Synthesis of cysteinyl leukotrienes in human endothelial cells: subcellular localization and autocrine signaling through the CysLT2 receptor

• Published in: The FASEB journal 2011-10, Vol.25 (10), p.3519-3528
• Main Authors: Carnini, Chiara, Accomazzo, Maria Rosa, Borroni, Emanuele, Vitellaro‐Zuccarello, Laura, Durand, Thierry, Folco, Giancarlo, Rovati, G. Enrico, Capra, Valerie, Sala, Angelo
• Format: Article
• Language: English
• Subjects: Animals; Autocrine Communication - physiology; Biological Transport - physiology; Blood Platelets - metabolism; Calcium Signaling - physiology; Cercopithecus aethiops; Chemical Sciences; COS Cells; Endothelial Cells - metabolism; Gene Expression Regulation - physiology; GTP-Binding Protein alpha Subunits, Gq-G11 - metabolism; Humans; intracellular Ca2; Leukotriene A4 - metabolism; Leukotriene C4 - biosynthesis; microsomal glutathione S‐transferase‐II; Organic chemistry; Receptors, Leukotriene - genetics; Receptors, Leukotriene - metabolism; signal transduction
• ISSN: 0892-6638; 1530-6860
• DOI: 10.1096/fj.10-177030

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) A4 into LTC4 were studied by repeated incubations with LTA4 and immunogold electron microscopy. The CysLT2 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 CysLT2 receptor antagonist (pA2 of 8.33 and 6.79 against CysLT2 and CysLT1 receptors, respectively). The results obtained provide evidence that the mGST‐II synthesizing LTC4 in HUVECs is pharmacologically distinguishable from the LTC4‐synthase (IC50 of MK886 30 μM for mGST‐II), is not suicide‐inactivated and is strategically located on endothelial transport vesicles. The CysLT2 receptor is responsible for the increase in intracellular Ca2+ following exposure of HUVECs to cys‐LTs and is coupled to a pertussis toxin‐insensitive Gq protein. The synthesis of cys‐LTs from LTA4 by endothelial cells is directly associated with the activation of the CysLT2 receptor (EC50 0.64 μM) in a typical autocrine fashion.—Carnini, C., Accomazzo, M. R., Borroni, E., Vitellaro‐Zuccarello, L., Durand, T., Folco, G., Rovati, G. E., Capra, V., Sala, S. Synthesis of cysteinyl leukotrienes in human endothelial cells: subcellular localization and autocrine signaling through the CysLT2 receptor. FASEB J. 25, 3519–3528 (2011). www.fasebj.org