Neumann Jana, Brandsch Matthias
Membrane Transport Group, Biozentrum of the Martin-Luther-University Halle-Wittenberg, Halle (Saale), Germany.
J Pharmacol Exp Ther. 2003 Apr;305(1):219-24. doi: 10.1124/jpet.102.046573.
This study was performed to characterize the transport of the endogenous photosensitizer delta-aminolevulinic acid in tumor cells of the extrahepatic biliary duct. Uptake of [(3)H]delta-aminolevulinic acid into human cholangiocarcinoma SK-ChA-1 cells was linear for up to 10 min, independent of a Na(+) gradient, but stimulated 3- to 4-fold by an inwardly directed H(+) gradient. Uptake of delta-aminolevulinic acid was mediated by a single transport system with an apparent affinity (K(t)) of 2.1 mM and a maximal velocity (V(max)) of 60.1 nmol. 10 min(-1). mg of protein(-1). Glycylsarcosine, alanylalanine, and cefadroxil strongly inhibited the [(3)H]delta-aminolevulinic acid uptake with K(i) values of 1.3, 0.2, and 3.6 mM, respectively. In contrast, gamma-aminobutyric acid, glycine, L-glutamic acid, and L-aspartic acid (all 10 mM) had no effect on the total [(3)H]delta-aminolevulinic acid uptake, neither at pH 6.0 nor at pH 7.5. Applying a Dixon type of experiment and the ABC test revealed that glycylsarcosine and delta-aminolevulinic acid are transported via the same system, PEPT1. Treatment of the cells with phorbol 12-myristate 13-acetate, a phorbol ester that activates protein kinase C, resulted in a significant inhibition of the transport rate. This inhibition could be blocked by cotreatment with staurosporine. We conclude that delta-aminolevulinic acid is transported by the H(+)/peptide cotransporter PEPT1 into epithelial cells of the extrahepatic biliary duct. delta-Aminolevulinic acid can be accumulated specifically in bile duct tumor cells before photodynamic therapy.
本研究旨在表征内源性光敏剂δ-氨基乙酰丙酸在肝外胆管肿瘤细胞中的转运情况。[(3)H]δ-氨基乙酰丙酸进入人胆管癌细胞SK-ChA-1的摄取在长达10分钟内呈线性，与Na(+)梯度无关，但由内向的H(+)梯度刺激3至4倍。δ-氨基乙酰丙酸的摄取由单一转运系统介导，其表观亲和力（K(t)）为2.1 mM，最大速度（V(max)）为60.1 nmol·10 min(-1)·mg蛋白(-1)。甘氨酰肌氨酸、丙氨酰丙氨酸和头孢羟氨苄分别以1.3、0.2和3.6 mM的K(i)值强烈抑制[(3)H]δ-氨基乙酰丙酸的摄取。相比之下，γ-氨基丁酸、甘氨酸、L-谷氨酸和L-天冬氨酸（均为10 mM）在pH 6.0和pH 7.5时对[(3)H]δ-氨基乙酰丙酸的总摄取均无影响。应用狄克逊类型实验和ABC测试表明，甘氨酰肌氨酸和δ-氨基乙酰丙酸通过同一系统PEPT1转运。用佛波醇12-肉豆蔻酸酯13-乙酸酯（一种激活蛋白激酶C的佛波酯）处理细胞，导致转运速率显著抑制。这种抑制可通过与星形孢菌素共同处理来阻断。我们得出结论，δ-氨基乙酰丙酸通过H(+)/肽共转运体PEPT1转运至肝外胆管的上皮细胞。在光动力治疗前，δ-氨基乙酰丙酸可特异性地积聚在胆管肿瘤细胞中。
