D. Bednarczyk et al., NBD-TMA: a novel fluorescent substrate of the peritubular organic cation transporter of renal proximal tubules, PFLUG ARCH, 440(1), 2000, pp. 184-192
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.