Rosenthal L, Zacchetti D, Madeddu L, Meldolesi J
Department of Pharmacology, University of Milan, Italy.
Mol Pharmacol. 1990 Dec;38(6):917-23.
The potent neurotoxin alpha-latrotoxin (alpha LTx), from black widow spider venom, induces neurotransmitter release in both Ca2(+)-containing and Ca2(+)-free medium, following interaction with a specific cell surface receptor. Binding studies revealed two populations of alpha LTx binding sites in bovine synaptosomal membranes, showing the same high affinity (Kd, 0.3 x 10(-10) M) for alpha LTx, with approximately 50% of the sites being Ca2+ sensitive and the rest being Ca2+ insensitive. In contrast, in PC12 cells alpha LTx binding was completely unaffected by the removal of extracellular Ca2+ (Kd, 5 x 10(-10) M). The use of La3+ as an inhibitor of alpha LTx action, previously shown in synaptosomes, was extended to PC12 cells. In this system, La3+ (100 microM) was shown to inhibit Ca2+ influx, both Ca2(+)-dependent and -independent dopamine release, and polyphosphoinositide (PPI) hydrolysis induced by alpha LTx. At the same time, La3+ did not block alpha LTx binding or dopamine release evoked by either the ionophore ionomycin (0.5 microM) or the phorbol ester tetradecanoylphorbol acetate (100 nM). La3+ also blocked the influx of Mn2+ ions through the alpha LTx-induced cation channel, as measured by quenching of fura-2 fluorescence. In this PC12 cell line, PPI hydrolysis could also be induced by ionomycin, but only when it was present at concentrations that caused an elevation of free intracellular Ca2+ ([Ca2+]i) that was not transient but was as persistent as that evoked by alpha LTx. Our conclusions with regard to the mode of action of alpha LTx are as follows. (i) All the effects of alpha LTx in PC12 cells (dopamine release, PPI hydrolysis, and Ca2+ influx) can be mediated via a single, Ca2(+)-insensitive alpha LTx receptor. (ii) alpha LTx-induced PPI hydrolysis is most likely due to the activation of a Ca2(+)-sensitive phospholipase C following the persistent rise in [Ca2+]i elicited by the toxin in Ca2(+)-containing medium, and not via direct coupling of the alpha LTx receptor to the enzyme. (iii) Toxin-evoked Ca2(+)-independent dopamine release can be blocked by La3+ at the extracellular level, most likely by prevention of the entry of divalent cations.
来自黑寡妇蜘蛛毒液的强效神经毒素α- latrotoxin（α-LTx），在与特定细胞表面受体相互作用后，能在含Ca2+和无Ca2+的培养基中诱导神经递质释放。结合研究表明，牛突触体膜中有两类α-LTx结合位点，对α-LTx表现出相同的高亲和力（Kd，0.3×10-10 M），其中约50%的位点对Ca2+敏感，其余位点对Ca2+不敏感。相比之下，在PC12细胞中，去除细胞外Ca2+对α-LTx结合完全没有影响（Kd，5×10-10 M）。先前在突触体中显示的镧系元素La3+作为α-LTx作用抑制剂的应用扩展到了PC12细胞。在这个系统中，已证明La3+（100 μM）可抑制Ca2+内流、Ca2+依赖性和非依赖性多巴胺释放以及α-LTx诱导的多磷酸肌醇（PPI）水解。同时，La3+并不阻断α-LTx结合或由离子载体离子霉素（0.5 μM）或佛波酯十四烷酰佛波醇乙酸酯（100 nM）诱发的多巴胺释放。通过fura-2荧光淬灭测量发现，La3+还可阻断Mn2+离子通过α-LTx诱导的阳离子通道内流。在这种PC12细胞系中，离子霉素也可诱导PPI水解，但只有当它以能引起细胞内游离Ca2+（[Ca2+]i）升高的浓度存在时才行，这种升高不是短暂的，而是与α-LTx诱发的一样持久。我们关于α-LTx作用方式的结论如下：（i）α-LTx在PC12细胞中的所有作用（多巴胺释放、PPI水解和Ca2+内流）都可通过单一的、对Ca2+不敏感的α-LTx受体介导。（ii）α-LTx诱导的PPI水解很可能是由于在含Ca2+培养基中毒素引起的[Ca2+]i持续升高后激活了对Ca2+敏感的磷脂酶C，而不是通过α-LTx受体与该酶的直接偶联。（iii）毒素诱发的非Ca2+依赖性多巴胺释放在细胞外水平可被La3+阻断，最可能是通过阻止二价阳离子的进入。
