Inositol 1,4,5-trisphosphate-induced release of sequestered Ca2+ from highly purified human platelet intracellular membranes.
Inositol 1,4,5-trisphosphate-induced release of sequestered Ca2+ from highly purified human platelet intracellular membranes.
复制标题
肌醇 1,4,5-三磷酸诱导高度纯化的人血小板细胞内膜中隔离的 Ca2 的释放。
作者：
K. Authi;N. Crawford
越来越多的证据表明，当不同的激素、神经递质、生长因子和其他刺激物作用于细胞表面时，细胞内产生的肌醇1，4，5-三磷酸[INS(1，4，5)P3]在提高胞浆[Ca~(2+)]方面发挥了作用。刺激剂-受体相互作用引起的[Ca~(2+)]升高伴随着肌醇磷脂的快速水解。磷脂酰肌醇4，5-二磷酸分解产生的一种产物Ins(1，4，5)P3被证明可以促进多种细胞中非线粒体存储的钙释放。尽管血小板胞内膜与胞浆[Ca~(2+)]的控制有关，并且我们以前描述了与之相关的钙隔离机制，但我们还不知道在刺激-受体相互作用后，这个钙储藏是如何被动员起来的。采用自由流动电泳法分离纯化人血小板胞内膜。它们表现出内质网标记NADH：细胞色素C还原酶的高度浓缩和独占定位，并通过依赖于ATP的过程隔离钙离子，在10-12分钟内达到稳定值。与钙离子的饱和发生在大约10-30微米的外部钙离子。当将Ins(1，4，5)P3加入到45Ca负载的囊泡中时，会发生钙离子的快速释放。35%在15-30秒内)。释放量取决于外部[Ca~(2+)]，在0.3-0.8微米范围内最大，当外部[Ca~(2+)]大于1微米时，释放量较低。释放后，钙离子迅速重新吸收，并在1分钟内恢复到原来的稳态值。半最大释药发生在大约。0.25微米/平方英寸(1，4，5)P3。离子载体A23187和花生四烯酸没有观察到这种释放和再摄取的模式，这两种离子载体都不可逆转地释放钙离子。1，4-二磷酸肌醇不能有效地释放细胞内钙离子。这些结果支持INS(1，4，5)P3作为一种特定的细胞内介质的作用，将作用于血小板表面的兴奋性激动剂的作用转化为已知的在血小板激活过程中发生的代谢、机械力化学和其他功能事件。
Evidence has accumulated in support of a role for intracellularly generated inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] in raising cytosol [Ca2+] when various hormones, neurotransmitters, growth factors and other stimulants act on cell surfaces. The increase in [Ca2+] that follows stimulant-receptor interaction is accompanied by rapid hydrolysis of phosphoinositides. One product, Ins(1,4,5)P3, arising from the breakdown of phosphatidylinositol 4,5-bisphosphate was shown to promote the release of Ca2+ from non-mitochondrial stores in a variety of cells. Although platelet intracellular membranes have been implicated in the control of cytosol [Ca2+] and we previously characterized a Ca2+-sequestering mechanism associated with them, we have as yet no knowledge of how this Ca2+ store is mobilized after a stimulus-receptor interaction at the platelet surface. Using free-flow electrophoresis, we isolated and purified human platelet intracellular membranes. They show high enrichment and exclusive localization of the endoplasmic-reticulum marker NADH:cytochrome c reductase, and they sequester Ca2+ by an ATP-dependent process, reaching steady-state values in 10-12 min. Saturation with Ca2+ occurs at around 10-30 microM external Ca2+. When Ins(1,4,5)P3 is added to the 45Ca-loaded vesicles, a rapid release of Ca2+ occurs (approx. 35% in 15-30s). The magnitude of the release depends upon external [Ca2+], being maximum in the range 0.3-0.8 microM and low at external [Ca2+] greater than 1 microM. After release there is a rapid re-uptake of Ca2+, with restoration of the former steady-state values within 1 min. Half-maximal release occurs at approx. 0.25 microM-Ins(1,4,5)P3. This release and re-uptake pattern is not observed with ionophore A23187 or arachidonic acid, both of which liberate Ca2+ irreversibly. Inositol 1,4-bisphosphate was ineffective in releasing Ca2+ from these intracellular membranes. The results support the role of Ins(1,4,5)P3 as a specific intracellular mediator, transducing the action of excitatory agonists acting on the platelet surface into metabolic, mechanochemical and other functional events, known to occur during platelet activation.