STUDIES OF THE BIOGENIC-AMINE TRANSPORTERS .5. DEMONSTRATION OF 2 BINDING-SITES FOR THE COCAINE ANALOG [I-125] RTI-55 ASSOCIATED WITH THE 5-HT TRANSPORTER IN RAT-BRAIN MEMBRANES

Earlier work characterized the binding of the high-affinity cocaine an alog 3 beta-(4-(125)iodophenyl)-tropane-2-carboxylic acid methyl ester ([I-125]RTI-55) to membranes prepared from rat caudate. That investig ation demonstrated that [I-125]RTI-55-labeled serotonin (5-HT) transpo rters in addition to dopamine (DA) transporters and resolved [I-125]RT I-55 binding to 5-HT transporters into two distinct components. In the present study, we characterized [I-125]RTI-55 binding to membranes pr epared from whole rat brain minus caudate. The first series of experim ents established that [I-125]RTI-55 labels both DA and 5-HT transporte rs and that 50 nM paroxetine and either 1000 nM 1-[2-(diphenyl methoxy )ethyl]-4-(3-phenylpropyl)homopiperazine (LR1111) or 500 nM (RTI-120) could be used to block [I-125]RTI-55 binding to the 5-HT and DA transp orters, thereby generating selective assay conditions for the DA and 5 -HT transporters, respectively. Selective lesioning of dopaminergic an d serotonergic neurons with intracerebroventricular 6-hydroxydopamine and 5,7-dihydroxytryptamine selectively decreased [I-125]RTI-55 bindin g to DA and 5-HT transporters, respectively, thereby confirming the se lectivity of the assay conditions. The ligand-selectivity pattern of t he whole brain minus caudate 5-HT transporter correlated significantly with that of the caudate 5-HT transporter, although there were some s triking differences for selected test agents. Additional experiments r esolved [I-125]RTI-55 binding to the 5-HT transporter into two compone nts. A ligand-selectivity analysis of the two components failed to ide ntify a highly selective test agent. In summary, the major findings of the present study are that [I-125]RTI-55 labels both DA and 5-HT tran sporters 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 [I-125]RTI-55 binding t o the 5-HT transporter can be resolved into two similar components.