Phenylarsine oxide inhibits alpha-latrotoxin-stimulated [3H]GABA release from rat brain synaptosomes.
Phosphatidylinositol 4,5-biphosphate has been implicated in a variety of membrane-trafficking processes, including exocytosis of neurotransmitters. However, there are contradictory findings concerned ability of phenylarsine oxide (PAO), an inhibitor of phosphatidylinositol 4-kinase, to affect exocytotic release of different types of neurotransmitters. We bent our efforts to a detailed analysis of action of PAO on Ca(2+)-dependent and Ca(2+)-independent [3H]GABA release produced by exposure of rat brain synaptosomes to different concentrations of alpha-latrotoxin. We also compared PAO action on alpha-latrotoxin- and 4-aminopyridine (4-AP)-evoked [3H]GABA release. The experiments have shown that release of [3H]GABA evoked by the depolarization with 4-AP was decreased by 80% as a result of action of 3 microM PAO and the complete inhibition of release was observed with 10 microM PAO. When alpha-latrotoxin as a stimulant was applied, release of [3H]GABA was increased as toxin concentration used was elevated from 0.5 to 3.0 nM, however, concomitantly, the response of the toxin-induced [3H]GABA release to PAO became attenuated: 10 microM PAO led to almost complete inhibition of the effect of 0.5 nM alpha-latrotoxin and only partly decreased (by 40%) the response to 3.0 nM alpha-latrotoxin. To test whether the efficacy of PAO depended on the toxin-induced outflow of cytosolic [3H]GABA, synaptosomes with depleted cytosolic [3H]GABA pool were also exploited. Depletion was performed by means of heteroexchange of cytosolic [3H]GABA with nipecotic acid. The experiments have shown that treatment of loaded synaptosomes with nipecotic acid resulted in some increase of [3H]GABA release evoked by 0.5 nM alpha-latrotoxin, but in the two-fold decrease of the response to 3.0 nM alpha-latrotoxin. PAO essentially inhibited [3H]GABA release from depleted synaptosomes irrespective of alpha-latrotoxin concentration used. Therefore, the amount of [3H]GABA released from cytosolic pool determined, in considerable degree, the insensitivity of alpha-latrotoxin action to PAO. Thus, our data show that subnanomolar concentrations of alpha-latrotoxin may be used for stimulation of exocytotic release of [3H]GABA. Exposure of synaptosomes with nanomolar toxin concentrations leads not only to stimulation of exocytosis, but also to leakage of [3H]GABA from cytosolic pool. PAO potently inhibits exocytotic release of [3H]GABA and its inhibitory effectiveness is diminished as far as the outflow of [3H]GABA is elevated.
Linetska
M V
MV
Department of Neurochemistry, Palladin Institute of Biochemistry, National Academy of Science of Ukraine, Leontovich Street 9, Kiev 01601, Ukraine.
Storchak
L G
LG
Himmelreich
N H
NH
D015761:4-Aminopyridine / Q000494:pharmacology; D000818:Animals; D001152:Arsenicals / Q000494:pharmacology*; D001921:Brain / Q000378:metabolism*; D002352:Carrier Proteins / Q000187:drug effects / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D018756:GABA Antagonists / Q000494:pharmacology*; D050485:GABA Plasma Membrane Transport Proteins; D008297:Male; D008565:Membrane Proteins / Q000187:drug effects / Q000378:metabolism; D026901:Membrane Transport Proteins; D027361:Organic Anion Transporters; D051381:Rats; D017208:Rats, Wistar; D013111:Spider Venoms / Q000494:pharmacology*; D013574:Synaptosomes / Q000378:metabolism*; D014316:Tritium; D005680:gamma-Aminobutyric Acid / Q000172:deficiency / Q000378:metabolism*
D001152:Arsenicals; D002352:Carrier Proteins; D018756:GABA Antagonists; D050485:GABA Plasma Membrane Transport Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D027361:Organic Anion Transporters; D013111:Spider Venoms; C029341:oxophenylarsine; D014316:Tritium; D005680:gamma-Aminobutyric Acid; C020763:alpha-latrotoxin; D015761:4-Aminopyridine