Properties and regulation of organic cation transport in freshly isolated human proximal tubules.
The kidney, and more specifically the proximal tubule, is the main site of elimination of cationic endogenous metabolites and xenobiotics. Although numerous studies exist on renal organic cation transport of rat and rabbit, no information is available from humans. Therefore, we examined organic cation transport and its regulation across the basolateral membrane of isolated human proximal tubules. mRNA for the cation transporters hOCT1 and hOCT2 as well as hOCTN1 and hOCTN2 was detected in these tubules. Organic cation transport across the basolateral membrane of isolated collapsed proximal tubules was recorded with the fluorescent dye 4-(4-dimethylamino)styryl-N-methylpyridinium (ASP(+)). Depolarization of the cells by rising extracellular K(+) concentration to 145 mm reduced ASP(+) uptake by 20 +/- 5% (n = 15), indicating its electrogeneity. The substrates of organic cation transport tetraethylammonium (K(i) = 63 microm) and cimetidine (K(i) = 11 microm) as well as the inhibitor quinine (K(i) = 2.9 microm) reduced ASP(+) uptake concentration dependently. Maximal inhibition reached with these substances was approximately 60%. Stimulation of protein kinase C with 1,2-dioctanoyl-sn-glycerol (DOG, 1 microm) or ATP (100 microm) inhibited ASP(+) uptake by 30 +/- 3 (n = 16) and 38 +/- 13% (n = 6), respectively. The effect of DOG could be reduced with calphostin C (0.1 microm, n = 7). Activation of adenylate cyclase by forskolin (1 microm) decreased ASP(+) uptake by 29 +/- 3% (n = 10). hANP (10 nm) or 8-bromo-cGMP (100 microm) also decreased ASP(+) uptake by 17 +/- 3 (n = 9) or 32 +/- 5% (n = 10), respectively. We show for the first time that organic cation transport across the basolateral membrane of isolated human proximal tubules, most likely mediated via hOCT2, is electrogenic and regulated by protein kinase C, the cAMP- and the cGMP-dependent protein kinases.
Pietig
G
G
Medizinische Klinik and Poliklinik D, Experimentelle Nephrologie, Universitätsklinikum Münster, D-48149 Münster, Germany.
Mehrens
T
T
Hirsch
J R
JR
Cetinkaya
I
I
Piechota
H
H
Schlatter
E
E
D001692:Biological Transport; D002412:Cations; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000378:metabolism; D004075:Diglycerides / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005456:Fluorescent Dyes / Q000494:pharmacology*; D006367:HeLa Cells; D006801:Humans; D007684:Kidney Tubules / Q000378:metabolism*; D007700:Kinetics; D011188:Potassium / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism; D011726:Pyridinium Compounds / Q000494:pharmacology*; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013050:Spectrometry, Fluorescence
C032315:4-(4-dimethylaminostyryl)-1-methylpyridinium; D002412:Cations; D004075:Diglycerides; D005456:Fluorescent Dyes; D011726:Pyridinium Compounds; D012333:RNA, Messenger; C044295:1,2-dioctanoylglycerol; C016276:8-bromocyclic GMP; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D006152:Cyclic GMP; D011188:Potassium