HYPEROSMOLARITY STIMULATES PROSTAGLANDIN SYNTHESIS AND CYCLOOXYGENASE-2 EXPRESSION IN ACTIVATED RAT-LIVER MACROPHAGES

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) E(2), D-2 and thromboxane B-2 synthesis in a time- and osmolarity-dependent manner. Increased prostanoid production was observed about sh 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 PGE(2) production was also seen when arachidonate was added exogenously. Hyperosmotic stimulation of PGE(2) 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 A(2) and cyclooxygenase-1 did not change significantly. Dexamethasone, indomethacin and the selective Cox-2 inhibitor, NS-398, abolished the hypertonicity-induced stimulation of PGE(2) 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 5kDa. Tunicamycin treatment also suppressed the hypertonicity-induced stimulation of PGE(2) 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.