AR(‘HIVES OF BIOCHEMISTRY AND BIOPHYSICS 126, 286-294 (1968) Adenosine Triphosphate-Dependent Calcium Accumulation by Brain Microsomes’ JOSEPH D. ROBINSON AND W. DAVID LUST2 Department of Pharmacology, State University of New York, Upstate Medical Center, Syracuse, New York lS210 Received September 19, 1967; accepted October 27, 1967 A microsomal fraction from rat brain accumulated calcium by a temperature- dependent process requiring both ATP and Mg 2+.After brief incubations the concen- tration of calcium in the microsomes reached levels several hundred times that in the medium. The addition of oxalate, phosphate, or acetate did not augment the accumu- lation. Univalent cations reduced the uptake, and ouabain was not inhibitory. A variety of enzyme inhibitors, including sulfhydryl reagents, amytal, and oligomycin, decreased calcium accumulation. Procaine reduced uptake by one-third, but a series of neurotropic drugs had little effect. The relationship between calcium accumula- tion and ATPase activity as well as the state of the accumulated calcium remained unclear. A possible homeostatic role in neural metabolism is suggested. The concentration of calcium ions on each side of the cellular membrane profoundly influences neural processes. Calcium ions are necessary in the external medium for axonal conduction (l), but injection of calcium into the axoplasm (2) or internal perfusion of the axon with calcium media (3) blocks excit- ability, although the axonal membrane has a significant permeability to calcium ions (4). At t’he synapse, calcium ions are required in the extracellular medium for transmission, and experiments indicate that a transient influx of calcium into the presynaptic end- ing is involved in the release of the neuro- transmitter (5). Thus it would seem that a vital homeostatic mechanism in nerve metabolism must regulate intracellular cal- cium levels. Recently, two preliminary reports (6, 7) have described an ATP-dependent accumu- lation of calcium by microsomal prepara- tions from brain, although in a third publica- tion no such accumulation could be found 1 This work was supported by U.S. Public Health Service grants NB-05430 and GM-00293. 2 Predoctoral fellow of the U.S. Public Health Service. (8). Since such a process might be relevant to basic neuronal function it seemed of interest to examine the properties of such a system, to explore the experimental dis- crepancies, and to augment the brief reports. These studies also must be considered in the light of two major and distinct calcium- accumulating systems: the substrate- or ATP-dependent uptake of calcium by mitochondria that seem related to the energy-transducing apparatus (9), and the ATP-dependent uptake of calcium by fragments of muscle sarcoplasmic reticulum that seem related to muscle contraction and relaxation (10). In addition, a recent report indicated that erythrocytes extrude calcium ions by an ATP-dependent process (11). METHODS Brain microsomes were prepared (12) by homog- enizing rat brains in 9 volumes of 0.25 M sucrose- 0.001 M EDTA (pH 7). The homogenate was centrifuged for 12 minutes at lS,OOOg, and the supernatant material was then centrifuged for 25 minutes at 30,OOOg. The pellet was washed with 0.25 M sucrose without EDTA and recentrifuged, and the final pellet was suspended in 0.25 M su- crose. Mitochondria were prepared from a crude 286