Nicotinic acetylcholine receptors of the neuronal type occur in the plasma membrane of sea urchin eggs.
Nicotinic acetylcholine receptors of the neuronal type occur in the plasma membrane of sea urchin eggs.
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神经元型烟碱乙酰胆碱受体存在于海胆卵的质膜中。
DOI：
10.1017/s0967199400003737
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Chambers,EL
中科院分区：
文献类型：
--
作者：
Ivonnet,PI;Chambers,EL
在悬浮在海水 (SW) 中的未受精海胆卵 (Lytechinus variegatus) 附近的浴槽中添加乙酰胆碱（ACh，100 μl，10 μM），可突然使卵的膜电位 (Vm) 从约 -70 mV 的静息值去极化。这导致卵子动作电位的激发，随后部分复极化。类似地，将 ACh 添加到钳位在 -70 mV 的鸡蛋电压中，会产生突然开始的内向电流，峰值幅度为 -1.26 ± 0.20 nA（SE，n = 81）。当鸡蛋被钳位在比-70 mV更正的Vm时，ACh诱导的内向电流的峰值幅度减小，在大约-20 mV的钳位Vm处变为0。 Vm 进一步正向移动不会引起电流反转。应用乙酰胆碱后，夹在-70 mV 的卵母细胞表现出类似的内向电流反应。由于ACh同时刺激烟碱和毒蕈碱受体（分别为nACh-R和mACh-R），因此检查了将鸡蛋暴露于每种受体的激动剂和拮抗剂的效果。对于钳位在-70 mV 的未受精卵，应用 100 μM (-)-尼古丁氢酒石酸盐（nACh-R 的激动剂）会诱导类似于 10 μM ACh 引起的内向电流响应，但峰值幅度较小。相反，使用 (+)-氯化毒蕈碱（mACh-R 的激动剂）无法诱导任何反应。 nACh-R 拮抗剂抑制神经元型 nACh-R 或骨骼肌型 nACh-R。拮抗剂对 ACh 诱导的内向电流响应幅度的影响通过以下方式确定：将单个卵夹在 -70 mV 处，所需拮抗剂溶解在 SW 中，然后在卵附近添加 100 μl 10 μM ACh。氯化美加明是神经元 nACh-R 的拮抗剂，浓度为 1 μM 时，可显着降低对 ACh 的反应，而浓度为 10 μM 时，对 ACh 的反应被消除。氯化六甲铵是神经元型 nACh-R 的另一种抑制剂，也可减弱 ACh 诱导的反应，但浓度为 10 μM 时，该反应并未完全消除。将鸡蛋暴露于浓度高达 250 nM 的 α-银环蛇毒素（一种骨骼肌 nACh-R 拮抗剂）30 分钟，对 ACh 诱导的反应没有影响。还检查了 mACh-R 的两种拮抗剂硫酸阿托品和 QNB (R-(−)-3-quinuclidinyl benzilate) 的作用。鸡蛋暴露于 1 μM 阿托品不会影响 ACh 诱导的反应，但在 10 μM 阿托品浓度下，ACh 诱导的内向电流的幅度显着降低。将鸡蛋暴露于 QNB（一种 mACh-R 的高度特异性拮抗剂）中，浓度高达 50 nM，对 ACh 诱导的反应没有影响。因此，对阿托品的抑制作用的可能解释是，高浓度的阿托品与 nACh-R 发生交叉反应。这些发现揭示了未受精海胆卵中存在类似于神经元 nACh-R 的 ACh-R。没有证据表明这些受体在精子进入、卵子激活或早期发育中发挥作用。
The addition of acetylcholine (ACh, 100 μl of 10 μM) to the bath in the vicinity of unfertilised sea urchin eggs (Lytechinus variegatus) suspended in sea water (SW) abruptly depolarises the membrane potential (Vm) of the eggs from the resting value of approximately −70 mV. This results in the firing of the egg's action potential, followed by partial repolarisation. Similar addition of ACh to eggs voltage clamped at −70 mV induces an inward current of abrupt onset with peak amplitude of −1.26 ± 0.20 nA (SE, n = 81). When the eggs are clamped at a Vm more positive than −70 mV, the peak amplitude of the ACh-induced inward current decreases, becoming 0 at a clamped Vm of approximately −20 mV. Further positive shift of the Vm fails to cause reversal of the current. Oocytes clamped at −70 mV exhibit similar inward current responses following application of ACh. Since ACh stimulates both nicotinic and muscarinic receptors (nACh-R and mACh-R, respectively), the effects of exposing eggs to the agonists and antagonists for each type of receptor were examined. For unfertilised eggs clamped at −70 mV the application of 100 μM (−)-nicotine hydrogen tartrate, an agonist of the nACh-R, induces an inward current response similar to that elicited by 10 μM ACh, but of smaller peak amplitude. In contrast, the application of (+)-muscarine chloride, an agonist of the mACh-R, fails to induce any response. Antagonists of the nACh-R inhibit either the neuronal type of nACh-R or the skeletal muscle type of nACh-R. The effect of the antagonists on the amplitudes of the ACh-induced inward current response was determined by supervising individual eggs clamped at −70 mV with the desired antagonist dissolved in SW, followed by the addition of 100 μl of 10 μM ACh in the vicinity of the egg. Mecamylamine chloride, an antagonist of the neuronal nACh-R at a concentration of 1 μM, markedly decreases the response to ACh, while at a concentration of 10 μM the response to ACh is abolished. Hexamethonium chloride, another inhibitor of the nACh-R of the neuronal type, also diminishes the ACh-induced response, but at a concentration of 10 μM the response is not completely abolished. Exposure of eggs to α-bungarotoxin, an antagonist of the skeletal muscle nACh-R at concentrations up to 250 nM for periods of 30 min, has no effect on the ACh-induced response. The effects of two antagonists of the mACh-R, atropine sulphate and QNB (R-(−)-3-quinuclidinyl benzilate) were also examined. Exposure of eggs to 1 μM atropine does not affect the ACh-induced response, but at concentrations of 10 μM atropine the amplitude of the ACh-induced inward current is significantly reduced. The exposure of eggs to QNB, a highly specific antagonist of the mACh-R, at concentrations up to 50 nM, has no effect on the ACh-induced response. Consequently, the likely explanation for the inhibitory effect of atropine is that at high concentrations atropine cross-reacts with the nACh-R. These findings reveal the presence in unfertilised sea urchin eggs of an ACh-R resembling the neuronal nACh-R. No evidence could be obtained that these receptors have a role in sperm entry, activation of the egg, or early development.