反应详情

[3H]DA uptake was performed according to a modification of the previously reported methods (Dwoskin et al., 1999). Striata were homogenized in 20 ml of ice-cold sucrose solution (0.32 M sucrose and 5 mM sodium bicarbonate, pH 7.4) with 12 passes of a teflon-pestle homogenizer (clearance approximately .003 in). The homogenate was centrifuged at 2,000 g, 4° C. for 10 min. The supernatant was centrifuged at 12,000 g, 4° C, for 20 minutes. The resulting pellet was resuspended in 1.5 ml ice-cold assay buffer (in mM; 125 NaCl, 5 KCl, 1.5 KH2PO4, 1.5 MgSO4, 1.25 CaCl2, 10 glucose, 0.1 L-ascorbate, 25 HEPES, 0.1 EDTA and 0.1 pargylin pH 7.4). The final protein concentration was 400 μg/ml. Assays were performed in duplicate in a total vol of 500 μl. Aliquots (50 μl synaptosomal suspension containing 20 pg of protein) were added to assay tubes containing 350 μl buffer and 50 μl of 1 of 9 concentrations (final concentration, 1 nM-1 mM) of analog or vehicle. Synaptosomes were preincubated at 34° C. for 10 min before the addition of 50 μl of [3H]DA (30.1 Ci/mmole, final concentration 10 nM) and accumulation proceeded for 10 min at 34° C. High affinity uptake was defined as the difference between accumulation in the absence and presence of 10 μM GBR 12935. Preliminary studies demonstrated that at 10 minutes [3H]DA uptake is within the linear range of the time-response curve when experiments are performed at 34° C. Accumulation was terminated by addition of 3 ml ice-cold assay buffer containing 1 mM pyrocatechol and rapid filtration through a Whatman GF/B glass fiber filter paper (presoaked with buffer containing 1 mM pyrocatecol) using a Brandel Cell Harvester. The filters were washed 3 times with 3 ml of 10 ml ice-cold buffer containing 2 mM pyrocatechol, and then transferred to scintillation vials and radioactivity determined (Packard Model B1600TR scintillation counter, Meriden, Conn.). Protein concentration was determined using bovine serum albumin as the standard (Bradford, 1976). Competition curves for analog inhibition of [3H]DA uptake were generated. Nonlinear regression analysis was used to fit curves either in the absence or presence of 9 concentrations of analog. IC50 values were corrected for concentration of [3H]DA (Cheng-Prusoff, 1973) to yield true inhibition constants (Ki=IC50/[1+c/Km]), where c is the concentration of free [3H]DA and Km is the concentration of analog at which half maximal [3H]DA uptake is achieved. These values (Ki) were converted to pKi before statistical analysis.

1. waitThe supernatant was centrifuged at 12,000 g, 4° C, for 20 minutes
2. additionAliquots (50 μl synaptosomal suspension containing 20 pg of protein)
3. additionwere added to assay tubes
4. additioncontaining 350 μl buffer
5. concentration50 μl of 1 of 9 concentrations (final
6. concentrationconcentration, 1 nM-1 mM) of analog or vehicle
7. additionSynaptosomes were preincubated at 34° C. for 10 min before the addition of 50 μl of [3H]DA (30.1 Ci/mmole, final concentration 10 nM) and accumulation
8. waitproceeded for 10 min at 34° C
9. customdemonstrated that at 10 minutes [3H]DA uptake
10. customare performed at 34° C
11. customAccumulation was terminated by addition of 3 ml ice-cold assay buffer
12. additioncontaining
13. filtration1 mM pyrocatechol and rapid filtration through a Whatman GF/B glass fiber
14. filtrationfilter
15. additionpaper (presoaked with buffer containing 1 mM pyrocatecol)
16. washThe filters were washed 3 times with 3 ml of 10 ml ice-cold
17. additionbuffer containing 2 mM pyrocatechol
18. concentrationProtein concentration
19. customto yield true inhibition constants (Ki=IC50/[1+c/Km]), where c