Table 6.
Dopamine uptake inhibition potency and binding affinity of cocaine at N2A neuroblastoma cells as a function of amount of WT DAT plasmid introduced
| WT DAT Plasmid (% of Optimal) | ||
|---|---|---|
| 25 | 100 | |
| IC50 (nM) | ||
| [3H]-DA uptake inhibition | ||
| Cocaine | 476 ± 46 | 789 ± 64a |
| IC50 (nM) | ||
| [3H]-WIN 35,428 inhibition | ||
| Cocaine | 229 ± 34 | 196 ± 11 |
| [3H]-WIN 35,428 binding | ||
| Kd (nM) | 25 ± 2 | 18 ± 3 |
| Bmax (pmol/mg) | 1.3 ± 0.4 | 2.8 ± 0.5a |
a
P < 0.05 versus "25% plasmid" cells for that assay (one-way ANOVA, Newman-Keuls post hoc test
Transient transfections were carried out using 25% or 100% of the optimal amount of WT DAT - pIRES plasmid in 35 mm wells containing confluent monolayers of N2A neuroblastoma cells controlled for passage number. Cocaine inhibition of [3H]-dopamine uptake and [3H]-WIN 35,428 binding, as well as direct [3H]-WIN 35,428 binding, were measured under identical assay conditions 48 hours after N2A cell transfections. Mean ± SEM for 3 experiments.