DINOFLAGELLATE TOXINS AS TOOLS FOR STUDYING MEMBRANE CHOLINERGIC RECEPTORS

乙酰胆碱（ACh）受体是最好的特征之一， 参与兴奋性现象的膜蛋白。的 这种受体的功能特性得到了 发现具有新作用和高受体的非常特异的毒素， 结合亲和力我们最近证明了胆碱能活性 在从雪卡毒梭鱼获得的提取物中以及在提取物中， 如从Ostreopsis获得的色谱分离的级分中 与雪卡毒有关的底栖甲藻， 加勒比海甲基苯丙胺提取物抑制突触电位， 神经放松或沐浴引起的收缩 青蛙缝匠肌中的乙酰胆碱这种影响似乎是 是由甲藻物质中的极性成分引起的， 能够降低烟碱型ACh受体的离子电导 培养的鸡胚心肌细胞中的通道。其他研究表明， 提取物和第二个色谱级分显示毒蕈碱 活性，与氚化毒蕈碱配体的结合竞争 对大鼠脑细胞膜的作用，并在青蛙中表现出类似激动剂的作用 胃肌层此应用程序旨在发现 这些甲藻毒素的胆碱能作用的机制 使用各种电生理学、药理学和 生化技术实验提出，以达到 以下具体目的：a）色谱分离 加勒比沟藻有毒提取物的组分 使用反相HPLC测定Ostreopsis aerogularis; B）测定 胆碱能成分的化学性质及结构;及 c）确定隔离的作用部位和作用机制 胆碱能受体活性化合物。拟采用的研究战略 在后一个阶段中是：i）使用神经元的结合实验 膜和膜从鱼雷发电器官检查 的放射性标记配体的置换和竞争 不同的受体亚型; ii）突触的细胞内记录 蛙式运动终板电位及其变化的测量 活性组分引起的强度和时间特征; 和iii）记录膜离子电流，使用单一的 通道和全细胞版本的膜片钳技术， 培养的鸡胚肌细胞和交感神经、睫状神经 和背根神经节。一旦发现并描述了这些新的 甲藻毒素可作为研究的分子探针 中枢和外周神经系统中的胆碱能受体。 参加这些研究的少数民族学生将学习现代 神经化学和电生理方法以及组织 培养技术

The acetylcholine (ACh) receptor is one of the best characterized of the membrane proteins involved in excitability phenomena. The functional characterization of this receptor has been helped by the discovery of very specific toxins with novel actions and high receptor- binding affinities. We have recently demonstrated cholinergic activity in extracts obtained from ciguatoxic barracuda and in extracts, as well as in chromatographically isolated fractions, obtained from Ostreopsis lenticularis - the benthic dinoflagellate associated with ciguatera in the Caribbean. Methanolic extracts inhibited synaptic potentials and the contractions elicited by nerve-released or bath-applied acetylcholine in frog sartorious muscles. This effect appeared to be caused by a polar fraction in the dinoflagellate material that was capable of reducing the ionic conductance of nicotinic ACh receptor channels in cultured chick embryo myocytes. Other studies showed that extracts and a second chromatographic fraction displayed muscarinic activity, competing with the binding of tritiated muscarinic ligands to rat brain membranes and exhibiting an agonist-like action in frog gastric muscularis muscles. This application aims to discover the mechanism of the cholinergic effects of these dinoflagellate toxins using a variety of electrophysiological, pharmacological and biochemical techniques. Experiments are proposed to attain the following specific aims: a) the chromatographic isolation of the fractions from toxic extracts of the Caribbean dinoglagellate Ostreopsis lenticularis using reverse phase HPLC; b) the determination of the chemical nature and structure of cholinoactive components; and c) identification of the sites and mechanisms of action of the isolated cholinergic - receptor active compounds. Research strategies to be used in this latter phase are: i) binding experiments using neuronal membranes and membranes from Torpedo electric organ to examine displacement of and competition with radiolabeled ligands of the different receptor subtypes; ii) intracellular recording of synaptic potentials in frog motor end plates and measurement of changes in their magnitude and temporal characteristics caused by the active fractions; and iii) recording of membrane ionic currents, using both the single channel and the whole cell versions of the patch clamp technique in cultured chick embryo myocytes and neurons from sympathetic, ciliary and dorsal root ganglia. Once identified and characterized these new dinoflagellate toxins should be useful molecular probes for the study of cholinergic receptors in the central and peripheral nervous systems. Minority students participating in these studies will learn modern neurochemical and electrophysiological methods as well as tissue culture techniques.