Involvement of prenylated proteins in calcium signaling induced by LTD4 in differentiated U937 cells
Introduction
It has long been accepted that cysteine-containing leukotrienes (Cys-LTs), LTC4, LTD4 and LTE4, play an important role in a number of inflammatory diseases, particularly in asthma, participating both to the bronchoconstriction and to the chronic inflammatory component of the disease. Cys-LTs originate from the oxidative metabolism of arachidonic acid through a key enzyme, 5-lipoxygenase, in a number of inflammatory cells including eosinophils, basophils, mast cells and macrophages [1].
Recently, the first two receptor isoforms have been cloned [2], [3], [4], [5], [6], with a distribution that clearly appears to be quite peculiar for each isoform. While CysLT1 receptor is more abundant in smooth muscle cells and lung macrophages, in agreement with the antibronchoconstrictive and antiinflammatory actions of CysLT1 receptor antagonists [1], it is also expressed in most peripheral blood eosinophils and basophils, and in subsets of monocytes and B lymphocytes [7]. At variance, CysLT2 is more abundant in heart and brain [4].
When activated by LTD4, CysLT1 receptor seems to activate at least two or even three signaling systems, including pertussis toxin (PTx)-sensitive and -insensitive G proteins [8]. It is widely recognized that the result of this interaction is the rise in cytosolic Ca2+ concentration [Ca2+]i, that is usually a complex phenomenon: a first, rapid and transient phase is due to discharge from the intracellular organelles, while a second, slower and more persistent elevation represents influx through the plasma membrane. However, differences exist between cell types with respect both to the role of Ca2+ and to the mechanisms of [Ca2+]i elevation in LTD4-induced response [9]. For instance, LTD4 is able to induce Ca2+ influx through the plasma membrane without any release from intracellular stores [10], possibly through a receptor-operated Ca2+ channel (ROC) [8]; or it may only discharge the ion from intracellular organelles by means of phospholipase C (PLC) activation which in turn induces phosphoinositol hydrolysis [11]; or, in other cell types, both Ca2+ influx and release have been reported to occur [11], [12]. Furthermore, in human epithelial cells, PTx inhibits only LTD4-induced Ca2+ influx across the plasma membrane [12] while LTD4-induced inositol 1,4,5-trisphosphate (IP3) formation involves a PLC of the γ1 isoform [13] instead of the usually expected β isoform for a Gq-coupled receptor.
The aim of this work was to study the signal transduction mechanisms responsible for LTD4-induced [Ca2+]i elevation in intact differentiated U937 (dU937) cells, a promonocytic leukemia cell line known, upon differentiation, to endogenously express a high density of LTD4 receptors [14]. We have investigated the role of both PTx-sensitive and -insensitive pathways and the involvement of isoprenylated proteins such as the βγ subunit of heterotrimeric G proteins and small GTP binding proteins in a physiologically relevant cell type closely related to the inflammatory cells responsible of many Cys-LT biological actions, especially in asthma.
Section snippets
Materials
Phosphate buffered saline (PBS), RPMI 1640, fetal calf serum (FCS), bovine serum albumin (BSA), EGTA, PMSF, aprotinin, penicillin, streptomycin, l-glutamine, DMSO, probenecid, penicillamine, Hepes, and Clostridium difficile toxin B were from Sigma (St. Louis, MO). All salts for saline and Tris solution were from Merck (Darmstadt, D). FPT II, U73122, FTS and PTx were from Calbiochem (La Jolla, CA); BAL 9504 was provided by Prof. M. Macchia; Clostridium sordellii lethal toxin was a generous gift
Characterization of CysLT1 receptor-mediated [Ca2+]i transient
In order to confirm that dU937 cells express an LTD4 receptor, we performed equilibrium binding studies in intact cells using -LTD4 as labeled ligand (Fig. 1a). Computer analysis of the mixed type curve [15] revealed the presence of a single high-affinity binding site with Kd=0.5 nM±35% CV and Bmax=5.8 fmol/106 cells±33% CV.
LTD4 was able to trigger a concentration-dependent increase in [Ca2+]i with an EC50 value of 3.4 nM±27% CV (Fig. 1b). Fig. 2a shows a representative trace of the [Ca2+]i
Discussion
In this study, we demonstrate that the receptor expressed in the promonocytic leukemia cell line U937 differentiated with DMSO is pharmacologically a CysLT1 receptor, and that part of the LTD4-generated signal in these cells is through a PTx-sensitive G protein. In addition, we report the involvement of isoprenylated proteins such as the βγ complex of heterotrimeric G protein and a member of the Ras GTPase family in LTD4-induced [Ca2+]i elevation.
Equilibrium binding data confirmed the presence
Acknowledgements
The authors would like to acknowledge Dr. M.R. Popoff for kindly providing the Clostridium sordellii lethal toxin. This work was partially supported by Grant MM05242257 from MURST (CoFin 2000) and by Grant 2002055453 (CoFin 2002) to G.E.R. and M.P.
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The first and second authors have equally contributed to the work.