Liberation of [3H]arachidonic acid and changes in cytosolic free calcium in fura-2-loaded human platelets stimulated by ionomycin and collagen.
Liberation of [3H]arachidonic acid and changes in cytosolic free calcium in fura-2-loaded human platelets stimulated by ionomycin and collagen.
复制标题
离子霉素和胶原蛋白刺激的装载 fura-2 的人血小板中 [3H] 花生四烯酸的释放和胞质游离钙的变化。
DOI：
10.1042/bj2350869
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发表时间：
1986
期刊：
影响因子：
--
通讯作者：
R. Irvine
中科院分区：
文献类型：
--
作者：
W. K. Pollock;T. Rink;R. Irvine
研究了负载荧光 Ca2+ 指示剂染料 fura-2 并用 [3H]花生四烯酸标记的血小板中的胞质 Ca2+ 水平和花生四烯酸释放。 Fura-2 优先于 quin2 使用，因为后者会干扰磷脂的 [3H]花生四烯酸标记。从大约 100 nM 的静息游离 Ca2+ 水平开始，离子霉素 (10-200 nM) 会引起胞质 Ca2+ 瞬时、浓度依赖性增加，仅在 Ca2+ 水平大于 1 µM 时才会导致 [3H]花生四烯酸释放（比对照高出 4 倍）。添加胶原蛋白 (10 微克/毫升) 引起 Ca2+ 升高高达 461 +/- 133 nM。 Ca2+的这些变化伴随着[3H]花生四烯酸增加2-4倍，同时[3H]磷脂酰胆碱消耗17+/-4%，[3H]磷脂酰肌醇消耗41+/-7%。吲哚美辛 (10 microM) 将胶原蛋白引起的 Ca2+ 升高降低至 115 +/- 18 nM，但没有显着抑制 [3H]花生四烯酸的 2-4 倍增加。在吲哚美辛存在下，胶原蛋白的[3H]磷脂酰胆碱和[3H]磷脂酰肌醇分别降低9+/-7%和10+/-6%。在用[32P]Pi预标记的细胞中形成[32P]磷脂表明在存在或不存在吲哚美辛的情况下胶原蛋白对磷酸肌醇周转的刺激。由于吲哚美辛的存在，这种磷脂酸盐的形成减少了（75%）。在吲哚美辛存在的情况下，单独的佛波醇肉豆蔻酸酯乙酸酯 (20 nM) 或与离子霉素 (30 nM) 组合，尽管显着刺激了聚集，但未能刺激花生四烯酸的释放。这些结果表明，离子霉素需要 microM 范围内的 Ca2+ 才能释放花生四烯酸，而胶原蛋白（尤其是在吲哚美辛存在下）则需要基础 Ca2+ 水平。胶原蛋白调节花生四烯酸释放的机制尚不清楚，但似乎不涉及蛋白激酶 C 的激活或胞质游离 Ca2+ 的升高。
Cytosolic Ca2+ levels and arachidonate liberation were investigated in platelets loaded with the fluorescent Ca2+ indicator dye fura-2, and labelled with [3H]arachidonate. Fura-2 was used in preference to quin2 because the latter interfered with [3H]arachidonate labelling of phospholipids. From a resting free Ca2+ level of around 100 nM, ionomycin (10-200 nM) evoked an instantaneous, concentration-dependent increase in cytosolic Ca2+ that only resulted in [3H]arachidonate liberation (up to 4-fold over control) at Ca2+ levels greater than 1 microM. Addition of collagen (10 micrograms/ml) evoked an elevation in Ca2+ up to 461 +/- 133 nM. These changes in Ca2+ were accompanied by a 2-4-fold elevation in [3H]arachidonate with depletion of [3H]phosphatidylcholine by 17 +/- 4% and [3H]phosphatidylinositol by 41 +/- 7%. Indomethacin (10 microM) reduced the elevation in Ca2+ by collagen to 115 +/- 18 nM but did not significantly inhibit the 2-4-fold increase in [3H]arachidonate. [3H]Phosphatidylcholine and [3H]phosphatidylinositol were decreased by 9 +/- 7% and 10 +/- 6%, respectively, with collagen in the presence of indomethacin. Stimulation of phosphoinositide turnover by collagen in the presence and absence of indomethacin was indicated by [32P]phosphatidate formation in cells prelabelled with [32P]Pi. This phosphatidate formation was decreased (75%) by the presence of indomethacin. In the presence of indomethacin, phorbol myristate acetate (20 nM) alone or in combination with ionomycin (30 nM) failed to stimulate arachidonate liberation despite a marked stimulation of aggregation. These results indicate that, whereas ionomycin requires Ca2+ in the microM range for arachidonate liberation, collagen, notably in the presence of indomethacin, does so at basal Ca2+ levels. The mechanisms underlying the regulation of arachidonate release by collagen are not clear, but do not appear to involve activation of protein kinase C, or an elevation of cytosolic free Ca2+.