Reaktion #76526

Reaktionsgleichung

NaCl

KCl

MgCl2

CaCl2

α-D-glucose

NaHCO3

L-ascorbic acid

disodium ethylenediaminetetraacetate

O2

[3H]NE

[3H]NE

→

[3H]Dopamine

Reaktionsbedingungen

Aufarbeitung

1. 1
   WaschenAfter rinsing
2. 2
   workup.ADDITIONbuffer containing pargyline (10 μM) and nomifensine (10 μL) or pargyline and desipramine (10 μL) in experiments
3. 3
   SonstigeAfter 60 min of superfusion, three 5-min samples (5 mL) were collected
4. 4
   SonstigeAfter collection of the third basal sample, slices from an individual rat
5. 5
   Einengenwere superfused in the absence or presence of a single concentration of analog, which
6. 6
   workup.WAITAfter 30 min
7. 7
   workup.ADDITIONS(−)-nicotine (10 μM) was added to the buffer containing analog, and superfusion
8. 8
   workup.WAITcontinued for an additional 60 min
9. 9
   Einengensuch that the analog concentration-effect

Vorschrift

Alkaloid effects on [3H]overflow from rat striatal slices preloaded with [3H]DA and hippocampal slices preloaded with [3H]NE were determined using modifications of a previously published method (Dwoskin and Zahniser, 1986). Briefly, coronal striatal or hippocampal slices (500 μm, 6-8 mg) were incubated in Krebs' buffer (118 mM NaCl, 4.7 mM KCl, 1.2 mM MgCl2, 1.0 mM NaH2PO4, 1.3 mM CaCl2, 11.1 mM α-D-glucose, 25 mM NaHCO3, 0.11 mM L-ascorbic acid and 4.0 mM disodium ethylenediaminetetraacetate; pH 7.4, and saturated with 95% O2/5% CO2) in a metabolic shaker at 34° C. for 30 min. Striatal or hippocampal slices were incubated in fresh buffer (6-8 slices/3 mL) containing 0.1 μM [3H]DA (3,4-[7-3H]-dihydroxyphenylethylamine; specific activity 28 Ci/mMol) or 0.1 μM [3H]NE (levo-[7-3H]-norepinephrine; specific activity 14.4 Ci/mMol), respectively, for an additional 30 min. After rinsing, each slice was transferred to a glass superfusion chamber maintained at 34° C. and was superfused at 1 mL/min with oxygenated Krebs' buffer containing pargyline (10 μM) and nomifensine (10 μL) or pargyline and desipramine (10 μL) in experiments assessing [3H]DA and [3H]NE overflow, respectively. After 60 min of superfusion, three 5-min samples (5 mL) were collected to determine basal [3H]outflow. After collection of the third basal sample, slices from an individual rat were superfused in the absence or presence of a single concentration of analog, which remained in the buffer until the end of the experiment. Each slice was exposed to only one concentration of analog. After 30 min, S(−)-nicotine (10 μM) was added to the buffer containing analog, and superfusion continued for an additional 60 min. These experiments utilized a repeated measures design, such that the analog concentration-effect was determined in both the absence and presence of S(−)-nicotine using striatal or hippocampal slices from a single rat. Additionally, one striatal or hippocampal slice was superfused in the absence of analog and constituted the control condition. At the end of the experiment, each slice was solubilized with TS-2. The pH and volume of the solubilized tissue samples were adjusted to those of the superfusate samples. Radioactivity in the superfusate and tissue samples was determined by liquid scintillation spectroscopy (Packard model B1600 TR Scintillation Counter, Downer's Grove, Ill.).

Quelle