Calcium Buffering and Free Ca2+ in Rat Brain Synaptosomes

Abstract: The effects of K+ depolarization and of stimulation by veratridine on apparent cytosolic free Ca2+ ([Ca2+]cyt) and net Ca2+ accumulation were measured in isolated rat brain presynaptic nerve terminals (synaptosomes). [Ca2+]cyt was determined with fura‐2, and Ca2+ accumulation was measured with tracer 45Ca. [Ca2+]cyt was ∼ 325 nM in synaptosomes incubated in the normal physiological salt solution under resting conditions. When [K+]0, was increased from the normal 5 mM to 30 or 50 mM, 45Ca uptake and [Ca2+]cyt both increased within 1 s. Both increases were directly related to [Ca2+]0 for [Ca2+]0= 0.02–1.2 mM; however, the increase in 45Ca uptake greatly exceeded the increase in [Ca2+]cyt. With small Ca2+ loads ≤100 μmol/L of cell water, equivalent to the Ca2+ entry during a train of ≤60 impulses), the 45Ca uptake exceeded the increase in [Ca2+]cyt by a factor of nearly 1,000. This indicates that ∼99.9% of the entering Ca2+ was buffered and/or sequestered within ∼ 1 s. With larger Ca2+ loads, a larger fraction of the entering Ca2+ was buffered; ∼99.97% of the load was buffered with loads of 250–425 μmol/L of cell water. The ratio between the total Ca2+ entry and the increase in [Ca2+]cyt, the “calcium buffer ratio”β, was therefore ∼ 3,500:1. This ratio was somewhat lower than the ratio of total intraterminal calcium: [Ca2+]cyt, which ranged between ∼7,300:1 and 12,800:1. When the synaptosomes were activated with 10 μM veratridine ([Ca2+]0= 0.2–0.6 mM), 45Ca influx and [Ca2+]cyt increased progressively for ∼10 s (β= 2,700:13,050:1) and then leveled off. Application of 10 μM tetrodotoxin before the introduction of veratridine prevented the increases in 45Ca influx and [Ca2+]cyt. Application of 10 μM tetrodotoxin after 5–10 s of exposure to veratridine caused both the [Ca2+]cyt and the veratridine‐stimulated 45Ca within the terminals to decline, thereby demonstrating that the Ca2+ loading is reversible in the presence of extracellular Ca2+. These data show that synaptosomes are capable of buffering and metabolizing Ca2+ in a manner expected for intact neurons.