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-isothiocyanatophenyl)ethyl]- piperazine} to inhibit [3H]-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 Vmax and KM. 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-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. 4. One day after intrastriatal injection, DEEP-NCS elicited similar dose-dependent decreases in ex vivo dopamine uptake and mazindol binding (ID50=6.9-8 ng striatum-1). Changes in KM and Vmax for ex vivo dopamine transport 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 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. 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 suggests that the physiological half-life of the rat striatal DAT is close to 6 days.
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UPRESA C.N.R.S. 6036, IFRMP 23, Site Universitaire du Madrillet U.F. R. de Médecine & Pharmacie, 76800, Saint Etienne du Rouvray, France.
Syringas
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Leblond
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Costentin
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Bonnet
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D000818:Animals; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D002794:Choline / Q000378:metabolism; D002997:Clomipramine / Q000494:pharmacology; D004298:Dopamine / Q000378:metabolism; D050483:Dopamine Plasma Membrane Transport Proteins; D018765:Dopamine Uptake Inhibitors / Q000008:administration & dosage / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D006207:Half-Life; D066298:In Vitro Techniques; D008297:Male; D008454:Mazindol / Q000378:metabolism; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D009627:Nomifensine / Q000494:pharmacology; D009638:Norepinephrine / Q000378:metabolism; D010879:Piperazines / Q000008:administration & dosage / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012701:Serotonin / Q000378:metabolism; D017367:Selective Serotonin Reuptake Inhibitors / Q000494:pharmacology; D013378:Substantia Nigra / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D013574:Synaptosomes / Q000187:drug effects / Q000378:metabolism
D002352:Carrier Proteins; D002794:Choline; D002997:Clomipramine; D004298:Dopamine; D050483:Dopamine Plasma Membrane Transport Proteins; D018765:Dopamine Uptake Inhibitors; D008454:Mazindol; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D009627:Nomifensine; D009638:Norepinephrine; D010879:Piperazines; D012333:RNA, Messenger; D012701:Serotonin; D017367:Selective Serotonin Reuptake Inhibitors; C400151:1-(2-(diphenylmethoxy)ethyl-4-2-(4-isothiocyanatophenyl)ethyl)piperazine; C493216:Slc6a3 protein, rat