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Expression of Rat Liver Cell Membrane Transporters for Thyroid Hormone in Xenopus laevis Oocytes¹

Departments of Internal Medicine III and Clinical Endocrinology (R.D., E.C.H.F., P.G.J.v.S., E.P.K., M.E.E., T.J.V., G.H.), and Nuclear Medicine (E.P.K.), Erasmus University Medical School, Rotterdam, The Netherlands

Address all correspondence and requests for reprints to: R. Docter, Ph.D., Laboratory for Endocrinology, University Hospital Dijkzigt, Dr. Molewaterplein 40, 3015 GD Rotterdam, The Netherlands.

The present study was conducted to explore the possible use of Xenopus laevis oocytes for the expression cloning of cell membrane transporters for iodothyronines. Injection of stage V–VI X. laevis oocytes with 23 ng Wistar rat liver polyadenylated RNA (mRNA) resulted after 3–4 days in a highly significant increase in [¹²⁵I]T₃ (5 nM) uptake from 6.4 ± 0.8 fmol/oocyte·h in water-injected oocytes to 9.2 ± 0.65 fmol/oocyte·h (mean ± SEM; n = 19). In contrast, [¹²⁵I]T₄ (4 nM) uptake was not significantly stimulated by injection of total liver mRNA. T₃ uptake induced by liver mRNA was significantly inhibited by replacement of Na⁺ in the incubation medium by choline⁺ or by simultaneous incubation with 1 µM unlabeled T₃. In contrast, T₃ uptake by water-injected oocytes was not Na⁺ dependent. Fractionation of liver mRNA on a 6–20% sucrose gradient showed that maximal stimulation of T₃ uptake was obtained with mRNA of 0.8–2.1 kilobases (kb). In contrast to unfractionated mRNA, the 0.7- to 2.1-kb fraction also significantly stimulated transport of T₄, and it was found to induce uptake of T₃ sulfate (T₃S). Because T₃S is a good substrate for type I deiodinase (D1), 2.3 ng rat D1 complementary RNA (cRNA) were injected either alone or together with 23 ng of the 0.8- to 2.1-kb fraction of rat liver mRNA. Compared with water-injected oocytes, injection of D1 cRNA alone did not stimulate uptake of [¹²⁵I]T₃S (1.25 nM). T₃S uptake in liver mRNA and D1 cRNA-injected oocytes was similar to that in oocytes injected with mRNA alone, showing that transport of T₃S is independent of the metabolic capacity of the oocyte. Furthermore, coinjection of liver mRNA and D1 cRNA strongly increased the production of ¹²⁵I^-, showing that the T₃S taken up by the oocyte is indeed transported to the cell interior.

In conclusion, injection of rat liver mRNA into X. laevis oocytes resulted in a stimulation of saturable, Na⁺-dependent T₄, T₃ and T₃S transport, indicating that rat liver contains mRNA(s) coding for plasma membrane transporters for these iodothyronine derivatives.

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