Carlotta E. Groves

Influence of Estrogen and Xenoestrogens on Basolateral Uptake of Tetraethylammonium by Opossum Kidney Cells in Culture

The sex steroid hormone estrogen down-regulates renal organic cation (OC) transport in animals, and it may contribute to sex-related differences in xenobiotic accumulation and excretion. Also, the presence of various endocrine-disrupting chemicals, i.e., environmental chemicals that possess estrogenic activity (e.g., xenoestrogens) may down-regulate various transporters involved in renal accumulation and excretion of xenobiotics. The present study characterizes the mechanism by which long-term (6-day) incubation with physiological concentrations of 17β-estradiol (E2) or the xenoestrogens diethylstilbestrol (DES) and bisphenol A (BPA) regulates the basolateral membrane transport of the OC tetraethylammonium (TEA) in opossum kidney (OK) cell renal cultures. Both 17β-E2 and the xenoestrogen DES produced a dose- and time-dependent inhibition of basolateral TEA uptake in OK cell cultures, whereas the weakly estrogenic BPA had no effect on TEA uptake. Treatment for 6 days with either 1 nM 17β-E2 or DES reduced TEA uptake by ∼30 and 40%, respectively. These effects were blocked completely by the estrogen receptor antagonist ICI 182780 (Faslodex, fulvestrant), suggesting that these estrogens regulate OC transport through the estrogen receptor, which was detected (estrogen receptor α) in OK cell cultures by reverse transcription-polymerase chain reaction. The Jmax value for TEA uptake in 17β-E2- and DES-treated OK cell cultures was ∼40 to 50% lower than for ethanol-treated cultures, whereas Kt was unaffected. This reduction in transport capacity was correlated with a reduction in OC transporter OCT1 protein expression following treatment with both agents.

Tetrapentylammonium (TPeA): slowly dissociating inhibitor of the renal peritubular organic cation transporter

The efflux of tetraethylammonium (TEA) from suspensions of rabbit renal proximal tubules is completely blocked by 500 microM tetrapentylammonium (TPeA) in the extracellular medium. The basis of this trans-inhibition of TEA transport by TPeA was examined in tubule suspensions. At TPeA concentrations < 10 microM, efflux of TEA was reduced by approximately 50%, whereas at concentrations > 10 microM, TPeA reduced efflux an additional 50% to produce a near complete block of TEA efflux. Increasing concentrations of TPeA from 0-500 microM were found to produce a biphasic, concentration-dependent trans-inhibition of TEA efflux from tubule suspensions suggesting that TPeA may block efflux by binding to both a high and low affinity TPeA binding site. The trans-inhibition of TEA efflux by TPeA at low concentrations (< 10 microM) may result from a slow carrier turnover when TPeA is bound to the carrier site. To determine whether the inhibitory effectiveness of TPeA was also associated with its slow dissociation from the carrier site, the effect of a 10 s preincubation with 1 microM TPeA on TEA uptake was examined. The uptake of TEA by tubules preincubated for 10 s with TPeA was reduced by approximately 30-50% compared to control tubules not preincubated with TPeA. A 10 s preincubation with 150 microM unlabeled TEA had no effect on TEA uptake compared to control tubules not preincubated with TEA. When the 10 s preincubation with 1 microM TPeA was followed by a 10 min recovery period, TEA uptake returned to control levels, indicating that the prolonged inhibition was reversible. This prolonged inhibition of TEA uptake after a 10 s preincubation with 1 microM TPeA, as suspected, may arise from a slow dissociation of TPeA from the OC transporter following a rapid association to the binding site. TPeA inhibition of TEA uptake into tubules was competitive in nature with a Ki of 1 microM. The ability of TEA to compete with TPeA for binding to the carrier suggests that the binding of TPeA to the carrier can be displaced by large concentrations of TEA. These observations suggest that the interactions of TPeA, and perhaps similarly large hydrophobic OCs, with the OC transporter are complex.