Ganapathy M E, Brandsch M, Prasad P D, Ganapathy V, Leibach F H
Department of Medicine, Medical College of Georgia, Augusta 30912, USA.
J Biol Chem. 1995 Oct 27;270(43):25672-7. doi: 10.1074/jbc.270.43.25672.
This study was initiated to determine if there are differences in the recognition of beta -lactam antibiotics as substrates between intestinal and renal peptide transporters, PEPT 1 and PEPT 2. Reverse transcription-coupled polymerase chain reaction and/or Northern blot analysis have established that the human intestinal cell line Caco-2 expresses PEPT 1 but not PEPT 2, whereas the rat proximal tubule cell line SKPT expresses PEPT 2 but not PEPT 1. Detailed kinetic analysis has provided unequivocal evidence for participation of PEPT 2 in SKPT cells in the transport of the dipeptide glycylsarcosine and the aminocephalosporin cephalexin. The substrate recognition pattern of PEPT 1 and PEPT 2 was studied with cefadroxil (a cephalosporin) and cyclacillin (a penicillin) as model substrates for the peptide transporters constitutively expressed in Caco-2 cells (PEPT 1) and SKPT cells (PEPT 2). Cyclacillin was 9-fold more potent than cefadroxil in competing with glycylsacosine for uptake via PEPT 1. In contrast, cefadroxil was 13-fold more potent than cyclacillin in competing with the dipeptide for uptake via PEPT 2. The substrate recognition pattern of PEPT 1 and PEPT 2 was also investigated using cloned human peptide transporters functionally expressed in HeLa cells. Expression of PEPT 1 or PEPT 2 in HeLa cells was found to induce H(+)-coupled cephalexin uptake in these cells. As was the case with Caco-2 cells and SKPT cells, the uptake of glycylsarcosine induced in HeLa cells by PEPT 1 cDNA and PEPT 2 cDNA was inhibitable by cyclacillin and cefadroxil. Again, the PEPT 1 cDNA-induced dipeptide uptake was inhibited more potently by cyclacillin than by cefadroxil, and the PEPT 2 cDNA-induced dipeptide uptake was inhibited more potently by cefadroxil than by cyclacillin. It is concluded that there are marked differences between the intestinal and renal peptide transporters in the recognition of beta -lactam antibiotics as substrates.
开展本研究是为了确定肠道和肾脏肽转运体PEPT 1和PEPT 2在将β-内酰胺类抗生素识别为底物方面是否存在差异。逆转录聚合酶链反应和/或Northern印迹分析已证实，人肠道细胞系Caco-2表达PEPT 1但不表达PEPT 2，而大鼠近端小管细胞系SKPT表达PEPT 2但不表达PEPT 1。详细的动力学分析为PEPT 2参与SKPT细胞中二肽甘氨酰肌氨酸和氨基头孢菌素头孢氨苄的转运提供了明确证据。以头孢羟氨苄（一种头孢菌素）和环青霉素（一种青霉素）作为在Caco-2细胞（PEPT 1）和SKPT细胞（PEPT 2）中组成性表达的肽转运体的模型底物，研究了PEPT 1和PEPT 2的底物识别模式。在通过PEPT 1与甘氨酰肌氨酸竞争摄取方面，环青霉素的效力比头孢羟氨苄高9倍。相反，在通过PEPT 2与二肽竞争摄取方面，头孢羟氨苄的效力比环青霉素高13倍。还使用在HeLa细胞中功能性表达的克隆人肽转运体研究了PEPT 1和PEPT 2的底物识别模式。发现HeLa细胞中PEPT 1或PEPT 2的表达可诱导这些细胞中H⁺偶联的头孢氨苄摄取。与Caco-2细胞和SKPT细胞的情况一样，PEPT 1 cDNA和PEPT 2 cDNA在HeLa细胞中诱导的甘氨酰肌氨酸摄取可被环青霉素和头孢羟氨苄抑制。同样，环青霉素对PEPT 1 cDNA诱导的二肽摄取的抑制作用比对头孢羟氨苄的抑制作用更强，而头孢羟氨苄对PEPT 2 cDNA诱导的二肽摄取的抑制作用比对环青霉素的抑制作用更强。结论是，肠道和肾脏肽转运体在将β-内酰胺类抗生素识别为底物方面存在显著差异。
