2-01401

In vitro test for inhibition of DOB binding to cloned human 5HT2a receptors. Introduction: Swiss-mouse 3T3 cells are stable transfected with cloned human 5HT2a receptors. The serotonin ligand 1-[4-bromo-2,5-dimethoxyphenyl]-2-aminopropane (DOB) binds reversibly to these receptors. Using labelled DOB the binding can be assessed by separation of bound and non-bound DOB. Specific binding of labelled DOB can be inhibited by 5HT2 agonists (e.g. serotonin) and 5HT2 antagonists (e.g. mianserine). This in vitro test is used to evaluate a test compound for binding activity to cloned human 5HT2a receptors by measuring the inhibition of DOB binding to these receptors. Test medium: 3T3 cell homogenate (Swiss-mouse 3T3 cells transfected with cloned human 5HT2a receptors) Ref. compound: Serotonin: 10-6 mol × l-1 induces and inhibition of approx 80%.  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 .6H2 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 1 l with water. The buffer has to be freshly prepared.  3. 3T3 cell homogenate: Swiss-mouse 3T3 cells are stably transfected with human 5HT2a clone 5.  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 was 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 Na2HPO4; 0,2 g × l-1 KH2PO4 ) containing EDTA (1 mmol × l-1). Following settlement of the carriers the supernatant, containing the cells, is collected. This removal step is repeated 4tiDOB 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) 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-MgCl2 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 3 Eppendorf tubes (sufficient for one 96-wells micromedia rack or 96 reaction tubes) is allowed to thaw and resuspended in 45 ml Tris-MgCl2 buffer using a Polytron homogenizer (15s, max speed).  4. Labelled DOB solution: A solution of [propyl-1,2-3H]DOB (specific activity approx 13,3Ci × mmol-1) in ethanol (1,00mCi ×ml-1; NET-943; New England Nuclear-Dupont, USA) is stored at -20 °C. Immediately before use 0,0025 ml of this solution is made up to 9,4 ml with Tris-MgCl2 buffer containing pargyline. An aliquot of 0,050 ml of this solution is used for each assay (final concentration 2 × 10-9 mol × l-1 reaction mixture). 5. Serotonin solution: Serotonin (0,405 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. 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 4 concentrations (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 DOB binding: Tris-MgCl 2 buffer (0,050 ml), labelled DOB 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, pre-soaked in Prosil 28 (1% v / v) for 1 h, into either:  1. the Skatron 96-wells Harvester. Each residue is washed for 10s with ice-cold Tris buffer (0,005 mol ×l-1). 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 ice--cold Tris buffer (0,005 mol ×l-1). The filters are transferred to scintillation vials containing 2,5 ml Ultima Gold MV scintillation fluid. Each sample is counted once for 3 min in a Packard-Tricarb liquid scintillation counter.  b. Estimation of non-specific DOB binding: Mianserin solution (0,050 ml), labelled DOB 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 DOB binding in the presence of test or reference compound: Test or reference compound solution (0,050 ml), labelled DOB solution (0,050 ml) and 3T3 cell homogenate (0,40 ml) are 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 DOB radioactivity: Labelled DOB 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 min (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 Ki for the binding of the test compound to the 5HT2a receptor is calculated using the formula:        IC 50 K i = -------------------        1 + c ×Kd -1 where: Kd = dissociation constant (mol × l-1) for the receptor-DOB equilibrium. The constant is determined at least once a year by processing different concentrations of labelled DOB solutions in a similar manner as described under Procedures, and calculated using non-linear regression analysis. c = concentration of labelled DOB 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 DOB in Ci × mmol-1 V = dilution factor for incubation volume to 1 ml  The final result is expressed as the pKi-value, the negative logarithm of Ki. Interpretation of results: the pKi-value is measured for the binding affinity of the test compound to the 5HT2a receptors present in 3T3 cell membrane homogenates. Test compound showing a pKi-value >6 are considered to have a biologically relevant binding activity.