The Aminosteroid Phospholipase C Antagonist U-73122 (1-[6-[[17-β-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-β-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 ≈2 to 4 μM), but it failed to suppress responses induced by ionomycin or thapsigargin. 5-Lipoxygenase (5-LO) is a Ca²⁺-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 ≈2 μM. Direct inhibition of 5-LO by U-73122 was evident in PMNL homogenates (IC₅₀ of ≈2.4 μM), and isolated human recombinant 5-LO enzyme was potently inhibited by U-73122 (IC₅₀ of ≈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.