Brammer M J, Weaver K
Department of Neuroscience, Institute of Psychiatry, London, U.K.
Biochem Pharmacol. 1990 Oct 15;40(8):1901-6. doi: 10.1016/0006-2952(90)90372-r.
The carbachol and adrenaline-mediated accumulation of inositol monophosphate isomers in rat cortical prisms has been studied using a commonly employed experimental protocol involving preincubation with myo-[2-3H]-inositol and subsequent incubation with agonists in the presence of 10 mM LiCl. Inositol phosphate isomers have been analysed by HPLC and identified by comparison of their elution characteristics with those of commercially available standards and the degradation products of authentic Ins 1,3,4-P3 and Ins 1,4,5-P3. Incubation of prelabelled cortical prisms for 1 hr with 10 mM LiCl alone gives rise to accumulation of radioactivity in two inositol monophosphate peaks which co-elute with Ins 1-P and Ins 4-P and one major bisphosphate peak which co-migrates with Ins 1,4-P2. Most of the monophosphate radioactivity is recovered in the Ins 4-P peak (Ins 1-P/Ins 4-P labelling ratio 0.68). Both carbachol and adrenaline produce dose-dependent increases in the labelling of Ins 1-P and Ins 4-P which are antagonized by atropine and prazosin respectively. However, carbachol produces a larger stimulation of accumulation of both monophosphates and also gives rise to a larger selective increase in the accumulation of Ins 1-P (Ins 1-P/4-P labelling ratio 1.40 in the presence of 1 mM carbachol, 0.98 in the presence of 1 mM adrenaline). Kinetic studies of the carbachol-stimulated increases in inositol mono- and bisphosphate labelling have revealed that, in the early period following carbachol addition (0-5 min), Ins 4-P and Ins 1,4-P2 are labelled more rapidly than Ins 1-P, whereas the reverse is true at later periods (15-60 min) of the incubations. These observations, coupled with the low levels of labelling of the major Ins 1,3,4-P3 breakdown products (Ins 1,3-P2 and Ins 3,4-P2) compared with that of Ins 1,4-P2, suggest that large-scale production of Ins 1-P is a comparatively late feature of carbachol-mediated inositol phospholipid metabolism and that, if the Ins 1-P is derived from breakdown of Ins 1,3,4,5-P4 via Ins 1,3,4-P3, the turnover of Ins 1,3-P2 + Ins 3,4-P2 must be approximately one order of magnitude greater than that of the Ins 4-P precursor, Ins 1,4-P2.
已使用一种常用的实验方案研究了卡巴胆碱和肾上腺素介导的大鼠皮质棱镜中肌醇单磷酸异构体的积累，该方案包括用肌醇-[2-³H]进行预孵育，随后在10 mM LiCl存在的情况下与激动剂一起孵育。通过高效液相色谱法（HPLC）分析肌醇磷酸异构体，并通过将其洗脱特性与市售标准品以及真实的Ins 1,3,4-P3和Ins 1,4,5-P3的降解产物的洗脱特性进行比较来鉴定。仅用10 mM LiCl将预先标记的皮质棱镜孵育1小时，会导致放射性在两个与Ins 1-P和Ins 4-P共洗脱的肌醇单磷酸峰以及一个与Ins 1,4-P2共迁移的主要双磷酸峰中积累。大部分单磷酸放射性在Ins 4-P峰中回收（Ins 1-P/Ins 4-P标记比为0.68）。卡巴胆碱和肾上腺素均使Ins 1-P和Ins 4-P的标记呈剂量依赖性增加，分别被阿托品和哌唑嗪拮抗。然而，卡巴胆碱对两种单磷酸积累的刺激更大，并且还导致Ins 1-P积累的选择性增加更大（在1 mM卡巴胆碱存在下，Ins 1-P/4-P标记比为1.40，在1 mM肾上腺素存在下为0.98）。对卡巴胆碱刺激的肌醇单磷酸和双磷酸标记增加的动力学研究表明，在添加卡巴胆碱后的早期（0-5分钟），Ins 4-P和Ins 1,4-P2的标记比Ins 1-P更快，而在孵育的后期（15-60分钟）则相反。这些观察结果，再加上主要的Ins 1,3,4-P3降解产物（Ins 1,3-P2和Ins 3,4-P2）的标记水平与Ins 1,4-P2相比很低，表明Ins 1-P的大规模产生是卡巴胆碱介导的肌醇磷脂代谢的一个相对较晚的特征，并且，如果Ins 1-P是通过Ins 1,3,4-P3从Ins 1,3,4,5-P4的分解产生的，那么Ins 1,3-P2 + Ins 3,4-P2的周转率必须比Ins 4-P前体Ins 1,4-P2的周转率大约高一个数量级。
