Uptake and release of calcium by rat brain synaptosomes

Rat brain synaptosomes, prepared by discontinuous Ficoll density gradient centrifugation, accumulated ⁴⁵Ca during brief incubations in modified Krebs-Ringer media. Uptake of ⁴⁵Ca was increased by 5 mM glutamate and 50 mM KCl, conditions that depolarize nerve cells; uptake of ²²Na was also increased by these agents. With 0.2 mM diphenylhydantoin, the increased ⁴⁵Ca uptake due to KCl was diminished, whereas that due to glutamate was less affected; conversely, with 0.15 μM tetrodotoxin the increased ⁴⁵Ca uptake due to glutamate was diminished, whereas that due to KCl was less affected. Both diphenylhydantoin and tetrodotoxin diminished the augmented uptake of ²²Na due to KCl and glutamate; thus the increased uptake of ⁴⁵Ca under depolarizing conditions may be dissociated from the increased influx of sodium. Ruthenium red decreased the uptake of ⁴⁵Ca under all conditions, as did procaine and the lanthanide Pr³⁺. Neither 5 mM lutamate nor 50 mM KCl increased ⁴⁵Ca uptake by brain mitochondria under comparable experimental conditions, whereas ATP increased the uptake by mitochondria but not that by these synaptosomes. Altering the sodium gradient by equimolar substitution of lithium or choline for sodium in the medium increased ⁴⁵Ca uptake, whereas ²²Na uptake was decreased. Inhibiting the sodium pump by ouabain or strophanthidin also increased ⁴⁵Ca uptake, and increased ²²Na uptake as well. The increased uptake of ⁴⁵Ca induced by ouabain was inhibited by diphenylhydantoin and tetrodotoxin. Measurements of the total calcium content showed that conditions producing an increased uptake of ⁴⁵Ca also produced a net uptake of calcium, rather than merely accelerating a ⁴⁵Ca⁴⁰Ca exchange. Experiments measuring the loss of previously accumulated ⁴⁵Ca showed that directly decreasing the sodium gradient or inhibiting the sodium pump slowed the loss of ⁴⁵Ca. These data are considered in terms of calcium influx through ‘leak’ pathways and gated channels (sensitive to membrane depolarization) and of net efflux dependent on a coupled sodium-calcium exchange mechanism.