Transport of estradiol-17β-glucuronide, estrone-3-sulfate and taurocholate across the endoplasmic reticulum membrane: evidence for different transport systems

Abstract

Important reactions of drug metabolism, including UGT mediated glucuronidation and steroidsulfatase mediated hydrolysis of sulfates, take place in the endoplasmic reticulum (ER) of hepatocytes. Consequently, UGT generated glucuronides, like estradiol-17β-glucuronide, have to be translocated back into the cytoplasm to reach their site of excretion. Also steroidsulfatase substrates, including estrone-3-sulfate, have to cross the ER membrane to reach their site of hydrolysis. Based on their physicochemical properties such compounds are not favored for passive diffusion and therefore likely necessitate transport system(s) to cross the ER membrane in either direction. The current study aims to investigate the transport of taurocholate, estradiol-17β-glucuronide, and estrone-3-sulfate in smooth (SER) and rough (RER) endoplasmic reticulum membrane vesicles isolated from Wistar and TR(-) rat liver. Time-dependent and bidirectional transport was demonstrated for taurocholate, showing higher uptake rates in SER than RER vesicles. For estradiol-17β-glucuronide a fast time-dependent efflux with similar efficiencies from SER and RER but no clear protein-mediated uptake was shown, indicating an asymmetric transport system for this substrate. Estrone-3-sulfate uptake was time-dependent and higher in SER than in RER vesicles. Inhibition of steroidsulfatase mediated estrone-3-sulfate hydrolysis decreased estrone-3-sulfate uptake but had no effect on taurocholate or estradiol-17β-glucuronide transport. Based on inhibition studies and transport characteristics, three different transport mechanisms are suggested to be involved in the transport of taurocholate, estrone-3-sulfate and estradiol-17β-glucuronide across the ER membrane.

Keywords: Biotransformation; Endoplasmic reticulum; Estradiol-17β-glucuronide; Estrone-3-sulfate; Liver; Transport.

Graphical abstract

Fig. 1

Western blot analysis of Cyp3a2, Sec61α, Oatp1a1, Ntcp, Mrp2, and PDI in SER and RER isolated from Wistar and TR⁻ rat livers. 5 μg (Cyp3a2, Mrp2), 50 μg (Sec61α) or 100 μg (Oatp1a1, Ntcp) protein was loaded per lane and separated by SDS-PAGE as given in section 2.4. Samples were probed against Cyp3a2, Sec61α, Oatp1a1, Ntcp, Mrp2, and PDI as described in 2.4. PDI was used as loading control in SER and RER fractions. Basolateral membrane fraction (BLM) isolated from Sprague Dawley rat liver was used as positive control for Oatp1a1 and Ntcp expression. Canalicular (CM) membrane fraction isolated from Sprague Dawley rat liver was used as positive control for Mrp2 expression. The figure shows a representative Western blot of at least two independent SER and RER isolations.

Fig. 2

TC uptake (A, B, C) and efflux (D) in SER and RER of Wistar and TR⁻ rat liver. A: Uptake of 1 μM TC was determined in SER and RER fractions after different time points of incubation at 37 °C at an inward positive membrane potential as described in section 2.5.1. Data are shown for a representative sample of 3 independent experiments where uptake is calculated as mean pmol/mg protein ± SD from measurements at least in triplicates. B: Uptake of 1 μM TC was determined in SER and RER fractions after 30 sec of incubation at an inward positive (+) or negative (-) membrane potential as described in section 2.5.1.1. Data are shown for a representative sample of 3 independent experiments where uptake is calculated as mean pmol/mg protein ± SD from measurements at least in triplicates. Statistically significant difference in TC uptake between an inward positive and negative membrane potential are indicated by asterisks as followed: ** for p < 0.01 and *** for p < 0.001. C: Uptake of 1 μM TC was determined in SER, RER fractions and basolateral membrane (BLM) after 30 sec of incubation at 37 °C in presence and absence of sodium as described in section 2.5.1.4. Data are shown for a representative sample of 2 independent experiments. Data show TC uptake in absence of sodium given as % of uptake measured in presence of sodium. Values are given as means ± SD from measurements at least in triplicates. Statistically significant difference in TC uptake between ER fractions and basolateral membrane vesicles are indicated by asterisks as followed: * for p < 0.05 and ** for p < 0.01. D: Efflux of TC out of SER and RER vesicles preloaded with 1 μM TC was measured at an inward positive membrane at 30 sec of incubation at 37 °C as described in 2.5.2. Data (mean ± SD from measurements in triplicates) are shown for a representative sample of 2 independent experiments.

Fig. 3

Osmolarity plot for TC (A) and E17βG (B) in SER of Wistar rat liver. Uptake of TC (1 μM) or E17βG (5 μM) was measured after an incubation time of 10 min at 37 °C at an inward positive membrane potential as described in 2.5.1.5. Outside osmolarity (Osm) was increased by adding sucrose ranging from 50 to 800 mM. Data (mean ± SD and linear regression analysis) are shown for a representative sample of 2 independent experiments.

Fig. 4

Time-dependent uptake (A) and efflux (B) of E17βG in SER and RER of Wistar and TR⁻ rat liver. A: Uptake of 5 μM E17βG was determined in SER and RER fractions after different time points of incubation at 37 °C at an inward positive membrane potential as described in 2.5.1. Data are shown for a representative sample of 3 independent experiments where uptake is calculated as mean pmol/mg protein ± SD from measurements at least in triplicates. B: Effux of E17βG out of E17βG (5 μM) preloaded vesicles was measured at an inward positive membrane potential after different time points at 37 °C as described in 2.5.2. Data (mean ± SD from measurements in triplicates) are shown for a representative sample of 2 independent experiments.

Fig. 5

Uptake (A, B) and osmolarity blot (C) of E3S. A: Time-dependent uptake of 5 μM E3S was determined in SER and RER fractions of Wistar and TR⁻ rat liver after different time points of incubation at 37 °C at an inward positive membrane potential as described in 2.5.1. Uptake is calculated as mean pmol/mg protein ± SD from measurements at least in triplicates. Data are shown for a representative sample of 2 independent experiments. B: Uptake of 5 μM E3S was determined in SER and RER fractions of Wistar and TR⁻ rat liver after 10 sec of incubation at an inward positive (+) or negative (-) membrane potential as described in section 2.5.1.1. Data are shown for a representative sample of 2 independent experiments where uptake is calculated as mean pmol/mg protein ± SD from measurements in triplicates. Statistically significant difference in TC uptake between an inward positive and negative membrane potential are indicated by asterisks as followed: * for p < 0.05 and ** for p < 0.01. C: Uptake of E3S (5 μM) was measured in SER of Wistar rat liver after an incubation time of 10 min at 37 °C at an inward positive membrane potential as described in 2.5.1.5. Outside osmolarity (Osm) was increased by adding sucrose ranging from 50 to 800 mM. Data (mean ± SD and linear regression analysis) are shown for a representative sample of 2 independent experiments.

Fig. 6

Autoradiogram (A-D) and time-dependent uptake (E) of E3S in presence (B, D, E) and absence (A, C, E) of the STS inhibitor Stx64. A-D: Wi SER (A, B) and RER (C, D), either not preincubated (A, C) or preincubated with 1 μM Stx64 (B, D), were incubated for 0, 15 sec, 30 sec, 2 min, 5 min and 10 min with 5 μM E3S and processed as described in section 2.6. After chromatography, X-ray film was exposed for 1 month. S, incubation buffer containing 5 μM E3S as standard. E: Uptake of 5 μM E3S was determined in SER and RER fractions either without (light grey) or with (dark grey) Stx64 (1 μM) preincubation after different time points of incubation at 37 °C at an inward positive membrane potential as described in 2.5.1. Data are shown for a representative sample of 2 independent experiments. Uptake is calculated as mean pmol/mg protein ± SD from measurements at least in triplicates.

Fig. 7

Schematic overview of postulated transport systems (T1, T2, T3) for E17βG, E3S, and TC in ER vesicles. Transport and inhibitory effect (⊥) of the different compounds are indicated by the different colors used. E, estradiol; E17βG, estradiol-17β-glucuronide; E3S, estrone-3-sulfate; TC, taurocholate; UDP-GA, UDP-glucuronic acid; CYPs, cytochrome P450s; UGTs, UDP-glucuronosyltransferases, STS, steroidsulfatase.