RTI-76, an isothiocyanate derivative of a phenyltropane cocaine analog, as a tool for irreversibly inactivating dopamine transporter function in vitro

Autor: Lijuan C. Wang, Maarten E.A. Reith, Frank I. Carroll, Michael J. Kuhar, Berfield Jl
Rok vydání: 2000
Předmět:
Zdroj: Naunyn-Schmiedeberg's Archives of Pharmacology. 362:238-247
ISSN: 1432-1912
0028-1298
DOI: 10.1007/s002100000289
Popis: Human dopamine transporters, stably expressed by human embryonic kidney-293 cells, were reacted with 3β-(3p-chlorophenyl)tropan-2β-carboxylic acid p-isothiocyanatophenylethyl ester (RTI-76) under varying conditions. Exposure to RTI-76 (1 µM) at 0°C, followed by extensive wash-out, reduced subsequent binding of the cocaine analog [3H]2β-carbomethoxy-3β-(4-fluorophenyl)tropane (WIN 35,428), which was caused by an increase in K d in the absence of a B max change. Exposure to RTI-76 (50 nM–1 µM) at 37°C, however, caused concentration-dependent reductions in binding B max; increases in K d were observed only at high levels of RTI-76 (0.5–1 µM). The reductions in B max are consonant with acylation of transporters, the increases in K d with incomplete wash-out observed also for the amine precursor of RTI-76 which lacks the isothiocyanate group for irreversible binding and which did not lower B max at 37°C. Reductions in binding B max generated by varying concentrations of RTI-76 up to 300 nM at 37°C correlated with reductions in [3H]dopamine uptake V max on a one-to-one basis, with K m values showing a tendency towards a small reduction as a function of transporter inactivation, but the potency of WIN 35,428 in inhibiting uptake not showing a change. The results are discussed in the context of possible oligomeric assemblies of dopamine transporters carrying multiple recognition sites for cocaine analogs and dopamine.
Databáze: OpenAIRE
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