Calcium Buffering and Free Ca2+ in Rat Brain Synaptosomes

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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 approximately 325 nM in synaptosomes incubated in the normal physiological salt solution under resting conditions. When [K+]o 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+]o for [Ca2+]o = 0.02-1.2 mM; however, the increase in 45Ca uptake greatly exceeded the increase in [Ca2+]cyt. With small Ca2+ loads (< or = 100 mumol/L of cell water, equivalent to the Ca2+ entry during a train of < or = 60 impulses), the 45Ca uptake exceeded the increase in [Ca2+]cyt by a factor of nearly 1,000. This indicates that approximately 99.9% of the entering Ca2+ was buffered and/or sequestered within approximately 1 s. With larger Ca2+ loads, a larger fraction of the entering Ca2+ was buffered; approximately 99.97% of the load was buffered with loads of 250-425 mumol/L of cell water. The ratio between the total Ca2+ entry and the increase in [Ca2+]cyt, the "calcium buffer ratio," beta, was therefore approximately 3,500:1. This ratio was somewhat lower than the ratio of total intraterminal calcium: [Ca2+]cyt, which ranged between approximately 7,300:1 and 12,800:1. When the synaptosomes were activated with 10 microM veratridine ([Ca2+]o = 0.2-0.6 mM), 45Ca influx and [Ca2+]cyt increased progressively for approximately 10 s (beta = 2,700:1-3,050:1) and then leveled off.(ABSTRACT TRUNCATED AT 250 WORDS)