Siddiqui R A, English D
Bone Marrow Transplantation Laboratory, Methodist Hospital of Indiana, Indianapolis 46202, USA.
Cell Signal. 1996 Aug;8(5):349-54. doi: 10.1016/0898-6568(96)00072-1.
Neutrophils provide an attractive model with which to characterize cellular effects of phosphatidic acid (PA) independently of effects triggered by lysophosphatidic acid (LPA), since these cells lack LPA receptors. We developed a novel method to quantitate binding of PA to neutrophils and neutrophil plasma membranes. Intact cells or subcellular fractions were immobilized on nitrocellulose membranes and incubated in a bath containing [32P]PA under various conditions, followed by rapid rinsing with a mild detergent (0.05% Tween 20) to minimize non-specific binding. With this method, dioctanoyl PA specifically ligated plasma-membrane binding sitesin a time- and temperature-dependent manner. Specific binding of (DiC8-PA was markedly potentiated by pre-treatment of cells or membranes with ecto-phosphatidic acid phosphohydrolase (PAPase) inhibitor dimethylsphingosine (DMS). Optimum binding of DiC8-PA to PAPase-inhibited cells occurred within 10 min at room temperature, increased linearly with the cell concentration used, and was not significantly affected by alteration of pH over the range of 5.5-8.5. Of several phosphatidic acid species examined, optimal specific binding to immobilized neutrophils was observed with DiC8-PA and dicapryl (DiC10) PA; dicaproyl (DiC6) PA bound weakly, whereas dimyristoyl (DiC14) PA and dipalmitoyl (DiC16) PA did not bind. Dioleoyl (DiC18:1) PA bound to immobilized cells, but this binding was essentially non-specific, in that it was not reduced by excess non-radioactive ligand. Various LPA preparations, including [32P] lyso-octanoyl (C8) PA and [32P] lyso-oleoyl (C18:1) PA, showed very low specific binding to neutrophils in this system. Specific binding of DiC8-PA and DiC10-PA preparations correlated well with the ability of each to effect the mobilization of intracellular Ca2+ in neutrophils. Ca2+ mobilization was characterized by two distinct phases; a rapid rise that was inhibited in the presence of the tyrosine kinase inhibitor herbimycin-A, followed by a sustained increase that was eliminated in the presence of EGTA. The results are consistent with the hypothesis that neutrophils have specific binding sites for phosphatidic acid, the occupation of which leads to rapid mobilization of intracellular free Ca2+ via activation of tyrosine kinases. The methods described in this report may facilitate the identification and characterization of functional phosphatidic acid receptors on neutrophil plasma membranes.
由于中性粒细胞缺乏溶血磷脂酸（LPA）受体，因此它们为独立于LPA触发效应来表征磷脂酸（PA）的细胞效应提供了一个有吸引力的模型。我们开发了一种新方法来定量PA与中性粒细胞及中性粒细胞质膜的结合。完整细胞或亚细胞组分固定在硝酸纤维素膜上，并在含有[32P]PA的浴中在各种条件下孵育，然后用温和的去污剂（0.05%吐温20）快速冲洗以尽量减少非特异性结合。用这种方法，二辛酰PA以时间和温度依赖性方式特异性连接质膜结合位点。用胞外磷脂酸磷酸水解酶（PAPase）抑制剂二甲基鞘氨醇（DMS）预处理细胞或膜可显著增强（DiC8-PA的特异性结合。DiC8-PA与PAPase抑制细胞的最佳结合在室温下10分钟内发生，随所用细胞浓度呈线性增加，并且在5.5-8.5的pH范围内改变pH对其没有显著影响。在所检测的几种磷脂酸种类中，观察到DiC8-PA和二癸酰（DiC10）PA与固定化中性粒细胞的最佳特异性结合；二己酰（DiC6）PA结合较弱，而二肉豆蔻酰（DiC14）PA和二棕榈酰（DiC16）PA不结合。二油酰（DiC18:1）PA与固定化细胞结合，但这种结合基本上是非特异性的，因为它不会被过量的非放射性配体减少。各种LPA制剂，包括[32P]溶血辛酰（C8）PA和[32P]溶血油酰（C18:1）PA，在该系统中与中性粒细胞的特异性结合非常低。DiC8-PA和DiC10-PA制剂的特异性结合与它们各自影响中性粒细胞内Ca2+动员的能力密切相关。Ca2+动员具有两个不同阶段；一个快速上升阶段在酪氨酸激酶抑制剂赫曲霉素-A存在时受到抑制，随后是一个持续增加阶段在EGTA存在时被消除。这些结果与中性粒细胞具有磷脂酸特异性结合位点的假设一致，其占据通过酪氨酸激酶的激活导致细胞内游离Ca2+的快速动员。本报告中描述的方法可能有助于鉴定和表征中性粒细胞质膜上功能性磷脂酸受体。
