title: Synthesis of cysteinyl leukotrienes in human endothelial cells: subcellular localization and autocrine signaling through the CysLT2 receptor.
abstract: The purpose of this study was to characterize enzyme, receptor, and signaling involved in the synthesis and the activity of cysteinyl leukotrienes (cys-LTs) in human umbilical vein endothelial cells (HUVECs). We used primary cultures of HUVECs and evaluated the formation of cys-LTs by RP-HPLC. Suicide inactivation and subcellular localization of the enzyme responsible for the conversion of leukotriene (LT) A(4) into LTC(4) were studied by repeated incubations with LTA(4) and immunogold electron microscopy. The CysLT(2) receptor in HUVECs was characterized by equilibrium binding studies, Western blot analysis, and immunohistochemistry. Concentration-response curves in HUVECs and in transfected COS-7 cells were used to characterize a novel specific CysLT(2) receptor antagonist (pA(2) of 8.33 and 6.79 against CysLT(2) and CysLT(1) receptors, respectively). The results obtained provide evidence that the mGST-II synthesizing LTC(4) in HUVECs is pharmacologically distinguishable from the LTC(4)-synthase (IC(50) of MK886 <5 μM for LTC(4)-synthase and >30 μM for mGST-II), is not suicide-inactivated and is strategically located on endothelial transport vesicles. The CysLT(2) receptor is responsible for the increase in intracellular Ca(2+) following exposure of HUVECs to cys-LTs and is coupled to a pertussis toxin-insensitive G(q) protein. The synthesis of cys-LTs from LTA(4) by endothelial cells is directly associated with the activation of the CysLT(2) receptor (EC(50) 0.64 μM) in a typical autocrine fashion.
authors.affiliation: Department of Pharmacological Sciences, University of Milan, 20133 Milan, Italy.
authors.forename: Chiara
authors.initials: C
authors.lastname: Carnini
authors.forename: Maria Rosa
authors.initials: MR
authors.lastname: Accomazzo
authors.forename: Emanuele
authors.initials: E
authors.lastname: Borroni
authors.forename: Laura
authors.initials: L
authors.lastname: Vitellaro-Zuccarello
authors.forename: Thierry
authors.initials: T
authors.lastname: Durand
authors.forename: Giancarlo
authors.initials: G
authors.lastname: Folco
authors.forename: G Enrico
authors.initials: GE
authors.lastname: Rovati
authors.forename: Valerie
authors.initials: V
authors.lastname: Capra
authors.forename: Angelo
authors.initials: A
authors.lastname: Sala
mesh_terms: D000818:Animals; D019898:Autocrine Communication / Q000502:physiology*; D001692:Biological Transport / Q000502:physiology; D001792:Blood Platelets / Q000378:metabolism; D019556:COS Cells; D020013:Calcium Signaling / Q000502:physiology; D002522:Chlorocebus aethiops; D042783:Endothelial Cells / Q000378:metabolism*; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000378:metabolism; D005786:Gene Expression Regulation / Q000502:physiology; D006801:Humans; D017572:Leukotriene A4 / Q000378:metabolism; D017997:Leukotriene C4 / Q000096:biosynthesis*; D018077:Receptors, Leukotriene / Q000235:genetics / Q000378:metabolism*
chemical_list: D017572:Leukotriene A4; D018077:Receptors, Leukotriene; D017997:Leukotriene C4; C412375:cysteinyl leukotriene receptor 2; D043802:GTP-Binding Protein alpha Subunits, Gq-G11