CALCIUM BUFFERING AND FREE CA-2+ IN RAT-BRAIN SYNAPTOSOMES

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 Ca-45. [Ca2+]cyt was approximately 325 nM in synaptosomes incubated in the normal physiological salt solution under resting conditions. When [K+]. was increased from the normal 5 mM to 30 or 50 mM, Ca-45 uptake and [Ca2+]. 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 Ca-45 uptake greatly exceeded the increase in [Ca2+]cyt. With small Ca2+ loads (less-than-or-equal-to 100 mumol/L of cell water, equivalent to the Ca2+ entry during a train of less-than-or-equal-to 60 impulses), the Ca-45 uptake exceeded the increase in [Ca2+]. 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 muM veratridine ([Ca2+]o = 0.2-0.6 mM), Ca-45 influx and [Ca2+]cyt increased progressively for approximately 10 s (beta = 2,700:1 -3,050:1) and then leveled off. Application of 10 muM tetrodotoxin before the introduction of veratridine prevented the increases in Ca-45 influx and [Ca2+]cyt. Application of 10 muM tetrodotoxin after 5-10 s of exposure to veratridine caused both the [Ca2+]cyt and the veratridine-stimulated Ca-45 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.