Studies of leukotriene C4 synthase isoenzymes and the cysteinyl leukotrine receptors in human endothelial- and mast cells

Abstract: Leukotriene (LT) C4, the parent compound of LTD4 and LTE4, is generated when LTA4 is conjugated with GSH by either leukotriene C4 synthase (LTC4S) or microsomal GSH S-transferase (MGST) type 2 or 3. Together these lipid mediators cause contractions particularly in airway smooth muscle and the microcirculation, leading to bronchconstriction, plasma extravasation and edema formation as well as increased mucus secretion from epithelial cells and recruitment of inflammatory cells. Human umbilical vein endothelial cells (HUVEC) were found to generate LTC4 via MGST2 while the levels of LTC4S protein and mRNA were below the detection limit in these cells. Human mast cells expressed two functional LTC4 producing enzymes; LTC4S and MGST2 but no MGST3. Activity assays showed that LTC4S was responsible for around 80 %, of the LTC4 production in these cells. Moreover, MGST2 was shown to prefer the free acid of LTA4, the naturally occurring form of substrate, over the non-natural methyl ester, which is opposite to the preferences of LTC4S. The rat orthologs of these enzymes were cloned and found to be highly similar to their human counterparts with amino acid identities of 86.7 %, 79.6 %, and 86.2 % for LTC4S, MGST2, and MGST3, respectively. LTC4 as well as the FLAP inhibitor MK-886 was shown to inhibit all enzyme activities tested in the three enzymes pointing to a common, or at least overlapping, active site(s) for the different enzyme activities. Rats injected intra-peritoneally with lipopolysaccharide (LPS), showed a transient up-regulation of LTC4S mRNA within one hour in almost all tissues. LTC4S protein expression in brain, heart, liver, and adrenal gland increased 4.9-, 4.0, 2.9- and 2.3-fold, respectively. No effects were detected for MGST 2 or -3. Together, the data from human and rat indicate that LTC4S and MGST2 both serve as LTC4 producers. However, their individual roles are not yet elucidated but possibly MGST2 exists for housekeeping purposes while LTC4S is more active in certain situations concerning allergic- and inflammatory responses. The effects induced by the cysteinyl leukotrienes are mediated via at least two G- protein coupled seven transmembrane spanning surface receptors, the CysLT1 and CysLT2 receptors. CysLT1 mRNA and protein expression was detected in HUVEC and mast cells. The localization of the CysLT1 receptor was determined in cord blood mast cells (CBMC) and cultured HMC-1 mainly to the surface membrane but also to some extent to granules and the cytoplasm. Calcium signalling induced by LTD4 and LTC4 indicated LTD4 as a more potent agonist and the response could be completely inhibited by I µM of the CysLT1 specific antagonist Zafirlukast, both results consistent with a CysLT1-dominated scenario. HUVEC, however, besides CysLT1, also expressed CysLT2. When examined by quantitative RT-PCR, the mRNA ratio of CysLT2 to CysLT1 was determined to > 4300:1. Further, calcium responses elicited by cys-LTs and BAYu9773, a selective partial agonist for CysLT2, indicated signalling mainly from this receptor. In addition, BAYu9773 completely blocked calcium signalling induced by leukotrienes while MK-571, a CysLT1 specific antagonist, gave poor, if any, inhibition. Together, these data suggest that CysLT2 is the dominant receptor in HUVEC. The expression of CysLT2 in HUVEC grown in the presence of LPS or cytokines was also studied. LPS induced a transient suppression of mRNA while the effects of IL-10 and TNFalpha were at least 20 % suppression lasting 120 min or more. In light of these data it seems likely that the cys-LTs produced, in endothelial cells (EC) and mast cells, as they are exported, will be present in the vicinity and may very well act on the CysLT1/2 surface receptors of these cells, or cells of the same type nearby, thereby creating an autocrine or paracrine signalling loop.