Ueno E, Rosenberg P
Department of Pharmaceutical Sciences, University of Connecticut, School of Pharmacy, Storrs 06269, USA.
Toxicon. 1996 Nov-Dec;34(11-12):1219-27. doi: 10.1016/s0041-0101(96)00113-4.
The snake venom phospholipase A2 neurotoxin, beta-bungarotoxin, acts presynaptically to alter acetylcholine release in both the peripheral and central nervous systems. In investigating the mechanism of this action, we found that beta-bungarotoxin inhibited phosphorylation of synapsin I, GAP-43 and MARCKS in rat brain synaptosomes. This inhibition was not due to the inhibition of ATP synthesis, action of arachidonic acid metabolites, or stimulation of phosphatase activities. Furthermore, the activities of Ca2+/calmodulin-kinase II, cAMP-kinase and protein kinase C were not altered by beta-bungarotoxin in either synaptic plasma membranes or cytosol. When synaptic plasma membranes were treated with beta-bungarotoxin, MARCKS phosphorylation was inhibited, and this inhibition was overcome by the addition of exogenous protein kinase C. These results suggest that the interaction between MARCKS and endogenous protein kinase C is altered by beta-bungarotoxin. In contrast, Naja naja atra phospholipase A2, a typical phospholipase A2 enzyme, had effects on phosphorylation which were different from those of beta-bungarotoxin: (1) inhibition of phosphorylation of synapsin I in intact synaptosomes was less potent than that by beta-bungarotoxin; (2) it stimulated basal phosphorylation of GAP-43 and MARCKS; and (3) it increased the activity of protein kinase C. The inhibition of synapsin I phosphorylation by N. n. atra phospholipase A2 in intact synaptosomes may be due to the inhibition of ATP synthesis. The stimulation of GAP-43 and MARCKS by N. n. atra phospholipase A2 can be explained by the production of arachidonic acid, which stimulated protein kinase C activity to a similar extent as that caused by N. n. atra phospholipase A2. Thus, the mechanism of action of beta-bungarotoxin appears to be quite different from that of a phospholipase A2 enzyme, suggesting that phospholipase A2 activity of beta-bungarotoxin may not be essential for its action. beta-Bungarotoxin may be a useful tool to study the physiological role of phosphorylation of synaptosomal proteins in neurotransmitter release.
蛇毒磷脂酶A2神经毒素β-银环蛇毒素，在外周和中枢神经系统中均作用于突触前，改变乙酰胆碱的释放。在研究其作用机制时，我们发现β-银环蛇毒素抑制大鼠脑突触体中突触素I、GAP-43和MARCKS的磷酸化。这种抑制并非由于ATP合成的抑制、花生四烯酸代谢产物的作用或磷酸酶活性的刺激。此外，β-银环蛇毒素在突触质膜或胞质溶胶中均未改变Ca2+/钙调蛋白激酶II、cAMP激酶和蛋白激酶C的活性。当用β-银环蛇毒素处理突触质膜时，MARCKS的磷酸化受到抑制，而添加外源性蛋白激酶C可克服这种抑制。这些结果表明，β-银环蛇毒素改变了MARCKS与内源性蛋白激酶C之间的相互作用。相比之下，眼镜蛇磷脂酶A2（一种典型的磷脂酶A2酶）对磷酸化的影响与β-银环蛇毒素不同：（1）在完整突触体中，其对突触素I磷酸化的抑制作用比β-银环蛇毒素弱；（2）它刺激GAP-43和MARCKS的基础磷酸化；（3）它增加蛋白激酶C的活性。眼镜蛇磷脂酶A2在完整突触体中对突触素I磷酸化的抑制可能是由于ATP合成的抑制。眼镜蛇磷脂酶A2对GAP-43和MARCKS的刺激作用可以用花生四烯酸的产生来解释，花生四烯酸对蛋白激酶C活性的刺激程度与眼镜蛇磷脂酶A2引起的刺激程度相似。因此，β-银环蛇毒素的作用机制似乎与磷脂酶A2酶的作用机制有很大不同，这表明β-银环蛇毒素的磷脂酶A2活性可能对其作用并非必不可少。β-银环蛇毒素可能是研究突触体蛋白磷酸化在神经递质释放中的生理作用的有用工具。
