Characterization of the interactions of gallamine with muscarinic receptors from brain.
Characterization of the interactions of gallamine with muscarinic receptors from brain.
复制标题
没食子胺与大脑毒蕈碱受体相互作用的表征。
DOI：
10.1016/0006-2952(85)90423-x
复制
发表时间：
1985
影响因子：
5.8
通讯作者：
Lenox,RH
中科院分区：
文献类型：
--
作者：
Ellis,J;Lenox,RH
Ellis和Hoss先前的报告[1]表明，胆碱胺抑制了强效和特异性的毒蕈碱拮抗剂苯基喹啉酯（QNB）的结合。虽然其他研究发现胆碱胺是毒蕈碱系统的拮抗剂[2-51]，但胆碱胺的结合特性与毒蕈碱激动剂相似。也就是说，胆碱的占用曲线更适合两种受体群体的模型，而不是单位点模型，它对脑干受体的总体亲和力比对前脑受体的亲和力更强[1]。最近，Stockton等人报道胆碱胺通过变构机制调节毒蕈碱受体。因此提出了一种可能性，即早期研究中由胆碱检测到的异质性可能是人为的，因为假设了不正确的作用机制。然而，很难用非竞争机制来解释Ellis和Hoss之前的所有发现，特别是消除对激动剂碳乙醇亲和力低的受体会导致对胆碱亲和力低的受体的损失111。本研究旨在试图调和这些明显不同的报道，并进一步评估胆碱胺与毒蕈碱受体相互作用的机制。氚化L-QNB （32.2 Ci/ mol）和[' H] n -甲基东莨菪碱(NMS。84.8 Ci/ mol)来自&新英格兰核公司（Boston， MA）。三硫代胆碱从k&k实验室（Plainsview， NY）获得。神经膜取材于先前描述的雄性Sprague-Dawley大鼠的前脑[1]，冷冻保存在70℃直到使用。“前脑”一词指的是间脑前面的那部分大脑，加上上面的皮层和海马组织。结合试验在40 mM磷酸钠钾缓冲液（除非另有说明）中25英寸处进行，pH为7.2。并通过GF/B玻璃纤维过滤器(Brandel, Gaithersburg；MD)。在所有情况下，通过标记配体包含1 nM未标记的QNB来确定非特异性结合。在低离子强度条件下，高浓度的胆碱可以降低[3H] QNB的非特异性结合。因此，通过将1 PM未标记的QNB与标记的QNB和gallamine分别放入一组独立的测定管中，来确定每种浓度的gallamine的非特异性结合。采用[IH] NMS的实验结果（图1A和2A）与Stockton等人的报告一致。结合和解离动力学均因胆碱的存在而显著减慢。单独的研究（数据未显示）发现，对13H1NMS脱出率的一半最大影响发生在下午30点的三聚氰胺。然而，当使用[' H] QNB进行类似研究时（图2）。lB， lC和2B)，没有发现变构效应。即使在100 uM下，胆碱胺也不能显著改变(3H] QNB的结合速率或先前结合的IjHlONB的解离速率。我们希望解决的下一个问题是，似乎对胆碱具有不同亲和力的毒蕈碱位点是否独立且不可相互转换。解决这个问题的一种方法是用不可逆或缓慢可逆的拮抗剂（如QNB）选择性地阻断低亲和力位点，就像过去对由毒蕈碱激动剂区分的位点所做的那样[1,8,91]。最初的研究（图3）发现，在低离子强度条件下，不同的位点表达了对胆碱的最广泛不同的亲和力。因此选择了这样的条件……
A previous report by Ellis and Hoss [l] demonstrated that gallamine inhibits the binding of the potent and specific muscarinic antagonist quinuclidinyl benzilate(QNB). Although other studies have found gallamine to be an antagonist in muscarinic systems [2-51, the binding properties of gallamine were shown to be similar to those of muscarinic agonists. That is, the occupancy curve for gallamine is better suited to a model of two populations of receptors than to a one-site model and it possesses greater overall affinity toward brainstem receptors than toward those of the forebrain[I]. Recently, Stockton et al.[6] reported that gallamine regulates muscarinic receptors by an allosteric mechanism. The possibility was therefore raised that the heterogeneity detected by gallamine in the earlier study might have been artifactual, due to the assumption of an incorrect mechanism of action. However, it is difficult to account for all of the previous findings of Ellis and Hoss by a noncompetitive mechanism, especially that the elimination of receptors with low affinity for the agonist carbachol results in-a loss of receptors with low affinitv for gallamine 111. The present studv was undertaken in an-attempt to reconcile &these apparently discrepant reports and to further evaluate the mechanism (s) by which gallamine interacts with muscarinic receptors. Tritiated L-QNB (32.2 Ci/mmole) and [‘H] N-methylscopolamine(NMS. 84.8 Ci/mmole) were obtained from the&New England Nuclear Corp.(Boston, MA). Gallamine triethiodide was obtained from K & K Laboratories (Plainsview, NY). Neural membranes were prepared from the forebrains of male Sprague-Dawley rats as described previously [l] and stored frozen at-70” until used. The term forebrain denotes the portion of the brain anterior to the diencephalon, plus overlying cortical and hippocampal tissue. Binding assays were conducted at 25” in 40 mM sodium-potassium phosphate buffer (except where otherwise indicated), pH 7.2. and were terminated by filtration through GF/B glass fiber filters (Brandel, Gaithersburg. MD). In all cases, nonspecific binding was determined by the inclusion of 1 nM unlabeled QNB with the labeled ligand. Under conditions of low ionic strength, high concentrations of gallamine were found to decrease the nonspecific binding of [3H] QNB. Therefore, nonspecific binding was determined for each concentration of gallamine by including 1 PM unlabeled QNB with the labeled QNB and gallamine in a separate set of assay tubes. The results of experiments which employed [IH] NMS (Figs. 1A and 2A) were consistent with the report by Stockton et al.[6]. Both the association and dissociation kinetics were dramatically slowed by the presence of gallamine. Separate studies (data not shown) found the halfmaximal effect on the off-rate of 13H1NMS to occur at 30 PM gallamine. However, when similar studies were carried out with [‘H] QNB (Figs. lB, lC, and 2B), no allosteric effects were noted. Even at 100 uM, gallamine failed to significantly alter the rate of associationof(3H] QNB or the rate of dissociation of oreviouslv bound IjHlONB. The next question that we wished to address was whether the muscarinic sites that appear to possess different affinities for gallamine are separate and non-interconvertible. One approach to this question is to selectively block the low-affinity sites with an irreversible or slowly reversible antagonist (eg QNB), as has been done in the past for the sites-that are differentiated by the muscarinic agonists [l, 8, 91. Initial studies (Fig. 3) found that the different sites expressed the most widely disparate affinities for gallamine under conditions of low ionic strength. Such conditions were therefore chosen …
没食子胺对胆碱能受体的影响
DOI：
10.1007/bf03004717
发表时间：
1970
期刊：
Canadian Anaesthetists’ Society Journal
影响因子：
--
作者：
Flora J. Rathbun;J. T. Hamilton
通讯作者：
J. T. Hamilton
影响因子：
4.7
作者：
BIRD, SJ;AGHAJANIAN, GK
通讯作者：
AGHAJANIAN, GK
没食子胺与多种毒蕈碱受体的竞争性相互作用。
影响因子：
5.8
作者：
J. Ellis;W. Hoss
通讯作者：
W. Hoss