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

Abstract：

: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.

journal_name

Neuroscience

journal_title

Neuroscience

authors

Storchak LG,Pozdnyakova NG,Himmelreich NH

doi

10.1016/s0306-4522(97)00599-x

subject

Has Abstract

pub_date

1998-08-01 00:00:00

pages

989-97

issue

3

eissn

0306-4522

issn

1873-7544

pii

S0306-4522(97)00599-X

journal_volume

85

pub_type

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