Prednisolone (PRED) is ineffective in treating adult respiratory distress syndrome (ARDS) (Bernard et al., 1987). ARDS is associated with the generation of superoxide (O₂^▪-), which negates nitric oxide (NO) bioavailability. In turn, NO down-regulates NADPH oxidase and inhibits O₂^▪- formation (Muzaffar et al., 2003; 2004). The lack of a therapeutic effect of PRED may be due to an inhibitory effect on the expression of eNOS. In order to test this proposal, the effect of PRED on O₂^▪- formation and the expression of eNOS and gp91^phox (catalytic subunit of NADPH oxidase) in pig pulmonary artery (PA) segments and endothelial cells (PAECs) was investigated.

PA segments and PAECs were incubated with PRED and tumour necrosis factor- (TNF) for 16 hrs. O₂^▪- was then measured spectrophometrically and gp91^phox and eNOS expression assessed using Western blotting. The role of the NO-cyclic GMP axis in mediating effects was studied using m orpholinosydnonimine hydrochloride (SIN-1), the guanylyl cyclase inhibitor, 1H-{1,2,4}oxadiazolo{4,3-a}quinoxalin-1-one (ODQ) and 8-bromo cyclic GMP. Data below are expressed as mean ± SEM, n = 6. Statistics were carried out using ANOVA and post hoc unpaired students t-test with a Bonferonni adjustment.

PRED elicited a concentration-dependent increase in the formation of O₂^▪- in intact pig pulmonary artery segments (with endothelium) (maximal response at 1 µM PRED; 120 ± 18 nmoles / hr compared t o 25 ± 5 nmoles in controls ) and PAECs (maximal response at 1 µM PRED: 7 ± 0.3 m moles / hr compared to 4 ± 0.7 m moles / hr in controls). PRED (1 µM) -induced increase in O 2 .- release was augmented by
TNF (10 ng / ml) in i ntact PA segments (175 ± 9 nmoles / hr ) and PAECs (15 ± 0.2 m moles / hr) . O ₂^▪- formation was completely inhibited by apocynin and DPI (NADPH oxidase inhibitors). The increase of O₂^▪- release from PAECs in response to PRED was blocked by SIN-1 and 8-Bromo cGMP. In turn, the effect of SIN-1 on PRED -induced O 2 .- release was blocked by ODQ. gp91^phox protein expression was significantly enhancedincreased by 1 µM PRED (40% increase compared to controls) whereas eNOS protein expression was significantly reduced (50% decrease compared to controls).

Prednisolone increases O₂^▪- formation in porcine PAECs through an upregulation of NADPH oxidase that is mediated, at least in part, through a down-regulation of eNOS expression. Since the NO-cGMP axis inhibits gp91^phox expression, the resultant decrease in endogenous NO formation augments NADPH oxidase activity and therefore O₂^▪- formation. Since O₂^▪- promotes inflammation, hypertension and further negation of NO bioavailability, this mechanism may explain, in part, why prednisolone is ineffective in treating ARDS. Thus, the co-administration of an NO donor may render prednisolone more effective in treating ARDS.

Bernard, G.R., et al. (1987) N. Engl. J. Med.317: 1565-1570
Muzaffar, S., et al. (2003) Thorax 58 : 598-604
Muzaffar, S., et al. (2004) Circulation: in press