In vitro test for inhibition
of n-propylnorapomorphine (NPA)-binding to dopaminergic receptors
in rat brain striatal membrane homogenates.
Introduction:
N-propylnorapomorphine (NPA) binds reversibly to dopaminergic receptors
present in rat brain striatal membranes.
Using labelled NPA, the binding can be assessed by separation of bound
and non-bound NPA.
Drugs with dopaminergic blocking properties, like the antagonist butaclamol
and the agonist apomorphine, can inhibit the binding of NPA.
This in vitro test is used to evaluate a test compound for inhibition
of NPA-binding to dopaminergic 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-EDTA
buffer containing ascorbic acid and nialamide.
Ref. compound:
d-Butaclamol HCl : 10-9 mol/l
induces an inhibition of approx. 70%.
Apomorphine HCl : 10-9 mol/l
induces an inhibition of approx. 33%.
Vehicle:
Test compounds should preferentially be dissolved in distilled water
or 0,1 mol/l HCl followed by neutralisation to pH 7 - 8.
If neutralisation is not possible, the pH of the incubation mixture
has to be checked (this pH should be 7 - 8).
Technique:
Reagents
1. Tris-EDTA buffer:
1,82 g (0,015 mol) tromethamine
(2-amino-2-hydroxymethyl-1,3-propanediol), Tris; analytical grade,
Baker Chemicals, Deventer, The Netherlands), 0,37 g (0,001 mol) disodium
ethylenediaminetetraacetate.2H
2
O (EDTA;
analytical grade Baker Chemicals) and 0,10 g (0,01%) ascorbic acid
(analytical grade, Baker Chemicals) are dissolved in approx. 950 ml
distilled water.
This solution is adjusted at pH = 7,5 with 4 mol/l HCl and made up
to 1 litre with distilled water.
This buffer solution can not be stored and has to be freshly prepared
before use and kept on ice or in a refrigerator.
2. Tris-EDTA buffer containing an enzyme blocker:
1,82 g (0,015 mol) Tris, 0,37 g
(0,001 mol) EDTA and 0,10 g (0,01%) ascorbic acid are dissolved in
approx. 950 ml distilled water. 3,72 mg nialamide HCl (Pfizer, New
York, N.Y., U.S.A.) or 2,45 mg pargyline HCl (Abbott Laboratories,
North Chicago, Ill., U.S.A.) dissolved in a few ml 4 mol/l HCl are
added.
This solution is adjusted at pH = 7,5 with 4 mol/l HCl and made up
to 1 litre with distilled water.
This buffer solution can not be
stored and has to be freshly prepared before use and kept on ice or
in a refrigerator.
3. Rat brain striatal membrane homogenate:
Male rats are killed by decapitation
and the whole brains are removed and placed on ice.
The corpora striate are dissected
free and, if relevant, stored at -70
°C
until required for use, but not longer than 14 days.
The corpora striate are, if relevant,
allowed to thaw, weighed and homogenised in 40 volumes (w/v) ice-cold
Tris-EDTA buffer for 10 sec using the Polytron homogenizer (setting:
half of the maximum speed).
The crude homogenate is centrifuged at 400000 N/kg for 10 min at 4
°
C using the cryocentrifuge.
The supernatant is discarded and the residual pellet is washed by
resuspending in 40 volumes (based on the original weight) Tris-EDTA
buffer and centrifugation as described above.
The pellet is resuspended in 40 volumes (based on the original weight)
Tris-EDTA buffer and the resulting homogenate is incubated at 37 °
C for 10 min. The incubation mixture is centrifuged and the
resulting pellet washed once as described above.
The final pellet can be stored at -70 °
C until required for use, but not
longer than 14 days.
Before use, the pellet is allowed to thaw and homogenised in 40 volumes
(based on the original weight) Tris-EDTA buffer containing an enzyme
blocker using the Potter-Elvehjem homogenizer (10 strokes up-and-down
at 500 rpm). This final solution containing 25 mg wet tissue/ml is
kept on ice.
4. Labelled NPA:
L-[N-propyl-3H]-N-propylnorapomorphine,
approx. 60 Ci/mmol in KH
2
PO4
, 0,01 mol/l distilled water containing
5% ethanol (pH = 3,0) (1,00 mCi/ml; Net-619, New England Nuclear,
Boston, Mass., U.S.A.) is stored under nitrogen and protected from
light in the original combi vial or polypropylene tubes at 0,4 °
C.
Just before use, 0,005 ml of this stock solution is made up to 1,00
ml with Tris-EDTA buffer containing an enzyme blocker and 0,15 ml
of this diluted
solution is made up to 4,00 ml again with this buffer.
0,05 ml of this final solution, containing approx. 9 nCi (0,15 x 10-12 mol)
NPA, is used for each assay (final concentration 0,15 x 10-9 mol/l
reaction mixture.
5. Butaclamol solution:
0,80 mg d-butaclamol HCl or 1,59 mg dl-butaclamol HCl (Ayerts
Laboratories, New York, N.Y., U.S.A.) is dissolved in 1,00 ml distilled
water and 0,10 ml of this solution is made up to 10,0 ml with distilled
water to give a solution containing 20 x 10-6 mol/l
d-butaclamol or 40 x 10-6
mol/l dl-butaclamol.
0,05 ml of this final solution is used for each assay (final concentration
10-6
mol d-butaclamol or 2 x 10-6 mol
dl-butaclamol per litre reaction mixture.
6. Picofluor 30 scintillation fluid (Packard Instruments, Downers
Grove, Ill., U.S.A.)
Equipment
1. Potter-Elvehjem homogenizer
(Braun-Melsungen, Melsungen, West Germany)
2. Polytron homogenizer (Model 6837, Kinematica, Kriens-Lucerne, Switzerland)
3. Cryocentrifuge (Model 20-3, Hereaus Christ, Osterode, West Germany)
4. Eppendorf thermostat 3401 (Eppendorf
Geratebau, Hamburg, West Germany)
5. Eppendorf rotation mixer 3300
6. Eppendorf reaction tube holders 3930
7. Eppendorf stoppered polypropylene reaction tubes 3810, 1,5 ml
8. Eppendorf pipettes, 50, 100, 200, 250 and 500
μ,
l
9. Packard-Tricarb liquid scintillation counter, model 2450
10. Glass scintillation vials (Amphabel, Lesine, Belgium)
11. Vortex whirlmixer (Wilten en Co., Etten-Leur, The Netherlands)
12. Whatman GF/B glass fibre filters, diameter 2,5 cm
13. Glass filter G/O-O support.
Procedure
a.
Estimation of total NPA-binding
0,75 ml Tris-EDTA buffer containing an enzyme blocker and 0,05 ml
labelled NPA 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 45-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-EDTA
buffer containing an enzyme blocker 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
NPA-binding
0,70 ml Tris-EDTA buffer containing an enzyme blocker, 0,05 ml butaclamol
solution and 0,05 ml labelled NPA are pipetted into a stoppered polypropylene
reaction tube.
0,20 ml rat brain striatal homogenate is added and the mixture is
processed as described under a.
c.
Estimation of NPA-binding in the presence of test compound
0,70 ml Tris-EDTA buffer containing an enzyme blocker, 0,05 ml test
compound and 0,05 ml labelled NPA are pipetted into a stoppered reaction
tube.
0,20 ml rat brain striatal homogenate is added and the mixture is
processed as described under a.
d.
Estimation of total NPA-radioactivity
0,05 ml of the incubated homogenate
obtained before the start of the filtration process described under
a., 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: 10-6, 10-7, 10-8, 10-9 and
10-10 mol/l reaction mixture, 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 with the help
of 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 mean percentage
change in specific binding is calculated using the formula:
(C - A) - (T - A)
C - T
-------------------- x 100 = --------- 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.
If relevant, an IC
50-value is calculated
using a linear regression method.
In addition, the results are also evaluated manually by plotting the
percentage change (ordinate) against the negative logarithm of the
concentration expressed in mol/l reaction mixture (abscissa).
In addition, the affinity constant K
i for
the binding of test compound to the receptor is calculated using the
formula:
IC50
K
i = -----------
1 + c/Kd
where:
c = concentration of NPA expressed in nmol/l reaction mixture and
calculated using the formula:
20 x D
c = ------------
2220 x S
where:
D = mean dpm found for total radioactivity
S = specific activity of labelled NPA expressed in Ci/mmol
Kd
= dissociation constant (nmol/l)
for the equilibrium: receptor-NPA.
This constant is determined at least twice a year by processing different
amounts (in the range of 0,05-3,20 nmol/l reaction mixture) of labelled
NPA in a similar manner as described under "Procedure".
The Kd-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 pKi-value is a measure for the binding affinity of the test compound
to the dopaminergic receptors present in rat brain striatal membrane
homogenates.
Test compounds showing a pKi-value of less than 7 are considered
to have no binding activity to the dopaminergic receptors present
in rat brain striatal membrane homogenate.
Quantities required:
5 mg for the investigation in one batch of rat brain striatal membrane
homogenate.
Reference:
Creese, I., Padgett, L., Fazzini, E. and Lopez, L., European
Journal of Pharma cology, 56 (1979) 411 - 412.
Leijsen, J.E. and Gommersen, W., Journal of Neurochemistry, 36 (1981) 201209.
|
|