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. 2007 Mar 20;151(2):237–252. doi: 10.1038/sj.bjp.0707158

Table 3.

Summary of methods for determination of functional responses at native rat and recombinant human monoamine receptor in vitro

Receptor Tissue/cell line Functional measure Incubation conditions Inc. time (min) & temp. (°C) Literature reference
rD2 Rat striatum [35S]GTPγS binding Buffer A 60, 37° Newman-Tancredi et al. (2001)
hD2L Sf9 cells [35S]GTPγS binding Buffer B 40, 30° Cosi et al. (2006)
hD2S CHO cells ERK1/2 phosphorylation Ham's F12 serum-free 5, 37° Bruins Slot et al. (2006)
hD3 COS cells Gαo [35S]GTPγS binding Buffer C 30, 30° Pauwels et al. (2003)
hD4 CHO cells [35S]GTPγS binding Buffer D 30, 23° Newman-Tancredi et al. (1997)
r5-HT1A Rat hippocampus [35S]GTPγS binding Buffer A 60, 37° Newman-Tancredi et al. (2005)
r5-HT1A Rat hippocampus Gαo [35S]GTPγS binding Buffer E 60, 23° Martel et al. (2007)
h5-HT1A HeLa cells [35S]GTPγS binding Buffer F 60, 30° Newman-Tancredi et al. (2005)
h5-HT1A HeLa cells cAMP formation Buffer G 10, 23° Newman-Tancredi et al. (2005)
h5-HT1A CHO cells ERK1/2 phosphorylation RPMI serum-free 5, 37° Bruins Slot et al. (2006)
h5-HT1D C6 glial cells [35S]GTPγS binding Buffer C 30, 23° Pauwels et al. (1997)
h5-HT2A CHO cells Gαq [35S]GTPγS binding Buffer H 60, 23° Cussac et al. (2002b)
h5-HT2B CHO cells Gαq [35S]GTPγS binding Buffer H 60, 23° Cussac et al. (2002b)
h5-HT2C CHO cells Gαq [35S]GTPγS binding Buffer H 60, 23° Cussac et al. (2002b)
h5-HT7A HEK293 cells cAMP formation Buffer I 5, 37° Rauly-Lestienne et al. (2004)
hα2A C6 glial cells [35S]GTPγS binding Buffer C 30, 23° Pauwels et al. (2003)
hα2C C6 glial cells [35S]GTPγS binding Buffer C 30, 23° Pauwels et al. (2003)

Abbreviations: CHO, Chinese hamster ovary cells; HeLa, human carcinoma cells; DTT, dithiothreitol.

Buffer A: 50 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM MgCl2, 100 μM GDP, 0.2 mM EDTA, 0.2 mM DTT; Buffer B: 20 mM HEPES, pH 7.4, 100 mM NaCl, 10 mM MgCl2, 1 μM GDP, 0.1 mM DTT; Buffer C: 20mM HEPES, pH 7.4, 100 mM NaCl, 3 mM MgCl2, 30 μM GDP; Buffer D: 20 mM HEPES, pH 7.4, 30 mM NaCl, 3 mM MgCl2, 3 μM GDP; Buffer E: 20 mM HEPES, pH 7.4, 100 mM NaCl, 5 mM MgCl2, 50 μM GDP, 0.2 mM EDTA, 0.2 mM DTT; Buffer F: 20 mM HEPES, pH 7.4, 100 mM NaCl, 10 mM MgCl2, 30 μM GDP, 10 μM pargyline; Buffer G: DMEM + 10 mM HEPES, pH 7.4, 100 μM forskolin, 100 μM isobutylmethylxanthine; Buffer H: 20 mM HEPES, pH 7.4, 150 mM NaCl, 50 mM MgCl2, 0.1 μM GDP; Buffer I: 25 mM Tris-hCl, pH 7.4, 120 mM NaCl, 5.4 mM KCl, 0.8 mM MgCl2, 5 mM glucose, 1 mM isobutylmethylxanthine.

When drugs were tested for antagonist properties, an additional 30 min pre-incubation was performed before addition of the agonist, except for ERK1/2 phosphorylation experiments (15 min pre-incubation).

G-protein activation experiments were carried out by [35S]GTPγS binding to cell membrane preparations. Other measures (cAMP formation and ERK1/2 phosphorylation) were carried out on whole cells.