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Functional coupling of a recombinant human 5-HT5A receptor to G-proteins in HEK-293 cells.

ABSTRACT: 1. We have cloned, expressed and pharmacologically characterized the Human 5-HT5A receptor. 2. We have shown that ligand activation of the Human 5-HT5A receptor results in functional coupling to G-proteins in HEK-293 cells. 3. Stimulation of the receptor with 5-CT (5-carboxamidotryptamine) resulted in a dose-dependent increase in the % [35S]-GTPgammaS binding over the basal level. This is the first study to describe such G-protein activation for the Human 5-HT5A receptor in any cell. 4. A dose-dependent inhibition of cyclic AMP accumulation was observed in the recombinant Human 5-HT5A receptor cell line, suggesting a functional coupling to a G alpha i, G-protein in the HEK-293 cell line. 5. A ligand-stimulated reduction in the detectable level of the catalytic domain of protein kinase A (PKA) in nuclear extracts isolated from Human 5-HT5A expressing cells was observed. This observation was consistent with the reduction in the level of cyclic AMP accumulation, in response to receptor activation.

SUBMITTER: Hurley PT

PROVIDER: S-EPMC1565485 | biostudies-other | 1998 Jul

REPOSITORIES: biostudies-other

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Functional coupling of a recombinant human 5-HT5A receptor to G-proteins in HEK-293 cells.

Hurley P T PT McMahon R A RA Fanning P P O'Boyle K M KM Rogers M M Martin F F

British journal of pharmacology 19980701 6

1. We have cloned, expressed and pharmacologically characterized the Human 5-HT5A receptor. 2. We have shown that ligand activation of the Human 5-HT5A receptor results in functional coupling to G-proteins in HEK-293 cells. 3. Stimulation of the receptor with 5-CT (5-carboxamidotryptamine) resulted in a dose-dependent increase in the % [35S]-GTPgammaS binding over the basal level. This is the first study to describe such G-protein activation for the Human 5-HT5A receptor in any cell. 4. A dose-d ...[more]

PMID: 9720796

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Project description:Background:Synthetic cannabinoids are a commonly used class of recreational drugs that can have significant adverse effects. There have been sporadic reports of co-consumption of illicit drugs with rodenticides such as warfarin and brodifacoum (BFC) over the past 20 years but recently, hundreds of people have been reported to have been poisoned with a mixture of synthetic cannabinoids and BFC. We have sought to establish whether BFC directly affects cannabinoid receptors, or their activation by the synthetic cannabinoid CP55940 or the phytocannabinoid ?9-tetrahydrocannabinol (?9-THC). Methods:The effects of BFC on the hyperpolarization of wild type AtT20 cells, or AtT20 cells stably expressing human CB1- or CB2- receptors, were studied using a fluorescent assay of membrane potential. The effect of BFC on CB1- and CB2-mediated inhibition of forskolin-stimulated adenylyl cyclase (AC) activation was measured using a BRET assay of cAMP levels in HEK 293 cells stably expressing human CB1 or CB2. Results:BFC did not activate CB1 or CB2 receptors, or affect the hyperpolarization of wild type AtT20 cells produced by somatostatin. BFC (1 µM) did not affect the hyperpolarization of AtT20-CB1 or AtT20-CB2 cells produced by CP55940 or ?9-THC. BFC (1 µM) did not affect the inhibition of forskolin-stimulated AC activity by CP55940 in HEK 293 cells expressing CB1 or CB2. BFC (1 µM) also failed to affect the desensitization of CB1 and CB2 signaling produced by prolonged (30 min) application of CP55940 or ?9-THC to AtT20 cells. Discussion:BFC is not a cannabinoid receptor agonist, and appeared not to affect cannabinoid receptor activation. Our data suggests there is no pharmacodynamic rationale for mixing BFC with synthetic cannabinoids; however, it does not speak to whether BFC may affect synthetic cannabinoid metabolism or biodistribution. The reasons underlying the mixing of BFC with synthetic cannabinoids are unknown, and it remains to be established whether the "contamination" was deliberate or accidental. However, the consequences for people who ingested the mixture were often serious, and sometimes fatal, but this seems unlikely to be due to BFC action at cannabinoid receptors.

Dataset Information

Functional coupling of a recombinant human 5-HT5A receptor to G-proteins in HEK-293 cells.

Publications

Functional coupling of a recombinant human 5-HT5A receptor to G-proteins in HEK-293 cells.

Similar Datasets

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