Evaluation of the Ca2+ Concentration in Purified Nerve Terminals: Relationship Between Ca2+ Homeostasis and Synaptosomal Preparation

Abstract

The presynaptic Ca²⁺ concentration ([Ca]_i) was evaluated by studying intracellular free Ca²⁺ with quin-2 and fura-2 in synaptosomal preparations. The synaptosomal preparations were purified with hyperosmotic (sucrose) and isoosmotic (Percoll) density gradient centrifugation. Synaptosomes are most viable in the heavier fractions of the density gradients. These synaptosomal fractions exhibit the lowest [Ca]_i, [204 ± 2 nM for Percoll (C-band) synaptosomes, loaded at 30°C with the acetoxymethyl ester of fura-2 (fura-2-AM)], a high stability during prolonged incubations at 37°C, and a more potent response to membrane depolarization by elevated extracellular [K⁺]. [Ca]_i measurement was critically dependent on dye loading, calibration, type of dye used, synaptosomal preparation, and incubation temperature (30° or 37°C). Loading quin-2 in synaptosomes inserts a considerable buffer component in the synaptosomal [Ca]_i regulation, and consequently there is a quin-2 dependency of [Ca]_i, independent of endogenous heavy metal ions. Use of fura-2 is preferable in synaptosomes, although above a critical fura2-AM/protein ratio during loading ester hydrolysis is not complete, giving rise to errors in [Ca]i determination. Ionomycin is a selective tool to detect the presence of partially hydrolyzed esters and saturate indicators in the cytosol with Ca²⁺ for calibration. Parallel studies on lactate dehydrogenase and fura-2 fluorescence indicate that synaptosomal viability is very sensitive to prolonged incubations at 37°C. This study shows the applicability of measuring steady-state [Ca]_i and dynamic [Ca]_i changes quantitatively in fura-2-loaded synaptosomes. The possible involvement of different synaptosomal pools to explain the divergence in [Ca]_i between different preparations and the interpretation in physiological terms of [Ca]_i measured in synaptosomes are discussed.