title: Nonselective effects of the putative phospholipase C inhibitor, U73122, on adenosine A1 receptor-mediated signal transduction events in Chinese hamster ovary cells.
abstract: Adenosine A1 receptors can signal, through Gi/o proteins, to inhibit adenylyl cyclase activity and also to stimulate phosphoinositide hydrolysis and the subsequent release of intracellular Ca2+ stores. The aminosteroid U73122 (1-[6-1[17beta-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1 H-pyrrole-2,5-dione) has been widely used as an inhibitor of phospholipase C, the enzyme mediating phosphoinositide hydrolysis. Using U73122, we sought to selectively block signalling through the phospholipase C pathway, in Chinese hamster ovary (CHO-K1) cells heterologously expressing human adenosine A1 receptors. U73122 inhibited A1 receptor-mediated phosphoinositide hydrolysis, as measured by total inositol phosphate accumulation, over the concentration range 1-15 microM. However, over the same concentration range, it also appeared to inhibit A1 receptor-mediated inhibition of forskolin-stimulated cyclic AMP accumulation, A1 receptor agonist-promoted [35S]GTP-gammaS binding, and at the higher concentrations (10-15 microM) produced marked morphological changes, leading to cytolysis. The structural analogue of U73122, U73343 (1-[6-[[17beta-3-methoxyestra-1,3,5(10-trien-17-yl]amino]hexyl]-2, 5-pyrrolidone-dione), typically used as an inactive control compound, had little effect on these events. The data suggest that U73122 is not a selective inhibitor of phospholipase C activity, interfering with adenosine A1 receptor signalling generally, either at the pre-effector level involving Gi/o proteins, or as a consequence of the morphological changes it induces.
authors.affiliation: Institute of Cell Signalling, School of Biomedical Sciences, Queen's Medical Centre, Nottingham, UK.
authors.forename: E M
authors.initials: EM
authors.lastname: Walker
authors.forename: J R
authors.initials: JR
authors.lastname: Bispham
authors.forename: S J
authors.initials: SJ
authors.lastname: Hill
mesh_terms: D000818:Animals; D016466:CHO Cells; D002470:Cell Survival / Q000187:drug effects; D005576:Colforsin / Q000494:pharmacology; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004963:Estrenes / Q000494:pharmacology*; D019204:GTP-Binding Proteins / Q000502:physiology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism; D007700:Kinetics; D010716:Phosphatidylinositols / Q000378:metabolism; D011760:Pyrrolidinones / Q000494:pharmacology*; D018047:Receptors, Purinergic P1 / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D011994:Recombinant Proteins / Q000096:biosynthesis; D015398:Signal Transduction / Q000187:drug effects*; D014162:Transfection; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors; D014970:Xanthines / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D004963:Estrenes; D007295:Inositol Phosphates; D010716:Phosphatidylinositols; D011760:Pyrrolidinones; D018047:Receptors, Purinergic P1; D011994:Recombinant Proteins; D014970:Xanthines; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; C074854:U 73343; D005576:Colforsin; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C051360:1,3-dipropyl-8-cyclopentylxanthine; D000242:Cyclic AMP; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins