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Tritium-labeled agonists as tools for studying adenosine A2B receptors.

ABSTRACT: A selective agonist radioligand for A2B adenosine receptors (A2BARs) is currently not available. Such a tool would be useful for labeling the active conformation of the receptors. Therefore, we prepared BAY 60-6583, a potent and functionally selective A2BAR (partial) agonist, in a tritium-labeled form. Despite extensive efforts, however, we have not been able to establish a radioligand binding assay using [3H]BAY 60-6583. This is probably due to its high non-specific binding and its moderate affinity, which had previously been overestimated based on functional data. As an alternative, we evaluated the non-selective A2BAR agonist [3H]NECA for its potential to label A2BARs. [3H]NECA showed specific, saturable, and reversible binding to membrane preparations of Chinese hamster ovary (CHO) or human embryonic kidney (HEK) cells stably expressing human, rat, or mouse A2BARs. In competition binding experiments, the AR agonists 2-chloroadenosine (CADO) and NECA displayed significantly higher affinity when tested versus [3H]NECA than versus the A2B-antagonist radioligand [3H]PSB-603 while structurally diverse AR antagonists showed the opposite effects. Although BAY 60-6583 is an A2BAR agonist, it displayed higher affinity versus [3H]PSB-603 than versus [3H]NECA. These results indicate that nucleoside and non-nucleoside agonists are binding to very different conformations of the A2BAR. In conclusion, [3H]NECA is currently the only useful radioligand for determining the affinity of ligands for an active A2BAR conformation.

SUBMITTER: Hinz S

PROVIDER: S-EPMC6107469 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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Tritium-labeled agonists as tools for studying adenosine A2B receptors.

Hinz Sonja S Alnouri Wessam M WM Pleiss Ulrich U Müller Christa E CE

Purinergic signalling 20180511 3

A selective agonist radioligand for A2B adenosine receptors (A2BARs) is currently not available. Such a tool would be useful for labeling the active conformation of the receptors. Therefore, we prepared BAY 60-6583, a potent and functionally selective A2BAR (partial) agonist, in a tritium-labeled form. Despite extensive efforts, however, we have not been able to establish a radioligand binding assay using [3H]BAY 60-6583. This is probably due to its hi ...[more]

PMID: 29752618

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Project description:To determine the mechanisms by which blockade of adenosine A(2B) receptors (A(2B)Rs) reduces insulin resistance.We investigated the effects of deleting or blocking the A(2B)R on insulin sensitivity using glucose tolerance tests (GTTs) and hyperinsulinemic-euglycemic clamps in mouse models of type 2 diabetes. The effects of diabetes on A(2B)R transcription and signaling were measured in human and mouse macrophages and mouse endothelial cells. In addition, tag single nucleotide polymorphisms (SNPs) in ~42 kb encompassing the A(2B)R gene, ADORA2B, were evaluated for associations with markers of diabetes and inflammation.Treatment of mice with the nonselective adenosine receptor agonist 5'-N-ethylcarboxamidoadensoine (NECA) increased fasting blood glucose and slowed glucose disposal during GTTs. These responses were inhibited by A(2B)R deletion or blockade and minimally affected by deletion of A(1)Rs or A(2A)Rs. During hyperinsulinemic-euglycemic clamp of diabetic KKA(Y) mice, A(2B)R antagonism increased glucose infusion rate, reduced hepatic glucose production, and increased glucose uptake into skeletal muscle and brown adipose tissue. Diabetes caused a four- to sixfold increase in A(2B)R mRNA in endothelial cells and macrophages and resulted in enhanced interleukin (IL)-6 production in response to NECA due to activation of protein kinases A and C. Five consecutive tag SNPs in ADORA2B were highly correlated with IL-6 and C-reactive protein (CRP). Diabetes had a highly significant independent effect on variation in inflammatory markers. The strength of associations between several ADORA2B SNPs and inflammatory markers was increased when accounting for diabetes status.Diabetes affects the production of adenosine and the expression of A(2B)Rs that stimulate IL-6 and CRP production, insulin resistance, and the association between ADORA2B SNPs and inflammatory markers. We hypothesize that increased A(2B)R signaling in diabetes increases insulin resistance in part by elevating proinflammatory mediators. Selective A(2B)R blockers may be useful to treat insulin resistance.

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Tritium-labeled agonists as tools for studying adenosine A2B receptors.

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Tritium-labeled agonists as tools for studying adenosine A<sub>2B</sub> receptors.

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