Study on mechanism of phospholipae A_2 activation.

磷脂A_2激活机制研究。

• 批准号: 63580159
• 负责人: SUGATANI Junko
• 金额: $ 1.22万
• 依托单位: Kansai Medical University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1988
• 资助国家: 日本
• 起止时间: 1988 至 1989
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-63580159/
• 关键词: phospholipase A_2; phospholipase A_2 inhibitory protein; ionophore A23187; platelet activation; arachidonic acid release; platelet-activating factor; gastric PAF; scintillation proximity radioimmunoassay; GC@MS; シンチレ-ション・プロキシミティ-・ラジオイムノアッセイ(SPRIA

(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

(1)血小板磷脂酶A_2抑制蛋白。1-酰基-2-[^4C<14>]花生四烯酰GPC在10 mM Ca^2+存在下不被牛血小板裂解物（冻融血小板）（作为酶）水解。在[^3H]花生四烯酸标记的血小板膜上加入10 mM Ca^&lt;2+&gt;可使[^3H]花生四烯酸释放，但即使在10 mM Ca^&lt;2+&gt;存在下，牛血小板裂解物也能抑制膜结合磷脂酶A_2的活性。本研究证实了牛血小板中存在血小板磷脂酶A_2抑制蛋白，该蛋白不同于脂皮质素。(2)在存在和不存在细胞外Ca^&lt;2+&gt;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激活的机制，我们用[^ P]磷酸和[^3H]花生四烯酸双标记的洗涤过的兔血小板<32>，在有和无细胞外Ca^&lt;2+&gt;的条件下，研究了A23187诱导的血小板激活。在没有 ...更多信息 细胞外Ca^&lt;2+&gt;，A23187增加细胞内Ca^&lt;2+&gt;的量，但低于细胞外Ca^&lt;2+&gt;存在时。TPA诱导40 kDa和20 kDa蛋白磷酸化，而与细胞外Ca^&lt;2+&gt;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^&lt;2+&gt;存在和不存在的情况下都增加，但在细胞外Ca^&lt;2+&gt;不存在的情况下，肌醇磷脂代谢不受A23187的影响。因此，我们阐明了在细胞外Ca^&lt;2+&gt;存在和不存在的情况下A23187诱导血小板活化的不同机制。(3)血小板活化因子在正常大鼠胃中的存在及水浸应激对其含量的影响为了阐明PAF生物合成与磷脂酶A_2激活有关的机制，我们在胃肠道中检测到血小板活化物质，并通过GC/MS分析证实。在正常大鼠胃中，PAF在胃窦粘膜中表达较高。PAF各分子种类的百分比组成为16：OPAF（34%）和18：OPAF（66%）。水浸应激不仅影响腺胃中PAF的含量，还影响其分子异质性。应激7 h后，随着损伤程度加重，18：OPAF含量明显增加（4倍）。这些变化可能与胃溃疡的致病性有关。(4)一种新的血小板活化因子闪烁近接放射免疫分析方法的建立。SPRIA测定系统适用于定量0.03至2 pmol的16：OPAF。与1-烷基-2-乙酰基GPC的交叉反应性高，但与PAF类似物和PAF拮抗剂的交叉反应性非常低。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

项目成果

Junko Sugatani: "Development of a novel scintillation proximity radioimmunoassay for platelet-activating factor measurement:comparison with bioassay and GC/ML techniques" Life Sciences. (1990)

Junko Sugatani：“开发一种用于血小板活化因子测量的新型闪烁邻近放射免疫测定法：与生物测定法和 GC/ML 技术的比较”生命科学。

Junko Sugatani: "Occurrence of platelet-activating factor (PAF) in normal rat stomach and alteration of PAF level by water immersion stress" FASEB J.3. 65-70 (1989)

Junko Sugatani：“正常大鼠胃中血小板激活因子（PAF）的出现以及水浸应激引起的 PAF 水平的改变”FASEB J.3。

菅谷純子: 臨床科学. 24. 1220-1228 (1988)

菅谷淳子：临床科学。24。1220-1228（1988）

Kazuyo Fujimura: "Serum Platelet-Activating Factor Acetylhydrolase Activity in Rats with Gastric Ulcers induced by Water-immersion Stress." Scand. J. Gastroenterol., 24(suppl 162), 59-62 (1989).

Kazuyo Fujimura：“水浸应激诱发胃溃疡大鼠的血清血小板激活因子乙酰水解酶活性。”

Kazuyo Fujimura: Scand.J.Gastroenterol.(Supplement). (1989)

Kazuyo Fujimura：Scand.J.Gastroenterol.（补充）。