title: The aminosteroid phospholipase C antagonist U-73122 (1-[6-[[17-beta-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione) potently inhibits human 5-lipoxygenase in vivo and in vitro.
abstract: U-73122 (1-[6-[[17-beta-3-methoxyestra-1,3,5(10)-trien-17-yl]amino] hexyl]-1H-pyrrole-2,5-dione) is a widely used antagonist of phosphoinositide-specific phospholipase C (PLC) and is frequently used to define a role of PLC in receptor-mediated elevation of intracellular calcium concentration ([Ca2+]i). In human polymorphonuclear leukocytes (PMNLs), U-73122 inhibited increases in [Ca2+]i induced by G protein-coupled receptor (GPCR) agonists (N-formyl-methionyl-leucyl-phenylalanine or platelet-activating factor; IC50 of approximately 2 to 4 microM), but it failed to suppress responses induced by ionomycin or thapsigargin. 5-Lipoxygenase (5-LO) is a Ca(2+)-regulated enzyme that can be activated in leukocytes by stimuli that elevate [Ca2+]i. Attempts to investigate the involvement of PLC in cellular 5-LO activation revealed that U-73122 suppresses 5-LO product synthesis regardless of the stimulus and independently of Ca2+. Thus, U-73122 blocked 5-LO product synthesis induced by cell stress, involving 5-LO phosphorylation pathways in the absence of Ca2+ with an IC50 of approximately 2 microM. Direct inhibition of 5-LO by U-73122 was evident in PMNL homogenates (IC50 of approximately 2.4 microM), and isolated human recombinant 5-LO enzyme was potently inhibited by U-73122 (IC50 of approximately 30 nM). Thiols (glutathione) strongly blunted the effect of U-73122 on isolated 5-LO. On the other hand, depletion of cellular thiols by N-ethylmaleimide strongly increased the efficacy of U-73122 to inhibit 5-LO in intact cells or corresponding homogenates, suggesting that U-73122 may interfere with sulfhydryl groups on 5-LO. Since 5-LO products induce increases in [Ca2+]i via GPCRs, caution should be used when interpreting data where U-73122 is used as tool to determine a direct role of PLC in receptor-mediated Ca2+ mobilization.
authors.affiliation: Institute of Pharmaceutical Chemistry, University of Frankfurt, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Feisst
authors.forename: Dana
authors.initials: D
authors.lastname: Albert
authors.forename: Dieter
authors.initials: D
authors.lastname: Steinhilber
authors.forename: Oliver
authors.initials: O
authors.lastname: Werz
mesh_terms: D001094:Arachidonate 5-Lipoxygenase / Q000378:metabolism; D001792:Blood Platelets / Q000187:drug effects / Q000201:enzymology; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D004963:Estrenes / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D016859:Lipoxygenase Inhibitors; D011760:Pyrrolidinones / Q000737:chemistry / Q000494:pharmacology*; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D004963:Estrenes; D016859:Lipoxygenase Inhibitors; D011760:Pyrrolidinones; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; D001094:Arachidonate 5-Lipoxygenase; D010738:Type C Phospholipases