H(+)-peptide cotransport in Madin-Darby canine kidney cells: expression and calmodulin-dependent regulation.
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-trifluoromethoxy-phenylhydrazone. 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Brandsch
M
M
Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta 30912-2100.
Ganapathy
V
V
Leibach
F H
FH
D000818:Animals; D001562:Benzimidazoles / Q000494:pharmacology; D001693:Biological Transport, Active / Q000187:drug effects; D002147:Calmodulin / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002460:Cell Line; D003470:Culture Media; D004151:Dipeptides / Q000378:metabolism; D004285:Dogs; D007668:Kidney / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D007930:Leucine / Q000378:metabolism; D010455:Peptides / Q000378:metabolism*; D011522:Protons; D013449:Sulfonamides / Q000494:pharmacology; D013654:Taurine / Q000378:metabolism
D001562:Benzimidazoles; D002147:Calmodulin; D003470:Culture Media; D004151:Dipeptides; D010455:Peptides; D011522:Protons; D013449:Sulfonamides; C052087:CGS 9343B; D013654:Taurine; C004194:glycylsarcosine; C017967:W 7; D007930:Leucine