Glushakov Alexander V, Voytenko Larysa P, Skok Marina V, Skok Vladimir
Department of Anesthesiology, College of Medicine, University of Florida, PO Box 100254, Gainesville, FL 32610-0254, USA.
Auton Neurosci. 2004 Jan 30;110(1):19-26. doi: 10.1016/j.autneu.2003.08.012.
The subunit composition and localisation of nicotinic acetylcholine receptors (nAChRs) in the submucosal plexus of the guinea-pig ileum were studied using both affinity-purified monoclonal and polyclonal antibodies against alpha3, alpha4, alpha5 and alpha7 nAChR subunits and specific alpha7-containing nAChRs blocker methyllycaconitine (MLA). By means of immunohistochemistry performed in non-dissociated preparations, it was found that only 4% of submucosal ganglia expressed nAChRs. Specific staining, associated with cell membranes, was found with alpha3-, alpha5- and alpha7-, but not alpha4-specific antibodies. Double staining using alpha5- and alpha7-specific antibodies demonstrated that about one-half of the nAChR-positive ganglia contained neurons immunoreactive to both antibodies, while the others possessed either alpha5- or alpha7-immunoreactivity. Nanomolar concentrations of MLA prevented alpha7-specific antibody binding and did not influence the alpha5-specific antibody binding even when applied in micromolar concentrations. In electrophysiological experiments performed using a patch-clamp 'whole-cell' recording method, the neurons were identified by their sensitivity to MLA. In conclusion, submucosal neurons of the guinea-pig ileum express nAChRs containing alpha3-, alpha5- and alpha7-subunits. The co localisation of alpha5- and alpha7-subunits found in immunohistochemical experiments as well as kinetic characteristics of MLA-blocked receptors found by electrophysiological experiments allow us to suggest the presence of heteromeric alpha7-containing nAChRs in the submucosal plexus of the guinea-pig ileum.
利用针对α3、α4、α5和α7烟碱型乙酰胆碱受体（nAChRs）亚基的亲和纯化单克隆和多克隆抗体以及特异性含α7的nAChRs阻滞剂甲基lycaconitine（MLA），研究了豚鼠回肠黏膜下神经丛中nAChRs的亚基组成和定位。通过在未解离制剂中进行免疫组织化学，发现只有4%的黏膜下神经节表达nAChRs。用α3、α5和α7特异性抗体，但不用α4特异性抗体，发现了与细胞膜相关的特异性染色。使用α5和α7特异性抗体的双重染色表明，约一半的nAChR阳性神经节含有对两种抗体均有免疫反应性的神经元，而其他神经节则具有α5或α7免疫反应性。纳摩尔浓度的MLA可阻止α7特异性抗体结合，即使以微摩尔浓度应用也不影响α5特异性抗体结合。在使用膜片钳“全细胞”记录方法进行的电生理实验中，根据神经元对MLA的敏感性来识别它们。总之，豚鼠回肠的黏膜下神经元表达含有α3、α5和α7亚基的nAChRs。免疫组织化学实验中发现的α5和α7亚基的共定位以及电生理实验中发现的MLA阻断受体的动力学特征，使我们推测在豚鼠回肠黏膜下神经丛中存在含α7的异聚体nAChRs。
