In vitro test for inhibition
of Serotonin-binding to 5-HT1B
receptors in rat brain striatal
membrane homogenates.
Introduction:
Serotonin (5-hydroxytryptamine; 5-HT) binds reversibly to 5-HT1B
receptors present in rat brain striatal membranes.
Using labelled 5-HT, The binding can be assessed by separation of
bound and non-bound 5-MT.
Specific binding of labelled 5-HT can be inhibited by 5-HT agonists
like 5-methoxytryptamine and 5-HT antagonists like Metergoline.
This in vitro test is used to evaluate a test compound for inhibition
of 5-HT - binding to 5-HT1B
receptors in rat brain striatal membrane homogenates.
Test medium:
Striatal membrane homogenates from male rats (150 - 200 g; strain:
Cpb: WU, SPF-bred by CPB-TNO, Zeist, The Netherlands) in Tris-CaCI2
buffer.
Ref. compound:
5-Methoxytryptamine HCI: 10-8 mol/l induces an inhibition
of approx. 75 %
Metergoline: 10-8 mol/l induces an inhibition of approx.
30 %.
Vehicle:
Test compounds should preferably be dissolved in water* or HCI (0,1
mol/l) followed by neutralisation to pH 7 - 8.
If neutralisation is not possible, the pH of the incubation mixture
has to be checked (this should be 7 - 8).
* For aqueous solutions water for injection (USP) or ultrapure water
(Milli-Q quality) is used.
Technique:
Reagents
1. Tris buffer
Tromethamine (Tris; 6,06 g; 0,05 mol; analytical grade) is dissolved
in approx. 950 ml ultrapure water.
This solution is adjusted to pH 7,7 with HCI (4 mol/l) and made up
to 1 I with ultrapure water.
The buffer has to be freshly prepared before use and placed on ice
or in a refrigerator.
2. Tris-CaCI2
buffer
Tris (6,06 g; 0,05 mol; analytical grade), CaCI2
.2H
2
O (0,59
g; 0,004 mol;
analytical grade), ascorbic acid (1,0 g; analytical grade) and the
enzyme blocker pargyline HCI (1,96 mg; 10 μ,
mol; Abbott Laboratories, North Chicago, III., USA) are dissolved
in approx. 950 ml ultrapure water.
This solution is adjusted to pH 7,7 with HCI (4 mol/l) and made up
to 1 I with ultrapure water.
The buffer has to be freshly prepared before use and placed on ice
or in a refrigerator.
3. Rat brain striatal membrane homogenate
In general, 15 rats are killed by decapitation using a guillotine
and the whole brains are removed and placed in ice.
The corpora striate are dissected free, weighed and homogenised in
20 volumes (m/v) of ice-cold Tris buffer for 10 s using the Polytron
homogenizer (setting: half of the maximum speed).
The crude homogenate is centrifuged at 200 000 N/kg for 10 min at
4
°C using the Centrikon
centrifuge.
The supernatant is discarded and the pellet is resuspended in 20 volumes
(based on the original weight) of Tris buffer and incubated for 10
min at 37
°C.
The incubation mixture is centrifuged as described above and the final
pellet can be stored at -70
°C (at most 3 weeks)
until required for use.
Several experiments can be performed with one batch of brain homogenate.
Before use the pellet is allowed to thaw and then homogenised in 40
volumes (based on the original weight) of Tris-CaCl2
buffer using the Potter-Elvehjem
homogenizer (10 strokes up and down at 500 rpm) giving a homogenate
of 25 mg wet tissue/ml which should be kept on ice.
4. Labelled 5-HT
A solution of 5-[1,2-3
H(N)]-hydroxytryptamine creatinine sulphate (specific activity
approx. 27 Ci/mmol) in a mixture of ethanol and water (2:98; 1 mCi/ml;
NET-498; New England Nuclear, Boston, Mass., USA) is stored under
argon and protected from light in the original combi vial or polypropylene
tubes at 0-5
°C.
Just before use, 0,005 ml of this stock solution is added to 6,00
ml Tris-CaCI2
buffer.
A sample of this final solution (0,05 ml), containing approx. 42 nCi
and 1,6 x 10-12 mol 5-HT, is used for each assay (final
concentration 1,6 x 10-9 mol/l reaction mixture).
5. 5-HT solution
5-Hydroxytryptamine creatinine sulphate (0,081 mg; Merck, Darmstadt,
West Germany) is dissolved in 1,00 ml water for injection (USP) or
ultrapure water to give a solution containing 2x10-4 mol/l.
A sample of this solution (0,05 ml) is used for the estimation of
non-specific binding (final concentration 10 mol/l reaction mixture).
6. Picofluor 30 scintillation
fluid (Packard Instruments, Downers Grove, Ill., USA).
Equipment
1. Guillotine
2. Potter-Elvehjam homogenizer (Braun-Melsungen,
Melsungen, West Germany) with Teflon pestle
3. Polytron homogenizer, model
6837 (Kinematica, Kriens-Lucerne, Switzerland)
4. Centrikon centrifuge, H401 (Kontron, Zürich, Switzerland)
5. Eppendorf thermostat, 3401 (Eppendorf Gerätebau, Hamburg, West
Germany)
6. Eppendorf rotation mixer, 3300
7. Eppendorf reaction tube holder, 3830
8. Eppendorf stoppered polypropylene reaction tubes 3810: 1.5 ml
9. Eppendorf pipettes, 50; 100; 200; 500; 700 and 1000
μ,l.
10. Eppendorf multipipette, 4780
11. Packard-Tricarb liquid scintillation counter, model 2450
12. Glass scintillation vials (Amphabel, Lesines, Belgium)
13. Vortex whirlmixer (WiIten en Co., Etten-Leur, The Netherlands)
14. Whatman GF/B glass fibre filters, diameter 2,5 cm
15. Glass filter G/O-O support.
Procedure
a.
Estimation of total 5-HT-binding
Tris-CaCl
2
buffer (0,75 ml) and 0,05 ml labelled 5-HT are pipetted into a stoppered
polypropylene reaction tube. Immediately after the addition of 0,20
ml rat brain striatal membrane homogenate, the incubation at 25 °
C is started in the Eppendorf thermostat.
During the 40-min incubation period, the tube is mixed a few times
using the Eppendorf rotation mixer.
The incubation is terminated by rapid filtering 0,50 ml of the incubated
homogenate through a pre-wetted glass fibre filter.
The residue is rapidly washed 3 times with 5 ml ice-cold Tris-CaCl
2 buffer
and the filter + residue is transferred to a scintillation vial containing
5 ml Picofluor 30.
Each sample is counted once for 4 min.
b.
Estimation of non-specific 5-HT-binding
Tris-CaCl
2
buffer (0,70 ml), 0,05 ml 5-HT solution and 0,05 ml labelled 5-HT
are pipetted into a stoppered polypropylene reaction tube.
Rat brain striatal membrane homogenate (0,20 ml) is added and the
mixture is processed as described in sub-heading a.
c.
Estimation of 5-HT-binding in
the presence of test compound
Tris-CaCl2
buffer (0,70 ml), 0,05 ml test
compound and 0,05 ml labelled 5-HT are pipetted into a stoppered polypropylene
reaction tube.
Rat brain striatal membrane homogenate (0,20 ml) is added and the
mixture is processed as described in sub-heading a.
d.
Estimation of total 5-HT - radioactivity
A sample of 0,05 ml of the incubated homogenate from sub-heading a.
(non-filtered) is pipetted into a scintillation vial containing 5
ml Picofluor 30.
Each sample is counted once for 4 min.
All radioactivity measurements are stored in a computer file.
Compound doses:
Test compounds are usually investigated in 5 concentrations: 10-9,
10-8, 10-7, 10-6 and 10-5
mol/l reaction mixture, each in triplicate using the same batch of
rat brain striatal membrane homogenate.
For estimation of a more reliable IC50
-value, 5 appropriate concentrations
differing by a factor of 10 are investigated in at least 2 different
batches of rat brain striatal membrane homogenate.
Evaluation
of responses:
All responses stored in the computer file, are processed using computer
programmes.
All counting figures are corrected for quenching and converted to
numbers of disintegration's per min (dpm) and, for each concentration,
the mean dpm-value is calculated.
For each concentration of each test compound, the percentage change
of specific binding is calculated using the formula:
(C - A) - (T - A)
x 100
=
C - T
x 100
T - A T
- A
where:
T = mean dpm found for total binding
A = mean dpm found for non-specific binding
C = mean dpm found for binding in the presence of test compound.
An IC50
-value is calculated using a linear regression method, where
the percentage change of specific binding is used as the dependent
variable and
the negative logarithm of the concentration as the independent variable.
In addition, the affinity constant K
i for
the binding of test compound to the receptor is calculated using the
formula:
K
i =
IC
50
1
+ c/Kd
where:
c = concentration of 5-HT expressed in nmol/l reaction mixture and
calculated using the formula:
c
= 20
x D
2220 x S
where:
D = mean dpm found for total radioactivity
S = specific activity of labelled
5-HT expressed in Ci/mmol
Kd
= dissociation constant (nmol/l)
for the equilibrium: receptor/ 5-HT.
This constant is determined at least
twice a year by processing different amounts (within the range of
1-100 nmol/l reaction mixture) of labelled 5-HT in a similar manner
as described under: Procedure.
The K
d value
is calculated using the Scatchard analysis.
The final result is expressed as the pKi
-value, the negative logarithm of
Ki
.
Interpretation
of results:
The pK
i-value
is a measure for the binding affinity of the test compound to the
5-HT1B
receptors present in rat brain striatal membrane homogenates.
The results can be interpreted as follows:
|
pKi |
conclusion |
|
> 7 |
highly
active |
|
6 - 7 |
active |
|
5 - 6 |
weakly
active |
|
< 5 |
inactive |
Quantities required:
5 mg for investigation in one batch of rat brain striatal membrane
homogenate.
Reference:
Peroutka, S.J., Brain Research,
344 (1985) 167-171 |
|