杰克豆脲酶调节昆虫神经肌肉接头处的神经递质释放,Pesticide Biochemistry and Physiology

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杰克豆脲酶调节昆虫神经肌肉接头处的神经递质释放
Pesticide Biochemistry and Physiology ( IF 4.2 ) Pub Date : 2018-04-01 , DOI: 10.1016/j.pestbp.2018.02.009
Thiago Carrazoni ₁ , Christine Nguyen ₂ , Lucas F Maciel ₃ , Andres Delgado-Cañedo ₃ , Bryan A Stewart ₂ , Angela B Lange ₂ , Chariston A Dal Belo ₃ , Celia R Carlini ₄ , Ian Orchard ₂

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背景植物已经发展出广泛的机制来与植食性昆虫竞争，包括昆虫毒性蛋白，例如脲酶。豆科植物 Canavalia ensiformis 产生几种脲酶异构体，其中更丰富的称为 Jack Bean Urease (JBU)。预览工作已经通过迄今为止尚未完全阐明的机制证明了 JBU 的潜在杀虫作用。在这项工作中，我们研究了 JBU 诱导昆虫神经肌肉接头上神经递质释放活动的机制。方法使用神经和肌肉动作电位的电生理记录和钙成像生物测定。结果与结论 JBU（0.28 mg/动物/天）在 Locusta migratoria 2 龄通过饲喂和注射未引起致死性，尽管它确实导致 168 小时结束时体重增加减少了 20%（n = 9，p ≤ 0.05）。将 JBU（0.014 和 0.14 毫克）直接注射到蝗虫后腿中，会导致肌肉动作电位幅度的剂量和时间依赖性降低，最高剂量时幅度最大降低 70%（n = 5，p ≤ 0.05)。在相同剂量下，JBU 不会改变运动神经元诱发的动作电位的幅度。使用果蝇 3 龄幼虫神经肌肉制剂，JBU (10-7 M) 在 1 mM CaCl2 (n = 5, p ≤ 0.05) 存在下增加了微型兴奋性连接电位 (mEJPs) 的发生。在低钙 (0.4 mM) 测定中，JBU (10-7 M) 无法调节事件的发生。在无 Ca2+ 条件下，使用 EGTA 或 CoCl2，JBU 导致 mEPJs 的发生显着减少 (n = 5, p ≤ 0.05)。注射到 Nauphoeta cinerea 蟑螂的第三个腹部神经节后，JBU (1 μM) 导致 Ca2+ 流入显着增加（n = 7，p ≤ 0.01），类似于高 KCl (35 mM) 条件下所见。总之，结果证实了 JBU 对昆虫神经肌肉接头和可能的中央突触的直接作用，可能是通过破坏神经元突触前区域的钙机制。

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Jack bean urease modulates neurotransmitter release at insect neuromuscular junctions

BACKGROUND Plants have developed a vast range of mechanisms to compete with phytophagous insects, including entomotoxic proteins such as ureases. The legume Canavalia ensiformis produces several urease isoforms, of which the more abundant is called Jack Bean Urease (JBU). Previews work has demonstrated the potential insecticidal effects of JBU, by mechanisms so far not entirely elucidated. In this work, we investigated the mechanisms involved in the JBU-induced activity upon neurotransmitter release on insect neuromuscular junctions. METHODS Electrophysiological recordings of nerve and muscle action potentials, and calcium imaging bioassays were employed. RESULTS AND CONCLUSION JBU (0.28 mg/animal/day) in Locusta migratoria 2nd instar through feeding and injection did not induce lethality, although it did result in a reduction of 20% in the weight gain at the end of 168 h (n = 9, p ≤ 0.05). JBU (0.014 and 0.14 mg) injected direct into the locust hind leg induced a dose and time-dependent decrease in the amplitude of muscle action potentials, with a maximum decrease of 70% in the amplitude at the highest dose (n = 5, p ≤ 0.05). At the same doses JBU did not alter the amplitude of action potentials evoked from motor neurons. Using Drosophila 3rd instar larvae neuromuscular preparations, JBU (10-7 M) increased the occurrence of miniature Excitatory Junctional Potentials (mEJPs) in the presence of 1 mM CaCl2 (n = 5, p ≤ 0.05). In low calcium (0.4 mM) assays, JBU (10-7 M) was not able to modulate the occurrence of the events. In Ca2+-free conditions, with EGTA or CoCl2, JBU induced a significant decrease in the occurrence of mEPJs (n = 5, p ≤ 0.05). Injected into the 3rd abdominal ganglion of Nauphoeta cinerea cockroaches, JBU (1 μM) induced a significant increase in Ca2+ influx (n = 7, p ≤ 0.01), similar to that seen for high KCl (35 mM) condition. Taken together the results confirm a direct action of JBU upon insect neuromuscular junctions and possibly central synapses, probably by disrupting the calcium machinery in the pre-synaptic region of the neurons.

更新日期：2018-04-01

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