Thyrotropin-releasing hormone mobilizes Ca2+ from endoplasmic reticulum and mitochondria of GH3 pituitary cells: characterization of cellular Ca2+ pools by a method based on digitonin permeabilization.
Thyrotropin-releasing hormone mobilizes Ca2+ from endoplasmic reticulum and mitochondria of GH3 pituitary cells: characterization of cellular Ca2+ pools by a method based on digitonin permeabilization.
复制标题
促甲状腺激素释放激素从 GH3 垂体细胞的内质网和线粒体动员 Ca2：通过基于毛地黄皂苷透化的方法表征细胞 Ca2 池。
DOI：
10.1073/pnas.79.20.6294
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发表时间：
1982
影响因子：
11.1
通讯作者：
Martin,TF
中科院分区：
文献类型：
--
作者：
Ronning,SA;Heatley,GA;Martin,TF
用不同浓度的洋地黄素处理含45Ca~(2+)的GH3细胞，发现由不同的去污剂敏感性所定义的细胞45Ca~(2+)离散池(I和II)。胞浆和胞内细胞器的标记表明，这两个45Ca~(2+)池与细胞内两个主要的钙隔离细胞器--内质网(I)和线粒体(II)相关。对不同抑制剂的研究与这些分配是一致的。线粒体解偶联剂优先耗尽45Ca~(2+)库II，而三氟拉嗪选择性耗尽45Ca~(2+)库I。对照实验表明，细胞器45Ca~(2+)在通透过程中和通透后的原位移位可以忽略不计。采用洋地黄素通透法观察促甲状腺激素释放激素(TRH)对GH3细胞内钙池的影响。TRH能迅速耗尽内质网和线粒体可交换钙离子25-30%。加入TRH后1min，线粒体45Ca~(2+)损失量最大，随后进入恢复期；线粒体45Ca~(2+)含量在30min时恢复到对照水平。先前用线粒体解偶联剂乙酰氰化物对三氟甲氧基苯肼处理细胞可阻断TRH诱导的45Ca~(2+)从线粒体外流，而先前用会耗尽两个45Ca~(2+)池的呋喃霉素处理则可阻断TRH对这些池的任何额外作用。我们的结论是，由于激素诱导内质网和线粒体的钙离子动员，TRH迅速促进了GH3细胞可交换钙的净损失。
Treatment of 45Ca2+-loaded GH3 pituitary cells with various concentrations of digitonin revealed discrete pools (I and II) of cellular 45Ca2+ defined by differing detergent sensitivities. Markers for cytosol and intracellular organelles indicated that the two 45Ca2+ pools were correlated with the two major cellular Ca2+-sequestering organelles, endoplasmic reticulum (I) and mitochondria (II). Studies with various inhibitors were consistent with these assignments. Mitochondrial uncouplers preferentially depleted 45Ca2+ pool II while trifluoperazine selectively depleted 45Ca2+ pool I. Control experiments indicated that translocation of in situ organellar 45Ca2+ during and after permeabilization was negligible. We used the digitonin-permeabilization method to examine the effect of thyrotropin-releasing hormone (TRH) treatment on intracellular Ca2+ pools of GH3 pituitary cells. TRH was found to rapidly deplete both endoplasmic reticulum and mitochondrial exchangeable Ca2+ by 25-30%. The 45Ca2+ loss from both pools was maximal by 1 min after TRH addition and was followed by a recovery phase; mitochondrial 45Ca2+ content returned to control levels by 30 min. Previous treatment of cells with the mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxy-phenylhydrazone blocked TRH-induced 45Ca2+ efflux from mitochondria, while previous treatment with valinomycin, an agent that depleted both 45Ca2+ pools, blocked any additional effect of TRH on these pools. We conclude that TRH rapidly promotes a net loss of exchangeable Ca2+ from GH3 cells as a result of hormone-induced mobilization of Ca2+ from endoplasmic reticulum and mitochondria.