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Functional characterization of agonists at recombinant human 5-HT2A, 5-HT2B and 5-HT2C receptors in CHO-K1 cells.

ABSTRACT: 1. The goal of this study was to characterize the agonist pharmacology of human 5-HT2A, 5-HT2B and 5-HT2C (VSV) receptors expressed in CHO-K1 (Chinese hamster ovary) cells. 2. We used a fluorometric imaging plate reader (FLIPR) which allows rapid detection of rises in intracellular calcium levels upon the addition of agonists. 3. Stimulation of all three receptors by 5-HT caused a robust concentration dependent increase in intracellular calcium levels. No such effect was observed from non-transfected control CHO-K1 cells. 4. The rank order of potency of agonists at the different receptor subtypes varied. Tryptamines, BW-723C86, d-norfenfluramine, Ro 60-0175 and LSD exhibited the following rank order of potency; 5-HT2B>5-HT2C>5-HT2A. Piperazines such as m-Chlorophenylpiperazine (mCPP), ORG-12962, MK-212 and also ORG-37684 exhibited a rank order of potency of 5-HT2C>5-HT2B>5-HT2A. The phenylisopropylamines DOI and DOB had a rank order of 5-HT2A>5-HT2B>5-HT2C. 5. Many agonists tested had partial agonist actions when compared to 5-HT, and a wide range of relative efficacies were exhibited, which was cell line dependent. For example, mCPP had a relative efficacy of 65% at 5-HT2C receptors but <25% at either 5-HT2A or 5-HT2B receptors. 6. Interpretation of literature values of functional assays using different cell lines, different receptor expression levels and different receptor isoforms, is complex. Species differences and the previous use of antagonist radioligands to characterize agonist potency in binding assays emphasizes the importance of studying agonists in the same experiment using the same assay conditions and parental cell lines.

SUBMITTER: Porter RH

PROVIDER: S-EPMC1571597 | biostudies-literature | 1999 Sep

REPOSITORIES: biostudies-literature

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Functional characterization of agonists at recombinant human 5-HT2A, 5-HT2B and 5-HT2C receptors in CHO-K1 cells.

Porter R H RH Benwell K R KR Lamb H H Malcolm C S CS Allen N H NH Revell D F DF Adams D R DR Sheardown M J MJ

British journal of pharmacology 19990901 1

1. The goal of this study was to characterize the agonist pharmacology of human 5-HT2A, 5-HT2B and 5-HT2C (VSV) receptors expressed in CHO-K1 (Chinese hamster ovary) cells. 2. We used a fluorometric imaging plate reader (FLIPR) which allows rapid detection of rises in intracellular calcium levels upon the addition of agonists. 3. Stimulation of all three receptors by 5-HT caused a robust concentration dependent increase in intracellular calcium levels. No such effect was observed from non-transf ...[more]

PMID: 10498829

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Project description:Timing deficits are observed in patients with schizophrenia. Serotonergic hallucinogens can also alter the subjective experience of time. Characterizing the mechanism through which the serotonergic system regulates timing will increase our understanding of the linkage between serotonin (5-HT) and schizophrenia, and will provide insight into the mechanism of action of hallucinogens. We investigated whether interval timing in mice is altered by hallucinogens and other 5-HT2 receptor ligands. C57BL/6J mice were trained to perform a discrete-trials temporal discrimination task. In the discrete-trials task, mice were presented with two levers after a variable interval. Responding on lever A was reinforced if the interval was <6.5 s, and responding on lever B was reinforced if the interval was >6.5 s. A 2-parameter logistic function was fitted to the proportional choice for lever B (%B responding), yielding estimates of the indifference point (T50) and the Weber fraction (a measure of timing precision). The 5-HT2A antagonist M100907 increased T50, whereas the 5-HT2C antagonist SB-242,084 reduced T50. The results indicate that 5-HT2A and 5-HT2C receptors have countervailing effects on the speed of the internal pacemaker. The hallucinogen 2,5-dimethoxy-4-iodoamphetamine (DOI; 3 mg/kg IP), a 5-HT2 agonist, flattened the response curve at long stimulus intervals and shifted it to the right, causing both T50 and the Weber fraction to increase. The effect of DOI was antagonized by M100907 (0.03 mg/kg SC) but was unaffected by SB-242,084 (0.1 mg/kg SC). Similar to DOI, the selective 5-HT2A agonist 25CN-NBOH (6 mg/kg SC) reduced %B responding at long stimulus intervals, and increased T50 and the Weber fraction. These results demonstrate that hallucinogens alter temporal perception in mice, effects that are mediated by the 5-HT2A receptor. It appears that 5-HT regulates temporal perception, suggesting that altered serotonergic signaling may contribute to the timing deficits observed in schizophrenia and other psychiatric disorders.

Project description:Though obesity is a global epidemic, the physiological mechanisms involved are little understood. Recent advances reveal that susceptibility to obesity can be programmed by maternal and neonatal nutrition. Specifically, a maternal low protein diet during pregnancy causes decreased intrauterine growth, rapid postnatal catch-up growth and increased risk for diet-induced obesity. Given that the synthesis of the neurotransmitter 5-hydroxytryptamine (5-HT) is nutritionally regulated and 5-HT is a trophic factor, we hypothesized that maternal diet influences fetal 5-HT exposure, which then influences central appetite network development and the subsequent efficacy of 5-HT to control energy balance in later life. Consistent with our hypothesis, pregnant low protein fed rat mothers exhibited elevated serum 5-HT, which was also evident in the placenta and fetal brains at E16.5. This increase was associated with reduced hypothalamic expression of 5-HT2CR - the primary 5-HT receptor influencing appetite. As expected, reduced 5-HT2CR expression was associated with impaired sensitivity to 5-HT-mediated appetite suppression. 5-HT primarily achieves effects on appetite via 5-HT2CR stimulation of pro-opiomelanocortin (POMC) peptides within the arcuate nucleus of the hypothalamus (ARC). We reveal that 5-HT2ARs are also anatomically positioned to influence the activity of ARC POMC and that 5-HT2AR mRNA is increased in the hypothalamus of in utero growth restricted offspring that underwent rapid postnatal catch-up growth. Furthermore, these animals are more sensitive to 5-HT2AR agonist-induced appetite suppression. These findings may not only reveal a 5-HT-mediated mechanism underlying programming of obesity susceptibility but also provide a promising means to correct it, via a 5-HT2AR agonist treatment. The study was carried out using male Wistar rats (Rattus norvegicus). On postnatal day 3, two experimental groups of offspring were established: controls (offspring of control dams) and recuperated (offspring of dams fed a low-protein diet (8% protein, w/v), but nursed by control dams. The animals were fed with standard chow until 3 months of age where the brains were collected for transcriptomic profiling

Dataset Information

Functional characterization of agonists at recombinant human 5-HT2A, 5-HT2B and 5-HT2C receptors in CHO-K1 cells.

Publications

Functional characterization of agonists at recombinant human 5-HT2A, 5-HT2B and 5-HT2C receptors in CHO-K1 cells.

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