Separation of carrier mediated and vesicular release of GABA from rat brain slices.
In this study the temperature dependence of [3H]GABA release from brain slices evoked by electrical field stimulation and the Na+/K+ ATPase inhibitor ouabain was investigated. [3H]GABA has been taken up and released from hippocampal slices at rest and in response to electrical field stimulation (20 V, 10 Hz, 3 msec, 180 pulses) at 37 degrees C. When the bath temperature was cooled to 7 degrees C, during the sample collection period, the tissue uptake and the resting outflow of [3H]GABA were not significantly changed. In contrast, the stimulation-induced tritium outflow increased both in absolute amount (Bq/g) and in fractional release and the S2/S1 ratio was also higher at 7 degrees C. Perfusion of the slices with tetrodotoxin (TTX, 1 microM) inhibited stimulation-induced [3H]GABA efflux indicating that exocytotic release of vesicular origin is maintained under these conditions. 15 min perfusion with ouabain (10-20 microM) induced massive tritium release both in hippocampal and in striatal slices. However, the fraction of [3H]GABA outflow evoked by ouabain was much higher in the hippocampus than in the striatum. Sequential lowering the bath temperature from 37 degrees C to 17 degrees C completely abolished ouabain-induced [3H]GABA release in both brain regions, indicating that it is a temperature-dependent, carrier-mediated process. When the same experiments were repeated under Ca2+ free conditions, cooling the bath temperature to 17 degrees C, although substantially decreased the release but failed to completely abolish the tritium outflow evoked by ouabain, a significant part was maintained. Our results show that vesicular (field stimulation-evoked) and carrier-mediated (ouabain-induced) release of GABA is differentially affected by low temperature: while vesicular release is unaffected, carrier-mediated release is abolished at low bath temperature. Therefore, lowering the temperature offers a reliable tool to separate these two kinds of release and makes possible to study exclusively the pure neuronal release of GABA of vesicular origin.
Vizi
E S
ES
Department of Pharmacology, Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest. esvizi@koki.hi
Sperlágh
B
B
D000818:Animals; D001921:Brain / Q000378:metabolism*; D003342:Corpus Striatum / Q000187:drug effects / Q000378:metabolism; D004558:Electric Stimulation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006624:Hippocampus / Q000187:drug effects / Q000378:metabolism; D007700:Kinetics; D008297:Male; D010042:Ouabain / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors; D013696:Temperature; D013779:Tetrodotoxin / Q000494:pharmacology; D014316:Tritium; D005680:gamma-Aminobutyric Acid / Q000378:metabolism*
D004791:Enzyme Inhibitors; D014316:Tritium; D013779:Tetrodotoxin; D005680:gamma-Aminobutyric Acid; D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase