Quantitative analysis of depolarization-induced ATP release from mouse brain synaptosomes: external calcium dependent and independent processes

Abstract

We and others have shown previously that ATP is secreted from mouse brain synaptosomes following depolarization of the membrane by high [K+]o and the time course can be monitored accurately by measuring the light emitted from luciferin-luciferase included in the reaction medium. In the present work we have evaluated the relative importance of [Ca2+]o and membrane potential on the ATP secretion process by modelling the time course of ATP release under different conditions. After correction of the records for destruction of released ATP by synaptosomal ecto-ATPase activity, we found that ATP secretion occurs by an apparent first order process. We also established that, in addition to the classical [Ca2+]o-dependent mode, ATP secretion also occurred in the absence of extracellular calcium ([Ca2+]o less than 1 microM). Upon lowering the extracellular Ca2+ concentration, both the rate and the extent of ATP secretion decreased. To assess the contribution of membrane potential to the release rate we measured ATP secretion at membrane potentials determined by extracellular [K+]o (or [Rb+]o) as defined by the distribution of the carbocyanine dye, diSC3(5). Rate constants computed from measured secretion curves revealed that this parameter was essentially independent of membrane potential in the absence of [Ca2+]o. Noise analysis of the light signal showed that the variance increased upon stimulation by high [K+]o, suggesting that both modes of secretion are quantal. Thus, we conclude that the rate of ATP secretion from nerve terminals depends upon Ca2+ entry but not on membrane potential, per se.