Evidence for a dual effect of IP receptor activation on E. coli-induced NOSII activity in murine macrophages

Lucy Bailey, Laura Moreno, Mark Paul-Clark, Jane Mitchell
Imperial College, London, United Kingdom

Prostacyclin (PGI2) is an endogenous hormone, synthesised by the endothelium, with important cardiovascular effects. It is increasingly recognised that PGI2 and other prostaglandins also have significant immunomodulatory roles (Rajakariar et al., 2006). The principle receptor for PGI2 is the cell surface IP receptor. In the current study we have investigated the effect of activation of IP receptors on E. coli-induced nitric oxide synthase (NOS) II activity in macrophages.

Murine macrophages (J774) were cultured using standard techniques (Paul-Clark et al., 2006) and plated into 96-well plates for treatment. Cells were treated with the PGI2 analogue treprostinil sodium at a range of concentrations (Figure 1) with or without an additional pre-treatment with the IP receptor antagonist CAY10441 (10 μM), and stimulated with whole heat-killed E. coli (1x106 or 3x107 CFU/ml). After an incubation period of 24 hours, NOSII activity was assessed by measuring the accumulation of nitrite in cell supernatants using the Griess reaction.

Figure 1. Effect of treprostinil sodium and an IP antagonist (CAY10441) on NOSII activity in murine macrophages stimulated with sub-maximal (A) and maximal (B) concentrations of whole heat-killed E. coli. Data is mean ± S.E.M. of n=4. Differences in treprostinil sodium vs. control (Con) treatments were analysed by Dunnett’s post-test following ANOVA. Statistical significance (*) was assumed at P<0.05.

Under control culture conditions, no nitrite was detected. E. coli at 106 CFU/ml induced a submaximal increase in NOSII activity which was inhibited by treprostinil sodium (Figure 1A). E. coli at 3x107 CFU/ml induced higher levels of NOSII activity which were enhanced by treprostinil sodium (Figure 1B). Both these inhibitory and stimulatory effects of treprostinil sodium were suppressed by an additional treatment with the IP antagonist CAY10441. The effects of pre-treatment with the IP receptor antagonist also indicate that endogenous IP receptor agonists are acting to enhance NOSII activity in these cells.

These observations suggest that treprostinil sodium has a dual effect on NOSII activity, illustrating the complex relationship between PGI2 signalling and immune responses

Paul-Clark, M.J. et al.(2006) British Journal of Pharmacology 148(8), 1067-1075

Rajakariar, R et al. (2006) Molecular Interventions 6(4), 199-207