Allan C J, Exton J H
Howard Hughes Medical Institute, Vanderbilt University School of Medicine, Nashville, TN 37232.
Biochem J. 1993 Mar 15;290 ( Pt 3)(Pt 3):865-72. doi: 10.1042/bj2900865.
The hydrolysis of inositol phospholipids induced by vasopressin in hepatocytes during 60 min was quantified chemically. There was a large release of myo-inositol which was abolished by Li+, indicating that it was derived from inositol phosphates and not from phospholipase D action on PtdIns. There was also a large release of inositol phosphates which was increased approx. 2-fold by Li+ at 30 min, but then remained constant, suggesting that inositol phospholipid breakdown declined substantially beyond this time. In cells prelabelled with myo-[3H]inositol and treated with Li+, [3H]PtdIns(4,5)P2 decreased maximally (50%) at 15 s and then recovered to a level at 5 min that was maintained at 25% below control for 40 min. [3H]PtdIns4P and [3H]PtdIns showed slower decreases to approx. 30% below control at 15 min, but with no further changes. Labelled Ins(1,4,5)P3 and Ins(1,3,4)P3 showed 2-4-fold increases within 30 s and then declined to values that were maintained at a constant level above the control, except for [3H]Ins(1,3,4)P3, which showed a second increase. [3H]Ins(1,4)P2 showed a very large increase over 10 min, whereas [3H]Ins4P and [3H]Ins1P showed little change before 6 and 15 min respectively. The total [3H]inositol phosphates showed little further increase after 20 min. These data are consistent with a rapid, but not sustained, hydrolysis of PtdIns-(4,5)P2, but not of PtdIns, by phospholipase C, but do not exclude PtdIns4P as a substrate. Phosphatidate was rapidly increased by vasopressin, whereas diacylglycerol was increased after a 1-2 min lag. Both were maintained at levels 2-3-fold above control for 60 min. The vasopressin-induced increase in inositol phosphates plus myo-inositol (approx. 120 nmol/100 mg) was greater than the increase in diacylglycerol plus phosphatidate (approx. 60 nmol/100 mg) between 10 and 40 min. This indicates that there was substantial further metabolism of these lipids. Addition of 75 mM ethanol resulted in rapid production of phosphatidylethanol in response to vasopressin and a 35% reduction in phosphatidate, but no decrease in diacylglycerol. In summary, the results indicate that inositol phospholipid hydrolysis by phospholipase C can account for most of the diacylglycerol and phosphatidate that accumulate during 60 min of vasopressin action, but that these phospholipids are probably not the major source of the phosphatidate that is formed during the first 2 min by phospholipase D, or of the diacylglycerol and phosphatidate that are formed beyond 30 min.
对血管加压素作用60分钟期间肝细胞中肌醇磷脂的水解进行了化学定量分析。有大量的肌醇释放出来，Li⁺可消除这种释放，这表明它来源于肌醇磷酸酯，而非磷脂酶D对磷脂酰肌醇（PtdIns）的作用。同时也有大量的肌醇磷酸酯释放出来，在30分钟时Li⁺使其释放量增加了约2倍，但随后保持恒定，这表明在此时间之后肌醇磷脂的分解大幅下降。在用肌醇-[³H]肌醇预标记并经Li⁺处理的细胞中，[³H]磷脂酰肌醇-4,5-二磷酸（PtdIns(4,5)P2）在15秒时最大程度下降（50%），然后在5分钟时恢复到一个水平，并在40分钟内维持在比对照低25%的水平。[³H]磷脂酰肌醇-4-磷酸（PtdIns4P）和[³H]磷脂酰肌醇（PtdIns）在15分钟时下降较慢，降至比对照低约30%，但之后没有进一步变化。标记的肌醇-1,4,5-三磷酸（Ins(1,4,5)P3）和肌醇-1,3,4-三磷酸（Ins(1,3,4)P3）在30秒内增加了2 - 4倍，然后下降至维持在对照水平之上的恒定值，但[³H]肌醇-1,3,4-三磷酸（Ins(1,3,4)P3）除外，它出现了第二次增加。[³H]肌醇-1,4-二磷酸（Ins(1,4)P2）在10分钟内大幅增加，而[³H]肌醇-4-磷酸（Ins4P）和[³H]肌醇-1-磷酸（Ins1P）分别在6分钟和15分钟之前变化不大。总的[³H]肌醇磷酸酯在20分钟后几乎没有进一步增加。这些数据与磷脂酶C对PtdIns-(4,5)P2的快速但非持续性水解一致，但不排除PtdIns4P作为底物。血管加压素可使磷脂酸迅速增加，而二酰甘油在延迟1 - 2分钟后增加。两者在60分钟内都维持在比对照高2 - 3倍的水平。在10到40分钟之间，血管加压素诱导的肌醇磷酸酯加肌醇的增加量（约120 nmol/100 mg）大于二酰甘油加磷脂酸的增加量（约60 nmol/100 mg）。这表明这些脂质有大量的进一步代谢。加入75 mM乙醇会导致血管加压素作用下迅速产生磷脂酰乙醇，磷脂酸减少35%，但二酰甘油没有减少。总之，结果表明磷脂酶C对肌醇磷脂的水解可解释血管加压素作用60分钟期间积累的大部分二酰甘油和磷脂酸，但这些磷脂可能不是磷脂酶D在最初2分钟形成的磷脂酸的主要来源，也不是30分钟后形成的二酰甘油和磷脂酸的主要来源。
