h5-HT1A receptor-mediated stimulation of [35S]GTPγ S binding: disruption by anti-Gαi1/3 antibodies
| Ligand | Emin | Emax | pEC50H | pEC50L | % High | nH | n |
|---|---|---|---|---|---|---|---|
| % | % | ||||||
| 5-HT | |||||||
| Control | 100 | 218 ± 12 | 9.09 ± 0.03 | 7.70 ± 0.05 | 59 ± 3 | 0.72 ± 0.04 | 7 |
| + anti-Gαi1/3 | 87 ± 3 | 181 ± 6 | 8.15 ± 0.05 1-a | 0.91 ± 0.03 | 5 | ||
| +anti-pERK | 107 ± 3 | 221 ± 16 | 9.16 ± 0.04 | 7.81 ± 0.16 | 55 ± 2 | 0.69 ± 0.05 | 3 |
| Pindolol | |||||||
| Control | 100 | 137 ± 2 | 7.88 ± 0.05 1-a | 1.16 ± 0.07 | 3 | ||
| + anti-Gαi1/3 | 82 ± 2 | 101 ± 3 | 7.79 ± 0.32 1-a | 0.98 ± 0.08 | 3 | ||
| +anti-pERK | 97 ± 2 | 133 ± 2 | 8.18 ± 0.17 1-a | 0.78 ± 0.08 | 3 |
Membranes were preincubated either with buffer (control conditions) or with an antibody against Gαi1/3 subunits or against pERK. The full agonist 5-HT or the partial agonist pindolol and [35S]GTPγS were then added and incubated for a further 1 h before filtering the membranes and scintillation counting. Isotherms were analyzed by nonlinear regression. Emin values are the basal [35S]GTPγ S binding observed in the absence of ligand, and are expressed as a percentage of control basal values (100%). Emax values are the maximal observed stimulation (relative to control basal values = 100%). pEC50H and pEC50L are effective concentration values for the high and low potency components. %High values are the percentage of sites in the high-potency component, andn H is the slope of the isotherms. Data are expressed as mean ± S.E.M. of n independent determinations performed in triplicate. In the case of 5-HT, isotherms were biphasic (P < 0.05, F-test), except when preincubated with anti-Gαi1/3. Pindolol yielded monophasic isotherms in all cases. Anti-Gαi1/3, but not anti-pERK, antibodies reduced basal [35S]GTPγ S binding from control values.
↵1-a pEC50 value (single site fit).