CORTISOL UPTAKE IN HUMAN OAT EXPRESSING Xenopus laevis OOCYTES

Although many reports have been published on the inhibitory effect of steroids on organic anion transporters, so far none of these transporters has been checked for a direct transport of steroids like cortisol (Beery et al. 2003; Hagenbuch et al. 2003;

Inaba et al. 1974; Whitehouse et al. 1971). Therefore we investigated, if human OAT1, OAT3, and OAT4 are capable of transporting cortisol. For this purpose, we expressed human OAT1, OAT2, OAT3, and OAT4 in oocytes and performed uptake experiments with radiolabeled cortisol.

4.5.1 Radiolabeled cortisol uptake in human OAT1 and OAT2 expressing Xenopus laevis oocytes

The human OAT1 expressing oocytes exhibited a 27-fold greater [³H]PAH uptake than water-injected oocytes: 100 ± 9.2% (P < 0.0001) versus 3.7 ± 0.1% as shown in figure 4.16, panel A. In contrast, hOAT1 expressing oocytes did not show any significant transport of [³H]cortisol as compared to water-injected control oocytes:

100 ± 2.7% versus 100.7 ± 5.5% (B). These results demonstrate that hOAT1 protein was functionally expressed in oocytes, but does not transport cortisol.

Human OAT2 expressing oocytes were subjected to [¹⁴C]salicylic acid uptake (a potent substrate of hOAT2) and [³H]cortisol. There was neither significant uptake for [¹⁴C]salicylic acid nor for [³H] cortisol. We conclude from the results that there was no functional protein expression by the available clone of hOAT2. Hence, we were not able to document the ability of hOAT2 to transport cortisol.

Figure 4.16 hOAT1 mediated uptake in Xenopus laevis oocytes. Three days after injection with either human OAT1 cRNA, or water, the uptake of (A) [³H]PAH (5 µCi/ml, 1 µM), and (B) [³H]cortisol (1.4 µCi/ml, 14 nM) was performed for 1 h in ORI. Data represent means ± SEM of three independent experiments with 7-11 oocytes/group in each experiment.

In each experiment, uptake of [³H]PAH or [³H]cortisol by hOAT1 cRNA-injected oocytes was set to (100%). The significance was calculated by student’s t-test against water-injected oocytes (***, P < 0.0001; NS = not significant).

4.5.2 Radiolabeled cortisol uptake experiments in human OAT4 expressing Xenopus laevis oocytes

RT-PCR signals for human OAT4 were present both in NCI-H295R cells as well as in

Figure 4.17 OAT4 mediated uptake experiments in Xenopus laevis oocytes. Three days after injection with either human OAT4 cRNA, or water, the uptake of (A) [³H]DHEAS (0.74 µCi/ml, 10 nM), and (B) [³H]cortisol (1.4 µCi/ml, 14 nM) was performed for 1 h in ORI. Data represent means ± SEM of four independent experiments with 6-10 oocytes/group in each experiment. In each experiment radiolabeled uptake in hOAT4 cRNA-injected oocytes was set to 100%. The significance was calculated by student’s t-test against water-injected oocytes (***, P < 0.0001; NS = not significant).

human adrenal tissues. Human OAT4 expressing oocytes were checked for the typical substrate [³H]DHEAS. hOAT4 showed a 22-fold greater [³H]DHEAS transport than water-injected oocytes: 100 ± 13.1% (P < 0.0001) versus 4.5 ± 0.3% (Figure 4.17, panel A). When human OAT4 oocytes were incubated with [³H]cortisol, no significant uptake was observed over water-injected oocytes: 100 ± 3.2% versus 109.4 ± 5.4% (B).

4.5.3 Radiolabeled cortisol uptake in human OAT3 expressing

Human OAT3 expressing Xenopus laevis oocytes showed a significant [³H]estrone sulfate uptake compared to water-injected oocytes: 100 ± 4.6% (P < 0.0001) versus 1.6 ± 0.1% (Figure 4.18, panel A). In comparison to hOAT1 and hOAT4, hOAT3 expressing oocytes exhibited a significant [³H]cortisol transport, almost fourfold over water-injected oocytes: 100 ± 2.8% (P < 0.0001) versus 26.9 ± 1.2% (Figure 4.18, panel B). These results demonstrate for the first time a hOAT3-mediated cortisol transport.

Figure 4.18 OAT3 mediated uptake experiments in Xenopus laevis oocytes. Three days after injection with either human OAT3 cRNA, or water, the uptake of (A) [³H]estrone sulfate (0.44 µCi/ml, 10 nM), and [³H]cortisol (1.4 µCi/ml, 14 nM) was performed for 1 h in ORI. Data represent means ± SEM of more than six independent experiments with 9-12 oocytes/group in each experiment. In each experiment uptake of [³H]estrone sulfate or [³H]cortisol by hOAT3 cRNA-injected oocytes was set to (100%). The significance was calculated by student’s t-test against water-injected oocytes (***, P < 0.0001).

4.5.4 Concentration dependent inhibition of radiolabeled cortisol uptake by unlabeled cortisol in hOAT3 expressing Xenopus

We checked whether this [³H]cortisol transport by hOAT3 can be inhibited by unlabeled cortisol. For this purpose, [³H]cortisol uptake into hOAT3 was carried out in the presence of different concentrations of unlabeled cortisol and control without exposure to unlabeled cortisol. The results are shown in figure 4.19. The apparent K_t value of hOAT3 for cortisol was 2.4 ± 0.6 µM in oocytes.

Figure 4.19 Inhibition of [³H]cortisol uptake by unlabeled cortisol in hOAT3 expressing Xenopus laevis oocytes. Three days after injection with either human OAT3 cRNA or water, the [³H]cortisol (1.4 µCi/ml, 14 nM) uptake was performed for 1 h in the presence of 0.1 µM, 1.0 µM, 10 µM, 25 µM, or 50 µM unlabeled cortisol. Data represent means ± SEM of four independent experiments with 8-10 oocytes/group in each experiment. Apparent Kt value was calculated according to the Hill equation using SigmaPlot 2001.

4.5.5 Inhibition of radiolabeled estrone sulfate uptake in human OAT3 expressing Xenopus laevis oocytes by unlabeled cortisol

As estrone sulfate is a good substrate of hOAT3, we checked if [³H]estrone sulfate uptake could be inhibited by unlabeled cortisol. [³H]estrone sulfate uptake was conducted in hOAT3 cRNA injected oocytes, in the presence of different concentrations of unlabeled cortisol or absence of unlabeled cortisol (control), as shown in the figure 4.20. The calculated IC50 value for cortisol was 15.6 ± 2.3 µM.

Figure 4.20 Inhibition of [³H]estrone sulfate uptake by unlabeled cortisol in hOAT3 expressing Xenopus laevis oocytes. Three days after injection with either human OAT3 cRNA or water, the [³H]estrone sulfate (0.44 µCi/ml, 10 nM) uptake was performed for 1 h in the presence of 0.1 µM, 0.5 µM, 1.0 µM, 10 µM, or 50 µM unlabeled cortisol. Data represent means ± SEM of four independent experiments with 8-10 oocytes/group in each experiment.

IC50 value was calculated according to the Hill equation using SigmaPlot 2001.

DHEAS is an important steroid hormone, which is also transported by OAT3, OAT4 and many other OATPs. Human OAT3-expressing Xenopus laevis oocytes showed a sixtyfold higher [³H]DHEAS uptake as compared to water-injected oocytes: 100 ± 5.0% (P < 0.0001) versus 1.6 ± 0.9%. The [³H]DHEAS uptake was inhibited by 1 mM unlabeled DHEAS to 1.8 ± 0.2% (P < 0.0001). The cis-inhibition of [³H]DHEAS uptake with 50 µM and 100 µM unlabeled cortisol showed significant reduction to 18.2 ± 1.8% (P < 0.0001), and 11.6 ± 0.8% (P < 0.0001), respectively. These results are similar to the results we obtained from [³H]estrone sulfate uptake and its inhibition with cortisol, confirming an affinity of hOAT3 for cortisol.

4.6 CORTISOL UPTAKE IN HUMAN OATP EXPRESSING