Yuan G, Ott R J, Salgado C, Giacomini K M
School of Pharmacy, University of California, San Francisco 94143.
J Biol Chem. 1991 May 15;266(14):8978-86.
The goal of this study was to determine the mechanisms involved in the transport of the organic cation, tetraethylammonium (TEA), across the apical membrane of OK cells. [14C]TEA accumulated in OK cell monolayers reaching equilibrium in 2 h. The uptake of [14C]TEA at equilibrium was dependent upon temperature and was inhibited by sodium azide and by various organic cations, including N1-methylnicotinamide (NMN), mepiperphenidol, and cimetidine but not by the organic anion, p-aminohippuric acid. The initial uptake of [14C]TEA was characterized by a saturable process. The mean +/- S.D. Km was 27.8 +/- 2.6 microM and the Vmax was 414 +/- 26.5 pmol/mg protein/min. Both an accelerated efflux and influx of [14C]TEA in the presence of a trans-gradient of unlabeled TEA and NMN was observed, whereas a deaccelerated influx and efflux was observed in the presence of a trans-gradient of mepiperphenidol. The mechanism of interaction between NMN and TEA was examined. NMN significantly increased the apparent Km (mean +/- S.D.) of TEA to 82.8 +/- 16.4 microM (p less than 0.001), whereas the Vmax (mean +/- S.D.) was only slightly affected (478 +/- 72 pmol/mg protein/min) suggesting a competitive inhibition. The stimulatory effect of trans-gradients of NMN on TEA transport was due to an increase in the Vmax of TEA suggesting that NMN trans-stimulates TEA transport by increasing the turnover rate of the exchanger. In the presence of an inwardly directed proton gradient, the efflux at 30 s of [14C]TEA from the OK cell monolayers was significantly accelerated (p less than 0.05). Studies with the pH-sensitive fluorescent probe, 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein, suggested that TEA could drive the countertransport of protons. In apical membrane vesicles prepared from OK cells, the uptake of [3H]NMN exhibited an apparent "overshoot phenomenon" in the presence of an initial outwardly directed proton gradient. Protons competitively inhibited TEA uptake suggesting that the proton/organic cation and the organic cation/organic cation self exchange mechanism are the same mechanism. This is the first report describing both TEA self-exchange and proton/TEA exchange in the apical membrane of a continuous cell line. OK cells are an excellent model for the study of organic cation transport across the apical membrane.
本研究的目的是确定有机阳离子四乙铵（TEA）跨OK细胞顶端膜转运的相关机制。[¹⁴C]TEA在OK细胞单层中积累，2小时达到平衡。平衡时[¹⁴C]TEA的摄取依赖于温度，并受到叠氮化钠和各种有机阳离子的抑制，包括N¹-甲基烟酰胺（NMN）、美哌隆和西咪替丁，但不受有机阴离子对氨基马尿酸的抑制。[¹⁴C]TEA的初始摄取具有饱和过程的特征。平均±标准差Km为27.8±2.6μM，Vmax为414±26.5 pmol/mg蛋白质/分钟。在未标记的TEA和NMN的跨梯度存在下，观察到[¹⁴C]TEA的外流和内流加速，而在美哌隆的跨梯度存在下，观察到内流和外流减速。研究了NMN与TEA之间的相互作用机制。NMN显著增加了TEA的表观Km（平均±标准差）至82.8±16.4μM（p＜0.001），而Vmax（平均±标准差）仅受到轻微影响（478±72 pmol/mg蛋白质/分钟），提示存在竞争性抑制。NMN跨梯度对TEA转运的刺激作用是由于TEA的Vmax增加，表明NMN通过增加交换体的周转率来反刺激TEA转运。在向内的质子梯度存在下，[¹⁴C]TEA从OK细胞单层在30秒时的外流显著加速（p＜0.05）。用pH敏感荧光探针2′,7′-双（羧乙基）-5（6）-羧基荧光素进行的研究表明，TEA可驱动质子的反向转运。在从OK细胞制备的顶端膜囊泡中，在初始向外的质子梯度存在下，[³H]NMN的摄取表现出明显的“过冲现象”。质子竞争性抑制TEA摄取，提示质子/有机阳离子和有机阳离子/有机阳离子自交换机制是相同的机制。这是第一份描述连续细胞系顶端膜中TEA自交换和质子/TEA交换的报告。OK细胞是研究有机阳离子跨顶端膜转运的优秀模型。
