Differential effect of protein kinase inhibitors on calcium-dependent and calcium-independent [ 14C]GABA release from rat brain synaptosomes

• Published in: Neuroscience 1998-08, Vol.85 (3), p.989-997
• Main Authors: Storchak, L.G, Pozdnyakova, N.G, Himmelreich, N.H
• Format: Article
• Language: English
• Subjects: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine - pharmacology; 4-aminopyridine; 4-Aminopyridine - pharmacology; [ 14C]GABA release; Animals; Biological and medical sciences; Biological Transport - drug effects; Brain Chemistry - drug effects; Calcium - metabolism; Carbon Radioisotopes; Central nervous system; Central neurotransmission. Neuromudulation. Pathways and receptors; Enzyme Inhibitors - pharmacology; Fundamental and applied biological sciences. Psychology; gamma-Aminobutyric Acid - pharmacokinetics; Male; Potassium Chloride - pharmacology; protein kinase inhibitors; Rats; Sphingosine - pharmacology; Spider Venoms - pharmacology; Staurosporine - pharmacology; Sulfonamides - pharmacology; synaptosome; Synaptosomes - drug effects; Synaptosomes - enzymology; Vertebrates: nervous system and sense organs; α-latrotoxin
• ISSN: 0306-4522; 1873-7544
• DOI: 10.1016/S0306-4522(97)00599-X

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 Ca 2+-dependent and Ca 2+-independent [ 14C]GABA release. The Ca 2+-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 Ca 2+ while K +-evoked [ 14C]GABA release only partly decreases in the absence of calcium. The substitution of sodium by choline in Ca 2+-free medium completely abolished Ca 2+-independent part of K +-evoked [ 14C]GABA release. So the main effect of 4-aminopyridine is the Ca 2+-dependent one while high K + is able to evoke [ 14C]GABA release in both a Ca 2+-dependent and Na +-dependent manner. In experiments with protein kinase inhibitors, 4-aminopyridine and high K + concentration were used to study the Ca 2+-dependent and the Ca 2+-independent [ 14C]GABA release, respectively. In addition, the Ca 2+-independent [ 14C]GABA release. was studied using α-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 Ca 2+-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 45Ca 2+ influx into synaptosomes. Only sphingosine (100 μM) reduced the 45Ca 2+ influx. All the inhibitors investigated were absolutely ineffective in blocking the Ca 2+-independent [ 14C]GABA release stimulated by α-latrotoxin. Three of them, except for sphingosine, did not affect the Ca 2+-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 Ca 2+-independent manner irrespective of whether α-latrotoxin or high K + stimulated this process, it was not inhibited by the drugs decreased the Ca 2+-dependent [ 14C] GABA release. Given the above points it is therefore not unreasonable to assume that the absence of Ca 2+ in the extracellular medium created the conditions in which the activation of neurotransmitter release was not accompanied by Ca 2+-dependent d