Molecular biology and electrophysiology of neuronal nicotinic receptors of rat chromaffin cells
Molecular biology and electrophysiology of neuronal nicotinic receptors of rat chromaffin cells
复制标题
DOI：
10.1046/j.1460-9568.2003.02669.x
复制
发表时间：
2003-06-01
影响因子：
3.4
通讯作者：
Nistri, A
中科院分区：
文献类型：
--
作者：
Di Angelantonio, S;Matteoni, C;Nistri, A
肾上腺髓质中嗜铬细胞的神经元烟碱乙酰胆碱受体在生理上被乙酰胆碱激活，以介导儿茶酚胺释放到血流中。本研究利用分子生物学、免疫细胞化学和全细胞膜片钳检查了大鼠嗜铬细胞神经元烟碱乙酰胆碱受体的亚基组成和功能特性。逆转录聚合酶链反应分析表明存在α2、α3、α4、α5、α7、β2和β4转录物（无法检测到α6和β3）。免疫细胞化学显示大多数细胞的 α3、β2、β4 和 α5 蛋白呈阳性。很少有细胞对 alpha2 和 alpha4 有免疫反应，而没有细胞对 alpha7 有免疫反应。在单细胞水平上，可以证明 alpha3alpha5 和 alpha4beta2 的共定位。蛋白质印迹分析证实了抗体对 alpha3、alpha4、alpha5、beta2 和 beta4 亚基的特异性。尼古丁脉冲引起的内向电流对 α-银环蛇毒素和低剂量的甲基乌头碱不敏感，表明缺乏功能性 α7 受体。使用 AuIB α-芋螺毒素（选择性针对 α3β4 受体）或 MII α-芋螺毒素（选择性针对 α3β2 受体）观察到尼古丁电流的部分阻断。随着高浓度的共同应用的毒素，拮抗闭塞的发展，表明亚基选择性的丧失。二氢-β-赤霉素的拮抗作用与 AuIB 和 MII 抑制呈非线性叠加，证实了神经元烟碱乙酰胆碱受体阻滞的异质性。目前的结果表明，大鼠嗜铬细胞最常见的受体应包含α3β4、α3β2 以及附加的α5 亚基。由于普遍的亚基组成，大鼠嗜铬细胞神经元烟碱乙酰胆碱受体是合适的模型，特别是对于最近在离散大脑区域中证明的哺乳动物大脑受体的α3β4亚类。
Neuronal nicotinic acetylcholine receptors of chromaffin cells in the adrenal medulla are physiologically activated by acetylcholine to mediate catecholamine release into the bloodstream. The present study examined the subunit composition and functional properties of rat chromaffin cell neuronal nicotinic acetylcholine receptors using molecular biology, immunocytochemistry and whole-cell patch-clamp. Reverse transcription-polymerase chain reaction analysis indicated the presence of alpha2, alpha3, alpha4, alpha5, alpha7, beta2 and beta4 transcripts (alpha6 and beta3 could not be detected). Immunocytochemistry revealed most cells positive for alpha3, beta2, beta4 and alpha5 proteins. Few cells were immunoreactive for alpha2 and alpha4, while none was for alpha7. At single-cell level, colocalization could be demonstrated for alpha3alpha5 and alpha4beta2. Western blot analysis confirmed antibody specificity for alpha3, alpha4, alpha5, beta2 and beta4 subunits. Inward currents elicited by nicotine pulses were insensitive to alpha-bungarotoxin and low doses of methyllycaconitine, demonstrating lack of functional alpha7 receptors. Partial block of nicotine currents was observed with either AuIB alpha-conotoxin (selective against alpha3beta4 receptors) or MII alpha-conotoxin (selective against alpha3beta2 receptors). With high concentrations of co-applied toxins, antagonism occlusion developed, suggesting loss of subunit selectivity. Antagonism by dihydro-beta-erythroidine summated nonlinearly with AuIB and MII inhibition, confirming heterogeneity of neuronal nicotinic acetylcholine receptor block. The present results suggest that the most frequently encountered receptors of rat chromaffin cells should comprise alpha3beta4, alpha3beta2 with the addition of alpha5 subunits. Because of the prevailing subunit composition, rat chromaffin cell neuronal nicotinic acetylcholine receptors are suitable models, particularly for the alpha3beta4 subclasses of mammalian brain receptors recently demonstrated in discrete cerebral areas.