title: Studies of the biogenic amine transporters. V. Demonstration of two binding sites for the cocaine analog [125I]RTI-55 associated with the 5-HT transporter in rat brain membranes.
abstract: Earlier work characterized the binding of the high-affinity cocaine analog 3 beta-(4-125iodophenyl)-tropane-2-carboxylic acid methyl ester ([125I]RTI-55) to membranes prepared from rat caudate. That investigation demonstrated that [125I]RTI-55-labeled serotonin (5-HT) transporters in addition to dopamine (DA) transporters and resolved [125I]RTI-55 binding to 5-HT transporters into two distinct components. In the present study, we characterized [125I]RTI-55 binding to membranes prepared from whole rat brain minus caudate. The first series of experiments established that [125I]RTI-55 labels both DA and 5-HT transporters and that 50 nM paroxetine and either 1000 nM 1-[2-(diphenylmethoxy)ethyl]-4-(3-phenylpropyl)homopiperazine (LR1111) or 500 nM (RTI-120) could be used to block [125I]RTI-55 binding to the 5-HT and DA transporters, thereby generating selective assay conditions for the DA and 5-HT transporters, respectively. Selective lesioning of dopaminergic and serotonergic neurons with intracerebroventricular 6-hydroxydopamine and 5,7-dihydroxytryptamine selectively decreased [125I]RTI-55 binding to DA and 5-HT transporters, respectively, thereby confirming the selectivity of the assay conditions. The ligand-selectivity pattern of the whole brain minus caudate 5-HT transporter correlated significantly with that of the caudate 5-HT transporter, although there were some striking differences for selected test agents. Additional experiments resolved [125I]RTI-55 binding to the 5-HT transporter into two components. A ligand-selectivity analysis of the two components failed to identify a highly selective test agent. In summary, the major findings of the present study are that [125I]RTI-55 labels both DA and 5-HT transporters in membranes prepared from whole brain minus caudate, that 50 nM paroxetine and either 1000 nM LR1111 or 500 nM RTI-120 can be used as a blocking agent to generate selective assay conditions for the DA and 5-HT transporters, respectively, and that [125I]RTI-55 binding to the 5-HT transporter can be resolved into two similar components.
authors.affiliation: Clinical Psychopharmacology Section, IRP, NIDA, NIH, Baltimore, Maryland, USA.
authors.forename: M L
authors.initials: ML
authors.lastname: Silverthorn
authors.forename: C M
authors.initials: CM
authors.lastname: Dersch
authors.forename: M H
authors.initials: MH
authors.lastname: Baumann
authors.forename: J L
authors.initials: JL
authors.lastname: Cadet
authors.forename: J S
authors.initials: JS
authors.lastname: Partilla
authors.forename: K C
authors.initials: KC
authors.lastname: Rice
authors.forename: F I
authors.initials: FI
authors.lastname: Carroll
authors.forename: K M
authors.initials: KM
authors.lastname: Becketts
authors.forename: A
authors.initials: A
authors.lastname: Brockington
authors.forename: R B
authors.initials: RB
authors.lastname: Rothman
mesh_terms: D000818:Animals; D001381:Azepines / Q000494:pharmacology; D001665:Binding Sites; D001923:Brain Chemistry; D002352:Carrier Proteins / Q000032:analysis*; D003042:Cocaine / Q000031:analogs & derivatives* / Q000378:metabolism; D004298:Dopamine / Q000378:metabolism*; D050483:Dopamine Plasma Membrane Transport Proteins; D007457:Iodine Radioisotopes; D008297:Male; D008562:Membrane Glycoproteins / Q000032:analysis*; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D051381:Rats; D017207:Rats, Sprague-Dawley; D012701:Serotonin / Q000378:metabolism*; D050486:Serotonin Plasma Membrane Transport Proteins
chemical_list: D001381:Azepines; D002352:Carrier Proteins; D050483:Dopamine Plasma Membrane Transport Proteins; D007457:Iodine Radioisotopes; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D050486:Serotonin Plasma Membrane Transport Proteins; C493232:Slc6a4 protein, rat; C081723:1-(2-(2-(diphenyl)methoxy)ethyl)-4-(3-phenylpropyl)homopiperazine; D012701:Serotonin; C069723:2beta-carbomethoxy-3beta-(4-iodophenyl)tropane; D003042:Cocaine; D004298:Dopamine