title: Effects of long-term treatment with the allosteric enhancer, PD81,723, on Chinese hamster ovary cells expressing recombinant human A1 adenosine receptors.
abstract: In this study, desensitization and down-regulation of A1 adenosine receptors (A1AR) by the allosteric enhancer PD81,723 (PD) and by N6-cyclopentyladenosine (CPA) were investigated after 24-hr pretreatment of CHO-K1 cells stably expressing recombinant human A1AR. Pretreatment with 20 microM PD and 10 microM CPA caused a 1.5- and 4.0-fold, respectively, desensitization (reduced potency of CPA to lower cAMP). Pretreatment with PD and/or CPA did not modify the acute effect of PD to increase (5-fold) the potency of CPA. Radioligand binding was used to measure receptor down-regulation in cell membranes and in intact cells. Pretreatment of cells with PD had no effect on the number of membrane binding sites for the agonist [125I] N6-(3-iodo-4-aminobenzyl) adenosine or for the antagonist, [3H]8-cyclopentyl-1,3-dipropylxanthine, but the binding of these radioligands to intact cells was modestly reduced (20-37%), possibly reflecting an effect of pretreatment on receptor subcellular distribution. Pretreatment of cells with CPA produced large ( > 40%) reductions in the binding of radioligands to both membranes and intact cells. Pretreatment of cells with CPA also increased the number of presumed internalized receptors measured as [3H]8-cyclopentyl-1,3-dipropylxanthine binding sites in intact cells insensitive to blockade by the charged antagonist 8-sulfophenyltheophylline. The relatively small degree of functional desensitization and down-regulation of A1AR caused by long term exposure of cells to PD is considered to be encouraging in terms of the therapeutic potential of the allosteric enhancer class of compounds.
authors.affiliation: Department of Internal Medicine, University of Virginia, Chariottesville 22908, USA.
authors.forename: S
authors.initials: S
authors.lastname: Bhattacharya
authors.forename: J
authors.initials: J
authors.lastname: Linden
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D000494:Allosteric Regulation; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D015536:Down-Regulation; D006801:Humans; D007700:Kinetics; D058906:Purinergic P1 Receptor Agonists; D011869:Radioligand Assay; D018047:Receptors, Purinergic P1 / Q000096:biosynthesis / Q000502:physiology*; D011994:Recombinant Proteins / Q000819:agonists / Q000378:metabolism; D013876:Thiophenes / Q000494:pharmacology*; D014162:Transfection
chemical_list: D058906:Purinergic P1 Receptor Agonists; D018047:Receptors, Purinergic P1; D011994:Recombinant Proteins; D013876:Thiophenes; C075004:PD 81723; C048599:N(6)-cyclopentyladenosine; D000242:Cyclic AMP; D000241:Adenosine