Influence of isolation media on synaptosomal properties: intracellular pH, pCa, and Ca2+ uptake

Preparations of synaptosomes isolated in sucrose or in Na(+)-rich media were compared with respect to internal pH (pHi), internal Ca2+ concentration ([Ca2+]i), membrane potential and 45Ca2+ 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 up 45Ca2+ both by VSCC'S and by Na+/Ca2+ exchange. The amiloride derivative 2',4'-dimethylbenzamil (DMB), at 30 microM, inhibits both mechanisms of Ca2+ influx, but 5-(N-4-chlorobenzyl)-2',4' dimethylbenzamil (CBZ-DMB), at 30 microM, 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.