title: Differential allosteric modulation by amiloride analogues of agonist and antagonist binding at A(1) and A(3) adenosine receptors.
abstract: The diuretic drug amiloride and its analogues were found previously to be allosteric modulators of antagonist binding to A(2A) adenosine receptors. In this study, the possibility of the allosteric modulation by amiloride analogues of antagonist binding at A(1) and A(3) receptors, as well as agonist binding at A(1), A(2A), and A(3) receptors, was explored. Amiloride analogues increased the dissociation rates of two antagonist radioligands, [3H]8-cyclopentyl-1,3-dipropylxanthine ([3H]DPCPX) and [3H]8-ethyl-4-methyl-2-phenyl-(8R)-4,5,7,8-tetrahydro-1H-imidazo[2,1-i]purin-5-one ([3H]PSB-11), from A(1) and A(3) receptors, respectively. Amiloride and 5-(N,N-dimethyl)amiloride (DMA) were more potent at A(1) receptors than at A(3) receptors, while 5-(N,N-hexamethylene)amiloride (HMA) was more potent at A(3) receptors. Thus, amiloride analogues are allosteric inhibitors of antagonist binding at A(1), A(2A), and A(3) adenosine receptor subtypes. In contrast to their effects on antagonist-occupied receptors, amiloride analogues did not affect the dissociation rates of the A(1) agonist [3H]N(6)-[(R)-phenylisopropyl]adenosine ([3H]R-PIA) from A(1) receptors or the A(2A) agonist [3H]2-[p-(2-carboxyethyl)phenyl-ethylamino]-5'-N-ethylcarboxamidoadenosine ([3H]CGS21680) from A(2A) receptors. The dissociation rate of the A(3) agonist radioligand [125I]N(6)-(4-amino-3-iodobenzyl)adenosine-5'-N-methyluronamide ([125I]I-AB-MECA) from A(3) receptors was decreased significantly by amiloride analogues. The binding modes of amiloride analogues at agonist-occupied and antagonist-occupied receptors differed markedly, which was demonstrated in all three subtypes of adenosine receptors tested in this study. The effects of the amiloride analogues on the action of the A(3) receptor agonist were explored further using a cyclic AMP functional assay in intact CHO cells expressing the human A(3) receptor. Both binding and functional assays support the allosteric interactions of amiloride analogues with A(3) receptors.
authors.affiliation: Molecular Recognition Section, Laboratory of Bioorganic Chemistry, NIDDK/National Institutes of Health, Bldg. 8A, Rm. B1A-19, Bethesda, MD 20892-0810, USA.
authors.forename: Zhan-Guo
authors.initials: ZG
authors.lastname: Gao
authors.forename: Neli
authors.initials: N
authors.lastname: Melman
authors.forename: Andreas
authors.initials: A
authors.lastname: Erdmann
authors.forename: Seong Gon
authors.initials: SG
authors.lastname: Kim
authors.forename: Christa E
authors.initials: CE
authors.lastname: Müller
authors.forename: Adriaan P
authors.initials: AP
authors.lastname: IJzerman
authors.forename: Kenneth A
authors.initials: KA
authors.lastname: Jacobson
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000494:Allosteric Regulation / Q000187:drug effects; D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology*; D000818:Animals; D016466:CHO Cells; D005576:Colforsin / Q000494:pharmacology; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D004232:Diuretics / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D007700:Kinetics; D010627:Phenethylamines / Q000494:pharmacology; D058906:Purinergic P1 Receptor Agonists; D058915:Purinergic P1 Receptor Antagonists; D011687:Purines / Q000494:pharmacology; D011869:Radioligand Assay; D043705:Receptor, Adenosine A2A; D043684:Receptor, Adenosine A3; D018047:Receptors, Purinergic P1 / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014316:Tritium; D014970:Xanthines / Q000494:pharmacology
chemical_list: C438613:8-ethyl-4-methyl-2-phenyl-4,5,7,8-tetrahydro-1H-imidazo(2,1-i)purin-5-one; D004232:Diuretics; D007093:Imidazoles; D010627:Phenethylamines; D058906:Purinergic P1 Receptor Agonists; D058915:Purinergic P1 Receptor Antagonists; D011687:Purines; D043705:Receptor, Adenosine A2A; D043684:Receptor, Adenosine A3; D018047:Receptors, Purinergic P1; D014970:Xanthines; D014316:Tritium; C061282:2-(4-(2-carboxyethyl)phenethylamino)-5'-N-ethylcarboxamidoadenosine; D005576:Colforsin; C047235:N-(1-methyl-2-phenylethyl)adenosine; D000584:Amiloride; C051360:1,3-dipropyl-8-cyclopentylxanthine; C089722:N(6)-(4-amino-3-iodobenzyl)adenosine-5'-N-methyluronamide; D000242:Cyclic AMP; D000241:Adenosine