Demonstration of a late amiloride-sensitive event as a necessary step in initiation of DNA synthesis by thrombin.
Demonstration of a late amiloride-sensitive event as a necessary step in initiation of DNA synthesis by thrombin.
复制标题
证明晚期阿米洛利敏感事件是凝血酶启动 DNA 合成的必要步骤。
DOI：
10.1002/jcp.1041170220
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发表时间：
1983
影响因子：
5.6
通讯作者：
Carney,DH
中科院分区：
文献类型：
--
作者：
Stiernberg,J;LaBelle,EF;Carney,DH
阿米洛利是一种Na+内流抑制剂，已显示可抑制小鼠胚胎成纤维细胞样细胞中凝血酶引发的DNA合成。长时间暴露（24小时）于高浓度阿米洛利抑制胸苷掺入凝血酶刺激和非刺激细胞的DNA中，表明这种抑制可能对凝血酶启动的DNA合成没有特异性。荧光显微镜和荧光分光光度法显示，阿米洛利被内化与一个明显的线粒体协会，并从培养基中去除阿米洛利后，很容易从细胞中释放的内化的阿米洛利。基于这种可逆性，在凝血酶处理期间将细胞短时间暴露于阿米洛利，以确定任何阿米洛利敏感事件与DNA合成起始之间的时间关系。阿米洛利（100 μM）在12小时暴露于凝血酶期间并未阻断凝血酶启动的DNA合成或细胞分裂，但确实延迟了DNA合成的开始和胸苷掺入DNA的峰值约3小时，表明在阿米洛利存在下可能会进行早期起始事件。86 Rb+转运研究表明，在该系统中，哇巴因敏感性K+通过Na，凝血酶在早期和晚期都能刺激K-ATP酶。这种刺激在用于生长实验的相同条件下是阿米洛利敏感的，表明阿米洛利抑制该系统中凝血酶刺激的Na+转运。另外的实验表明，仅在加入凝血酶后的前8小时内将细胞暴露于阿米洛利并不抑制起始。在加入凝血酶后8-12小时，阿米洛利的存在最大程度地抑制了凝血酶刺激的DNA合成。总之，这些结果表明，阿米洛利抑制凝血酶启动的DNA合成不是通过抑制前8小时内发生的早期事件，而是通过抑制加入凝血酶后8-12小时的一些后期事件。
Amiloride, a Na+influx inhibitor, has been shown to inhibit initiation of DNA synthesis by thrombin in mouse embryo fibroblast‐like cells. Long exoosures (24 hr) to high concentrations of amiloride inhibited incorporation of thymidine into the DNA of both thrombin‐stimulated and nonstimulated cells, suggesting that this inhibition might not be specific for thrombin‐initiated DNA synthesis. Fluorescence microscopy and spectrofluorimetry showed that amiloride was internalized with an apparent mitochondrial association and that the internalized amiloride was readily released from the cells after removing amiloride from the medium. Based on this reversibility, cells were exposed to amiloride for short periods of time during thrombin treatment to determine the temporal relationship between any amiloride‐sensitive event(s) and initiation of DNA synthesis. The presence of amiloride (100 μM) during a 12‐hr exposure to thrombin did not block thrombin‐initiated DNA synthesis or cell division but did delay the onset of DNA synthesis and the peak of thymidine incorporation into DNA by approximately 3 hr, suggesting that early initiation events might proceed in the presence of amiloride.86Rb+transport studies demonstrated that in this system ouabain‐sensitive K+uptake via the Na, K‐ATPase was stimulated by thrombin during both an early and a late period. This stimulation was amiloride‐sensitive under the same conditions used for growth experiments, suggesting that amiloride was inhibiting thrombin‐stimulated Na+transport in this system. Additional experiments showed that exposing cells to amiloride only during the first 8 hr after thrombin addition did not inhibit initiation. The presence of amiloride from 8–12 hr after thrombin addition maximally inhibited thrombin‐stimulated DNA synthesis. Together these results demonstrate that amiloride inhibits thrombin‐initiated DNA synthesis not by inhibiting an early event occurring during the first 8 hr, but rather by inhibiting some later event 8–12 hr after thrombin addition.