NBD-TMA: a novel fluorescent substrate of the peritubular organic cation transporter of renal proximal tubules

Traditionally, the measurement of transport activity has employed radiolabe led compounds. The resulting experimental procedures do not measure transpo rt in real time and are limited in temporal and spatial resolution. The use of epifluorescence microscopy provides the ability to measure transport ac tivity in real time with high temporal and spatial resolution. Using epiflu orescence microscopy we characterized the transport of the fluorescent orga nic cation, [2-(4-nitro-2,1,3-benzoxadiazol-7-yl)aminoethyl]trimethylammoni um (NBD-TMA(+), MW 266). NBD-TMA(+) has structural characteristics common t o other secreted organic cations and is fluorescent (lambda(ex)=458 nm; lam bda(em)=530 nm). The excitation and emission spectra are insensitive to cha nges in [Cl-] and minimally sensitive to pH in the physiologically relevant range (pH 5.0-7.4). A microscope equipped with a photon-detection system w as used to measure accumulation of NBD-TMA(+) by isolated rabbit renal prox imal tubules. Accumulation of NBD-TMA(+) by proximal tubules was time depen dent and saturable (Michaelis-Menten constant K-m 12 mu M). Proximal tubule accumulation of NBD-TMA(+) was inhibited by the organic cations tetraethyl ammonium (TEA(+)) (apparent inhibitory constant K-app(TEA) 134 mu M), cimet idine, and N-1-methylnicotinamide (NMN). Our experimental results provide s trong evidence that NBD-TMA(+) is transported by one or more of the basolat eral organic cation transporters involved in the renal secretion of this ch emical class of compound. This fluorescent substrate provides a sensitive m eans of investigating organic cation transport.