Characterization of K(+)-evoked [3H]D-aspartate outflow in the rat hippocampus in vitro.
The characteristics of K(+)-evoked outflow of [3H]D-aspartate, a glutamate release marker, were systematically investigated in the rat hippocampus, using 35 mM K(+)-evoked [3H]noradrenaline outflow as a reference. Elevation of external K+ concentrations increased [3H]D-aspartate outflow in a concentration-dependent manner both in slices and synaptosomes. In the absence of external Ca2+, K(+)-evoked [3H]D-aspartate outflow was decreased by approx 60% in synaptosomes and 80% in slices. However, elimination of external Ca2+ in the presence of 2 mM EGTA significantly reduced only 100 mM K(+)-evoked outflow, both in slices and synaptosomes. In the absence of external Ca2+, 35 mM K(+)-evoked [3H]noradrenaline outflow was abolished even when EGTA was present in the solution. Furthermore, the Ca(2+)-channel blockers omega-conotoxin (10 nM) and nifedipine (0.5 microM) did not significantly reduce K(+)-evoked [3H]D-aspartate outflow; [3H]noradrenaline outflow, however, was reduced by more than one third by omega-conotoxin. Finally [3H]D-aspartate overflow was insensitive to tetrodotoxin (0.5 microM) both in synaptosomes and in slices, while that of [3H]noradrenaline was significantly reduced in slices. It is concluded that (1) [3H]D-aspartate outflow is partly Ca(2+)-dependent; (2) differences between K(+)-evoked [3H]D-aspartate and [3H]noradrenaline outflow include sensitivity to stimulation by EGTA, to Ca(2+)-channel blockers and to tetrodotoxin. Some of these discrepancies may be ascribed to the existence of a cytosolic, Ca(2+)-independent pool of releasable glutamate and [3H]D-aspartate. These observations pose some problems as to the experimental approach for the study of Ca(2+)-dependent [3H]D-aspartate release.
Simonato
M
M
Institute of Pharmacology, University of Ferrara, Italy.
Bregola
G
G
Muzzolini
A
A
Bianchi
C
C
Beani
L
L
D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism*; D002118:Calcium / Q000494:pharmacology; D006624:Hippocampus / Q000378:metabolism* / Q000648:ultrastructure; D066298:In Vitro Techniques; D008297:Male; D009638:Norepinephrine / Q000378:metabolism; D011188:Potassium / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012964:Sodium / Q000494:pharmacology; D013574:Synaptosomes / Q000378:metabolism
D001224:Aspartic Acid; D012964:Sodium; D011188:Potassium; D002118:Calcium; D009638:Norepinephrine