Intra- vs extracellular calcium regulation of neurotransmitter-stimulated phosphoinositide breakdown

Abstract

The dependence on Ca 2+ of basal, glutamate- and carbachol-stimulated phosphoinositide (PI) turnover was studied on 8-day old rat brain synaptoneurosomes. For that purpose, intracellular and extracellular Ca 2+ concentrations were buffered by bis-(α-aminophenoxy)-ethane- N,N,N′,N′-tetraacetic acid, in its tetra(acetoxymethyl)-ester form (BAPTA-AM) and in its free acid form (BAPTA), respectively. The effects of both forms of the calcium chelator intracellular and extracellular Ca 2+ buffering on intracellular and extracellular Ca 2+ concentration ([Ca 2+] i and [Ca 2+] e) were determined with fluorimetric assay using fura2, either in its acetoxymethyl ester form (fura2-AM) or in its free acid form. Intracellular chelation of Ca 2+ ions with BAPTA-AM induced a dose-dependent reduction of the [Ca 2+] i. Basal inositol phosphate (IP) formation was slightly affected by this [Ca 2+] i buffering, while glutamate and carbachol stimulations of PI hydrolysis were similarly diminished. Chelation of extracellular Ca 2+ ions with BAPTA produced a reduction of both [Ca 2+] e and [Ca 2+] i. Basal IP accumulation was maximally inhibited by 50%. The carbachol-induced PI hydrolysis was completely inhibited in the presence of 200 μM BAPTA, while a substantial residual glutamate-elicited IP response remained (40% of the control response). It is concluded that [Ca 2+] i of synaptoneurosomes is not critical for basal and neurotransmitter-stimulated IP formation, whilst [Ca 2+] e is critical. Glutamate may, in part, stimulate PI breakdown in a Ca 2+-insensitive way.