title: Evidence for the sequential formation of two complexes between an uptake inhibitor, GBR 12783 [1-[2-(diphenylmethoxy)ethyl]-4-(3-phenyl-2-propenyl)piperazine], and the neuronal transporter of dopamine.
abstract: Incubation of a crude synaptosomal fraction from rat striatum with GBR 12783 at 37 degrees C produced an inhibition of the specific uptake of [3H]dopamine that increased with time. The inhibition increased when GBR 12783 was present during preincubation and incubation (IC50 = 1.85+/-0.1 nM) instead of incubation alone (IC50 = 25+/-3.5 nM). Time-course studies of uptake inhibition demonstrated that a first collision transporter-inhibitor complex (TI) was formed immediately after addition of GBR 12783 so that the initial uptake velocity (V0) decreased for increasing concentrations of inhibitor (Ki > or = 20 nM). TI slowly isomerized to a more stable complex TI* (Ki* < or = 5 nM) with a value of t1/2 = 20-270 s. Fits of data to model 2 in which the steady-state uptake (VS) is set to zero were generally preferred, suggesting that formation of TI* could tend to irreversibility, as a consequence of a very low reverse isomerization. As expected, k, V0, and VS tended to steady-state values in an asymptotic manner for high concentrations of GBR 12783. GBR 12783 at 2.5 nM produced a mixed inhibition of the uptake, with an increase in KM and a decrease in Vmax; these effects were improved for 10 nM GBR 12783 and at 20 degrees C. These results are discussed in relation to previous data concerning [3H]GBR 12783 binding. The present work gives the first experimental demonstration that dopamine uptake blockers can act according to a two-step mechanism of inhibition; this is of great interest, because these inhibitors can oppose the effects of cocaine or amphetamine on the transporter according to a reaction that is partly nondependent on the concentration of the abused agent.
authors.affiliation: UPRESA CNRS 6036, IFRMP 23, UFR de Médecine et Pharmacie, Saint Etienne du Rouvray, France.
authors.forename: J C
authors.initials: JC
authors.lastname: Do-Régo
authors.forename: H
authors.initials: H
authors.lastname: Hue
authors.forename: J
authors.initials: J
authors.lastname: Costentin
authors.forename: J J
authors.initials: JJ
authors.lastname: Bonnet
mesh_terms: D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002352:Carrier Proteins / Q000378:metabolism*; D050483:Dopamine Plasma Membrane Transport Proteins; D018765:Dopamine Uptake Inhibitors / Q000378:metabolism / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D007700:Kinetics; D008297:Male; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D009474:Neurons / Q000737:chemistry* / Q000378:metabolism*; D010879:Piperazines / Q000378:metabolism / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013574:Synaptosomes / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D013997:Time Factors
chemical_list: D002352:Carrier Proteins; D050483:Dopamine Plasma Membrane Transport Proteins; D018765:Dopamine Uptake Inhibitors; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D010879:Piperazines; C039737:1-(2-(diphenylmethoxy)ethyl)-4-(3-phenyl-2-propenyl)piperazine