BRAIN PHOSPHOINOSITIDES AND BARBITURATES

脑磷脂和巴比妥酸盐

• 批准号: 3297819
• 负责人: DIWAKAR S DESHMUKH
• 金额: $ 8.55万
• 依托单位: INSTITUTE FOR BASIC RES IN DEV DISABIL
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-05-01 至 1994-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3297819
• 关键词: Xenopus; alcohol phosphotransferase; anesthesia; anticonvulsants; calcium flux; calcium indicator; cerebral cortex; cholinergic agents; convulsants; diffusion; enzyme induction /repression; enzyme inhibitors; enzyme substrate; fluorescence spectrometry; hydrolase; inositol phosphates; laboratory rat; lipid metabolism; neuropharmacology; phenobarbital; phosphatidylinositols; phosphodiesterases; phosphomonoesterases; phosphorylation; phosphotransferases; platelets; protein kinase C; reflex; second messengers; sedative /hypnotic; statistics /biometry; synaptosomes; thin layer chromatography; thrombin

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.

麻醉药和抗惊厥药的作用方式尚未明确 尽管最有用的抗惊厥药物苯巴比妥已经被 使用了75年。 在亲脂性药物中，巴比妥类药物是 在结构上与肌醇最接近;都含有一个六元环， 多个氢键位点（O、OH或NH基团）。 因为酶 代谢磷酸肌醇无疑识别肌醇部分， 可能怀疑巴比妥酸盐会抑制聚磷酸肌醇， 新陈代谢. 我们发现苯巴比妥是一种有效的抑制剂， 激酶，尤其是磷脂酰肌醇-4-磷酸（PIP）激酶。 它 抑制磷酸水解酶，特别是磷脂酰-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郁症有关， PIP 2合成。 这种脂质在细胞刺激下产生第二种 信使，肌醇-1，4，5-三磷酸（IP 3），这反过来又动员 细胞内钙;因此，肌醇-钙反应可能是 被打湿了 我们计划调查这种可能性和相关问题。 将用突触体和突触神经体进行实验 大脑部分--包含所有相关的酶--如果合适的话， 血小板 将测定苯巴比妥麻醉的ED 50 （蝌蚪翻正反射），以证实体外PIP-激酶抑制 与体内（临床）浓度一致。 其他巴比妥类药物（非 抗惊厥药）以及巴比妥酸盐对其他 酶（例如CDP-二酰基甘油-肌醇转移酶、肌醇 磷酸水解酶，磷脂酶A2，甘油二酯激酶）;结果 将表明对巴比妥类药物最敏感的反应是否确实是 PIP-激酶。 苯巴比妥抑制PIP激酶的机制 将被确定（是否具有竞争力？）。 PIP 2的抑制（以及 PIP）合成苯巴比妥应导致这些脂质的减少 磷脂酰肌醇（PI）增加;这将通过以下方法进行测试： 孵育后肌醇的定量分析（突触体， 突触神经体和血小板）。 预期 苯巴比妥对钙动员的抑制作用， 胆碱能刺激突触体或突触神经体 毒蕈碱激动剂和凝血酶对血小板的影响将被测量为 细胞内膜钙释放减少， 荧光分析为Ca-fura-2形成。