Carbon monoxide formation in the ductus arteriosus in the lamb: Implications for the regulation of muscle tone

1 We have previously shown that carbon monoxide (GO) potently relaxes the lamb ductus arteriosus and have ascribed this response to inhibition of a cytochrome P450-based mono-oxygenase reaction controlling the formation of endothelin-1 (ET-1). In the present study, we have examined whether CO is formed naturally in the vessel. 2 The CO-forming enzyme, haem oxygenase (HO), was identified in ductal tissue in its constitutive (HO-2) and inducible (HO-1) isoforms by Western immunoblotting and immunological staining procedures (both light and electron microscopy). HO-1 was localized to endothelial and muscle cells, while HO-2 was found only in muscle cells. Inside the muscle cells, HO-1 and HO-2 immunoreactivity was limited to the perinuclear region, and the Golgi apparatus in particular. However, upon exposure to endotoxin, HO-1 became more abundant, and both HO isoforms migrated towards the outer region of the cytoplasm close to the sarcolemma. 3 CO was formed enzymatically from added substrate (hemin, 50 mu M) in the 10,000 g supernatant of the ductus and its formation was inhibited by zinc protoporphyrin IX (ZnPP, 200 mu M). 4 ZnPP (10 mu M) had no effect on the tone of the ductus under normal conditions (2.5 to 95% O-2), but it contracted the endotoxin-treated ductus (at 2.5% O-2) At the same concentration, ZnPP also tended to contract the hypoxic vessel (zero O-2) 5 ZnPP (10 mu M) curtailed the relaxant response of the oxygen (30%)/indomethacin (2.8 mu M)-contracted ductus to bradykinin (35 nM), while it left the sodium nitroprusside (35 nM) relaxation unchanged. 6 We conclude that CO is formed in the ductus and may exert a relaxing influence when its synthesis is upregulated by an appropriate stimulus.