Differential effect of protein kinase inhibitors on calcium-dependent and calcium-independent [14C]GABA release from rat brain synaptosomes.
Rat brain synaptosomes were isolated to study the effects of protein kinase inhibitors (sphingosine, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride, N-(6-aminohexyl)-5-chloro-1-naphtalenesulfonamide, staurosporine) on Ca2+-dependent and Ca2+-independent [14C]GABA release. The Ca2+-dependent [14C]GABA release was stimulated by depolarization with a K+-channel blocker, 4-aminopyridine, or high K+ concentration. It has been shown that 4-aminopyridine-evoked [14C]GABA release strongly depends on extracellular Ca2+ while K+-evoked [14C]GABA release only partly decreases in the absence of calcium. The substitution of sodium by choline in Ca2+-free medium completely abolished Ca2+-independent part of K+-evoked [14C]GABA release. So the main effect of 4-aminopyridine is the Ca2+-dependent one while high K+ is able to evoke [14C]GABA release in both a Ca2+-dependent and Na+-dependent manner. In experiments with protein kinase inhibitors, 4-aminopyridine and high K+ concentration were used to study the Ca2+-dependent and the Ca2+-independent [14C]GABA release, respectively. In addition, the Ca2+-independent [14C]GABA release was studied using alpha-latrotoxin as a tool. Pretreatment of synaptosomes with protein kinase inhibitors tested, except of 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride, resulted in a marked inhibition of 4-aminopyridine-stimulated Ca2+-dependent [14C]GABA release. The inhibitory effects of N-(6-aminohexyl)-5-chloro-1-naphtalenesulfonamide and staurosporine on [14C]GABA release were not due to their effects on 4-aminopyridine-promoted 45Ca2+ influx into synaptosomes. Only sphingosine (100 microM) reduced the 45Ca2+ influx. All the inhibitors investigated were absolutely ineffective in blocking the Ca2+-independent [14C]GABA release stimulated by alpha-latrotoxin. Three of them, except for sphingosine, did not affect the Ca2+-independent [14C]GABA release stimulated by high potassium. The inhibitory effect of sphingosine was equal to 30%. Thus, if [14C]GABA release occurred in a Ca2+-independent manner irrespective of whether alpha-latrotoxin or high K+ stimulated this process, it was not inhibited by the drugs decreased the Ca2+-dependent [14C] GABA release. Given the above points it is therefore not unreasonable to assume that the absence of Ca2+ in the extracellular medium created the conditions in which the activation of neurotransmitter release was not accompanied by Ca2+-dependent dephosphorylation of neuronal phosphoproteins, and as a consequence the regulation of exocytotic process was modulated so that the inhibition of protein kinases did not disturb the exocytosis.
Storchak
L G
LG
Department of Neurochemistry, Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kiev, St Leontovich.
Pozdnyakova
N G
NG
Himmelreich
N H
NH
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000494:pharmacology; D015761:4-Aminopyridine / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D001923:Brain Chemistry / Q000187:drug effects*; D002118:Calcium / Q000378:metabolism; D002250:Carbon Radioisotopes; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D008297:Male; D011189:Potassium Chloride / Q000494:pharmacology; D051381:Rats; D013110:Sphingosine / Q000494:pharmacology; D013111:Spider Venoms / Q000494:pharmacology; D019311:Staurosporine / Q000494:pharmacology; D013449:Sulfonamides / Q000494:pharmacology; D013574:Synaptosomes / Q000187:drug effects / Q000201:enzymology*; D005680:gamma-Aminobutyric Acid / Q000493:pharmacokinetics*
D002250:Carbon Radioisotopes; D004791:Enzyme Inhibitors; D013111:Spider Venoms; D013449:Sulfonamides; D005680:gamma-Aminobutyric Acid; C017967:W 7; C020763:alpha-latrotoxin; D011189:Potassium Chloride; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D015761:4-Aminopyridine; D019311:Staurosporine; D013110:Sphingosine; D002118:Calcium