Studies on molecular properties and functional regulation of terminal leukotriene C4 synthases and cysteinyl-leukotriene receptor signalling in human endothelium

Abstract

The transformation of the unstable intermediate leukotriene (LT) A4 into the glutathione conjugate LTC4, the parent compound of LTD4 and LTE4, is catalysed by leukotriene C4 synthase (LTC4S) as well as microsomal glutathione S-transferase type 2 (MGST2) and type 3 (MGST3). Together, these eicosanoids also known as cysteinyl leukotrienes (cys-LT) are key mediators of immediate hypersensitivity reactions. However, recent evidence also supports their pivotal role in adaptive immune response as well as in the progression of inflammatory disease.

The rat orthologs of these LTC4S isoenzymes were successfully cloned and functionally expressed in Spodoptera frugiperda insect cells. The rat enzymes were found to be highly similar to their human counterparts with amino acid identities of 87%, 80%, and 86% for LTC4S, MGST2, and MGST3, respectively. As might be expected from the structural similarities between the human and rat enzyme, rat LTC4S also showed a high degree of analogy to the human ortholog regarding catalytic features. Thus, Km for the recombinant enzyme, using the free acid and the methyl ester of LTA4 as substrate, were calculated to 18.8 muM and 19.8 muM, respectively. In contrast, rat MGST2 converted the free acid of LTA4 more efficiently than the methyl ester which is in accordance with the human counterpart. Whereas the LTC4S capacity was preserved in rat MGST2, rat MGST3 failed to show any significant LTC4S activity. Both, LTC4 and the 5-lipoxygenase activating protein inhibitor MK-886 inhibited all respective enzymatic activities of the terminal LTC4S isoenzymes, i.e. LTC4S activity (LTC4S and MGST2), GSH transferase activity (MGST2), and peroxidase activity (MGST2 and MGST3), suggesting that the catalytic centres originate from structurally related overlapping active site(s).

Intraperitoneal injection of lipopolysaccharide (LPS) in rats lead to a transient increase of LTC4S mRNA in several tissues, particularly heart, brain, adrenal glands, and liver, within one hour followed by a 4.9-, 4.0-, 2.9, and 2.3-fold induction of LTC4S protein expression at six hours in brain, heart, liver, and adrenal gland, respectively, indicating that up-regulation of LTC4S might be triggered by systemic inflammatory signals and prime certain tissues for increased cys-LT biosynthesis. In contrast, no effects were detected for MGST2 and MGST3 suggesting that these enzymes do not contribute to LTC4 formation during host-defence reactions, but may be involved in cys-LT biosynthesis for other, basal house-keeping purposes.

Using in situ hybridization histochemistry and reverse transcription polymerase chain reaction (RT-PCR) the expression of MGST3 in the rat central nervous system (CNS) was investigated both, under normal conditions and after intraperitoneal injection of LPS. The broad distribution in the CNS was characterized by a strong signal in the hippocampal formation, the nuclei of the cranial nerves as well as the motor neurons in the spinal cord and sensory neurons in the dorsal root ganglia. A moderate signal was found in the cortex, thalamus, amygdala, and substantia nigra and a weak signal in the hypothalamus. However, no changes in the level of MGST3 mRNA expression in the CNS were found one, three, or six hours after LPS administration which do not support a role for MGST3 in the biosynthesis of pro-inflammatory cys-LT but rather suggest other functions, e.g. metabolic detoxication and neuroprotection.

Human umbilical vein endothelial cells (HUVEC) were found to abundantly express CysLT2 mRNA in vast excess (>4000-fold) of CysLT1 mRNA when examined by quantitative RT-PCR. Pro-inflammatory stimuli (LPS, Tumor necrosis factor alpha, and Interleukin-1beta) caused a rapid (within 30 minutes) and partially reversible suppression of CysLT2 mRNA levels. Challenge of HUVEC with BAY u9773, a partial CysLT2 agonist, triggered diagnostic Ca2+ transients. LTC4 and LTD4 were demonstrated to be equipotent agonists, and their actions could be blocked by BAY u9773, which is also a dual CysLT1 and CysLT2 receptor antagonist, but not by the CysLT1-selective antagonist MK571. Together, these data indicate that signalling events involving CysLT2 might trigger functional responses involved in critical components of cys-LT dependent vascular reactions, which in turn have implications for ischemic heart disease and myocardial infarction.