Study on mechanism of phospholipae A_2 activation.

血小板磷脂A_2抑制蛋白。在10 mM Ca^<2+>存在下，1-酰基-2-[^<14>]花生四烯酰基GPC不能被牛肉血小板裂解液（冻融血小板）（作为酶）水解。在[^3H]花生四烯酸标记的血小板膜中添加10 mM Ca^<2+>使[^3H]-花生四烯酸释放，但即使存在10 mM Ca^<2+>，膜结合磷脂酶A_2活性也被牛肉血小板裂解液抑制。结果表明，牛肉血小板中存在不同于脂皮素的血小板磷脂酶A_2抑制蛋白。(2)在细胞外Ca^<2+>存在和不存在的情况下，离子载体a23187诱导血小板活化的不同机制。为了研究血小板磷脂酶A_2活化的机制，我们用[^<32>P]磷酸和[^3H]花生四烯酸双标记的洗涤兔血小板，在细胞外Ca^<2+>存在和不存在的情况下，研究了a23187诱导的血小板活化。在没有Ca^<2+>的情况下，A23187增加了细胞内Ca^<2+>的数量，但低于存在Ca^<2+>的情况。TPA诱导40 kDa和20 kDa蛋白磷酸化，与细胞外Ca^<2+>无关。在tpa处理的血小板中，A23187诱导的花生四烯酸释放在细胞外Ca^<2+>存在和不存在的情况下都增加，但在细胞外Ca^<2+>不存在的情况下，A23187不影响肌醇磷脂代谢。因此，我们阐明了a23187在细胞外Ca^<2+>存在和不存在时诱导血小板活化的不同机制。(3)正常大鼠胃中血小板活化因子（PAF）的发生及水浸应激对PAF水平的影响。为了阐明PAF生物合成与磷脂酶A_2活化相关的机制，我们在胃肠道区域检测了血小板活化物质，并通过GC/MS分析证实了这一点。正常大鼠胃胃窦黏膜PAF水平较高。PAF各分子种的组成比例分别为16:OPAF（34%）和18:OPAF（66%）。水浸胁迫不仅影响腺胃中PAF的含量，而且影响其分子异质性。应激7小时后，随着严重病变，体中18:OPAF的数量明显增加（4倍），这些变化可能与胃溃疡的致病性有关。(4)血小板活化因子测量的新型闪烁接近放射免疫分析法的发展。SPRIA分析系统适用于0.03 ~ 2 pmol的16:OPAF的定量。与1-烷基-2-乙酰基GPC的交叉反应性较高，而与PAF类似物和PAF拮抗剂的交叉反应性很低。SPRIA的特异性高于生物测定法，与血小板受体的特异性有较大差异。少

(1) Platelet phospholipae A_2 inhibitory protein. 1-Acyl-2-[^<14>4C]arachidonyl GPC was not hydrolyzed by beef platelet lysate (freeze-thawing platelet)(as an enzyme) in the presence of 10 mM Ca^<2+>. The addition of 10 mM Ca^<2+> to [^3H]arachidonic acid-labelled platelet membrane made [^3H]-arachidonic acid released, but the membrane-bound phospholipase A_2 activity was suppressed by beef platelet lysate even in the presence of 10 mM Ca^<2+>. We elucidated the presence of platelet phospholipase A_2 inhibitory protein in beef platelet, which was distinct from lipocortin.(2) Distinct mechanism of ionophore A23187-induced platelet activation in the presence and absence of extracellular Ca^<2+>. In order to study the mechanism of platelet phospholipase A_2 activation, we investigated A23187-induced platelet activation using washed rabbit platelets double-labelled with [^<32>P]phosphoric acid and [^3H]arachidonic acid in the presence and absence of extracellular Ca^<2+>. In the absence of … More extracellular Ca^<2+>, A23187 increased the amount of intracellular Ca^<2+> which was lower than that in the presence of extracellular Ca^<2+>. TPA induced 40 kDa and 20 kDa protein phosphorylation, regardless of extracellular Ca^<2+>. In TPA-treated platelets, A23187-induced arachidonic acid release increased in both the presence and absence of extracellular Ca^<2+>, but inositol phospholipid metabolism was not affected by A23187 in the absence of extracellular Ca^<2+>. We elucidated thus distinct mechanism of A23187-induced platelet activation in the presence and absence of extracellular Ca^<2+>.(3) Occurrence of platelet-activating factor (PAF) in normal rat stomach and alteration of PA level by water-immersion stress. In order to elucidate the mechanism of PAF biosynthesis connected with phospholipase A_2 activation, we detected platelet-activating substance in gastrointestinal areas, which was confirmed by GC/MS analysis. In the normal rat stomach, the level of PAF was high in the antral mucosa. The percentage composition of each molecular species of PAF was 16:OPAF (34%) and 18:OPAF (66%). Application of water-immersion stress affected not only the amounts of PAF but also their molecular heterogeneity in the glandular stomach. The amount of 18:OPAF increased markedly (to 4-fold) in the corpus along with severe lesions after stress for 7 h. These changes might be associated with the pathogenicity of gastric ulcer.(4) Development of a novel scintillation proximity radioimmunoassay for platelet-activating factor measurement. The SPRIA assay system was suitable for the quantitation of 0.03 to 2 pmol of 16:OPAF. The cross-reactivity was high with 1- alkyl-2-acetyl GPC but was very low with PAF analogs and PAF antagonists. The specificity of SPRIA was higher than that of bioassay, quite different from that of the platelet receptor. Less