Co‐induction of nitric oxide synthase and cyclo‐oxygenase: interactions between nitric oxide and prostanoids
Authors: Swierkosz Tomasz A. , Mitchell Jane A. , Warner Timothy D. , Botting Regina M. , Vane John R. 
Published: July 2012

1 Lipopolysaccharide (LPS) co‐induces nitric oxide synthase (iNOS) and cyclo‐oxygenase (COX‐2) in J774.2 macrophages. Here we have used LPS‐activated J774.2 macrophages to investigate the effects of exogenous or endogenous nitric oxide (NO) on COX‐2 in both intact and broken cell preparations. NOS activity was assessed by measuring the accumulation of nitrite using the Griess reaction. COX‐2 activity was assessed by measuring the formation of 6‐keto‐prostaglandin F1α (6‐keto‐PGF1α) by radioimmunoassay. Western blot analysis was used to determine the expression of COX‐2 protein. We have also investigated whether endogenous NO regulates the activity and/or expression of COX in vivo by measuring NOS and COX activity in the lung and kidney, as well as release of prostanoids from the perfused lung of normal and LPS‐treated rats.

2 Incubation of cultured murine macrophages (J774.2 cells) with LPS (1 μg ml−1) for 24 h caused a time‐dependent accumulation of nitrite and 6‐keto‐PGF1α in the cell culture medium which was first significant after 6h. The formation of both 6‐keto‐PGF1α and nitrite elicited by LPS was inhibited by cycloheximide (1 μm) or dexamethasone (1 μm). Western blot analysis showed that J774.2 macrophages contained COX‐2 protein after LPS administration, whereas untreated cells contained no COX‐2.

3 The accumulation of 6‐keto‐PGF1α in the medium of LPS‐activated J774.2 macrophages was concentration‐dependently inhibited by chronic (24 h) exposure to sodium nitroprusside (SNP; 1–1000 μm). Sodium nitroprusside (l‐1000μm) also acutely (30 min) inhibited COX‐2 activity in broken cell preparations of LPS‐activated (12 h) J774.2 macrophages, in a similar concentration‐dependent manner. Addition of adrenaline (5 mM) and glutathione (0.1 mM) increased the activity of COX‐2 in broken cell preparations. In the presence of these co‐factors, SNP inhibited prostanoid production only at the highest concentration used (1 mM). When J774.2 cells were incubated in the presence of LPS (1 μg ml−1) and NG‐monomethyl‐L‐arginine (L‐NMMA: 1 mM) for 12 h, SNP at the highest concentration used (1 mM) acutely (30 min) inhibited the activity of COX‐2 in cell homogenates with co‐factors. However, when J774.2 macrophages were incubated for 24 or 12 h with LPS (1 μg ml−1) and L‐NMMA (1 mM), the addition of SNP (0.001–1000 μm) increased in a concentration‐dependent manner the accumulation of 6‐keto‐PGF1α in intact cells (measured at 24 h) and COX‐2 activity in cell homogenates in the presence of co‐factors (determined at 12 h). SNP (1 mM; together with LPS for 12 h) decreased the amount of COX‐2 protein induced by LPS in J774.2 macrophages.

4 Indomethacin (30 μm) abolished the formation of 6‐keto‐PGF1α by LPS‐activated macrophages, but had no effect on the release of nitrite. Conversely, L‐NMMA, at the highest concentrations used (1 and 10 mM), increased the release of 6‐keto‐PGF1α, an effect which was reversed by excess L‐arginine (3 mM) but not by D‐arginine. Similarly, the decrease in nitrite formation caused by L‐NMMA was partially reversed by L‐arginine (3 mM), but not by D‐arginine. L‐NMMA (10 mM; together with LPS for 12 h) increased the amount of COX‐2 protein induced by LPS in J774.2 macrophages.

5 In separate experiments, J774.2 macrophages were activated with LPS (1 μg ml−1), and L‐NMMA (10 mM) was added for various times (0.5–24 h) before the collection of medium at 24 h. L‐NMMA enhanced the release of 6‐keto‐PGF1α in a time‐dependent manner, with the maximal enhancement seen when the NOS inhibitor was incubated with the cells for 24 h.

6 In experiments on male Wistar rats, we investigated the effect of L‐NMMA on the release of prostanoids (6‐keto‐PGF1α, prostaglandin E2, thromboxane B2) elicited by arachidonic acid (AA, 30 nmol) from ex vivo perfused kidneys and lungs. The release from the organs from normal and LPS‐treated rats was unaffected by L‐NMMA intraperitoneally (30 mgkg−1) for 6 h together with LPS (5 mgkg−1) or LPS vehicle. Similarly, acute (15 min) in vitro exposure to L‐NMMA (1 mM) of the perfused organs from control and LPS‐treated animals did not change the release of prostanoids elicited by AA (30 nmol).

7 These results show that LPS causes the induction of iNOS and COX‐2 in J774.2 macrophages. The co‐release of NO and PGI2 induced by LPS is dependent on protein synthesis and occurs after a lag‐time of 6–12 h. The formation of COX metabolites has no effect on NOS activity whereas NO inhibits both COX‐2 activity and induction. These results demonstrate that NOS and COX can be co‐induced in vitro and that under these conditions large amounts of NO inhibit the degree of COX expression and activity. In the absence of endogenous NO, lesser amounts of exogenous NO increase the activity of COX‐2. In those situations in vivo when the level of NO induction is relatively low, NO does not regulate the increased activity of COX.