2 research outputs found
Expression of rat liver cell membrane transporters for thyroid hormone in Xenopus laevis oocytes
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 x h in water-injected oocytes to 9.2 +/- 0.65 fmol/oocyte x 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
microM 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