反応 #80685

反応方程式

HEPES

Dopamine

HEPES

dopamine

→

(S)-(-)-nicotine

溶媒

sucrose

反応条件

後処理

1. 1
   workup.ADDITIONThe homogenate was diluted to 5 ml with additional homogenization solution
2. 2
   workup.WAITthe resulting superatant was centrifuged at 12,000×g for 20 min
3. 3
   workup.ADDITIONdispersed in the top layer
4. 4
   workup.WAITAfter centrifugation at 15,000×g for 20 min.
5. 5
   その他the synaptosomes were recovered above the 16 percent layer with a Pasteur pipette
6. 6
   workup.ADDITIONdiluted with 8 ml of perfusion buffer (128 mM NaCl, 2.4 mM KCl, 3.2 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 25 mM HEPES pH 7.4, 10 mM dextrose, 1 mM ascorbate, 0.01 mM pargyline)
7. 7
   workup.WAITcentrifuged at 15,000×g for 20 min
8. 8
   その他The new pellet was collected
9. 9
   workup.WAITThe synaptosome suspension was incubated for 10 min. at 37° C.
10. 10
    workup.ADDITION[3H]-Dopamine (Amersham, 40-60 Ci/mmol) was added to the suspension
11. 11
    その他to give a final concentration of 0.1 uM
12. 12
    workup.WAITthe suspension was incubated for another 5 min
13. 13
    ろ過filtration through glass fiber filters
14. 14
    洗浄Synaptosomes were washed with perfusion buffer for a minimum of 20 min. before addition of the ligand
15. 15
    workup.ADDITIONAfter the addition of 0.2 ml of a solution
16. 16
    workup.ADDITIONcontaining various
17. 17
    濃縮concentrations of ligand
18. 18
    その他the perfusate was collected into scintillation vials at 1 min. intervals

実験手順

Dopamine release was measured by preparing synaptosomes from the striatal area of rat brain obtained from Sprague-Dawley rats generally according to the procedures set forth by Nagy et al., J. Neurochem., Vol. 43, pp. 1114-1123 (1984). Striata from 4 rats were homogenized in 2 ml of 0.32M sucrose buffered with 5 mM HEPES (pH 7.5), using a glass-Teflon tissue grinder. The homogenate was diluted to 5 ml with additional homogenization solution and centrifuged at 1,000×g for 10 min. This procedure was repeated on the new pellet and the resulting superatant was centrifuged at 12,000×g for 20 min. A 3 layer discontinuous Percoll gradient consisting of 16 percent, 10 percent and 7.5 percent Percoll in HEPES-buffered sucrose was made with the final pellet dispersed in the top layer. After centrifugation at 15,000×g for 20 min., the synaptosomes were recovered above the 16 percent layer with a Pasteur pipette, diluted with 8 ml of perfusion buffer (128 mM NaCl, 2.4 mM KCl, 3.2 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 25 mM HEPES pH 7.4, 10 mM dextrose, 1 mM ascorbate, 0.01 mM pargyline), and centrifuged at 15,000×g for 20 min. The new pellet was collected and re-suspended in perfusion buffer. The synaptosome suspension was incubated for 10 min. at 37° C. [3H]-Dopamine (Amersham, 40-60 Ci/mmol) was added to the suspension to give a final concentration of 0.1 uM, and the suspension was incubated for another 5 min. Using this method, 30 to 90 percent of the dopamine was taken up into the synaptosomes, as determined by scintillation counting following filtration through glass fiber filters soaked with 0.5 percent polyethyleneimine. A continuous perfusion system was used to monitor release following exposure to each ligand. Synaptosomes were loaded onto glass fiber filters (Gelman typc A/E). Perfusion buffer was dripped onto the filters (0.2-0.3 ml/min.) and pulled through the filters with a peristaltic pump. Synaptosomes were washed with perfusion buffer for a minimum of 20 min. before addition of the ligand. After the addition of 0.2 ml of a solution containing various concentrations of ligand, the perfusate was collected into scintillation vials at 1 min. intervals and the dopamine released was quantified by scintillation counting. Peaks of radioactivity released above background were summed and the average basal release during that time was subtracted from the total. Release was expressed as a percentage of release obtained with an equal concentration of (S)-(-)-nicotine.

出典