Distribution of rat organic anion transporting polypeptide-E (oatp-E) in the rat eye.
OBJECTIVE
To examine the protein and mRNA expression levels of the recently cloned rat multifunctional Na+-independent organic anion transporting polypeptide (rat oatp-E), which is involved in the transport of thyroid hormone in the rat, the distribution and function of this transporter were investigated in the retina.


METHODS
Real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) was performed with gene-specific primers for oatp-E in rat ocular tissues. Western blot analysis was performed by raising a specific antibody against oatp-E in rat ocular tissues. Immunohistochemistry was performed with a specific antibody for oatp-E in paraffin sections of rat eyes. The expression of oatp-E in isolated and cultured rat retinal pigment epithelial (RPE) cells was confirmed by RT-PCR, Western blot analysis, and immunohistochemistry. In addition, oatp-E function was analyzed in cultured rat RPE cells by measuring the uptake of triiodothyronine (T3), which is a known substrate for oatp-E.


RESULTS
Using real-time quantitative RT-PCR, oatp-E mRNA was detected, in order of highest to lowest concentration, in the rat retina, cornea, and ciliary body-iris. A single band for oatp-E was observed by Western blot analysis in the rat brain, retina, cornea, and ciliary body-iris. oatp-E immunostaining was predominantly expressed in the corneal epithelium, in the pigmented and nonpigmented epithelium of the ciliary body, and in the iris of the rat eye. In the rat retina, intense immunostaining was detected in the RPE, inner and outer nuclear layers, ganglion cell layer, and nerve fiber layer. In addition, oatp-E immunoreactivity in cultured rat RPE cells was expressed in the cell membrane and cytoplasm of RPE cells, a finding that was also confirmed by RT-PCR and Western blot analysis. RPE cells, which were shown to express high levels of oatp-E, transported T3 in a saturable and dose-dependent manner. Moreover, this uptake was significantly inhibited by sulfobromophthalein (BSP), an inhibitor of oatp, suggesting that oatp-E may in part contribute to this uptake.


CONCLUSIONS
Results from the present study revealed that rat oatp-E is localized mainly to the corneal epithelium, ciliary body, iris, and retina. Furthermore, the findings appear to suggest that transport of T3 in the RPE may have a functional role for organic anion (i.e., thyroid hormone) transport in the rat eye.
Ito
Aki
A
Department of Ophthalmology, Tohoku University Graduate School of Medicine, Sendai, Japan.
Yamaguchi
Katsuhiro
K
Tomita
Hiroshi
H
Suzuki
Takehiro
T
Onogawa
Tohru
T
Sato
Takeaki
T
Mizutamari
Hiroya
H
Mikkaichi
Tsuyoshi
T
Nishio
Toshiyuki
T
Suzuki
Takashi
T
Unno
Michiaki
M
Sasano
Hironobu
H
Abe
Takaaki
T
Tamai
Makoto
M
D000818:Animals; D017920:Antiporters / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism; D002478:Cells, Cultured; D002924:Ciliary Body; D003315:Cornea / Q000378:metabolism; D005123:Eye / Q000378:metabolism*; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism*; D007150:Immunohistochemistry; D007498:Iris / Q000378:metabolism; D008297:Male; D027361:Organic Anion Transporters; D010857:Pigment Epithelium of Eye / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D012160:Retina / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014284:Triiodothyronine / Q000378:metabolism
D017920:Antiporters; D005136:Eye Proteins; D027361:Organic Anion Transporters; D012333:RNA, Messenger; C478703:Slco4a1 protein, rat; D014284:Triiodothyronine