Martin T W, Michaelis K C
Department of Pathology, St. Louis University School of Medicine, MO 63104.
Biochim Biophys Acta. 1990 Sep 1;1054(2):159-68. doi: 10.1016/0167-4889(90)90237-8.
The feasibility of using saponin as a permeabilization agent to study the effect of free Ca2+ concentration ([Ca2+]f) on prostaglandin I2 (PGI2) synthesis and mobilization of arachidonic acid from membrane phospholipids was investigated in cultured bovine pulmonary artery endothelial cells (BPAEC). Treatment of BPAEC with 20 micrograms/ml saponin caused selective permeabilization of the plasma membrane as determined by measurements of the release of lactate dehydrogenase and beta-hexosaminidase. In cells prelabeled with [3H]arachidonic acid for 22 h, permeabilization with 20 micrograms/ml saponin induced PGI2 synthesis and release of [3H]arachidonic acid from membrane phospholipids. These effects were dependent upon [Ca2+]f in the range 72 nM to 5 microM. Release of [3H]arachidonic acid from phospholipid classes was determined in suspensions of BPAEC prelabeled with [3H]arachidonic acid and permeabilized with 20 micrograms/ml saponin. At [Ca2+]f optimal for PGI2 synthesis, 16.2% of the total incorporated [3H]arachidonic acid was released from phosphatidylinositol (3.4%), phosphatidylethanolamine (3.5%) and phosphatidylcholine (9.3%). The time course and dependence upon [Ca2+]f of [3H]arachidonic acid release from phospholipids correlated with PGI2 synthesis. The amount of PGI2 synthesized in permeabilized BPAEC was similar to that in cell cultures treated with the calcium ionophore A23187. In comparison, however, PGI2 synthesis induced by A23187 was associated with less release of [3H]arachidonic acid from membrane phospholipids, e.g., 2.3% versus 16.2%. The greater loss of [3H]arachidonic acid from phospholipids in saponin-permeabilized BPAEC was most likely due to the loss of cell integrity and/or nonspecific effects of the detergent on phospholipases. Despite these limitations, the Ca2+ dependence observed for PGI2 synthesis and [3H]arachidonic acid mobilization suggest that saponin-permeabilization may provide a useful system for studies of the intracellular events triggered by the rise in intracellular Ca2+ which culminate in PGI2 synthesis.
在培养的牛肺动脉内皮细胞（BPAEC）中，研究了使用皂苷作为通透剂来研究游离钙离子浓度（[Ca2+]f）对前列腺素I2（PGI2）合成以及花生四烯酸从膜磷脂中释放的影响的可行性。用20微克/毫升皂苷处理BPAEC，通过测量乳酸脱氢酶和β-己糖胺酶的释放来确定质膜的选择性通透。在用[3H]花生四烯酸预标记22小时的细胞中，用20微克/毫升皂苷通透可诱导PGI2合成以及[3H]花生四烯酸从膜磷脂中释放。这些效应在72 nM至5 microM的[Ca2+]f范围内依赖于[Ca2+]f。在用[3H]花生四烯酸预标记并用20微克/毫升皂苷通透的BPAEC悬浮液中，测定了磷脂类中[3H]花生四烯酸的释放。在对PGI2合成最适宜的[Ca2+]f下，总掺入的[3H]花生四烯酸中有16.2%从磷脂酰肌醇（3.4%）、磷脂酰乙醇胺（3.5%）和磷脂酰胆碱（9.3%）中释放出来。[3H]花生四烯酸从磷脂中释放的时间进程和对[Ca2+]f的依赖性与PGI2合成相关。通透的BPAEC中合成的PGI2量与用钙离子载体A23187处理的细胞培养物中的量相似。然而，相比之下，A23187诱导的PGI2合成与较少的[3H]花生四烯酸从膜磷脂中释放相关，例如，分别为2.3%和16.2%。皂苷通透的BPAEC中磷脂中[3H]花生四烯酸的更大损失很可能是由于细胞完整性的丧失和/或去污剂对磷脂酶的非特异性作用。尽管有这些局限性，但观察到的PGI2合成和[3H]花生四烯酸释放对Ca2+的依赖性表明，皂苷通透可能为研究由细胞内Ca2+升高引发并最终导致PGI2合成的细胞内事件提供一个有用的系统。
