TNF-stimulated arginine transport by human vascular endothelium requires activation of protein kinase C.
TNF-stimulated arginine transport by human vascular endothelium requires activation of protein kinase C.
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DOI：
10.1097/00000658-199505000-00017
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发表时间：
1995-05
影响因子：
9
通讯作者：
M. Pan;M. Wasa;D. Lind;J. Gertler;W. Abbott;W. Souba
中科院分区：
文献类型：
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作者：
M. Pan;M. Wasa;D. Lind;J. Gertler;W. Abbott;W. Souba
目的：作者确定了内皮精氨酸转运机制和肿瘤坏死因子（TNF）-α介导的涉及蛋白激酶C（PKC）的信号转导途径在调节培养的内皮细胞中这种转运的潜在作用。血管内皮细胞代谢精氨酸产生一氧化氮（NO），并且NO产生的增加可以被几种细胞因子刺激。负责精氨酸转运加速的机制知之甚少。方法在TNF +/-PKC抑制剂chelerythrine chloride存在下，测定人脐静脉内皮细胞的精氨酸转运。结果载体介导的精氨酸转运由两个Na（+）非依赖性转运体完成，系统y+（占总转运量的80%）和系统b 0，+（占总转运量的20%）。肿瘤坏死因子（0.1-2 ng/mL）通过增加系统y+转运最大容量，以时间和剂量依赖性方式增加系统y（+）介导的精氨酸转运（对照Vmax = 1325 +/-60 pmol/mg蛋白质/分钟对比TNF Vmax = 3015 +/-110 pmol/mg蛋白质/分钟，p < 0.01），而不影响转运蛋白亲和力（对照Km = 30 +/-1.4微M对34 +/-1.3微M精氨酸，p = NS）。刺激在8小时时间点达到最大，并被放线菌素D和放线菌酮抑制。此外，白屈菜红碱对PKC的抑制消除了TNF-精氨酸增强的转运。类似地，将细胞与直接PKC激活剂TPA（佛波醇酯12-肉豆蔻酸酯13-乙酸酯）一起孵育刺激了系统y（+）介导的精氨酸转运近5倍，继发于转运蛋白Vmax的增加（TPA Vmax = 5349 +/- 310 pmol/mg蛋白/分钟，p < 0.001 vs.对照），而Km没有变化。这种TPA诱导的精氨酸转运刺激也被白屈菜红碱CI，放线菌素D和放线菌酮阻断。孵育TNF刺激的细胞与两个NO合酶抑制剂没有降低运输活性，这表明精氨酸转运蛋白和NO合酶的酶可能，在一定程度上，独立调节。
OBJECTIVE The authors determined the endothelial arginine transport mechanism and the potential role of a tumor necrosis factor (TNF)-alpha-mediated signal transduction pathway involving protein kinase C (PKC) in regulating this transport in cultured endothelial cells. SUMMARY BACKGROUND DATA The vascular endothelium metabolizes arginine to generate nitric oxide (NO), and an increase in NO production can be stimulated by several cytokines. The mechanism(s) responsible for the accelerated arginine transport are poorly understood. METHODS Arginine transport was assayed in confluent human umbilical vein endothelial cells in the presence of TNF +/- the PKC inhibitor chelerythrine chloride. RESULTS Carrier-mediated arginine transport was accomplished by two Na(+)-independent transporters, System y+ (80% of total transport) and System b0,+ (20% of transport). Tumor necrosis factor (0.1-2 ng/mL) increased System y(+)-mediated arginine transport in a time- and dose-dependent manner by augmenting System y+ transport maximal capacity (control Vmax = 1325 +/- 60 pmol/mg protein/minute vs. TNF Vmax = 3015 +/- 110 pmol/mg protein/minute, p < 0.01) without affecting transporter affinity (control Km = 30 +/- 1.4 microM vs. 34 +/- 1.3 microM arginine, p = NS). Stimulation was maximal at the 8-hour time point and was inhibited by both actinomycin D and cycloheximide. In addition, inhibition of PKC with chelerythrine abrogated the TNF-augmented arginine transport. Similarly, incubation of cells with the direct PKC activator TPA (phorbol ester 12-myristate 13-acetate) stimulated System y(+)-mediated arginine transport nearly fivefold, secondary to an increase in transporter Vmax (TPA Vmax = 5349 +/- 310 pmol/mg protein/minute, p < 0.001 vs. control), with no change in Km. This TPA-induced stimulation of arginine transport also was blocked by chelerythrine CI, actinomycin D, and cycloheximide. Incubation of TNF-stimulated cells with two NO synthase inhibitors did not reduce transport activity, suggesting that the arginine transporter and the NO synthase enzyme may, in part, be independently regulated.