In vitro test for inhibition
of serotonin binding to cloned human 5HT2C receptors.
Introduction:
NIH/ 3T3 cells are stably transfected with cloned human 5-hydroxytryptamine
2C (5HT
2C
) receptors (previously called 5HT
1C
receptors). 5-Hydroxytryptamine (5HT; serotonin) binds reversibly
to these receptors. Using labelled 5HT the binding can be assessed
by separation of bound and non-bound 5HT. Specific binding of labelled
5HT can be inhibited by 5HT agonists (e.g. serotonin) and 5HT antagonists
(e.g. Mianserin).
This in vitro test is used to evaluate a test compound for binding
activity to cloned human 5HT2C
receptors by measuring the inhibition
of 5HT binding to these receptors.
Test medium:
3T3 cell homogenate (NIH/ 3T3 cells transfected with cloned human
5HT
2C
receptors)
Ref. compound:
Mianserin HCl: 10-8 mol
×
l-1 induces an inhibition of approx 70%.
Vehicle:
Ultrapure water (Milli-Q quality) or HCl (0,1 mol
×
l-1) followed by neutralization to pH 7 - 8.
Technique:
Reagents
All chemicals used are of analytical grade, for aqueous solutions
ultrapure water (Milli-Q Quality) is used.
1. Tris MgCl
2
buffer containing pargyline:
Tris(hydroxymethyl)aminomethane (Tris; 6,06g; 0,05 mol), ethylenediaminetetraacetic
acid (EDTA; 0,19 g; 0,5 mmol), MgCl2
.6H
2
O (2,033g; 10 mmol), ascorbic acid (1,0 g) and pargyline
HCl (1,96 mg; 0,01 mmol) are dissolved in approx 990 ml
water. This solution is adjusted to pH 7,4 with HCl (4 mol ×
l-1) and made up to
1l with water. The buffer has to be freshly prepared.
2. Tris buffer:
Tris(hydroxymethyl)aminomethane (Tris; 0,6g; 0,005 mol) is dissolved
in approx 950 ml water. This solution is adjusted to pH 7,4 with
HCl (4 mol
×
l-1) and made up to 1l with water.
The buffer has to be freshly prepared.
3. 3T3 cell homogenate:
NIH/ 3T3 cells are stably transfected with human 5HT2C
clone 9. The cells are grown in
3,6l DMEM / HAM F12 (1:1) + 5% (v / v) newborn-bovine
serum (Hyclone) on Cultispher-G (5 g ×
l-1) microcarriers in
a Celligen bioreactor. The cells are cultivated under stirring at
37 °C, pH 7- 7,4. Fresh medium is added continuously by perfusion
at a rate of 1,8 l DMEM / HAM per day and after two
weeks a final cell density of approx 5,5 x 106 cells ×
ml-1 is obtained.
After termination of the stirring and consequent settlement of the
microcarriers the supernatant is sucked up and the cells are removed
from the microcarriers by incubation under stirring in 800ml PBS (8
g
×l-1 NaCl;
0,2 g ×
l-1 KCl; 1,15 g ×
l-1 Na
2
HPO4
; 0,2 g
×
l-1 KH
2
PO4
) containing EDTA (1 mmol ×
l-1).
Following settlement of the carriers the supernatant, containing the
cells, is collected. This removal step is repeated 4times to give
approx 1 x 1010 cells which are centrifuged at 30.000 N ×
kg-1 for 5 min.
The supernatant is removed and the cells are suspended in 250 ml Tris
buffer (0,05 mol
×l-1), without
pargyline and ascorbic acid, using a Polytron homogenizer (15s, max
speed). The homogenate is incubated for 10 min at room-temperature
and then centrifuged at 400.000 N ×
kg-1 for 20 min. The
pellets are resuspended in Tris-MgCl
2
buffer using the Potter-Elvehjem homogenizer to give a concentration
of 2 x 107 cells
×
ml-1
per Eppendorf tube.
The homogenate is frozen and stored at -70 °C.
Before use the homogenate of 1 Eppendorf tube (sufficient for one
96-wells micromedia rack or 96 reaction tubes) is allowed to thaw
and resuspended in 40ml Tris MgCl2 buffer using a Polytron
homogenizer (15s, max speed).
4. Labelled 5HT solution:
A solution of 5-[1,2-3H(N)] hydroxytryptamine creatinine
sulphate (specific activity approx 25,4Ci ×
mmol-1) in ethanol (1,00mCi
×ml-1; NET-498;
New England Nuclear-Dupont, USA) is stored under protection from light
in sealed polypropylene tubes at 0-4 °C.
Immediately before use 0,005 ml of this solution is made up to 3,95 ml
with Tris-MgCl
2
buffer containing pargyline.
An aliquot of 0,050 ml of this solution is used for each assay (final
concentration 5 ×
10-9 mol
×
l-1 reaction mixture).
5. Mianserin solution:
Mianserin HCl (0,301 mg) is dissolved in 10 ml water (concentration
10-4 mol ×
l-1).
An aliquot of 0,050 ml is used for the determination of non-specific
binding (final concentration 10-5 mol
×
l-1 reaction mixture).
6. Prosil 28 solution:
2 ml of Prosil 28 solution (Johnson Matthey Alfa products) is added
to 198 ml water.
7. Ultima Gold MV scintillation fluid (Packard Instruments, USA).
Equipment
1. Polytron homogeniser, model 6837
(Kinematica, Switzerland)
2. Centrikon centrifuge, H401 (Kontron, Switzerland)
3. Adjustable Finn pipettes, 5- 50 µl, 50- 200 µl,
200- 1000 µl and 1- 5 ml.
4. Eppendorf multipipette, 4780
5. Robotic dispenser (Wilten, The Netherlands)
6. Micromedia glass test tubes, 10mm x 55 mm (Micromedia,
USA)
7. Whatman GF/ B glass fibre filters (Semat Technical Ltd., England)
8. Printed filtermat B, glass fibre filter, double thickness (Pharmacia)
9. Scatron 96-wells Harvester (Costar, The Netherlands)
10 Brandell 24-wells Harvester
11. Plastic scintillation vials (Pony vials, Packard Instruments,
USA)
12. Micromedia racks (96-wells, deepwell format 1,3cm; Beckman, USA)
13. Meltilex dry scintillation plate (Pharmacia)
14. LKB Betaplate counter (Pharmacia)
15. Packard Tricarb liquid scintillation counter, model 2200 CA (Packard
Instruments)
16. Heatsealer (Pharmacia)
Compound concentrations
Test compounds are dissolved in the vehicle and usually investigated
in triplicate at 4concentrations (10-5; 10-6; 10-7 and 10-8 mol
×l-1 reaction mixture) using the same batch of 3T3 cell homogenate.
Procedures
a. Estimation of total 5HT binding:
Tris-MgCl
2
buffer (0,050 ml), labelled 5HT solution (0,050 ml) and
3T3 cell homogenate (0,40 ml) are successively pipetted in triplicate
into wells of a micromedia rack or into glass test tubes. Immediately
after the addition of the homogenate, the mixture is incubated by
shaking for 60 min at room temperature.
The incubation is terminated by filtration of the whole volume through
the Whatman GF/ B glass fibre into either:
1. the Skatron 96-wells Harvester.
Each residue is washed for 10s with about 10 ml ice-cold Tris buffer.
×Each filter + residue
is sealed with a Meltilex dry scintillation plate, using a heatsealer,
and transferred to a LKB-Betaplate counter.
The samples are counted once for 2min, or:
2. the Brandell 24-wells Harvester. Each residue is washed for 10s
with about 10 ml ice-cold Tris buffer. The filters are transferred
to scintillation vials containing 2,5 ml Ultima Gold MV scintillation
fluid. Each sample is counted once for 4 min in a Packard-Tricarb
liquid scintillation counter.
b. Estimation of non-specific serotonin
binding:
Mianserin solution (0,050 ml), labelled 5HT solution (0,050 ml)
and 3T3 cell homogenate (0,40 ml) are successively pipetted in
triplicate into wells of a micromedia rack or into glass test tubes.
The procedure is further processed as described under a.
c. Estimation of serotonin binding in the presence of test or reference
compound:
Test or reference compound solution (0,050 ml), labelled 5HT solution
(0,050 ml) and 3T3 cell homogenate (0,40 ml) are successively
pipetted in triplicate into wells of a micromedia rack or into glass
test tubes.
The procedure is further processed as described under a.
d. Estimation of total serotonin radioactivity:
Labelled serotonin solution (0,050 ml) is pipetted into a scintillation
vial and 2,5 ml Ultima Gold MV scintillation fluid is added.
The sample is counted once for 2,5 min using a liquid scintillation
counter.
All radioactivity measurements obtained
are stored in a computer file.
Evaluation of responses:
All responses are processed using a computer program. Counting figures
can be expressed as counts per minute (cpm) or can be corrected for
quenching and converted as numbers of disintegration's per min (dpm).
For each concentration of test compound the mean cpm- or dpm value
is calculated.
The mean percentage change of specific binding for each concentration
is calculated using the formula:
(C -A) - (T - A) C
- T
-------------------- x 100 = ---------- x 100
T - A T
- A
where:
T= mean cpm or dpm for total binding
A= mean cpm or dpm for non-specific binding
C= mean cpm or dpm for binding in the presence of a test compound
If relevant, an IC50-value is calculated
using the four-parameter logistic fitting procedure according to Rodbard.
The mean percentage change of specific binding is used as the dependent
variable and the negative logarithm of the compound concentration
as the independent variable.
In addition the affinity constant K
i for the binding of
the test compound to the 5HT
2C receptor is calculated
using the formula:
IC
50
K
i = -------------------
1 + c ×
K
d-1
where:
K
d = dissociation constant
(mol ×
l-1) for the receptor-serotonin
equilibrium.
The constant is determined at least once a year by processing different
concentrations of labelled serotonin solutions in a similar manner
as described under Procedure, and calculated using non-linear regression
analysis.
c = concentration of labelled serotonin in mol
×l-1 reaction mixture which is calculated using the formula:
V
x D
c =
------------------
2220
x S
where:
D = mean dpm for total radioactivity
S = specific activity for labelled serotonin in Ci ×
mmol
-1
V = dilution factor for incubation volume to 1ml
The final result is expressed as the pK
i
-value, the negative logarithm of K
i
.
Interpretation of results:
The pK
i
-value is measured for the binding affinity of the test compound
to the 5HT2C
receptors present in 3T3 cell membrane
homogenates.
Test compound showing a pK
i
-value >6 are considered to have a biologically relevant
binding activity.
Reference:
Havlick S., Peroutka S.J., Differential radioligand properties of
[3
H]-hydroxytryptamine and [3
H]-mesulergine in a clonal 5-hydroxytryptamine
--1C cell line. Brain Research. 1992; 584 (191- 6).
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