Hawel L, Tjandrawinata R R, Byus C V
Department of Biochemistry, University of California, Riverside 92521.
Biochim Biophys Acta. 1994 May 26;1222(1):15-26. doi: 10.1016/0167-4889(94)90020-5.
Cultured Reuber H35 rat hepatoma cells under highly viable serum-free conditions were found to selectively export putrescine from inside the cell into the culture medium, but not spermidine, spermine, or their acetylated derivatives. Even untreated cells, with very low intracellular putrescine levels, constitutively exported significant amounts of only putrescine for a 12 h period. Administration of the phorbol ester TPA (12-O-tetradecanoylphorbol 13-acetate) which markedly elevates ornithine decarboxylase (ODC), did not potentiate putrescine export over what was measured in the unstimulated cultures. However, addition of 1 mM ornithine to the cultures resulted in increased intracellular putrescine (maximum at 4 h) with a marked concomitant increase in putrescine export between 0 and 8 h, after which putrescine export again stopped. Treatment with 10(-7) M insulin yielded intracellular putrescine levels that remained elevated for 36 along with a continuous and more rapid export of putrescine over the same 36 h time period. When insulin and ornithine were administered together, even higher levels of intracellular putrescine and putrescine export were observed, with putrescine efflux proceeding over the 36 h time-course at the highest observed rates of 1.5 (0-12 h) and 1.0 (12-36 h) nmol/mg total protein per h. Exposure to DFMO, an inhibitor of ODC, depleted intracellular putrescine stores and effectively suppressed putrescine export. There was not a positive correlation between the time-dependent decreases in the intracellular putrescine concentrations and the respective alterations in the rate of putrescine export under a variety of conditions. Furthermore, the drug verapamil was capable of completely inhibiting putrescine export (IC50 approx. 1 microM) without any change in the level of intracellular putrescine. This data was not consistent with the involvement of simple diffusion of putrescine through the membrane as the major mechanism for putrescine export. The potential mechanisms involved in putrescine export and the role of this process in regulating intracellular polyamine levels, as well as, possible functions of extracellular putrescine are discussed.
发现在高活力无血清条件下培养的鲁伯H35大鼠肝癌细胞会选择性地将细胞内的腐胺转运到培养基中，但不会转运亚精胺、精胺或它们的乙酰化衍生物。即使是未处理的细胞，细胞内腐胺水平很低，在12小时内也会持续输出大量的腐胺。施用能显著提高鸟氨酸脱羧酶（ODC）活性的佛波酯TPA（12-O-十四酰佛波醇13-乙酸酯），并不会使腐胺输出量比未刺激培养物中的测量值增加。然而，向培养物中添加1 mM鸟氨酸会导致细胞内腐胺增加（4小时时达到最大值），同时在0至8小时内腐胺输出显著增加，之后腐胺输出再次停止。用10^(-7) M胰岛素处理会使细胞内腐胺水平在36小时内持续升高，并且在相同的36小时时间段内腐胺输出持续且更快。当胰岛素和鸟氨酸一起施用时，会观察到细胞内腐胺和腐胺输出水平更高，腐胺流出在36小时时间进程中以每小时1.5（0 - 12小时）和1.0（12 - 36小时）nmol/mg总蛋白的最高观察速率进行。暴露于ODC抑制剂DFMO会耗尽细胞内腐胺储备并有效抑制腐胺输出。在各种条件下，细胞内腐胺浓度随时间的下降与腐胺输出速率的相应变化之间没有正相关。此外，药物维拉帕米能够完全抑制腐胺输出（IC50约为1 microM），而细胞内腐胺水平没有任何变化。这些数据与腐胺通过膜的简单扩散作为腐胺输出的主要机制不一致。本文讨论了腐胺输出所涉及的潜在机制、该过程在调节细胞内多胺水平中的作用以及细胞外腐胺的可能功能。
