反応 #1369417

反応方程式

glutamate

CO2

glucose

KH2PO4

KCl

MgSO4

CaCl2

pargyline HCl

NaCl

Ascorbic acid

HEPES

→

(S)-(−)-nicotine

溶媒

sucrose

反応条件

後処理

1. 1
   その他Rat brain synaptosomes were prepared
2. 2
   その他hippocampus, striatum, and thalamus isolated
3. 3
   workup.WAITg for 20 minutes
4. 4
   workup.WAITcentrifuged for 15 minutes at 25000×g
5. 5
   その他placed in a water bath
6. 6
   workup.WAIT(37° C.) for 10 minutes
7. 7
   workup.WAITplaced in a water bath for additional 10 minutes
8. 8
   洗浄After a 10-minute wash period
9. 9
   その他fractions are collected
10. 10
    その他Further fractions were collected after agonist application
11. 11
    その他The perfusate was collected directly into scintillation vials

実験手順

Rat brain synaptosomes were prepared as follows: Female Sprague Dawley rats (100-200 g) were killed by decapitation after anesthesia with 70% CO2. Brains are dissected, and hippocampus, striatum, and thalamus isolated, and homogenized in 0.32 M sucrose containing 5 mM HEPES pH 7.4 using a glass/glass homogenizer. The tissue was then centrifuged for 1000×g for 10 minutes and the pellet discarded. The supernatant was centrifuged at 12000×g for 20 minutes. The resultant pellet was re-suspended in perfusion buffer (128 mM NaCl, 1.2 mM KH2PO4, 2.4 mM KCl, 3.2 mM CaCl2, 1.2 mM MgSO4, 25 mM HEPES, 1 mM Ascorbic acid, 0.01 mM pargyline HCl and 10 mM glucose pH 7.4) and centrifuged for 15 minutes at 25000×g. The final pellet was resuspended in perfusion buffer and placed in a water bath (37° C.) for 10 minutes. Radiolabeled neurotransmitter is added (30 L 3H DA, 20 L 3H NE, 10 L 3H glutamate) to achieve a final concentration of 100 nM, vortexed and placed in a water bath for additional 10 minutes. Tissue-loaded filters is placed onto 11-mm diameter Gelman A/E filters on an open-air support. After a 10-minute wash period, fractions are collected to establish the basal release and agonist applied in the perfusion stream. Further fractions were collected after agonist application to re-establish the baseline. The perfusate was collected directly into scintillation vials and released radioactivity was quantified using conventional liquid scintillation techniques. Release of neurotransmitter was determined in the presence of 10 M of various ligands and was expressed as a percentage of release obtained with a concentration of 10 M (S)-(−)-nicotine or 300 MTMA resulting in maximal effects.

出典