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Regulation of the 5-hydroxytryptamine1B receptor in opossum kidney cells after exposure to agonists
1Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia 19104-6084.
Abstract:
The density of 5-hydroxytryptamine (5-HT)1B receptors and their coupling to the inhibition of cAMP accumulation were investigated in opossum kidney cells maintained in culture. The density and properties of the receptor were determined using [125I] iodocyanopindolol as the radioligand. The pharmacological specificity of the binding site was consistent with that expected for a 5-HT1B receptor. Serotonin inhibited forskolin-stimulated cAMP accumulation with an EC50 of 4-8 nM. Compounds known to show selectivity at the 5-HT1B receptor, such as trifluoromethyl-phenylpiperazine and CGS-12066B, also inhibited forskolin-stimulated cAMP accumulation, acting as full agonists with efficacies comparable to that of serotonin. Other beta-adrenergic receptor antagonists, including (-)-pindolol and (-)-alprenolol, bound to the receptor with high affinity and acted as partial agonists capable of inhibiting forskolin-stimulated cAMP accumulation. Exposure of cells to 5-HT resulted in a time- and dose-dependent decrease in the density of 5-HT1B receptors that was not accompanied by a change in the Kd of the binding site for [125I] iodocyanopindolol. A maximum decrease of 60% in the number of 5-HT1B receptors was evident after a 16-hr treatment with 1 microM 5-HT. Concomitant with the observed decrease in the density of receptors was a marked increase in the EC50 for 5-HT-mediated inhibition of forskolin-stimulated cAMP accumulation. The EC50 was increased 4-5-fold after a 16-hr exposure to 1 microM 5-HT, and the maximal level of inhibition was markedly decreased. Whereas pretreatment with moderate concentrations of 5-HT (100-300 nM) for 16 hr produced significant decreases in the density of 5-HT1B receptors and increases in the EC50 for inhibition of forskolin-stimulated cAMP formation, there was little change in the maximal level of inhibition that could be attained. Such a combination of changes could be explained by the presence of "spare" 5-HT1B receptors on these cells.
Insights
This study investigated 5-hydroxytryptamine (5-HT)1B receptors in opossum kidney cells, finding that serotonin exposure decreases receptor density and impairs cAMP inhibition. This suggests a potential role for spare 5-HT1B receptors in cellular response regulation.
Area of Science:
- Pharmacology
- Cell Biology
- Neuroscience
Background:
- 5-hydroxytryptamine (5-HT)1B receptors are crucial in cellular signaling pathways.
- Understanding receptor density and function is key to elucidating cellular responses to serotonin.
- Opossum kidney cells provide a model system for studying G protein-coupled receptor regulation.
Purpose of the Study:
- To investigate the density and functional coupling of 5-HT1B receptors in cultured opossum kidney cells.
- To determine the impact of serotonin exposure on 5-HT1B receptor number and signaling efficacy.
- To explore the pharmacological properties and regulatory mechanisms of 5-HT1B receptors.
Main Methods:
- Utilized [125I] iodocyanopindolol radioligand binding assays to quantify 5-HT1B receptor density and affinity.
- Measured serotonin-induced inhibition of forskolin-stimulated cAMP accumulation to assess receptor function.
- Administered varying concentrations and durations of 5-HT exposure to evaluate receptor downregulation and functional changes.
Main Results:
- Pharmacological characterization confirmed the presence of specific 5-HT1B binding sites.
- Serotonin, along with selective 5-HT1B agonists and partial agonists, inhibited cAMP accumulation.
- 5-HT exposure led to a time- and dose-dependent decrease in 5-HT1B receptor density (up to 60%) and a 4-5 fold increase in the EC50 for cAMP inhibition, with reduced maximal inhibition.
Conclusions:
- Serotonin treatment induces significant downregulation of 5-HT1B receptors in opossum kidney cells.
- Receptor downregulation is associated with reduced signaling efficacy, indicated by increased EC50 and diminished maximal response.
- The observed pattern of changes suggests the presence of spare 5-HT1B receptors, contributing to the complex regulation of cellular responses.