The mechanism of the 3H-noradrenaline releasing effect of various substrates of uptake1: multifactorial induction of outward transport

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Abstract
Summary The mechanism of action of indirectly acting sympathomimetic amines was studied in the rat vas deferens, after inhibition of vesicular uptake (by reserpine), of MAO (by pargyline) and of COMT (by U-0521). 1. K m-values for the neuronal uptake of 12 substrates were determined as the IC50 of the unlabelled substrate inhibiting the initial rate of neuronal uptake of 0.2 μmol/l 3H-(−)-noradrenaline. The IC50 ranged from 0.35 μmol/l (for (+)-amphetamine) to 44.3 μmol/l (for 5-HT). The V max (determined for 8 substrates) was substrate-dependent. 2. Tissues were loaded with 0.2 μmol/l 3H-(−)-noradrenaline and then washed out with amine-free solution. All 12 substrates of uptake1, induced an outward transport of 3H-noradrenaline, and equieffective concentrations were positively correlated with K m. Moreover, the EC50 for release greatly exceeded K m. It is proposed that this discrepancy between EC50 and K m is indicative of the fact that at least four factors (each one in strict dependence on K m) contribute to the initiation of outward transport of 3H-noradrenaline: a) the appearance of the carrier on the inside of the axonal membrane (facilitated exchange diffusion), b) the co-transport of Na+, c) the co-transport of Cl− (both lowering the K m for 3H-noradrenaline at the inside carrier), and d) inhibition of the re-uptake of released 3H-noradrenaline (through competition for the outside carrier). 3. At least for amezinium, V max. appears to limit the maximum rate of outward transport. 4. For some substrates (especially for the highly lipophilic ones) bell-shaped concentration-release curves were obtained. Apparently, inward diffusion of the substrates can lead to partial saturation of the inside carrier. Moreover, if release is expressed as a FRL (fractional rate of loss), loading with 37 μmol/l 3H-(−)-noradrenaline decreased the releasing effect of various substrates. In this case the inside carrier appears to be partially saturated by the high axoplasmic concentration of 3H-noradrenaline. 5. Very high concentrations (especially of highly lipophilic substrates) were able to induce an additional intraneuronal release mechanism, presumably by increasing the pH inside storage vesicles.

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