Intra- vs extracellular calcium regulation of neurotransmitter-stimulated phosphoinositide breakdown.
The dependence on Ca2+ of basal, glutamate- and carbachol-stimulated phosphoinositide (PI) turnover was studied on 8-day old rat brain synaptoneurosomes. For that purpose, intracellular and extracellular Ca2+ concentrations were buffered by bis-(alpha-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 Ca2+ buffering on intracellular and extracellular Ca2+ concentration ([Ca2+]i and [Ca2+]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 Ca2+ ions with BAPTA-AM induced a dose-dependent reduction of the [Ca2+]i. Basal inositol phosphate (IP) formation was slightly affected by this [Ca2+]i buffering, while glutamate and carbachol stimulations of PI hydrolysis were similarly diminished. Chelation of extracellular Ca2+ ions with BAPTA produced a reduction of both [Ca2+]e and [Ca2+]i. Basal IP accumulation was maximally inhibited by 50%. The carbachol-induced PI hydrolysis was completely inhibited in the presence of 200 microM BAPTA, while a substantial residual glutamate-elicited IP response remained (40% of the control response). It is concluded that [Ca2+]i of synaptoneurosomes is not critical for basal and neurotransmitter-stimulated IP formation, whilst [Ca2+]e is critical. Glutamate may, in part, stimulate PI breakdown in a Ca(2+)-insensitive way.
Vignes
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INSERM U 254, Hôpital Saint Charles, Montpellier, France.
Blanc
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E
Sassetti
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I
Récasens
M
M
D000818:Animals; D002118:Calcium / Q000502:physiology*; D002217:Carbachol / Q000494:pharmacology; D002614:Chelating Agents / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D005110:Extracellular Space / Q000187:drug effects / Q000378:metabolism*; D018698:Glutamic Acid / Q000494:pharmacology; D066298:In Vitro Techniques; D018721:Muscarinic Agonists / Q000494:pharmacology; D018377:Neurotransmitter Agents / Q000494:pharmacology*; D010716:Phosphatidylinositols / Q000378:metabolism*; D051381:Rats; D013050:Spectrometry, Fluorescence; D013574:Synaptosomes / Q000378:metabolism
D002614:Chelating Agents; D018721:Muscarinic Agonists; D018377:Neurotransmitter Agents; D010716:Phosphatidylinositols; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D018698:Glutamic Acid; D004533:Egtazic Acid; D002217:Carbachol; D002118:Calcium