H+-PEPTIDE COTRANSPORT IN MADIN-DARBY CANINE KIDNEY-CELLS - EXPRESSION AND CALMODULIN-DEPENDENT REGULATION
H+-PEPTIDE COTRANSPORT IN MADIN-DARBY CANINE KIDNEY-CELLS - EXPRESSION AND CALMODULIN-DEPENDENT REGULATION
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DOI：
10.1152/ajprenal.1995.268.3.f391
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发表时间：
1995-03-01
期刊：
影响因子：
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通讯作者：
LEIBACH, FH
中科院分区：
文献类型：
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作者：
BRANDSCH, M;GANAPATHY, V;LEIBACH, FH
研究了二肽甘氨酰肌氨酸在肾细胞系OK、LLC-PK 1和Madin-Darby犬肾（MDCK）中的转运，这些细胞系在不可渗透的塑料支持物上生长为融合单层。在OK和LLC-PK 1细胞中的二肽的摄取是缓慢的，不受其他肽的抑制，并且不受向内定向的H+梯度的影响，这表明在我们的条件下在这些细胞中缺乏H+-肽共转运系统的表达。相反，在MDCK细胞中二肽的摄取是快速的，并且发现被向内定向的H+梯度刺激。这种刺激显着减少质子载体羰基氰化物对三氟甲氧基苯腙。二肽和三肽以及β-内酰胺类抗生素头孢氨苄可抑制甘氨酰肌氨酸的H+梯度依赖性摄取，但氨基酸甘氨酸和亮氨酸不抑制。摄取是饱和的，显然是通过一个单一的运输系统发生。该系统的米氏常数为1.3 +/- 0.1 mM，最大速度为13.3 +/- 0.7 nmol。30 min（-1）。mg蛋白（-1）。用钙调素拮抗剂N-（6-氨基己基）-5-氯-1-萘磺酰胺（W-7）、CGS-9343 B或calmidazolium处理MDCK细胞，以时间和剂量依赖性方式抑制甘氨酰肌氨酸摄取40-50%。方式与此相反，丙氨酸，亮氨酸，葡萄糖和牛磺酸的摄取被发现刺激治疗与W-7。动力学分析表明，肽转运活性的抑制主要与系统的最大速度的降低有关。它的结论是H+-肽共转运系统，这是已知存在于正常肾小管上皮细胞，在MDCK细胞中表达，并调节钙调素依赖的过程。
The transport of the dipeptide glycylsarcosine was studied in the kidney cell lines OK, LLC-PK1, and Madin-Darby canine kidney (MDCK), grown as confluent monolayers on impermeable plastic supports. Uptake of the dipeptide in OK and LLC-PK1 cells was slow, was not inhibited by other peptides, and was not influenced by an inwardly directed H+ gradient, indicating lack of expression of the H+-peptide cotransport system in these cells under our conditions. In contrast., uptake of the dipeptide in MDCK cells was rapid and was found to be stimulated by an inwardly directed H+ gradient. This stimulation was markedly reduced by the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone. The H+ gradient-dependent uptake of glycylsarcosine was inhibited by dipeptides and tripeptides and by the beta-lactam antibiotic cephalexin but not by the amino acids glycine and leucine. The uptake was saturable and apparently occurred via a single transport system. The Michaelis-Menten constant for the system was 1.3 +/- 0.1 mM, and the maximal velocity was 13.3 +/- 0.7 nmol . 30 min(-1) . mg protein(-1). Treatment of MDCK cells with the calmodulin antagonists N-(6-aminohexyl)-5-chloro-1-napthalenesulfonamide (W-7), CGS-9343B, or calmidazolium inhibited the glycylsarcosine uptake by 40-50% in a time- and dose-dependent. manner. In contrast, the uptake of alanine, leucine, glucose, and taurine was found to be stimulated by treatment with W-7. Kinetic analysis revealed that the inhibition of the peptide transport activity was mainly associated with a decrease of the maximal velocity of the system. It is concluded that the H+-peptide cotransport system, which is known to exist in normal renal tubular epithelial cells, is expressed in MDCK cells and is regulated by calmodulin-dependent processes.