In mammalian cells, active sodium transport and its derived functions (e.g., plasma membrane potential) are dictated by the activity of the Na ؉ ,K ؉ -ATPase (NK), whose regulation is essential for maintaining cell volume and composition, as well as other vital cell functions. Here we report the existence of a salt-inducible kinase-1 (SIK1) that associates constitutively with the NK regulatory complex and is responsible for increases in its catalytic activity following small elevations in intracellular sodium concentrations. Increases in intracellular sodium are paralleled by elevations in intracellular calcium through the reversible Na ؉ /Ca 2؉ exchanger, leading to the activation of SIK1 (Thr-322 phosphorylation) by a calcium calmodulin-dependent kinase. Activation of SIK1 results in the dephosphorylation of the NK ␣-subunit and an increase in its catalytic activity. A protein phosphatase 2A/phosphatase methylesterase-1 (PME-1) complex, which constitutively associates with the NK ␣-subunit, is activated by SIK1 through phosphorylation of PME-1 and its dissociation from the complex. These observations illustrate the existence of a distinct intracellular signaling network, with SIK1 at its core, which is triggered by a monovalent cation (Na ؉ ) and links sodium permeability to its active transport.cell volume ͉ Na ϩ /Ca2 ϩ exchanger ͉ Na ϩ ,K ϩ -ATPase ͉ protein phosphatase 2A

Leukotriene (LT) B4 is a powerful chemotactic and immune modulating agent that signals via two receptors denoted BLT1 and BLT2.Here we report that BLT1 and BLT2 are expressed at low levels in an apparently silent state in human umbilical vein endothelial cells (HUVEC). However, treatment with LPS leads to a >10 fold increase in the levels of BLT1 mRNA without any significant effects on BLT2 mRNA. In parallel, LPS also increases the amounts of BLT1 protein.Tumor necrosis factor-␣ (TNF-␣) increases the expression of BLT2 mRNA Ϸ6 times above basal levels with only a modest increase in BLT1 mRNA. Interleukin-1␤ causes variable and parallel increases of both BLT1 and BLT2 mRNA. The natural ligand LTB4 also increases BLT1, but not BLT2, mRNA and protein expression. Along with the induction of BLT1 and͞or BLT2, HUVEC acquire the capacity to respond to LTB4 with increased levels of intracellular calcium and these signals can be blocked by isotype selective BLT antagonists, CP-105696 and LY-255283. In addition, treatment of HUVEC with LTB4 causes increased release of both nitrite, presumably reflecting nitric oxide (NO), and monocyte chemoattractant protein-1. Our data indicate that expression of functional BLT receptors may occur at the surface of endothelial cells in response to LPS, cytokines, and ligand, which in turn may have functional consequences during the early vascular responses to inflammation. Moreover, the results point to BLT receptors as potential targets for pharmacological intervention in LT-dependent inflammatory diseases such as asthma, rheumatoid arthritis, and arteriosclerosis.inflammation ͉ arteriosclerosis ͉ rheumatoid arthritis ͉ asthma ͉ nitric oxide T he leukotrienes (LTs) are a family of lipid mediators that play important roles in a variety of allergic and inflammatory reactions (1, 2). These molecules are divided into two classes, the spasmogenic cysteinyl-LTs (cys-LT) and LTB 4 , which is a classical nM chemotactic agent produced by neutrophils, macrophages, and mast cells. Thus, LTB 4 is a potent chemoattractant for polymorphonuclear leukocytes, comparable to complement peptide C5a and fMet-Leu-Phe (3, 4). Recent data also indicate that LTB 4 is a strong chemoattractant for T cells, creating a functional link between early innate and late adaptive immune responses (5-7). Because of these biological effects, LTB 4 is regarded as an important chemical mediator in a variety of acute and chronic inflammatory diseases and only recently, genetic and biochemical evidence strongly implicate LTB 4 as a mediator of vascular inflammation and arteriosclerosis (8-10). LTB 4 , is synthesized from arachidonic acid via the concerted action of 5-lipoxygenase, assisted by 5-lipoxygenase-activating protein and the terminal LTA 4 hydrolase (11), usually in a single cell or via transcellular routes; this mechanism has been shown to occur in vivo (12,13). LTB 4 signals primarily via a specific, high-affinity, G proteincoupled seven-transmembrane receptor, termed BLT 1 (14). The BLT 1 gene is located on the huma...

Certain immunocompetent myeloid cells, such as eosinophils, basophils and mast cells, have a large capacity to synthesize the potent proinflammatory and spasmogenic mediator leukotriene (LT) C 4 via a specific microsomal glutathione S-transferase (MGST) termed LTC 4 synthase (LTC4S). Here, we report that MGST2, a distant homologue of LTC4S, is abundantly expressed in Human umbilical vein endothelial cells (HUVEC) and converts LTA 4 into a single product, LTC 4 . Thus, using Northern blot, RT-PCR, Western blot, and enzyme activity assays, we show that MGST2 is the main, if not the only, enzyme that converts LTA 4 into LTC 4 in membrane preparations of HUVEC. In fact, we failed to detect any expression of LTC4S, MGST1 or MGST3 in these cells, indicating that MGST2 is a critical enzyme for transcellular LTC 4 biosynthesis in the vascular wall. Unlike LTC4S, MGST2 prefers the naturally occurring free acid of LTA 4 over the methyl ester as substrate and is also susceptible to product inhibition with an IC 50 of about 1 mm for LTC 4 . Moreover, HUVEC were found to express the CysLT 1 receptor in line with a paracrine and autocrine role for cysteinyl-leukotrienes in endothelial cell function.Keywords: leukotriene C 4 synthase; microsomal GSH S-transferase; endothelial cells; inflammation; leukotriene receptor.Leukotrienes (LTs) are a family of lipid mediators involved in acute and chronic inflammatory and allergic diseases [1]. In the biosynthesis of LTs, the enzyme 5-lipoxygenase (5-LO) oxygenates arachidonic acid into the unstable epoxide LTA 4 , a central intermediate in LT biosynthesis. LTA 4 may be further converted into the potent chemoattractant LTB 4 , via the enzyme LTA 4 hydrolase, or conjugated with glutathione (GSH) to produce LTC 4 by a specific microsomal GSH S-transferase (MGST) termed LTC 4 synthase (LTC4S). LTC 4 is the parent compound of the cysteinyl-leukotrienes (cys-LTs) that include LTC 4 , LTD 4 , and LTE 4 . The cys-LTs are potent smooth musclecontracting agents, particularly in the respiratory tract and microcirculation and these effects are mediated via at least two cell surface receptors, CysLT 1 and CysLT 2 . The CysLT 1 receptor, which appears to signal most of the biological responses, was recently cloned and characterized as a putative G-protein-coupled receptor with seven transmembrane regions [2,3]. A large body of experimental evidence has demonstrated that cys-LTs play important roles in inflammatory and allergic diseases, particularly asthma [4±6]. It has also long been known that these lipid mediators have profound hemodynamic effects and constrict coronary vessels resulting in reduced cardiac output [7], probably a result of their spasmogenic properties. However, cys-LTs have also been shown to exert a variety of effects on endothelial cells. Thus, addition of LTC 4 or LTD 4 to human umbilical vein endothelial cells (HUVEC) stimulated synthesis of platelet-activating factor (PAF) and, concomitantly, the adherence of human polymorphonuclear leukocytes [8]. Furthermore, cys-LTs have ...

HUVECs almost exclusively express the CysLT2R. Furthermore, Ca2+ fluxes elicited by CysLT in these cells emanate from perturbation of the CysLT2R, rather than the expected CysLT1R. Hence, signaling events involving CysLT2R might trigger functional responses involved in the critical components of LT-dependent vascular reactions, which in turn have implications for ischemic heart disease and myocardial infarction.