Influence of isolation media on synaptosomal properties: Intracellular pH,pCa, and Ca2+ uptake |
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Authors: | C Bandeira-Duarte C A M Carvalho E J Cragoe Jr A P Carvalho |
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Institution: | (1) Center for Cell Biology, Department of Zoology and Institute of Histology and Embriology, University of Coimbra, 3049 Coimbra Codex, Portugal;(2) Present address: 2211 Oak Terrace Dr., 19446 Lansdale, PA |
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Abstract: | Preparations of synaptosomes isolated in sucrose or in Na+-rich media were compared with respect to internal pH (pH1), internal Ca2+ concentration (Ca2+]i), membrane potential and45Ca2+ uptake due to K+ depolarization and Na+/Ca2+ exchange. We found that synaptosomes isolated in sucrose media have a pHi of 6.77±0.04 and a Ca2+]i of about 260 nM, whereas synaptosomes isolated in Na+-rich ionic media have a pHi of 6.96±0.07 and a Ca2+]i of 463 nM, but both types of preparations have similar membrane potentials of about –50 mV when placed in choline media. The sucrose preparation takes up Ca2+ only by voltage sensitive calcium channels (VSCC'S) when K+-depolarized, while the Na+-rich synaptosomes take up45Ca2+ both by VSCC'S and by Na+/Ca2+ exchange. The amiloride derivative 2, 4 dimethylbenzamil (DMB), at 30 M, inhibits both mechanisms of Ca2+ influx, but 5-(N-4-chlorobenzyl)-2, 4 dimethylbenzamil (CBZ-DMB), at 30 M, inhibits the Ca2+ uptake by VSCC'S, but not by Na+/Ca2+ exchange. Thus, DMB and CBZ-DMB permit distinguishing between Ca2+ flux through channels and through Na+/Ca2+ exchange. We point out that the different properties of the two types of synaptosomes studied account for some of the discrepancies in results reported in the literature for studies of Ca2+ fluxes and neurotransmitter release by different types of preparations of synaptosomes.Abbreviations used BCECF
2,7-Biscarboxyethyl-5(6)-carboxyfluorescein
- BCECF/AM
acetoxymethyl ester of BCECF
- Ca2+]i
Internal free calcium ion concentration
- CBZ-DMB
5-(N-4-chlorobenzyl)-2,4-dimethylbenzamil
- DMB
2, 4-dimethylbenzamil
- DMSO
dimethyl sulfoxide
- Indo-1/AM
acetoxymethyl ester of Indo-1
- MES
2-|N-Morpholino|ethanesulfonic acid
- NMG
N-methyl-D-glucamine
- pHi
internal pH
- TPP+
tetraphenylphosphonium
- p
plasma membrane potential |
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Keywords: | Synaptosomes pH pCa2+ membrane potential K+ depolarization amiloride derivatives |
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