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

Abstract

Human dopamine transporters, stably expressed by human embryonic kidney-293 cells, were reacted with 3beta-(3p-chlorophenyl)tropan-2beta-carboxylic acid p-isothiocyanatophenylethyl ester (RTI-76) under varying conditions. Exposure to RTI-76 (1 microM) at 0 degrees C, followed by extensive wash-out, reduced subsequent binding of the cocaine analog [3H]2beta-carbomethoxy-3beta-(4-fluorophenyl) tropane (WIN 35,428), which was caused by an increase in Kd in the absence of a Bmax change. Exposure to RTI-76 (50 nM(-1) microM) at 37 degrees C, however, caused concentration-dependent reductions in binding Bmax; increases in Kd were observed only at high levels of RTI-76 (0.5-1 microM). The reductions in Bmax are consonant with acylation of transporters, the increases in Kd 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 Bmax at 37 degrees C. Reductions in binding Bmax generated by varying concentrations of RTI-76 up to 300 nM at 37 degrees C correlated with reductions in [3H]dopamine uptake Vmax on a one-to-one basis, with Km 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.

Preferential Increases in Nucleus Accumbens Dopamine after Systemic Cocaine Administration Are Caused by Unique Characteristics of Dopamine Neurotransmission

“…3) (Wightman et al, 1988). Thus, the dominant effect of RTI-76 on DA dynamics was a decrease in V max , which is consistent with the quantitative analysis of the evoked DA signals (Table 2) and binding and uptake assays (Fleckenstein et al, 1996;Wang et al, 2000).…”

“…The effects of RTI-76 were examined with the second experimental design. Because RTI-76 was used in the present study as a noncompetitive inhibitor of DA uptake (Fleckenstein et al, 1996;Wang et al, 2000), it was necessary to perform the voltammetric experiment either 1 or 2 d after drug administration. As a result, predrug data are not available.…”

Pharmacological profile of radioligand binding to the norepinephrine transporter: instances of poor indication of functional activity

“…Condition 4 was as condition 3 but the intact cells were used in the assay instead of broken membrane preparations. Intact NET cell suspensions were prepared as described by us previously for DAT cells (Wang et al, 2000). Briefly, confluent cells in their flasks were rinsed with Hank's solution and suspended in 0.25% (w/v) trypsin-EDTA for less than 2 min.…”

“…The hNET cDNA was cloned by Pacholczyk et al (1991) and the NET cell line was graciously provided by Randy Blakely (Vanderbilt University School of Medicine, Nashville, TN, USA). The general methods for working up and handling the cell preparations have been described by us previously (Li and Reith, 2000;Reith et al, 1996a;Wang et al, 2000). Briefly, NET cells were grown in DMEM medium containing 10% fetal bovine serum, 2 mM l-glutamine, 100 units/ml penicillin/streptomycin, and 250 g/ml G-418 at 37 • C and 5% CO 2 .…”

“…Uptake of tritiated substrate into suspended, intact HEK-293 cells (condition 5) was measured with the overall approach as described by us previously (Wang et al, 2000;Xu et al, 1995). Briefly, cells and inhibitor were preincubated for 5 min at room temperature, [ 3 H]norepinephrine (49.7 Ci/mmol, Dupont New England Nuclear) was added, incubation was continued for exactly 4 min, and the assay was terminated by addition of ice-cold buffer and rapid filtration through Whatman GF/C glass fiber filters, pre-soaked in 0.05% (w/v) poly-l-lysine, with a Brandel 24-pin harvester.…”