Hyperosmolarity stimulates prostaglandin synthesis and cyclooxygenase-2 expression in activated rat liver macrophages

Abstract

The effect of aniso-osmotic exposure on the level of inducible cyclooxygenase (Cox-2) and on prostanoid synthesis was studied in cultured rat liver macrophages (Kupffer cells). In lipopolysaccharide (LPS)- or phorbol 12-myristate 13-acetate-stimulated Kupffer cells, hyperosmotic (355 mosmol/l) exposure, due to addition of NaCl or impermeant sugars, markedly increased prostaglandin (PG) E2, D2 and thromboxane B2 synthesis in a time- and osmolarity-dependent manner. Increased prostanoid production was observed about 8 h after exposure to LPS in hyperosmotic medium compared to Kupffer cells treated with LPS under normotonic (305 mosmol/l) conditions. A similar stimulatory effect of hyperosmolarity on PGE2 production was also seen when arachidonate was added exogenously. Hyperosmotic stimulation of PGE2 production was accompanied by a strong induction of Cox-2 mRNA levels and an increase in immunoreactive Cox-2, whereas the levels of immunoreactive phospholipase A2 and cyclooxygenase-1 did not change significantly. Dexamethasone, indomethacin and the selective Cox-2 inhibitor, NS-398, abolished the hypertonicity-induced stimulation of PGE2 formation; dexamethasone also prevented the increase in Cox-2 mRNA and protein. The increase of immunoreactive Cox-2 lasted for about 24 h and was also blocked by actinomycin D or cycloheximide, but not by brefeldin A. Tunicamycin or treatment with endoglucosidase H reduced the molecular mass of hypertonicity-induced Cox-2 by 5 kDa. Tunicamycin treatment also suppressed the hypertonicity-induced stimulation of PGE2 production. The hyperosmolarity/LPS-induced stimulation of prostaglandin formation was partly sensitive to protein kinase C inhibition but was not accompanied by an increase in the cytosolic free Ca2+ concentration. The data suggest that osmolarity may be a critical factor in the regulation of Cox-2 expression and prostanoid production in activated rat liver macrophages.

Betaine is an osmolyte in RAW 264.7 mouse macrophages

“…organs facing either extreme osmotic stresses or being encapsulated by rigid bone. However, recent studies have indicated that eicosanoid formation by LPS-stimulated rat liver macrophages (Kupffer cells) is critically dependent upon the ambient osmolarity and probably cell volume within a narrow range [2]. Thus, betaine availability and/or regulation of betaine transporter expression by osmolarity and other factors could be a potential site for the regulation of macrophages cell function.…”

“…Upon stimulation with endotoxin they produce a variety of cytokines, lipid mediators and radicals. In rat liver macrophages, lipopolysaccharide (LPS)-induced prostaglandin E and thromboxane B formation and cyclooxygenase-2-expression is stimulated about 7-fold when ambient osmolarity increases from 300 to 350 mosmol/1 [2]. This sensitive and potent osmoregulation of prostanoid synthesis suggests that cell volume homeostasis may be a critical factor for macrophage function.…”

Modulation of phagocytosis by anisoosmolarity and betaine in rat liver macrophages (Kupffer cells) and RAW 264.7 mouse macrophages

“…Rat Kupffer cells were prepared by collagenase-pronase perfusion and separated by a single Nycodenz gradient and centrifugal elution as described previously [3,16]. Cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum (FCS) for 48 h. The experiments were performed during the following 24 h using Krebs-Henseleit hydrogen carbonate buffer (pH 7.4) containing 10 mmol/l glucose.…”

“…Recent studies have indicated that endotoxin enhances the expression of inducible cyclooxygenase-2 (Cox-2) [2][3][4][5][6], which results in the increased formation of prostanoids by Kupffer cells and other macrophages [3,[7][8][9]. Interestingly, lipopolysaccharide (LPS)-induced prostaglandin E2, D2 and thromboxane B2 formation and Cox-2 expression are stimulated up to 10-fold when ambient osmolarity increases from 300 to 350 mosmol/1 [3]. This remarkably sensitive and potent osmoregulation suggested that cell volume homeostasis is critical for Kupffer cell function.…”

Anisoosmotic regulation of the Nopp140 mRNA in H4IIE rat hepatoma cells and primary hepatocytes

“…This involves not only the regulation of osmoregulatory genes, such as the expression of aldose reductase [2] or speci¢c sodium-coupled transporters for myo-inositol [3,4], betaine [4,5] and taurine [4,6], but also the expression of genes encoding proteins that are not linked to osmoregulation. Examples of the latter are the rapid increases in mRNA levels for L-actin [7], inducible nitric oxide synthetase [8], cyclooxygenase-2 [9], c-jun [10] and phosphoenolpyruvate carboxykinase [11].…”