On the usefulness of Fura-2 measurements of intrasynaptosomal calcium levels in rat cortical synaptosomes to study mechanisms of presynaptic function.

ABSTRACT

Levels of [Ca2+]i in rat cortex synaptosomes were measured using the Ca2+ indicator Fura-2. Ca2+ influx was induced by veratridine in a concentration-dependent manner (1-10 microM). The resulting increase in [Ca2+]i was inhibited by tetrodotoxin (TTX). K+ (18 mM) increased the [Ca2+]i which was not influenced by TTX. K(+)-channel blockers such as 4-aminopyridine, alpha- and delta-dendrotoxin pre se were ineffective. The veratridine-induced Ca2+ influx in synaptosomes was reduced by L-type Ca(2+)-channel blockers, such as felodipine, nifedipine and PN-200-110, verapamil and diltiazem. omega-Conotoxin, and N-type Ca(2+)-channel blocker, did not inhibit the veratridine-stimulated [Ca2+]i increase. Bay K 8644, and L-channel agonist, stimulated an increase of [Ca2+]i in synaptosomes which was not sensitive to TTX. R-N6-Phenyl-isopropyl-adenosine (R-PIA) and clonidine, agonists at adenosine A1-receptors and alpha 2-adrenoceptors, respectively, did not influence the veratridine-stimulated [Ca2+]i increase. R-PIA did not interact with Bay K 8644-stimulated [Ca2+]i increase in synaptosomes. The results for all the substances used show major differences between the effects on Ca2+ influx in synaptosomes and on the electrically evoked neurotransmitter release in slice preparations. Thus, the synaptosome preparation is not a generally applicable experimental model for the study of Ca2+ mechanisms of presynaptic neuromodulation.