Lee C, Fisher S K, Agranoff B W, Hajra A K
Neuroscience Laboratory, University of Michigan, Ann Arbor, Michigan 48104-1687.
J Biol Chem. 1991 Dec 5;266(34):22837-46.
Quantitative changes in the total mass and the molecular species of 1,2-diacyl-sn-glycerol (DAG) and phosphatidic acid (PA) formed upon muscarinic receptor activation were studied in cultured human SK-N-SH neuroblastoma cells. DAG was isolated from the total lipid extracts of carbachol (CCh)-stimulated and unstimulated cells and after benzoylation, was subjected to reverse phase high performance liquid chromatography to separate the component species. The molecular species of DAG were identified by analyzing the fatty acid composition of each separated fraction by gas chromatography, and their total and individual masses were quantified from the known amount of an internal standard, 1,2-distearoyl-sn-glycerol, added during the extraction of the lipid. Relatively high basal levels of DAG (1.5 nmol/mg protein) are present in these cells, and addition of CCh elicited a 50-60% increase in the total amounts of DAG within 5 min. The increase was biphasic: an initial major peak at 5 min was followed by a sustained increase that persisted for at least 30 min. An increase in DAG was elicited by both full and partial muscarinic agonists and was blocked by atropine. The presence of extracellular Ca2+ was necessary for muscarinic receptor-activated formation of DAG. To determine the source of the DAG, the molecular species of the major phospholipids present in SK-N-SH cells were also analyzed. The phospholipids were first enzymatically hydrolyzed to DAGs which were then analyzed as described above. A number of unusual fatty acids, the major one being 20:3 (n-9), were present in these lipids especially in the phosphoinositides and also in the DAG formed after CCh stimulation. Within 5 s of CCh stimulation there were transient increases in the DAG species representative of phosphoinositides. By 5 min the newly formed molecular species of DAG resembled a mixture of phosphoinositides and phosphatidylcholine (PC). Quantitative comparison of the molecular species compositions of phosphoinositides, PC, and newly formed DAGs indicated that at time periods up to 10 min, approximately 30% of the DAG originated from the phosphoinositides and the rest from PC. At longer intervals (greater than 20 min), most (85%) of DAGs originated from PC. Activation of muscarinic receptors in SK-N-SH cells also elicited an increase in PA (200% in 5 min). A quantitative molecular species analysis, using 1,2-distearoyl-sn-glycerol-3-P as internal standard, was performed by enzymatic (alkaline phosphatase) hydrolysis of PA to DAG and subsequent analysis.(ABSTRACT TRUNCATED AT 400 WORDS)
在培养的人SK-N-SH神经母细胞瘤细胞中，研究了毒蕈碱受体激活后形成的1,2-二酰基-sn-甘油（DAG）和磷脂酸（PA）的总量及分子种类的定量变化。从卡巴胆碱（CCh）刺激和未刺激的细胞的总脂质提取物中分离出DAG，经苯甲酰化后，进行反相高效液相色谱以分离各组分。通过气相色谱分析每个分离组分的脂肪酸组成来鉴定DAG的分子种类，并根据脂质提取过程中添加的内标1,2-二硬脂酰-sn-甘油的已知量对其总量和单个质量进行定量。这些细胞中存在相对较高的基础水平的DAG（1.5 nmol/mg蛋白质），添加CCh后5分钟内DAG总量增加了50 - 60%。这种增加是双相的：5分钟时出现一个初始主峰，随后持续增加至少30分钟。完全和部分毒蕈碱激动剂均可引起DAG增加，并被阿托品阻断。毒蕈碱受体激活形成DAG需要细胞外Ca2+的存在。为了确定DAG的来源，还分析了SK-N-SH细胞中主要磷脂的分子种类。首先将磷脂酶解为DAG，然后按上述方法进行分析。这些脂质中存在许多不寻常的脂肪酸，主要的一种是20:3（n-9），尤其在磷酸肌醇以及CCh刺激后形成的DAG中。CCh刺激后5秒内，代表磷酸肌醇的DAG种类出现短暂增加。到5分钟时，新形成的DAG分子种类类似于磷酸肌醇和磷脂酰胆碱（PC）的混合物。磷酸肌醇、PC和新形成的DAG的分子种类组成的定量比较表明，在长达10分钟的时间段内，约30%的DAG源自磷酸肌醇，其余源自PC。在更长的时间间隔（大于20分钟），大多数（85%）的DAG源自PC。SK-N-SH细胞中毒蕈碱受体的激活也引起PA增加（5分钟内增加200%）。以1,2-二硬脂酰-sn-甘油-3-P为内标，通过将PA酶解（碱性磷酸酶）为DAG并随后进行分析来进行定量分子种类分析。（摘要截断于400字）
