Organic anion transporter 1 (OAT1/SLC22A6) enhances bioluminescence based on d-luciferin–luciferase reaction in living cells by facilitating the intracellular accumulation of d-luciferin

Bioluminescence (BL) imaging based on d-luciferin (d-luc)–luciferase reaction allows noninvasive and real-time monitoring of luciferase-expressing cells. Because BL intensity depends on photons generated through the d-luc–luciferase reaction, an approach to increase intracellular levels of d-luc could improve the detection sensitivity. In the present study, we showed that organic anion transporter 1 (OAT1) is useful, as a d-luc transporter, in boosting the BL intensity in luciferase-expressing cells. Functional screening of several transporters showed that the expression of OAT1 in HEK293?cells stably expressing Pyrearinus termitilluminans luciferase (HEK293/eLuc) markedly enhanced BL intensity in the presence of d-luc. When OAT1 was transiently expressed in HEK293?cells, intracellular accumulation of d-luc was higher than that in control cells, and the specific d-luc uptake mediated by OAT1 was saturable with a Michaelis constant (K_m) of 0.23?μM. The interaction between OAT1 and d-luc was verified using 6-carboxyfluorescein, a typical substrate of OAT1, which showed that d-luc inhibited the uptake of 6-carboxyfluorescein mediated by OAT1. BL intensity was concentration-dependent at steady states in HEK293/eLuc cells stably expressing OAT1, and followed Michaelis–Menten kinetics with an apparent K_m of 0.36?μM. In addition, the enhanced BL was significantly inhibited by OAT1-specific inhibitors. Thus, OAT1-mediated transport of d-luc could be a rate-limiting step in the d-luc–luciferase reaction. Furthermore, we found that expressing OAT1 in HEK293/eLuc cells implanted subcutaneously in mice also significantly increased the BL after intraperitoneal injection of d-luc. Our findings suggest that because OAT1 is capable of transporting d-luc, it can also be used to improve visualization and monitoring of luciferase-expressing cells.     

Identification of Brassinosteroids That Appear to Be Derived from Campesterol and Cholesterol in Tomato Shoots

To obtain information about the biosynthesis of brassinosteroids(BRs) in tomato shoots, we examined endogenous BRs by gas chromatography-massspectrometry. We identified two C²⁸ BRs, namely, castasteroneand 6-deoxocastasterone, and a C²⁷ BR, 28-norcastasterone. Ourfindings suggest that the major BRs in tomato are derived fromcampesterol and cholesterol. ⁴Present address: National Research Institute of Vegetables,Ornamental Plants and Tea, 360 Kusawa, Ano, Mie, 514-23 Japan.