Influx of extracellular calcium is required for the membrane translocation of 5-lipoxygenase and leukotriene synthesis

Abstract

Our studies assessed the effects of increases in intracellular calcium concentrations [( Ca2+]i) on leukotriene synthesis and membrane translocation of 5-lipoxygenase (5LO). The calcium ionophore ionomycin and the tumor promoter thapsigargin stimulated leukotriene production and translocation of 5-lipoxygenase to the membrane. Both agents elicited prolonged rises in [Ca2+]i. Leukotriene C4 production associated with [Ca2+]i in cells stimulated with various concentrations of ionomycin and thapsigargin suggests that a threshold [Ca2+]i level of approximately 300-400 nM is required. In the absence of extracellular Ca2+, both the ionomycin- and thapsigargin-induced rises in [Ca2+]i were transient, indicating that the prolonged [Ca2+]i elevation is due to an influx of extracellular Ca2+. Addition of EGTA to the external medium before, or at different times during, the treatment with ionomycin or thapsigargin instantaneously inhibited 5LO translocation and leukotriene synthesis, indicating that Ca2+ influx plays an essential role in 5LO membrane translocation and leukotriene synthesis. No leukotriene production was detected when cells were stimulated by a physiological stimulus of leukotriene D4. The addition of 100 nM leukotriene D4 triggered peak rises in [Ca2+]i that were comparable to those achieved by the ionomycin and thapsigargin. However, the leukotriene D4 induced rise was transient and rapidly declined to a lower but still elevated steady-state level, which was attributed to Ca2+ influx. Stimulation with 100 nM leukotriene D4 for 15 s increased the cellular levels of 1,4,5-inositol triphosphate (IP3), 1,3,4-IP3, and 1,3,4,5-inositol tetraphosphate (IP4).(ABSTRACT TRUNCATED AT 250 WORDS)

Time‐resolvedin situassembly of the leukotriene‐synthetic 5‐lipoxygenase/5‐lipoxygenase‐activating protein complex in blood leukocytes

“…Mouse anti-5-LO monoclonal antibody was a generous gift by Dr. Dieter Steinhilber (Goethe University, Frankfurt, Germany). Rabbit anti-CLP polyclonal antibody was from Acris Antibodies (San Diego, CA, USA); rabbit anti-FLAP polyclonal antibody, Abcam (Cambridge, United Kingdom); mouse anti-actin monoclonal antibody, Santa Cruz (Heidelberg, Germany); tritium-labeled [5,6,8,9,11,12,14, H]AA, Biotrend Chemicals GmbH (Cologne, Germany); HPLC solvents, Merck (Darmstadt, Germany); poly-D-lysin coated glass coverslips, neuVitro (El Monte, CA, USA). Duolink detection reagents red, Duolink PLA (proximity ligation assay) probe anti-rabbit PLUS, Duolink PLA probe antimouse MINUS, as well as Duolink wash buffers and all other chemicals were from Sigma-Aldrich (Deisenhofen, Germany) unless stated otherwise.…”

“…First, 5-LO catalyzes the incorporation of molecular oxygen at C5 of arachidonic acid yielding the intermediate 5(S)-hydroperoxy-6-trans-8,11,14-cis-eicosatetraenoic acid (5-HPETE) that in the second step is dehydrated by 5-LO to LTA 4 (5). The cellular capacity to generate 5-HPETE and LTA 4 depends on the translocation of 5-LO from a soluble compartment to the nuclear membrane, regulated by a fine-tuned machinery involving Ca 2+ -mediated binding to phosphatidylcholine (PC) in the nuclear membrane, phosphorylation at serine residues (6)(7)(8)(9)(10), and interaction of 5-LO with coactosin-like protein (CLP) (11,12).…”

Resolvin D1 limits 5-lipoxygenase nuclear localization and leukotriene B 4 synthesis by inhibiting a calcium-activated kinase pathway

“…Subsequent hydrolysis of LTA 4 by LTA 4 hydrolase yields LTB 4 (10,11). During inflammation, 5-LOX is phosphorylated and translocates to the nuclear membrane, which favors the biosynthesis of LTB 4 (12)(13)(14)(15)(16)(17). However, major gaps remain in our understanding of the relevance of this pathway to primary cells and animal models and how they are regulated by SPMs.…”

Regulating leukotriene synthesis: The role of nuclear 5‐lipoxygenase

“…Early studies on the source of calcium for 5-LO activation stressed the importance of extracellular calcium, rather than calcium released from internal stores [Schatz-Munding et al, 1991;Wong et al, 1991]. In these and similar studies, extracellular calcium was thought to move into the cytoplasm, where it activated 5-LO and cPLA 2 , consequently leading to LT synthesis.…”