Characterization of a 5‐HT1B receptor on CHO cells: functional responses in the absence of radioligand binding
Open Access
- 1 March 1996
- journal article
- research article
- Published by Wiley in British Journal of Pharmacology
- Vol. 117 (6), 1119-1126
- https://doi.org/10.1111/j.1476-5381.1996.tb16705.x
Abstract
1 Chinese hamster ovary (CHO) cells have been reported to be devoid of 5‐HT receptors and have frequently been used as hosts for the expression of cloned 5‐HT receptors. Unexpectedly, 5‐HT was found to induce profound inhibition of forskolin‐stimulated cyclic AMP production in these cells and the aim of this study was to classify the 5‐HT receptor involved. 1 In CHO(dhfr‐) cells 5‐HT was a potent agonist and caused 80–100% inhibition of forskolin stimulated cyclic AMP production. A study using several 5‐HT1 receptor agonists revealed the following potencies (p[A50]): RU24969 (9.09±0.17) > 5‐carboxamidotryptamine (8.86±0.20) > 5‐HT (8.07±0.05) > CP‐93,129 (7.74±0.10) > sumatriptan (5.93±0.04). All five agonists achieved a similar maximum effect. Irreversible receptor alkylation studies yielded a pKA estimate of 7.04±0.34 for 5‐HT. 3 The 5‐HT1A/1B antagonist, (±)‐cyanopindolol (4–100 nM), caused parallel rightward shifts of the 5‐HT concentration‐effect curve with no change in asymptote. Schild analysis yielded a pKB estimate of 8.69±0.09 (Schild slope 1.13±0.10). (±)‐Cyanopindolol actually behaved as a partial agonist with an intrinsic activity of 0.2‐0.5 and a p[A50] of 8.55. 4 5‐HT (0.01–10 μm) also elicited a concentration‐dependent increase in intracellular [Ca2+] in CHO(dhfr‐) cells thus demonstrating that dual coupling is not a phenomenon restricted to systems in which there is overexpression of transfected receptors. 5 This agonist and antagonist profile is consistent with the presence of a 5‐HT1B receptor. 8‐OH‐DPAT (1 μm) and renzapride (3 μm) were without effect on forskolin‐stimulated cyclic AMP production and ketanserin (0.3 μm) did not antagonize the inhibition produced by 5‐HT, thus excluding the involvement of 5‐HT1A, 5‐HT4, and 5‐HT2 receptors. 6 The possibility that expression of a 5‐HT1B receptor was associated with the dhfr‐ mutation was excluded since RU24969, 5‐HT and CP‐93,129 were also potent agonists in unmutated, CHO‐K1 cells: p[A50] 9.03±0.03, 8.34±0.05, 7.69±0.07 respectively, and (±)‐cyanopindolol (0.1 μm) shifted the 5‐HT curve to the right and yielded a pA2 estimate of 8.70±0.06. 7 Little or no specific binding of [3H]‐5‐HT (0.1–200 nM) or of the high affinity ligand [125I]‐iodocyanopindolol (0.01–3 nM) to CHO(dhfr‐) cell membranes could be detected. 5‐HT also failed to elicit any increase in the binding of [35S]‐GTPγS to CHO membranes. 8 In conclusion, cultured CHO cells express 5‐HT1B receptors which are negatively coupled to adenylyl cyclase and positively coupled to increases in intracellular calcium. The absence of radioligand binding was unexpected in view of the high potency of 5‐HT and the partial agonist activity of the normally ‘silent’ competitive antagonist, (±)‐cyanopindolol. This implies very efficient receptor‐effector coupling of a low density of 5‐HT1B receptors. Clearly, the absence of detectable radioligand binding cannot be assumed to mean the absence of receptors capable of eliciting a significant functional response.Keywords
This publication has 28 references indexed in Scilit:
- Pharmacological Characterisation of [35S]-GTPγS Binding to Chinese Hamster Ovary Cell Membranes Stably Expressing Cloned Human 5-HT1DReceptor SubtypesJournal of Receptors and Signal Transduction, 1995
- The human and dog 5-HT1D receptors can both activate and inhibit adenylate cyclase in transfected cellsEuropean Journal of Pharmacology: Molecular Pharmacology, 1993
- Distinct 5-HT1B and 5-HT1D serotonin receptors in rat: Structural and pharmacological comparison of the two cloned receptorsMolecular and Cellular Neuroscience, 1992
- 3-(1,2,5,6-Tetrahydropyrid-4-yl)pyrrolo[3,2-b]pyrid-5-one: a potent and selective serotonin (5-HT1*) agonist and rotationally restricted phenolic analog of 5-methoxy-3-(1,2,5,6-tetrahydropyrid-4-yl)indoleJournal of Medicinal Chemistry, 1990
- Estimation of agonist affinity and efficacy by direct, operational model-fittingJournal of Pharmacological Methods, 1990
- 5-HT1B receptors are negatively coupled with adenylate cyclase in rat substantia nigraEuropean Journal of Pharmacology, 1988
- (−)-Propranolol and (±)-cyanopindolol are mixed agonists-antagonists at serotonin autoreceptors in the hippocampus of the rat brainNeuropharmacology, 1987
- Further analysis of anomalous pKB values for histamine H2‐receptor antagonists on the mouse isolated stomach assayBritish Journal of Pharmacology, 1985
- Characterization of the 5-HTIB recognition site in rat brain: Binding studies with (−)[125I]IodocyanopindololEuropean Journal of Pharmacology, 1985
- Operational models of pharmacological agonismProceedings of the Royal Society of London. B. Biological Sciences, 1983