title: U73122 and U73343 inhibit receptor-mediated phospholipase D activation downstream of phospholipase C in CHO cells.
abstract: The aminosteroid 1-(6-¿[17beta-3-methoxyestra- 1,3,5(10)-trien- 17-yl]-amino¿hexyl)- 1H-pyrrole-2,5-dione (U73122) and its inactive analogue 1-(6-¿[17beta-3-methoxyestra-1,3,5(10)-trien- 17-yl]-amino¿hexyl-2,5-pyrrolidine-dione (U73343) are widely used to study the involvement of G protein-coupled 1-phosphatidylinositol-phosphodiesterase, or phospholipase C, in receptor-mediated cell activation. The present work shows that both aminosteroids inhibit cholecystokinin-(26-33)-peptide amide (CCK-8)-induced phospholipase D activation equipotently in Chinese hamster ovary cells expressing the cholecystokinin-A receptor (CHO-CCK(A) cells). In addition, the two aminosteroids virtually completely inhibited thapsigargin- and 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced phospholipase D activation. Since the latter two drugs mimic inositol 1,4,5-trisphosphate-mediated Ca2+ mobilisation and 1,2-diacylglycerol-mediated protein kinase C activation. respectively, this suggests that both U73122 and U73343 act downstream of phospholipase C to inhibit receptor-mediated phospholipase D activation. U73122, but not U73343. effectively inhibited both TPA/Ca2+-stimulated phospholipase D activation and TPA/phosphatidylserine-stimulated protein kinase C activation in a homogenate of CHO-CCK(A) cells. The data presented suggest that U73122 may act at the level of protein kinase C to inhibit activation of phospholipase D. The exact site of action of U73343 is presently unknown.
authors.affiliation: Department of Biochemistry, University of Nijmegen, Netherlands.
authors.forename: R R
authors.initials: RR
authors.lastname: Bosch
authors.forename: A M
authors.initials: AM
authors.lastname: Patel
authors.forename: S E
authors.initials: SE
authors.lastname: Van Emst-de Vries
authors.forename: R L
authors.initials: RL
authors.lastname: Smeets
authors.forename: J J
authors.initials: JJ
authors.lastname: De Pont
authors.forename: P H
authors.initials: PH
authors.lastname: Willems
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004963:Estrenes / Q000494:pharmacology*; D010739:Phospholipase D / Q000037:antagonists & inhibitors* / Q000378:metabolism; D010743:Phospholipids / Q000096:biosynthesis / Q000302:isolation & purification; D011760:Pyrrolidinones / Q000494:pharmacology*; D011949:Receptors, Cholecystokinin / Q000037:antagonists & inhibitors / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; D004963:Estrenes; D010743:Phospholipids; D011760:Pyrrolidinones; D011949:Receptors, Cholecystokinin; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; C074854:U 73343; D010738:Type C Phospholipases; D010739:Phospholipase D