Shikano Naoto, Kanai Yoshikatsu, Kawai Keiichi, Ishikawa Nobuyoshi, Endou Hitoshi
Department of Radiological Sciences, Ibaraki Prefectural University of Health Sciences, Ibaraki, Japan.
J Nucl Med. 2004 Jan;45(1):80-5.
Recently, complementary DNA (cDNA) encoding a p-aminohippurate (PAH) transporter designated rat organic anion transporter 1 (OAT1) was isolated. OAT1, a multispecific organic anion transporter at the basolateral membrane, is exclusively expressed in the middle segment of the proximal tubule in the rat kidney. It has been proposed that OAT1 is indirectly involved in PAH uptake via the Na(+) dicarboxylate cotransporter. In this study, in molecular biologic experiments using OAT1-expressing Xenopus laevis oocytes, we obtained evidence that (99m)Tc-mercaptoacetylglycylglycylglycine (MAG3) is transported via OAT1.
Capped OAT1 complementary RNA (cRNA) was synthesized from library plasmid cDNA linearized with BamHI using in vitro transcription. Defolliculated oocytes were injected with 10 ng of OAT1 cRNA. Two to 3 d after injection, uptake of (99m)Tc-MAG3 was measured using ND96 solution containing 18.5 kBq of (99m)Tc-MAG3. Before the uptake experiments, OAT1-expressing oocytes were preincubated for 2 h with 1 mmol/L glutarate (a dicarboxylate), to generate an outwardly directed glutarate gradient. Then, after incubation for 60 min at room temperature, radioactivity of oocytes was determined. For the inhibition experiments, uptake was assessed in the absence or presence of inhibitor: 2 mmol/L of PAH, o-iodohippurate (OIH), probenecid, 3,5-diiodo-4-pyridone-N-acetate (iodopyracet), furosemide, ethacrynic acid, glucoheptonate, maleic acid, L-Tyr, or tetraethylammonium (TEA) or 0.1 mmol/L of 2,4-dinitrophenol (DNP).
Na(+) had a significant effect on (99m)Tc-MAG3 uptake (P < 0.05). Accumulated glutarate stimulated simultaneous (99m)Tc-MAG3 uptake and glutarate excretion (P < 0.001). The following compounds significantly inhibited (99m)Tc-MAG3 uptake: PAH, 8.5% +/- 16.2% of (99m)Tc-MAG3 uptake in the absence of an inhibitor; OIH, 26.4% +/- 21.7%; probenecid, 29.1% +/- 12.4%; iodopyracet, 15.8% +/- 7.9%; furosemide, 30.5% +/- 15.7%; ethacrynic acid, 21.6% +/- 10.6%; glucoheptonate, 35.6% +/- 22.6%; and maleic acid, 60.1% +/- 18.7%. (99m)Tc-MAG3 accumulation in Xenopus laevis oocytes was not significantly inhibited by TEA, L-Tyr, or DNP.
The following substances had a cis-inhibitory effect on (99m)Tc-MAG3 transport: PAH, OIH, probenecid, iodopyracet, furosemide, ethacrynic acid, and glucoheptonate. Glutarate had a trans-stimulative effect on (99m)Tc-MAG3 transport. (99m)Tc-MAG3 acts as a substrate of OAT1, an organic anion/dicarboxylate exchanger.
最近，编码一种名为大鼠有机阴离子转运体1（OAT1）的对氨基马尿酸（PAH）转运体的互补DNA（cDNA）被分离出来。OAT1是一种位于基底外侧膜的多特异性有机阴离子转运体，仅在大鼠肾脏近端小管的中段表达。有人提出，OAT1通过Na(+)二羧酸共转运体间接参与PAH的摄取。在这项研究中，在使用表达OAT1的非洲爪蟾卵母细胞进行的分子生物学实验中，我们获得了证据，表明（99m）锝-巯基乙酰甘氨酰甘氨酰甘氨酸（MAG3）通过OAT1转运。
使用体外转录从用BamHI线性化的文库质粒cDNA合成带帽的OAT1互补RNA（cRNA）。将去滤泡的卵母细胞注射10 ng的OAT1 cRNA。注射后2至3天，使用含有18.5 kBq（99m）Tc-MAG3的ND96溶液测量（99m）Tc-MAG3的摄取。在摄取实验之前，将表达OAT1的卵母细胞与1 mmol/L戊二酸（一种二羧酸）预孵育2小时，以产生外向的戊二酸梯度。然后，在室温下孵育60分钟后，测定卵母细胞的放射性。对于抑制实验，在不存在或存在抑制剂的情况下评估摄取：2 mmol/L的PAH、邻碘马尿酸（OIH）、丙磺舒、3,5-二碘-4-吡啶酮-N-乙酸（碘吡啦啥）、呋塞米、依他尼酸、葡庚糖酸盐、马来酸、L-酪氨酸或四乙铵（TEA）或0.1 mmol/L的2,4-二硝基苯酚（DNP）。
Na(+)对（99m）Tc-MAG3的摄取有显著影响（P < 0.05）。积累的戊二酸刺激同时的（99m）Tc-MAG3摄取和戊二酸排泄（P < 0.001）。以下化合物显著抑制（99m）Tc-MAG3的摄取：PAH，在不存在抑制剂的情况下为（99m）Tc-MAG3摄取的8.5% +/- 16.2%；OIH，26.4% +/- 21.7%；丙磺舒，29.1% +/- 12.4%；碘吡啦啥，15.8% +/- 7.9%；呋塞米，30.5% +/- 15.7%；依他尼酸，
