Reaktion #5022

Reaktionsgleichung

HEPES

Dopamine

HEPES

dopamine

→

(S)-(-)-nicotine

Lösungsmittel

sucrose

Reaktionsbedingungen

Aufarbeitung

1. 1
   workup.ADDITIONThe homogenate was diluted to 5 ml with additional homogenization solution
2. 2
   workup.WAITthe resulting supernatant 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
   Sonstigethe 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
   SonstigeThe new pellet was collected
9. 9
   workup.WAITThe synaptosome suspension was incubated for 10 min. at 37° C
10. 10
    workup.ADDITIONThen [3H]-dopamine (Amersham, 40-60 Ci/mmol) was added to the suspension
11. 11
    Sonstigeto give a final concentration of 0.1 μM in suspension
12. 12
    workup.WAITthe suspension was incubated for another 5 min
13. 13
    Filtrationfiltration through glass fiber filters
14. 14
    WaschenSynaptosomes were washed with perfusion buffer for a minimum of 20 min. before addition of the ligand
15. 15
    workup.ADDITIONAfter the addition of a 0.2 ml of a 10 μM solution of ligand
16. 16
    Sonstigethe perfusate was collected into scintillation vials at 1 min. intervals

Vorschrift

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 1000×g for 10 min. This procedure was repeated on the new pellet and the resulting supernatant 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. Then [3H]-dopamine (Amersham, 40-60 Ci/mmol) was added to the suspension to give a final concentration of 0.1 μM in suspension, 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 (i.e., cytisine and N-methylcytisine). Synaptosomes were loaded onto glass fiber filters (Gelman type 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 a 0.2 ml of a 10 μM solution 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.

Quelle