Inhibition of norepinephrine transport into synaptic vesicles by amphetamine analogs

The abilities of several amphetamine analogs with restricted conformations to inhibit uptake of [3H]norepinephrine into synaptic vesicles isolated from rat brain cerebral cortex were compared. [3H]Norepinephrine was accumulated in the vesicles with a Km of 3.5 microM and a Vmax of 7.6 pmol/g of tissue per min. This uptake was inhibited by reserpine (IC50, 6.4 nM), amphetamine (IC50, 2.5 microM) and eight amphetamine analogs. 2-Aminotetralin, the most flexible of the analogs (capable of assuming both gauche and anticonformations), was the most potent (IC50, 22 microM). The side chain of amphetamine was held in one of its two low energy conformations [transantiperiplanar (extended) and gauche (folded)]. This was accomplished by using the benzobicyclo[2.2.1]heptane, benzobicylco[2.2.2]octane, or tetrahydroisoquinoline ring systems. The potencies of all of the conformationally defined analogs were reduced with IC50 values of 120 to 370 microM and the potency differences between anti- and gauche conformations were small. These results are in contrast to those obtained by us earlier for inhibition of neuronal reuptake and suggest that vesicular uptake may be more conformationally restrictive than neuronal reuptake. It is possible that: 1) the amphetamine pharmacophore must retain some conformational flexibility for vesicular uptake (hence activity for 2-aminotetralin but not for the rigid analogs); 2) there is another higher energy conformation of amphetamine not present in any of the rigid analogs evaluated that is required for optimal interaction with the vesicular uptake site; or 3) the extra steric bulk of the bridging atoms in the conformational analogs severely interferes with binding at the vesicular uptake site.