Storchak L G, Pozdnyakova N G, Himmelreich N H
Department of Neurochemistry, Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kiev, St Leontovich.
Neuroscience. 1998 Aug;85(3):989-97. 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 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.
分离大鼠脑突触体以研究蛋白激酶抑制剂（鞘氨醇、1 -（5 - 异喹啉磺酰基）- 2 - 甲基哌嗪二盐酸盐、N -（6 - 氨基己基）- 5 - 氯 - 1 - 萘磺酰胺、星形孢菌素）对钙离子依赖性和非钙离子依赖性[14C]γ-氨基丁酸（GABA）释放的影响。钙离子依赖性[14C]GABA释放可通过用钾通道阻滞剂4 - 氨基吡啶或高钾浓度进行去极化来刺激。已表明，4 - 氨基吡啶诱发的[14C]GABA释放强烈依赖于细胞外钙离子，而钾诱发的[14C]GABA释放在无钙情况下仅部分降低。在无钙培养基中用胆碱替代钠完全消除了钾诱发的[14C]GABA释放的非钙离子依赖性部分。因此，4 - 氨基吡啶的主要作用是钙离子依赖性的，而高钾能够以钙离子依赖性和钠离子依赖性方式诱发[14C]GABA释放。在蛋白激酶抑制剂实验中，4 - 氨基吡啶和高钾浓度分别用于研究钙离子依赖性和非钙离子依赖性[14C]GABA释放。此外，使用α- 银环蛇毒素作为工具研究非钙离子依赖性[14C]GABA释放。除1 -（5 - 异喹啉磺酰基）- 2 - 甲基哌嗪二盐酸盐外，用所测试的蛋白激酶抑制剂预处理突触体导致4 - 氨基吡啶刺激的钙离子依赖性[14C]GABA释放受到显著抑制。N -（6 - 氨基己基）- 5 - 氯 - 1 - 萘磺酰胺和星形孢菌素对[14C]GABA释放的抑制作用并非因其对4 - 氨基吡啶促进的45Ca2+流入突触体的影响。只有鞘氨醇（100微摩尔）减少了45Ca2+流入。所研究的所有抑制剂在阻断α- 银环蛇毒素刺激的非钙离子依赖性[14C]GABA释放方面完全无效。其中三种抑制剂（除鞘氨醇外）不影响高钾刺激的非钙离子依赖性[14C]GABA释放。鞘氨醇的抑制作用为30%。因此，如果[14C]GABA释放以非钙离子依赖性方式发生，无论α- 银环蛇毒素还是高钾刺激该过程，其都不会被降低钙离子依赖性[14C]GABA释放的药物所抑制。鉴于上述情况，因此可以合理推测，细胞外培养基中无钙离子创造了这样的条件，即神经递质释放的激活不伴随神经元磷酸蛋白的钙离子依赖性去磷酸化，结果是胞吐过程的调节被调制，使得蛋白激酶的抑制不会干扰胞吐作用。
