THE EXTRANEURONAL TRANSPORT MECHANISM FOR NORADRENALINE (UPTAKE2) AVIDLY TRANSPORTS 1-METHYL-4-PHENYLPYRIDINIUM (MPP+)

The corticosterone-sensitive extraneuronal transport mechanism for noradrenaline (uptake2) removes the neurotransmitter from the extracellular space. Recently, an experimental model for uptake2 has been introduced which is based on tissue culture techniques (human Caki-1 cells). The present study describes some properties of uptake2 in Caki-1 cells and introduces a new substrate, i.e., 1-methyl-4-phenylpyridinium (MPP+). Experiments on Caki-1 cells disclosed disadvantages of tritiated noradrenaline as substrate for the investigation of uptake2. The initial rate of H-3-noradrenaline transport [k(in) = 0.58-mu-l/(mg protein . min)] was low compared with other cellular transport systems and intracellular noradrenaline was subject to rapid metabolism (k(O-methylation) = 0.54 min-1). The neurotoxin MPP+ was found to be a good substrate of uptake2. Initial rates of specific H-3-MPP+ transport into Caki-1 cells were saturable, the K(m) being 24-mu-mol/l and the V(max) being 420 pmol/(mg protein . min). The rate constant of specific inward transport was 34 times higher [19.6-mu-l/(mg protein . min)] than that of H-3-noradrenaline. The ratio specific over non-specific transport was considerably higher for H-3-MPP+ (12.6) than for H-3-noradrenaline (3.0). H-3-MPP+ transport into Caki-1 cells was inhibited by various inhibitors of uptake2. The highly significant positive correlation (p < 0.001, r = 0.986, n = 7) between the IC50's for the inhibition of the transport of H-3-noradrenaline and H-3-MPP+, respectively, proves the hypothesis that MPP+ enters Caki-1 cells via uptake2. H-3-MPP+ is taken up via uptake2 not only by Caki-1 cells but also by the isolated perfused rat heart which is another established model of uptake2. Tritiated MPP+ is a new and convenient tool for the investigation of uptake2. The rate constant for inward transport, the factor of accumulation and the ratio specific over non-specific transport are considerably higher for H-3-MPP+ than for H-3-noradrenaline. In uptake studies with H-3-MPP+ inhibition of intracellular noradrenaline-metabolizing enzymes is not necessary. In tissues and tissue cultures which possess fewer uptake2 carriers than Caki-1 cells or the rat heart, the identification and characterization of uptake2 can be expected to be greatly facilitated by the use of H-3-MPP+.