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Apparent heterogeneity of cardiac A 1 adenosine receptors as revealed by radioligand binding experiments on N-ethylmaleimide-treated membranes

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Summary

While G protein-coupled receptors are often studied by analyzing antagonist radioligand: “cold” agonist inhibition curves using an independent site model, it is now clear that KL and KH values determined in these analyses are not reliable estimates of the affinities of the agonists for “free” and G protein-coupled forms of the receptor. Thus, such experiments cannot be used to contrast the characteristics of a given type of receptor in different tissues, i.e., to probe for the existence of receptor subtypes. Since treatment with N-ethylmaleimide treatment blocks receptor: Gi/G0 protein interactions, such analyses on N-ethylmaleimide-pretreated membranes should allow direct assessment of the affinities of competing ligands for the free receptor or for multiple receptor subtypes.

As A1 adenosine receptors couple to Gi, and perhaps to Go, we have performed A1 adenosine receptor radio-ligand “competition” studies first on control, then on N-ethylmaleimide-pretreated bovine cardiac and cerebral cortical membranes. Results of experiments with the antagonist radioligand [3H]xanthine amine congener appeared to be confounded by ligand binding to A2 adenosine receptors present in the cardiac membrane preparations. Further experiments utilized the A1-specific radioligand [3H] 1,3-dipropyl-8-cyclopentylxanthine. These experiments confirmed once more that the KL values determined by computer analysis of “competition” curves performed on control membranes are not reliable estimates of the affinities of the competing ligand for free receptors. Furthermore the results supported the hypothesis that similar analyses on NEM-treated membranes provide reliable estimates of the affinity(s) of competing ligands for free receptors. Lastly, the results suggest that cardiac membranes contain two subtypes of A1 adenosine receptors that are differentiated by 5′-modified but not N6-modified adenosine analogs. One of these receptor subtypes appears to be the same as the A1 receptor detected in cortical membranes.

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Abbreviations

[125I]ABA:

[1251](N6-p-aminobenzyl)adenosine

[3H]CPX:

[3H]8-cyclopentyl-1,3-dipropylxanthine

[3H]R-PIA:

[3H]N6-R-phenylisopropyladenosine

[6H]XAC:

[6H]xanthine amine congener

CHA:

N6-cyclohexyladenosine

EDTA:

ethylenediaminetetraacetic acid

[125I]BW-A844U:

[125I]3-(4-amino)phenethyl-l-propyl-8-cyclopentylxanthine

NCCA:

5′-N-cyclopropylcarboxamide adenosine

NECA:

5′-N-ethylcarboxamide adenosine

PMSF:

phenylmethylsulphonyl fluoride

NEM:

N-ethylmaleimide

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Authors and Affiliations

  1. Department of Pharmacology, University of Illinois at Chicago, College of Medicine, Box 6998, 60680, Chicago, IL, USA

    E. Leung & R. D. Green

  2. Laboratory of Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 20892, Bethesda, MD, USA

    K. A. Jacobson

Authors
  1. E. Leung
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  2. K. A. Jacobson
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  3. R. D. Green
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Recipient of a Postdoctoral Fellowship from the Chicago Heart Association

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Leung, E., Jacobson, K.A. & Green, R.D. Apparent heterogeneity of cardiac A 1 adenosine receptors as revealed by radioligand binding experiments on N-ethylmaleimide-treated membranes. Naunyn-Schmiedeberg's Arch Pharmacol 344, 639–644 (1991). https://doi.org/10.1007/BF00174747

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  • DOI: https://doi.org/10.1007/BF00174747

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