several microsomal preparations tested, the maximal binding of Ca2+ was found between ATP concentrations of 0.20 to 0.50 mM. In the absence of Mg2+, the amount of Ca2+ bound with or without ATP was essentially the same, thus showing that Mg2+ is required for ATP supported Ca2+ binding.
The differences be- tween total Ca2+ and adsorbed Ca2+ will be referred to as "transported Cati." In experiments in which microsomes were incubated for different time intervals with or without ATP, it was observed that the amount of Ca2+ transported increased progressively with the incubation time.   Otsuka et al. (8), and Lieberman et al. (9), in dog brain and crustacean peripheral nerve microsomes. Species differences could account for this discrepancy. Thus, the effects of Tris-oxalate and KzHP04 were tested with brain microsomes. Addition of Tris-oxalate in concentrations varying from 1 to 4 mM did not modify Ca2+ adsorbed or Ca2+ transported. No accumulation of electron-opaque material could be detected in the brain preparation after incubation with oxalate. This could be due to the impermeability of brain microsomes to oxalate. Table II shows that, in contrast with oxalate, the addition of 4 mM KzHPOd to the preparation decreases the amount of Ca2+ adsorbed and increases Ca2+ transported. E$ect of NaCl and KC1 on Ca2+ UptaJce-Lieberman et al. (9), with the use of crustacean peripheral nerve microsomes, observed that substitution of 115 mM KC1 by NaCl in the assay medium lowered the total amount of Ca2+ bound. Similar results were observed in cardiac muscle microsomes by Palmer and Posey (16). Recently, Rubin and Katz (17), with skeletal muscle microsomes, reported that NaCl or KC1 increases Ca2+ uptake. Thus, the effect of these ions on brain microsomal Ca2f uptake was tested. In these experiments, microsomes were washed with 240 mM sucrose as reported under "Methods." Fig. 5 and Table I show that the amount of Ca2f adsorbed was essentially the same when microsomes were incubated without monovalent cations or with 120 mM KCI. However, less Ca2+ was adsorbed upon incubation with 120 MM NaCl. Ca2+ transport was higher in the presence of 120 mM KC1 or NaCl than in a sucrose medium. Since cation contamination of the sucrose and other solutions was not measured, the possibility of contamination by small amounts of Na+ or K+ cannot be excluded. In media containing various proportions of NaCl and KCl, at a fixed concentration of 120 mM, the Ca2+ transported was progressively higher as the proportion of NaCl increased. Addition of 1 mM ouabain in a medium containing either NaCl or KC1 did not modify the Ca2+ adsorbed or total Ca2+ bound.
Acetyl Phosphate and Cazf Uptake-It has been previously shown (18, 19) that skeletal muscle microsomes can use acetyl phosphate as a substrate for Ca2f transport. Although brain microsomes hydrolyze acetyl phosphate (20-22), no Ca2+ transport was observed when acetyl phosphate was used as substrate under various experimental conditions. Thus, the acetyl phosphatase found in brain microsomes seems not to be an integral part of the Ca2+ transport system. Activation of Microsomal A TPase Activity by Mg2+ and Ca2'-The following experiments were designed to study a possible correlation between brain microsomal ATPase and Ca2f transport. Table III and Figs. 6 and 7 show that Mg2+ enhances microsomal ATPase. With 2 rnM ATP maximum activation was observed at MgS04 concentrations between 2.0 and 2.5 InM. If Ca2+ was added to a medium containing 4 mM Mg2+, further activation of ATPase was observed in all the microsomal preparations tested. However, the amount of increase was subject to marked variability (see Standard error, Table III). Maximum activation was observed with 0.05 and 0.10 mM CaC12. No ATPase activation was observed with 0.10 mM CaC12 in the absence of Mg2+. These data are essentially the same as those described for skeletal muscle microsomes (1, 17, 18, 23). Subsequently, ATPase activity in the presence of MgS04 will be referred to as "Mg2+-dependent" ATPase; that observed in the presence of MgS04 and CaC12, as "total" ATPase, and the extra activity induced by Ca2+ will be identified as "C&activated" ATPase. Effect of Na+ and K+ on Microsomal A TPase Activity- Fig.   8 and Table III show that NaCl and KC1 modify Mg2+-dependent ATPase. The difference in activity in KCl, as compared to NaCl, was statistically significant at the level (p < 0.001). Ca2+-activated ATPase is higher in the presence of 120 mM KC1 than in 120 mM NaCl (p < 0.05). Ouabain (1 mM) failed to inhibit either Mg2+-or Ca 2+-activated ATPase, with or without the high salt media. In order to study a possible correlation between (Na+ + K+)-ATPase (9, 17, 24) and Ca2+ transport, ATPase activity and Ca2f uptake were simultaneously measured in media containing different proportions of NaCl and KC1 at a total concentration of 120 rnw Fig. 9 shows that Ca2+ transport and Ca2+-dependent ATPase activity did not correlate with the activation pattern of ATPase. Addition of 1 mM ouabain inhibited (Na+ + K+)-ATPase, but not Ca2+ transport. Ca2+ Transport and ATPase Activity in Brain and Skeletal Muscle illicrosomes-In order to ascertain the efficiency of Ca2+ transport in brain and skeletal muscle, Ca2+ uptake and ATPase activity were measured simultaneously in media containing different concentrations of 45CaC12. Oxalate was not added to the assay medium since, as shown previously, this ion activated Ca2f uptake only in muscle microsomes.
The amount of microsomal protein added to the assay medium was adjusted to prevent Ca2+ exhaustion, i.e. 100% Ca2+ bound. Fig. 10 shows one of these experiments.
In 10 different experiments, with protein concentrations of 0.4 to 0.8 mg per ml, maximum Ca2+ uptake was obtained with CaClz concentrations varying between 0.1 and 0.2 mM for brain microsomes and 0.2 to 0.3 mrvr for muscle microsomes.
In terms of micromoles of Ca2+ per mg of protein, muscle microsomes bound 2 to 4 times more Ca2f than brain microsomes.
Different reports have correlated Ca2+ transport and CaQf-activated ATPase in skeletal muscle microsomes. As shown in Fig. 10, Ca2+-bound and Ca2+-activated ATPase reached a maximum at different Ca2+ concentrations.
This inhibition is in agreement with other reports (11, 23). Consequently, different ratios between the amounts of Ca2+ bound and of ATP hydrolyzed (Ca2+ :Pi ratios) can be obtained.
This ratio varied between 0.15 and 0.30 at Ca2+saturating concentrations both in muscle and brain microsomes. In control experiments with skeletal muscle microsomes, with the use of 4 mM Tris-oxalate, 120 mM KCl, 4 mM ATP and Mg"+, 0.1 mM 45CaC12, and microsomal protein concentrations to ensure complete Ca2+ removal, Cazf :Pi ratios of 1.0 to 1.2 were obtained.
This agrees with the values reported by Hasselbach (1) and Ebashi and Yamanouchi (25).

DISCUSSION
Ca2+ Transport in Excitable Tissues-It is not possible to determine whether microsomes derived from Schwann or neuronal cells are responsible for the Ca" uptake observed in brain microsomal fractions.
Nevertheless, the data presented suggest that Ca2+ transport is a general feature of excitable tissues, regardless of their origin. 10. Ca* transport and ATPase activity in brain and muscle microsomes. Incubation medium was as described under "Methods" plus KCl, 120 mM and 0.8 mg per ml of microsomal protein.

significant
Ca2+ uptake (8). As in skeletal muscle, brain microsomal Ca2f transport may represent a system for depleting the cells of free Ca2f. However, it is premature to speculate on a possible functional role of Ca2+ transport in neural membranes.
Alonso and Walser (10) did not observe ATP-dependent Ca2+ uptake in rat brain microsomes.
The use of deoxycholate in their procedure for microsomal preparation may have impaired the Ca2+ transport system.
Effect of Na+ and K+ on Ca2+-activated ATPase and Ca2+ Transport-In skeletal muscle microsomes, Ca2+-activated ATPase has been associated with Ca2+ transport. In brain microsomes, Ca2+ transport was higher in the presence of 120 mM NaCl or 120 mrvr KCl.
However, Ca2f-activated ATPase was higher only in a medium containing KCI. It is difficult then to correlate Ca2+ transport and Ca2f-activated ATPase in this preparation.
The presence of other phosphatases, contaminant or microsomal, cannot be ruled out.
It is interesting to note that, in contrast with skeletal muscle microsomes, acetyl phosphate cannot be used as substrate for Ca2f transport in brain microsomes. This may suggest different characterist,ics in the Ca2f transport system of these tissues.
Eficiency of Ca2+ Transport in Brain and &%elefal &fuscle Microsomes-Ca2+ transport in brain and skeletal muscle cannot be rigorously compared in terms of specific transport capacity. More significant measurements would be obtained with purified enzyme preparations.
The efficiency of the Ca2+ transport system is also related to the concentration of ionic Ca2f in the vesicles. Even on a unit protein basis, different results can be obtained depending on the vesicular volumes.
Thus, with the same amount of microsomal protein, and two systems, one of which has vesicles 4 times larger in volume than the other, the amount of Ca2+ bound required to achieve the same ionic Ca2+ concentration within the vesicles will be 4 times larger for the system with the larger vesicles. Our brain microsomal preparation was not pure, and vesicles of different diameters were observed in the electron microscope.
Thus, the 2-to 4-fold increase in bound Ca2f observed for muscle microsomes is only an approximate value.
Ca2f:Pi Rat&-The Caz+:Pi ratio obtained was similar for brain and muscle microsomes when the vesicles reach saturation with Ca?+. These data suggest the same energy efficiency in Caz+ transport in both systems. The Ca2f: Pi ratio for muscle microsomes reported in the literature varies from 1 to 3.5 (1, 17, 25). By using oxalate and microsomal protein concentrations sufficient to ensure complete removal of Ca2+ from the assay medium, the Ca2+:Pi ratio found was 1.0 to 1.2, in agreement with Hasselbach (1) and Ebashi and Yamanouchi (25). However, in the absence of oxalate, and with incomplete removal of Calf from the assay medium, the value found was 0.15 to 0.30. This again raises the question about the presence of other phosphatases.
In muscle microsomes, actomyosin is the most likely contaminant phosphatase (26). Several muscle microsomal preparations show no decrease in Ca2f-dependent ATPase after washing for 90 min in 20, 120, or 600 mu KCL2 This seems to exclude the possibility of actomyosin contamination. The low Ca2+:Pi ratios found could also result from the occurrence of a rapid turnover of Ca2f. When equilibrium is reached, part of the Ca2+ may leak out of the vesicles by diffusion, so that pumping of corresponding amount would be needed in order to maintain a stable Ca2+ level within the microsomes.
However, different authors (1, 23) have shown that muscle microsomes previously filled with Ca 2f have no Ca2+-dependent ATPase activity when incubated in a medium with ATP and Mg2+, thus excluding this possibility.
On the other hand, Ebashi and Yamanouchi (25) observed in rabbit skeletal muscle that Ca2+dependent ATPase activity varied upon the presence or absence of oxalate in the assay medium.
A possible explanation for the low Ca2+:Pi ratios we obtained with brain microsomes could be related to the ineffectiveness of oxalate in the assay medium and different free Ca2+ concentrations within the vesicles.