Uridine transport in novikoff rat hepatoma cells and other cell lines and its relationship to uridine phosphorylation and phosphorolysis
Uridine transport in novikoff rat hepatoma cells and other cell lines and its relationship to uridine phosphorylation and phosphorolysis
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诺维科夫大鼠肝癌细胞和其他细胞系中的尿苷转运及其与尿苷磷酸化和磷酸解的关系
DOI：
10.1002/jcp.1040970107
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发表时间：
1978
影响因子：
5.6
通讯作者：
R. Wohlhueter
中科院分区：
文献类型：
--
作者：
P. Plagemann;R. Marz;R. Wohlhueter
在未经处理和 ATP 耗尽的野生型和尿苷激酶缺陷的 Novikoff 细胞以及小鼠 L 和 P388 细胞、中国仓鼠卵巢细胞和人 HeLa 细胞中，在 25° 下测定了 [3H] 尿苷摄取的时间进程作为尿苷浓度的函数。短期吸收是通过快速采样技术测量的，该技术允许以短至一秒半的时间间隔对细胞悬浮液进行采样。在未处理的野生型细胞中，尿苷迅速磷酸化，在细胞中，尿苷磷酸化由于缺乏 ATP 或尿苷激酶而被阻止，时间进程的初始部分是相同的。因此，初始摄取速率反映了尿苷转运速率。然而，在尿苷浓度为 20-160 μM 时，尿苷吸收仅在 5 至 10 秒内呈近似线性，而在较高浓度下则时间更长。在磷酸化细胞中，尿苷摄取速率（80 μM）随后下降至初始速率的约 20-30%，并且该速率很大程度上取决于磷酸化速率而不是转运速率。然而，当尿苷浓度低于 1 μM 时，Novikoff 细胞的细胞内磷酸化速率接近转运速率。磷酸化的表观底物饱和表明这些细胞中存在低 Km 尿苷磷酸化系统。根据初始摄取率估计，所有检查的细胞系的尿苷促进转运的“零反式”(zt) Km 均落在 50 至 240 μM 之间。所有细胞系的零反式 Vmax 值也相似（4-15 pmoles/μ1 细胞 H2O.sec）。无论细胞是在悬浮培养物还是单层培养物中繁殖（并分析尿苷吸收），CHO 细胞吸收尿苷的时间过程和转运动力学常数大致相同。当 Novikoff 细胞预载 10 μM 尿苷时，表观 Km 和 Vmax 值（无限反式）比相应的零反式值高两到三倍。尿苷转运被其他几种核糖核苷和脱氧核糖核苷以简单的竞争性方式抑制。所有核苷似乎都由同一系统运输，但效率不同。次黄嘌呤、腺嘌呤、胸腺嘧啶、Persantin、罂粟碱和邻硝基苄基硫肌苷以及用对氯汞苯甲酸盐预处理细胞也会抑制尿苷转运，但高浓度的胞嘧啶、D-核糖或安息香素不会抑制尿苷转运。 Persantin 对尿苷转运的抑制涉及 V maxzt 和 K mzt 的变化。由于运输速度快，即使在 0° 下，当在缓冲溶液中冲洗细胞以去除细胞外底物时，细胞内尿苷也会发生一些损失。漂洗液中存在转运抑制剂Persantin，或者通过油离心将悬浮细胞从培养基中分离出来，可以防止这种损失。还发现细胞内尿苷的代谢转化在冲洗期间继续进行。漂洗过程中由于流出和新陈代谢造成的伪影程度随着漂洗持续时间的增加而增加。
Time courses of [3H]uridine uptake as a function of uridine concentration were determined at 25° in untreated and ATP‐depleted wild‐type and uridine kinase‐deficient Novikoff cells and in mouse L and P388 cells, Chinese hamster ovary cells and human HeLa cells. Short term uptake was measured by a rapid sampling technique which allows sampling of cell suspensions in intervals as short as one and one‐half seconds. The initial segments of the time courses were the same in untreated, wild‐type cells in which uridine is rapidly phosphorylated and in cells in which uridine phosphorylation was prevented due to lack of ATP or uridine kinase. The initial rates of uptake, therefore, reflected the rate of uridine transport. Uridine uptake, however, was approximately linear for only five to ten seconds at uridine concentrations from 20–160 μM and somewhat longer at higher concentrations. In phosphorylating cells the rate of uridine uptake (at 80 μM) then decreased to about 20–30% of the initial rate and this rate was largely determined by the rate of phosphorylation rather than transport. At uridine concentrations below 1 μM, however, the rate of intracellular phosphorylation in Novikoff cells approached the transport rate. The apparent substrate saturation of phosphorylation suggests the presence of a low Km uridine phosphorylation system in these cells. The “zero‐trans” (zt) Km for the facilitated transport of uridine as estimated from initial uptake rates fell between 50 and 240 μM for all cell lines examined. The zero‐trans Vmax values were also similar for all the lines (4–15 pmoles/μ1 cell H2O.sec). The time courses of uridine uptake by CHO cells and the kinetic constants for transport were about the same whether the cells were propagated (and analyzed for uridine uptake) in suspension or monolayer culture. When Novikoff cells were preloaded with 10 μM uridine the apparent Km and Vmax values (infinite‐trans) were two to three times higher than the corresponding zero‐trans values. Uridine transport was inhibited in a simple competitive manner by several other ribo‐ and deoxyribonucleosides. All nucleosides seem to be transported by the same system, but with different efficiencies. Uridine transport was also inhibited by hypoxanthine, adenine, thymine, Persantin, papaverin, and o‐nitrobenzylthioinosine, and by pretreatment of the cells with p‐chloromercuri‐benzoate, but not by high concentrations of cytosine, D‐ribose or acronycin. The inhibition of uridine transport by Persantin involved changes in both V maxzt and K mzt . Because of the rapidity of transport, some loss of intracellular uridine occurred when cells were rinsed in buffer solution to remove extracellular substrate, even at 0°. This loss was prevented by the presence of a transport inhibitor, Persantin, in the rinse fluid or by separating suspended cells from the medium by centrifugation through oil. Metabolic conversion of intracellular uridine were also found to continue during the rinse period. The extent of artifacts due to efflux and metabolism during rinsing increased with duration of the rinse.