Mechanism of prostaglandin E2 transport across the plasma membrane of HeLa cells and Xenopus oocytes expressing the prostaglandin transporter "PGT"
Mechanism of prostaglandin E2 transport across the plasma membrane of HeLa cells and Xenopus oocytes expressing the prostaglandin transporter "PGT"
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DOI：
10.1074/jbc.273.12.6689
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发表时间：
1998-03-20
影响因子：
4.8
通讯作者：
Schuster, VL
中科院分区：
文献类型：
--
作者：
Chan, BS;Satriano, JA;Schuster, VL
我们最近鉴定了一种称为PGT的新型前列腺素转运蛋白（Banal，N.，卢，R.，萨特里亚诺，J.A.，鲍，Y.，Wolkoff，A. W，and Schuster，V. L.（1995）Science 268，866-869）。基于最初的功能研究，我们假设PGT可能介导新合成的前列腺素（PG）的释放、PG的上皮转运或PG的代谢清除。在这里，我们研究了PGT运输的机制，表达在HeLa细胞和非洲爪蟾卵母细胞，使用同位素PG流入和流出的研究。在天然HeLa细胞和卵母细胞中，细胞膜对PG的渗透性较差。相反，在注射PGT mRNA的卵母细胞中，PG内流渗透系数是注射水的卵母细胞的90-157倍。PGF（2 α）底物的等级顺序接近PGE（2）> TXB_2，远大于6酮-PGF（1 α）。PG内流显示出示踪剂PGE（2）快速积累的超调，随后逐渐恢复至基线。基于估计的卵母细胞体积，PGT介导的PGE（2）积累在卵母细胞内浓度比外部培养基高25倍时达到稳态。PG的积累不是由于细胞内结合或代谢引起的。PGT介导的摄取是ATP和温度依赖性的，但不是钠依赖性的，并抑制二磺酸芪，尼氟灭酸，和硫醇反应性阴离子MTSES（钠（2-磺酰乙基）甲硫基磺酸盐）。外源性（反式）PGE（2）以剂量依赖方式刺激PGT转染的HeLa细胞的[H-3]PGE（2）外排，并被溴甲酚绿色和4，4 '-二异硫氰基二苯乙烯-2，2'-二磺酸盐抑制。膜去极化抑制[H-3]PGE（2）的摄取，与易位事件期间负电荷净向外移动的模型一致。这些结果表明，PGT介导的[H-3]PGE（2）积累通过强制性，产电阴离子交换。
We recently identified a novel prostaglandin transporter called PGT (Banal, N., Lu, R., Satriano, J.A., Bao, Y., Wolkoff, A. W, and Schuster, V. L. (1995) Science 268, 866-869). Eased on initial functional studies, we have hypothesized that PGT might mediate the release of newly synthesized prostaglandins (PG), epithelial transport of PGs, or metabolic clearance of PGs. Here we examined the mechanism of PGT transport as expressed in HeLa cells and Xenopus oocytes, using isotopic PG influx and efflux studies. In both native HeLa cells and oocytes, cell membranes were poorly permeable to PGs. In contrast, in oocytes injected with PGT mRNA, the PG influx permeability coefficient was 90-157 times that of oocytes injected with water. The rank order substrate profile was PGF(2 alpha) approximate to PGE(2) > TXB2 much greater than 6 keto-PGF(1 alpha). PG influx displayed an overshoot with rapid accumulation of tracer PGE(2), followed by a gradual return to baseline. Based on estimated oocyte volumes, the PGT-mediated accumulation of PGE(2) reached steady state at intra-oocyte concentrations 25-fold higher than the external media. The accumulation of PG was not due to intracellular binding or metabolism. PGT-mediated uptake was ATP-and temperature-dependent, but not sodium-dependent, and was inhibited by disulfonic stilbenes, niflumic acid, and the thiol reactive anion MTSES (Na(2-sulfonatoethyl)methanethiosulfonate). [H-3]PGE(2) efflux from PGT-transfected HeLa cells was stimulated by external (trans) PGE(2) in a dose-dependent fashion and was inhibited by bromcresol green and 4,4'-diisothiocyana tostilbene-2,2'-disulfonate. Membrane depolarization inhibited uptake of [H-3]PGE(2), consistent with a model of net outward movement of negative charge during the translocation event. These findings suggest that PGT mediates [H-3]PGE(2) accumulation via obligatory, electrogenic anion exchange.