Cyclo-oxigenase-2 expression in the lung in the period of pulmonary development after birth A. Somuyiwa, S. Stanford, J. Gitlin, I. Vojnovic, T. D. Warner, A. Hislop, S. Howarth and J. A. Mitchell. Unit of Critical Care, Imperial College School of Medicine, London SW3 6LY.

Print abstract Search PubMed for: Somuyiwa A Stanford S Gitlin J Vojnovic I Warner TD Hislop A Howarth S Mitchell JA

Cyclo-oxygenase (COX) is the enzyme responsible for the synthesis of prostacyclin (PGI₂), a product with important roles in the pulmonary vasculature. COX exists in two forms, COX-1 and COX-2, whose respective roles in PGI₂ production in pulmonary tissue before and after birth are not known. Previously, it has been shown that at birth there is an increase in PGI₂ production from the pulmonary vasculature, and that this is involved in the adaptation to extra-uterine life and the transition from placental to air breathing (Leffler et al., 1984; Velvis et al., 1991). When adaptation fails, persistent pulmonary hypertension of the new born (PPHN) occurs, which is associated with high levels of morbidity and mortality. Thus, in this study we have investigated how COX-2 levels change in the lung just before and just after birth.

Lung tissue was obtained from full term foetal through to adult pigs according to home office regulations. The extracted tissue was then homogenised, centrifuged (12,000RPM) for 10min at 4°C and protein content measured by a standard Lowry procedure. Gel loading buffer was added to the sample in a 1:1 ratio; the mixture was boiled at 100°C for 5 minutes, and centrifuged at 4°C for 10 min as we have described previously (Mitchell et al., 1993). After loading (50µg) samples onto SDS-polyacrylamide (12%) gels they were subjected to electrophoresis (20 mins at 100V, 1 hr at 150V), and the proteins were transferred to nitrocellulose filters (100V, 1 hr). The filters were blocked with PBS with 0.25% Tween 20 and 5% dried milk for 1 hr before the application of COX-2 primary antibody (raised in mouse 1:1000) for 1 hour, after washing, blots were incubated for 1 hr with horseradish-peroxidase conjugated anti-mouse secondary antibody.

The bands were visualised by chemiluminescence. In separate experiments, lung tissue from foetal and 14 day old pigs were incubated over night in culture in the presence or absence of the highly selective COX-2 inhibitor, DFU (10µM) or indomethacin (10µM), which inhibits both COX-1 and COX-2 (Mitchell et al., 1993; Warner et al., 1999) and PGE₂ measured as an index of COX activity. Lung tissue from each age group tested contained measurable COX-2 immunoreactivity. However, lungs from foetal and 14 day old pigs contained higher levels. Lungs from 1 and 3 day old pigs contained reduced levels of COX-2. COX activity in lung tissue from either foetal or 14 day old pigs was inhibited by more than 90% by either DFU or indomethacin (PGE2: control foetal 1.95±0.53ng mg^-1 ml^-1 n=8, 14 day 0.49±0.13ng mg^-1 ml^-1, n=6; after DFU: foetal 0.14±0.1, n=8; 14 day 0.39±0.15, n=6).

Fig 1. Representative Western blot of COX-2 protein expression in porcine whole lung tissue (1 - foetal, 2 - new born, 3 - 1 day, 4 - 3 day, 5 - 14 day, 6 - adult). Similar results were obtained in blots using tissue from two other animals from each time point.

Here we show that COX-2 is expressed in the lungs of healthy pigs and that its levels are temporally down regulated in the first few days after birth. These results have implications for our understanding of conditions such as PPHN as well as lung physiology in the adult. The mechanisms regulating COX-2 suppression at days 1-3 in the lung are the subject of further investigation.

This work was funded by the British Heart Foundation.

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