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transfected with rat 5-HT2C receptors (generously donated by
Lippincott-Raven: New York, 1998.
Dr. Beth Hoffman) were grown to confluence, suspended in
(3) Van Wijngaarden, I.; Soudijn, W. 5-HT2A, 5-HT2B, and 5-HT2C
50 mM TRIS-HCl and centrifuged at 12 000g for 30 min. The
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pellet was resuspended in buffer and centrifuged for an
Olivier, B., van Wijngaarden, I., Soudijn, W., Eds.; Elsevier:
additional 20 min. Assay buffer used in the experiments
Amsterdam, 1997; pp 161-197.
consisted of 50 mM TRIS-HCl, 0.5 mM EDTA, 10 mM MgCl2,
(4) Glennon, R. A.; Dukat, M. Novel serotonergic agents: 5-HT2 -
and 0.1% ascorbate (pH 7.4). After resuspension in assay
Update 1997. ID Res. Alerts 1997, 2, 107-113.
buffer, 1 mL membrane aliquots (
(5) Glennon, R. A.; Titeler, M.; McKenney, J. D. Evidence for 5-HT2
by bicinchoninic assay) were added to each tube containing 1
involvement in the mechanism of action of hallucinogenic agents.
mL of assay buffer with either 0.5 nM [3H]ketanserin (5-HT2A)
Life Sci. 1984, 35, 2505-2511.
or 2.0 nM [3H]mesulergine (5-HT2C) and competing test agent.
(6) Shulgin, A.; Shulgin, A. PIHKAL, Transform Press: Berlkley,
Ketanserin (10 µM, 5-HT2A) and mesulergine (1 µM, 5-HT2C) CA, 1991.
were used to determine nonspecific binding. Competition (7) Seggel, M. R.; Yousif, M. Y.; Lyon, R. A.; Titeler, M.; Roth, B.
L.; Suba, E. A.; Glennon, R. A. A structure-affinity study of the
experiments were performed in triplicate in a 2.0 mL volume.
binding of 4-substituted analogues of 1-(2,5-dimethoxyphenyl)-
Membranes were incubated for 30 min at 37 °C, then filtered
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on Schleicher and Schuell (Keene, NH) glass fiber filters
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(presoaked in 0.1% polyethyleneimine), and washed with 10
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mL of buffer. The filters were counted in an Ecoscint liquid
Comparisons of hallucinogenic phenylisopropylamine binding
scintillation counter at 40% efficiency. Competition experi-
affinities at cloned human 5-HT2A, 5-HT2B and 5-HT2C receptors.
ments were plotted and analyzed using Graphpad Prism. Ki
Pharmacol. 1999, 359, 1-6.
values were determined from the Cheng-Prusoff equation: Ki (9) Naunyn-Schmeideberg s Arch.D.; Lyon, R. A.; Titeler, M. 5-HT1
Glennon, R. A.; McKenney, J.
) IC50/(1 + [D]/KD).19 The results reflect a minimum of three
and 5-HT2 binding characteristics of 1-(2,5-dimethoxy-4-bro-
assays.
mophenyl)-2-aminopropane analogues. J. Med. Chem. 1986, 29,
C. PI Hydrolysis Assay. Inositol phosphate (IP) production
194-199.
was measured as previously described,20 with minor modifica-
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tions. Briefly, NIH-3T3 cells stably transfected with rat 5HT2A
serotonergic mechanisms. NIDA Res. Monogr. 1992, 119, 131-
receptors were washed with phosphate-buffered saline (PBS)
135.
and labeled with 0.25 µCi/well of myo-[3H]inositol (New (11) Roth, B. L.; Willins, D. L.; Kristiansen, K.; Kroeze, W. K.
England Nuclear) in inositol free/serum-free DMEM (GIBCO) 5-Hydroxytryptamine2-family receptors (5-hydroxytryptamine2A,
5-hydroxytryptamine2B, 5-hydroxytryptamine2C): Where struc-
for 18 h at 37 °C. After labeling, cells were washed with PBS
ture meets function. Pharmacol. Ther. 1998, 79, 231-257.
and preincubated in inositol-free/serum-free DMEM with 10
(12) Standridge, R. T.; Howell, H. G.; Tilson, H. A.; Chamberlain, J.
mM LiCl and 10 µM pargyline (assay medium) for 10 min at
H.; Holava, H. M.; Gylys, J. A.; Partyka, R. A.; Shulgin, A. T.
37 °C. When antagonists were used, they were added during
Phenylalkylamines with potential psychotherapeutic utility. 2.
the 10 min preincubation period. Agonists were added to each
Nuclear substituted 2-amino-1-phenylbutanes. J. Med. Chem.
well and incubation continued for an additional 30 min. Assay
1980, 23, 154-162 and references therein.
medium was removed and cells were lysed in 200 µL of stop
(13) Glennon, R. A.; Seggel, M. R.; Soine, W. H.; Herrick-Davis, K.;
solution (1 M KOH/18 mM sodium borate/3.8 mM EDTA) and
Lyon, R. A.; Titeler, M. [125I]-1-(2,5-Dimethoxy-4-iodophenyl)-2-
neutralized by adding 200 µL of 7.5% HCl. The contents of
aminopropane: An iodinated radioligand that specifically labels
each well were extracted with 3 volumes of CHCl3/MeOH (1:
the agonist high-affinity state of 5-HT2 serotonin receptors. J.
2), centrifuged 5 min at 10 000g, and the upper layer loaded
Med. Chem. 1988, 31, 5-7.
onto a 1 mLAG1-X8 resin (100-200 mesh, Bio-Rad) column. [ Pobierz całość w formacie PDF ]