Vanoxerine → Dopamine

Temperatur

37 °C

[3H]DA uptake was measured in cultures at day 7, and all the solutions were maintained at 37° C. The growth medium was removed, and the cultures were rinsed twice with 0.25 ml of uptake buffer, which consists of HBSS (Gibco 11201-092) supplemented with 28 mM glucose, 15 mM HEPES, 1 mM ascorbic acid (an antioxidant), and 0.5 nM pargyline (a monoamine oxidase inhibitor). The cultures were then incubated with 0.25 ml of fresh uptake buffer containing 50 nM [3H]DA (NEN/DuPont) for 20 min at 37° C. [3H]DA uptake was stopped by removing the incubation mixture, and cells were then washed twice with 0.5 ml of the uptake buffer at room temperature. To release [3H]DA from the cells, the cultures were lysed with 0.2 ml of 0.1 N NaOH for 1 h at room temperature, the lysate was then added to 1 ml of Microscint-20 (Packard 6013621), and counted for radioactivity in a microplate scintillation counter (Packard TopCount, NXT). Background values were obtained by adding to the uptake buffer 0.5 mM GBR-12909, a specific inhibitor of the high-affinity uptake pump of the dopaminergic neurons, and were usually <5% of the 3H uptake in untreated control cultures. Forskolin (Fk, 25 μM) was employed as a positive control.