Relation of exocytotic release of gamma-aminobutyric acid to Ca2+ entry through Ca2+ channels or by reversal of the Na+/Ca2+ exchanger in synaptosomes.
The specific inhibitor of the gamma-aminobutyric acid (GABA) carrier, NNC-711, (1-[(2-diphenylmethylene)amino]oxyethyl)- 1,2,5,6-tetrahydro-3-pyridine-carboxylic acid hydrochloride, blocks the Ca(2+)-independent release of [3H]GABA from rat brain synaptosomes induced by 50 mM K+ depolarization. Thus, in the presence of this inhibitor, it was possible to study the Ca(2+)-dependent release of [3H]GABA in the total absence of carrier-mediated release. Reversal of the Na+/Ca2+ exchanger was used to increase the intracellular free Ca2+ concentration ([Ca2+]i) to test whether an increase in [Ca2+]i alone is sufficient to induce exocytosis in the absence of depolarization. We found that the [Ca2+]i may rise to values above 400 nM, as a result of Na+/Ca2+ exchange, without inducing release of [3H]GABA, but subsequent K+ depolarization immediately induced [3H]GABA release. Thus, a rise of only a few nanomolar Ca2+ in the cytoplasm induced by 50 mM K+ depolarization, after loading the synaptosomes with Ca2+ by Na+/Ca2+ exchange, induced exocytotic [3H]GABA release, whereas the rise in cytoplasmic [Ca2+] caused by reversal of the Na+/Ca2+ exchanger was insufficient to induce exocytosis, although the value for [Ca2+]i attained was higher than that required for exocytosis induced by K+ depolarization. The voltage-dependent Ca2+ entry due to K+ depolarization, after maximal Ca2+ loading of the synaptosomes by Na+/Ca2+ exchange, and the consequent [3H]GABA release could be blocked by 50 microM verapamil. Although preloading the synaptosomes with Ca2+ by Na+/Ca2+ exchange did not cause [3H]GABA release under any conditions studied, the rise in cytoplasmic [Ca2+] due to Na+/Ca2+ exchange increased the sensitivity to external Ca2+ of the exocytotic release of [3H]GABA induced by subsequent K+ depolarization. Thus, our results show that the vesicular release of [3H]GABA is rather insensitive to bulk cytoplasmic [Ca2+] and are compatible with the view that GABA exocytosis is triggered very effectively by Ca2+ entry through Ca2+ channels near the active zones.
Duarte
C B
CB
Centre for Neurosciences of Coimbra, University of Coimbra, Portugal.
Ferreira
I L
IL
Carvalho
A P
AP
Carvalho
C M
CM
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism*; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002540:Cerebral Cortex / Q000378:metabolism; D004533:Egtazic Acid / Q000494:pharmacology; D005089:Exocytosis; D050485:GABA Plasma Membrane Transport Proteins; D008565:Membrane Proteins / Q000037:antagonists & inhibitors; D026901:Membrane Transport Proteins; D009539:Nicotinic Acids / Q000494:pharmacology; D009557:Nipecotic Acids; D027361:Organic Anion Transporters; D010091:Oximes / Q000494:pharmacology; D011189:Potassium Chloride / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D019831:Sodium-Calcium Exchanger; D013574:Synaptosomes / Q000378:metabolism*; D005680:gamma-Aminobutyric Acid / Q000378:metabolism*
D015220:Calcium Channels; D002352:Carrier Proteins; D050485:GABA Plasma Membrane Transport Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009539:Nicotinic Acids; D009557:Nipecotic Acids; D027361:Organic Anion Transporters; D010091:Oximes; D019831:Sodium-Calcium Exchanger; C078793:NNC 711; D004533:Egtazic Acid; D005680:gamma-Aminobutyric Acid; D011189:Potassium Chloride; D002118:Calcium