H (+) -peptide cotransport in Madin-Darby canine kidney cells: expression and calmodulin-dependent regulation
Brandsch, M.; Ganapathy, V.; Leibach, F.H.
American Journal of Physiology 268(3 Pt 2): F391-F397
1995
ISSN/ISBN: 0002-9513 PMID: 7900838 Document Number: 442063
The transport of the dipeptide glycylsarcosine was studied in the kidney cell lines OK, LLC-PK-1, and Madin-Darby canine kidney (MDCK), grown as confluent monolayers on impermeable plastic supports. Uptake of the dipeptide in OK and LLC-PK-1 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 cntdot 30 min-1 cntdot mg protein-1. Treatment of MDCK cells with the calmodulin antagonists N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), CGS9343B, 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.