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.
Bandeira-Duarte
C
C
Department of Zoology, University of Coimbra, Portugal.
Carvalho
C A
CA
Cragoe Júnior
E J
EJ
Carvalho
A P
AP
D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism; D002132:Calcium Radioisotopes; D002352:Carrier Proteins / Q000378:metabolism; D002458:Cell Fractionation; D002540:Cerebral Cortex / Q000648:ultrastructure*; D005260:Female; D006863:Hydrogen-Ion Concentration; D008297:Male; D008564:Membrane Potentials; D011188:Potassium / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D012964:Sodium; D019831:Sodium-Calcium Exchanger; D013395:Sucrose; D013574:Synaptosomes / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure
D015220:Calcium Channels; D002132:Calcium Radioisotopes; D002352:Carrier Proteins; D019831:Sodium-Calcium Exchanger; C054391:5-(N-4-chlorobenzyl)-N-(2',4'-dimethyl)benzamil; C053494:2',4'-dimethylbenzamil; D013395:Sucrose; D000584:Amiloride; D012964:Sodium; D011188:Potassium; D002118:Calcium