The organic cation transporters rOCT1 and hOCT2 are inhibited by cGMP.
The electrogenic cation transporters OCT1 and OCT2 in the basolateral membrane of renal proximal tubules mediate the first step during secretion of organic cations. Previously we demonstrated stimulation and change of selectivity for rat OCT1 (rOCT1) by protein kinase C. Here we investigated the effect of cGMP on cation transport by rOCT1 or human OCT2 (hOCT2) after expression in human embryonic kidney cells (HEK293) or oocytes of Xenopus laevis. In HEK293 cells, uptake was measured by microfluorimetry using the fluorescent cation 4-(4-(dimethyl-amino)styryl)-N-methylpyridinium iodide (ASP + ) as substrate, whereas uptake into Xenopus laevis oocytes was measured with radioactively labelled cations. In addition, ASP +-induced depolarizations of membrane voltages (Vm) were measured in HEK293 cells using the slow whole-cell patch-clamp method. Incubation of rOCT1-expressing HEK293 cells for 10 min with 100 mM 8-Br-cGMP reduced initial ASP + uptake by maximally 78% with an IC50 value of 24 +/- 16 mM. This effect was not abolished by the specific PKG inhibitor KT5823, indicating that a cGMP-dependent kinase is not involved. An inhibition of ASP + uptake by rOCT1 in HEK293 cells was also obtained when the cells were incubated for 10 min with 100 mM cGMP, whereas no effect was obtained when cGMP was given together with ASP +. ASP + (100 mM)-induced depolarizations of Vm were reduced in the presence of 8-Br-cGMP (100 mM) by 44 +/- 11% (n = 6). Since it could be demonstrated that [3H]cGMP is taken up by an endogeneous cyanine863-inhibitable transporter, the effect of cGMP is probably mediated from inside the cell. Uptake measurements with [14C]tetraethylammonium and [3H]2-methyl-4-phenylpyridinium in Xenopus laevis oocytes expressing rOCT1 performed in the absence and presence of 8-Br-cGMP showed that cGMP does not interact directly with the transporter. The data suggest that the inhibition mediated by cGMP observed in HEK293 cells occurs most likely via a mammalian cGMP-binding protein that interacts with OCT1-2 transporters.
Schlatter
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Universitätsklinikum Münster, Medizinische Klinik und Poliklinik D, Experimentelle Nephrologie, Domagkstr. 3a, D-48149 Münster, Germany. eberhard.schlatter@uni-muenster.de
Mönnich
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V
Cetinkaya
I
I
Mehrens
T
T
Ciarimboli
G
G
Hirsch
J R
JR
Popp
C
C
Koepsell
H
H
D000818:Animals; D050482:Catecholamine Plasma Membrane Transport Proteins; D002460:Cell Line; D006152:Cyclic GMP / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D007668:Kidney / Q000187:drug effects / Q000378:metabolism*; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism*; D027701:Organic Cation Transport Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D027702:Organic Cation Transporter 1 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000075102:Organic Cation Transporter 2; D051381:Rats; D014982:Xenopus laevis
D050482:Catecholamine Plasma Membrane Transport Proteins; D027701:Organic Cation Transport Proteins; D027702:Organic Cation Transporter 1; D000075102:Organic Cation Transporter 2; C491013:SLC22A2 protein, human; C493212:Slc22a1 protein, rat; C100590:Slc22a2 protein, rat; D006152:Cyclic GMP