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

1 In vitro, the ability of DEEP-NCS {1-[2-(diphenylmethoxy)ethyl]-4-[2-(4-i sothiocyanatophenyl)ethyl]-piperazine} to inhibit [H-3]-dopamine uptake by rat striatal synaptosomes was concentration-dependent and inversely related to the protein concentration. This inhibition was irreversible and resulte d from changes in V-max and K-M. DEEP-NCS was less potent on noradrenaline, serotonin and choline transport. 2 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. 3 In similar conditions, 300 pmol DEEP-NCS in 45% 2 hydroxypropyl-gamma-cyc lodextrin - 0.5% dimethylsulphoxide solution sub-totally reduced ex vivo do pamine uptake and mazindol binding, and moderately decreased choline and se rotonin transport. These reductions were specific to DEEP-NCS-injected stri ata. A clomipramine pretreatment (16 mg kg(-1) i.p. 1 h before) was perform ed in following experiments, since it reduced the DEEP-NCS-elicited decreas e in serotonin uptake without affecting other indices. 4 One day after intrastriatal injection, DEEP-NCS elicited similar dose-dep endent decreases in ex vivo dopamine uptake and mazindol binding (ID50 = 6. 9-8 ng striatum(-1)). Changes in K-M and V-max for ex vivo dopamine transpo rt produced by DEEP-NCS disappeared according to similar time-courses. 5 The t(1/2) for transporter recovery was 6.1 days. This value should corre spond to its actual turnover rate in vivo, since no change in transporter m RNA level was observed in substantia nigra ipsilateral to 300 pmol DEEP-NCS -injected striatum. 6 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 su ggests that the physiological half-life of the rat striatal DAT is close to 6 days.