ABSTRACT
We compared the characteristics of several cloned rabbit organic electrolyte (OE) transporters expressed in cultured cells with their behavior in intact rabbit renal proximal tubules (RPT) to determine the contribution of each to basolateral uptake of the weak acid ochratoxin A (OTA) and the weak base cimetidine (CIM). The activity of organic anion transporters OAT1 and OAT3 proved to be distinguishable because OAT1 had a high affinity for PAH (K(t) of 20 microM) and did not support estrone sulfate (ES) transport, whereas OAT3 had a high affinity for ES (K(t) of 4.5 microM) and a weak interaction with PAH (IC(50) > 1 mM). In contrast, both transporters robustly accumulated OTA. Intact RPT also accumulated OTA, with OAT1 and OAT3 each responsible for approximately 50%: ES and PAH each reduced uptake by approximately 50%, and the combination of the two eliminated mediated OTA uptake. The weak base CIM was transported by OAT3 (K(t) of 80 microM) and OCT2 (K(t) of 2 microM); OCT1 had a comparatively low affinity for CIM, and CIM uptake by OAT1 was equivocal. Intact RPT accumulated CIM, with TEA and ES reducing CIM uptake by 20 and 75%, respectively, suggesting that OAT3 plays a quantitatively more significant role in CIM uptake in the early proximal tubule than OCT1/2. In single S2 segments of RPT, ES and TEA each blocked approximately 50% of CIM uptake. Thus the fractional contribution of different OE transporters to renal secretion is influenced by their affinity for substrate and relative expression level in RPT.
Subject(s)
Electrolytes/metabolism , Estrone/analogs & derivatives , Kidney Tubules, Proximal/metabolism , Organic Anion Transport Protein 1/metabolism , Organic Anion Transporters, Sodium-Independent/metabolism , Organic Cation Transporter 1/metabolism , Animals , Cells, Cultured , Cimetidine/metabolism , Cloning, Molecular , Dicarboxylic Acids/metabolism , Estrone/pharmacology , Histamine H2 Antagonists/metabolism , Kidney Tubules/drug effects , Kidney Tubules/metabolism , Kinetics , Ochratoxins/pharmacology , RNA, Messenger/biosynthesis , Rabbits , Recombinant Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Substrate Specificity , Uric Acid/metabolismABSTRACT
The organic cation transporter, OCT2, plays a role in renal secretion of a broad array of weak bases. To determine whether the degree of ionization of these compounds plays a role in their interaction with OCT2, we examined the influence of external pH values on the activity of the human ortholog of OCT2, as expressed in Chinese hamster ovary-K1 cells. Importantly, changing the pH value from 7.0 to 8.0 had no effect on the rate of transport of the fixed cations, tetraethylammonium and 1-methyl-4-phenylpyridinium, i.e. the pH value did not have an effect upon the transporter itself. Cimetidine (pK(a) 6.92), a competitive inhibitor of hOCT2, displayed a 3.5-fold increase in IC(50) as pH values increased from 7 to 8. hOCT2-mediated cimetidine transport decreased over this pH range, the consequence of an increase in K(t) and decrease in J(max) at the higher pH value. The weak bases trimethoprim and 4-phenylpyridine showed a similar pattern of pH-sensitive interaction with hOCT2. The non-ionizable sterol, corticosterone, also inhibited hOCT2 activity, although it was neither competitive in nature nor was it sensitive to pH in the manner observed with weak bases. We conclude that the degree of ionization plays a critical role in binding of substrate to organic cation transporters.
