Recovery of dopamine neuronal transporter but lack of change of its mRNA in substantia nigra after inactivation by a new irreversible inhibitor characterized in vitro and ex vivo in the rat

In vitro , the ability of DEEP‐NCS {1‐[2‐(diphenylmethoxy)ethyl]‐4‐[2‐(4‐isothiocyanatophenyl)ethyl]‐piperazine} to inhibit [ 3 H]‐dopamine uptake by rat striatal synaptosomes was concentration‐dependent and inversely related to the protein concentration. This inhibition was irreversible and resulted from changes in V max and K M . DEEP‐NCS was less potent on noradrenaline, serotonin and choline transport. One day after intrastriatal injections of DEEP‐NCS (100 and 1000 pmol) in 20% dimethylsulphoxide, moderate decreases in the ex vivo dopamine uptake were observed in synaptosomes obtained from striatum injected with DEEP‐NCS or solvent, and the contralateral uninjected striatum. In similar conditions, 300 pmol DEEP‐NCS in 45% 2 hydroxypropyl‐γ‐cyclodextrin–0.5% dimethylsulphoxide solution sub‐totally reduced ex vivo dopamine uptake and mazindol binding, and moderately decreased choline and serotonin transport. These reductions were specific to DEEP‐NCS‐injected striata. A clomipramine pretreatment (16 mg kg −1 i.p. 1 h before) was performed in following experiments, since it reduced the DEEP‐NCS‐elicited decrease in serotonin uptake without affecting other indices. One day after intrastriatal injection, DEEP‐NCS elicited similar dose‐dependent decreases in ex vivo dopamine uptake and mazindol binding (ID 50 =6.9‐8 ng striatum −1 ). Changes in K M and V max for ex vivo dopamine transport produced by DEEP‐NCS disappeared according to similar time‐courses. The t ½ for transporter recovery was 6.1 days. This value should correspond to its actual turnover rate in vivo , since no change in transporter mRNA level was observed in substantia nigra ipsilateral to 300 pmol DEEP‐NCS‐injected striatum. The results indicate that DEEP‐NCS behaves as a potent, quite selective, irreversible inhibitor of the DAT, in vitro and in vivo . Its use in vivo suggests that the physiological half‐life of the rat striatal DAT is close to 6 days.British Journal of Pharmacology (1999) 128 , 51–60; doi: 10.1038/sj.bjp.0702784