Nitric Oxide (NO), Vasa Vasorum (v.v.), and the Ductus Arteriosus. • 845

Prior reports have shown that drugs that relax other blood vessels, by increasing NO and cGMP within the vessel, do not relax the ductus arteriosus; similarly, inhibitors of NO synthase (NOS), like L-NAME, do not constrict the fetal ductus in vivo. This suggests that NO may not have a role in ductus regulation. Using isolated ductus arteriosus from fetal lambs, we found that SNP and SNAP (which spontaneously release NO) are potent dilators of the ductus and their effects increase 6-fold with decreasing gestation (<100 d vs >125 d, term = 145 d). In the presence of PO₂ = 180 mm Hg, L-NAME constricts the ductus (15 ± 5% of maximal active tension). This is nearly as potent as indomethacin (21 ± 12% maximal active tension). The action of L-NAME is not mediated through prostaglandins and can be completely reversed by L-arg. Similarly, methylene blue (MB) and LY83583 (LY), inhibitors of cGMP release, contract the ductus to the same extent as L-NAME. These work through the same pathway since MB or LY has no effect after L-NAME contraction and vice versa. When the ductus is exposed to fetal PO₂ (28 mm Hg), L-NAME no longer affects the ductus. This appears to be due to decreased NO in the tissue, since SNP and SNAP relaxation is unaffected by low PO₂. In other blood vessels, removal of the luminal endothelium eliminates NO production in the vessel. In contrast, complete removal of the luminal endothelium decreases the effects of L-NAME, MB, and LY on the ductus by only 30%. We used RT-PCR to identify which of the 3 NOS isoforms are found in the fetal sheep ductus. Both eNOS and iNOS (but not bNOS) mRNA are present. By immunohistochemistry, iNOS is expressed only weakly by luminal endothelial cells, while eNOS is heavily expressed by both luminal and v.v. endothelial cells. After denuding the lumen, the only NOS isoform in the ductus is eNOS in the v.v. We conclude that endogenous NO does not regulate the ductus at fetal PO₂; in contrast, it may be as important as prostaglandins at postnatal PO₂. The rapid proliferation of v.v. postnatally in the ductus may make NO the most important inhibitor of postnatal closure.