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.