Evaluation of the Ca2+ concentration in purified nerve terminals: relationship between Ca2+ homeostasis and synaptosomal preparation.
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 degrees C with the acetoxymethyl ester of fura-2 (fura-2-AM)], a high stability during prolonged incubations at 37 degrees 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 degrees or 37 degrees 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 fura-2-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 degrees C. This study shows the applicability of measuring steady-state [Ca]i and dynamic [Ca]i changes quantitatively in fura-2-loaded synaptosomes.(ABSTRACT TRUNCATED AT 250 WORDS)
Verhage
M
M
Department of General Zoology, University of Amsterdam, The Netherlands.
Besselsen
E
E
Lopes Da Silva
F H
FH
Ghijsen
W E
WE
D000634:Aminoquinolines; D000818:Animals; D001572:Benzofurans; D001921:Brain / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D004987:Ethers / Q000494:pharmacology; D005456:Fluorescent Dyes; D016257:Fura-2; D006706:Homeostasis; D015759:Ionomycin; D008297:Male; D009411:Nerve Endings / Q000378:metabolism*; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000494:pharmacology; D011188:Potassium / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D013050:Spectrometry, Fluorescence; D013574:Synaptosomes / Q000187:drug effects / Q000378:metabolism*
D000634:Aminoquinolines; D001572:Benzofurans; D004987:Ethers; D005456:Fluorescent Dyes; D011092:Polyethylene Glycols; D015759:Ionomycin; D017830:Octoxynol; C035739:Quin2; D011188:Potassium; D002118:Calcium; D016257:Fura-2