In vitro assessment of the agonist properties of the novel 5-HT1A receptor ligand, CUMI-101 (MMP), in rat brain tissue

https://doi.org/10.1016/j.nucmedbio.2010.08.003Get rights and content

Abstract

Introduction

Development of agonist positron emission tomography (PET) radioligands for the 5-HT neurotransmitter system is an important target to enable the understanding of human 5-HT function in vivo. [11C]CUMI-101, proposed as the first 5-HT1A receptor agonist PET ligand, has been reported to behave as a potent 5-HT1A agonist in a cellular system stably expressing human recombinant 5-HT1A receptors. In this study, we investigate the agonist properties of CUMI-101 in rat brain tissue.

Methods

[35S]-GTPγS binding studies were used to determine receptor function in HEK (human embryonic kidney) 293 cells transfected with human recombinant 5-HT1A receptors and in rat cortex and rat hippocampal tissue, following administration of CUMI-101 and standard 5-HT1A antagonists (5-HT, 5-CT and 8-OH-DPAT).

Results

CUMI-101 behaved as an agonist at human recombinant 5-HT1A receptors (pEC50 9.2). However, CUMI-101 did not show agonist activity in either rat cortex or hippocampus at concentrations up to 10 μM. In these tissues, CUMI-behaved as an antagonist with pKBs of 9.2 and 9.3, respectively.

Conclusions

Our studies demonstrate that as opposed to its behavior in human recombinant system, in rat brain tissue CUMI-101 behaves as a potent 5-HT1A receptor antagonist.

Introduction

In vivo examination of serotonin (5-HT) neurotransmission is an important aspect of understanding the pathophysiology of neuropsychiatric disorders [1], [2], [3], [4]. Seven 5-HT receptor subtypes have been identified (5-HT1-7) of which 5-HT1A is best characterized [5], [6]. The 5-HT1A receptor belongs to the G protein-coupled receptor family and is coupled to Gi/o G-proteins which, following agonist stimulation, inhibit adenylyl cyclase [6]. G-protein receptors can exist in a “high-affinity” (or G-protein coupled) state and a “low-affinity” (or G-protein uncoupled) state, with agonists binding preferentially to the G-protein coupled receptor (‘high affinity’) state whereas antagonists bind to both “high-affinity” and “low-affinity” states with equal affinity [7], [8], [9]. Investigation of dopaminergic neurotransmission has been greatly aided by our ability to investigate the fluctuations in endogenous dopamine release, via an interaction of synaptic dopamine with PET ligands such as [11C]raclopride at the D2/D3 receptors [10]. Conversely, investigation of fluctuations of endogenous 5-HT levels using existing 5-HT1A PET radioligands, such as [11C]WAY-100635, has not proved fruitful [11]. The purported reasons for this lack of success include the hypothesis that the high-affinity state of the 5-HT1A receptor is relatively rare; hence, physiologically relevant increases in 5-HT (the binding of which is predominantly to the high-affinity receptor state) are not sufficient to significantly alter the binding potential of antagonist radioligands such as [11C]WAY-100635. If this hypothesis is correct, an agonist PET ligand for the 5-HT system should be more successful in detecting 5-HT fluctuations.

[11C]CUMI-101 (previously known as [11C]MMP) has been developed as the first putative 5-HT1A agonist PET radioligand and has been reported to behave as a potent agonist in a cellular system expressing human recombinant 5-HT1A receptors [12], [13]. As agonist-related behavior of a compound can be affected significantly by the system within which it is tested, we further investigated the pharmacology of CUMI-101 in rat brain tissue. Detailed understanding of the mechanism of action in rat tissue may allow for more confident extrapolation to human native systems, given that the 5-HT1A receptor shows 89% receptor homology between rat and human [14], [15]. Such characterization is an important step in the utilization of novel PET ligands.

Section snippets

Recombinant h5-HT1A receptor membrane preparation

Human embryonic kidney (HEK) 293 cells stably expressing human recombinant (h) 5-HT1A receptor were re-suspended in 10 volumes of 50 mM HEPES pH 7.4 with 1 mM EDTA, 100 μM leupeptin, 25 μg/ml bacitracin, 1 mM Phenyl-methyl-sulfonyl-fluoride (PMSF) and 2 μM pepstatin A and then homogenized using an Ultraturrex. The homogenate was centrifuged at 500×g for 20 min at 4°C and the resultant supernatant (S1) was removed and stored on ice. The S1 fraction was then centrifuged at 48,000×g for 30 min at

HEK 293 cells expressing h5-HT1A receptors

In a HEK 293 recombinant system expressing h5-HT1A receptors, CUMI-101 displayed a profile consistent with that of an agonist with a pEC50 of 9.2±0.07 and an IA of 0.7±0.05.

Rat native tissue–agonist studies

In rat cortex and hippocampus 5-HT stimulated [35S]-GTPγS binding with pEC50s of 6.9±0.2 and 6.7±0.2, respectively (Fig. 1A and B). In rat cortex and hippocampus, CUMI-101 had no effect on [35S]-GTPγS binding at concentrations up to 10 μM.

5-CT stimulated binding with a greater potency than 5-HT (pEC50 7.9±0.1 and 7.6±0.2 for

Conclusions

In this study, we investigated the effects of CUMI-101 on [35S]-GTPγS binding in rat native brain tissue. We replicated data previously published by Kumar et al., indicating that CUMI-101 is a potent agonist at the human recombinant 5-HT1A receptor. It is generally recognised that divergence in terms of agonist responses occurs often between recombinant and native tissue systems. We therefore investigated the pharmacology of CUMI-101 in rat cortex and hippocampus, brain regions associated with

Acknowledgments

The authors would like to thank Laurie Gordon for the data generated against human recombinant 5-HT1A receptors.

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