BRAIN PHOSPHOINOSITIDES AND BARBITURATES

麻醉药和抗惊厥药的作用机制尚不清楚。 尽管最有用的抗惊厥药物苯巴比妥一直是 已经使用了75年。在亲脂性药物中，巴比妥酸盐是 在结构上与肌醇最接近；两者都含有一个六元环，具有 几个氢键位置(O、OH或NH基团)。因为这些酶 新陈代谢的肌醇无疑识别肌醇部分， 可能会怀疑巴比妥酸盐会抑制多磷肌醇 新陈代谢。我们发现苯巴比妥是一种有效的抑制 激酶，尤其是磷脂酰肌醇-4-磷酸(PIP)激酶。它 抑制磷酸水解酶，尤其是磷脂酰-4，5-二磷酸 (PIP)2磷酸二酯酶(磷脂酶C)，仅弱。因此， 苯巴比妥的药理作用可能与抑制 PIP2合成。这种脂类在细胞刺激下产生第二种 信使，肌醇-1，4，5-三磷酸(IP3)，进而动员 细胞内钙；因此，肌醇磷脂-钙反应可能是 受潮了。我们计划调查这种可能性和相关问题。 将对突触体和突触神经体进行实验 大脑部分--含有所有相关酶--如果合适的话， 血小板。苯巴比妥麻醉的ED50将被测定 (蝌蚪翻正反射)证实PIP-K在体外有抑制作用 与体内(临床)浓度一致。其他巴比妥酸盐(非 抗惊厥剂)将被测试，以及巴比妥酸盐对其他 酶(例如CD-P-二甘油-肌醇转移酶、肌醇 磷酸(S)水解酶、磷脂酶A2、二甘油酸激酶)； 将表明对巴比妥酸盐最敏感的反应是否真的是 PIP-激酶的活性。苯巴比妥抑制PIP-激酶的作用机制 将被确定(它是竞争性的吗？)。PIP2的压抑(也 苯巴比妥合成PIP)应能降低这些脂类 以及磷脂酰肌醇(PI)的增加；这将通过以下方式进行测试 孵育后肌苷的定量分析(突触体， 突触、神经小体和血小板)。预期中的 苯巴比妥对钙离子动员的抑制作用 胆碱能刺激突触体或突触神经体 毒鼠碱激动剂和凝血酶对血小板的作用将被测量为 随之而来的是细胞内膜钙释放减少 荧光计量为Ca-Fura-2形成。

The mode of action of anesthetics and anticonvulsants is not yet clarified although the most useful of anticonvulsant drugs, phenobarbital, has been in use for 75 years. Among lipophilic drugs, the barbiturates are the nearest in structure to inositol; both contain a six-membered ring with several hydrogen bonding sites (O, OH, or NH groups). Since the enzymes metabolizing phosphoinositides undoubtedly recognize the inositol moiety, it may be suspected that barbiturates will inhibit polyphosphoinositide metabolism. We find that phenobarbital is an effective inhibitor of the kinases, especially the phosphatidylinositol-4-phosphate (PIP) kinase. It inhibits the phosphohydrolases, especially phosphatidyl-4,5-bisphosphate (PIP)2 phosphodiesterase (phospholipase C), only weakly. Therefore, the pharmacological action of phenobarbital may be connected to a depression of PIP2 synthesis. This lipid, on cell stimulation, yields the second messenger, inositol-1,4,5-trisphosphate (IP3), which in turn mobilizes intracellular calcium; thus, the inositide-calcium response may be dampened. We plan to investigate this possibility and related issues. Experiments will be carried out with synaptosomal and synaptoneurosomal brain fractions - which contain all relevant enzymes - and, if appropriate, platelets. The ED50 of phenobarbital anaesthesia will be determined (tadpole righting reflex) to confirm that PIP-kinase inhibition in vitro coincides with in vivo (clinical) concentration. Other barbiturates (non- anticonvulsant) will be tested, as well as barbiturate inhibition of other enzymes (e.g. CD P-diacylglycerol-inositoltransferase, inositol phosphate(s) hydrolases, phospholipase A2, diglyceride kinase); the results will indicate if the most sensitive response to barbiturates is indeed that of PIP-kinase. The mechanism of PIP-kinase inhibition by phenobarbital will be determined (is it competitive?). The depression of PIP2 (and also PIP) synthesis by phenobarbital should lead to a decrease in these lipids and an increase in phosphatidylinositol (PI); this will be tested by quantitative analysis of the inositides after incubation (synaptosomes, synaptoneurosomes, and platelets) with and without the drug. The expected dampening effect of phenobarbital on the mobilization of calcium resulting from stimulation of synaptosomes or synaptoneurosomes by cholinergic muscarinic agonists, and of platelets by thrombin will be measured as diminished release of Ca from the intracellular membranes, followed fluorimetrically as Ca-fura-2 formation.