Abstract 1742 Pulmonary Vascular Biology Platform, Monday, 5/3

Inhaled nitric oxide (NO) is a useful adjunct therapy in some neonates and children with pulmonary hypertension. However, increasing experience with inhaled NO has demonstrated a life threatening rapid increase in pulmonary vascular resistance (PVR) upon acute withdrawal. Recent data suggests that this "rebound pulmonary hypertension" is mediated by a reduction in the endogenous production of NO. However, its mechanisms and possible prevention strategies are unclear. We are interested in understanding this process. We have demonstrated that the treatment of 4-week-old lambs for 24 hours with inhaled NO produces a significant decrease in endogenous endothelial NO synthase (ENOS) activity in lung protein homogenates. Upon inhaled NO withdrawal this is associated with an acute increase in PVR. Similarly, the exposure of cultured endothelial cells to excess levels of NO attenuates ENOS activity. This attenuation is associated with an increase in cellular production of superoxide. The superoxide anion can be directly toxic but its oxidant reactivity is limited compared to other free radicals. However, NO contains an unpaired electron and can react rapidly with superoxide to form peroxynitrite. Peroxynitrite is a strong oxidizing agent which can react with biological molecules, including proteins. To investigate the possible roles and mechanisms of action of each of these species (NO, superoxide, and peroxynitrite) in the inhibition of ENOS activity we have used recombinant human ENOS purified from an an E.coli expression system. When exposed to either NO, superoxide, or peroxynitrite ENOS activity was significantly reduced. However, the mechanism by which this occurred was different in each case. NO was found to increase the levels of, inactive, monomeric, ENOS. Superoxide exposure resulted in the cleaving of the ENOS protein into two fragments of approximately 75kD and 60kD respectively. Peroxynitrite exposure was shown to induce a nitrosylation of the ENOS protein which was localized to the NADPH binding region within the reductase domain of the protein. This was shown to decrease the ability of the ENOS protein to accept electrons from NADPH. Thus, their appear to be multiple mechanisms by which reactive oxygen species can inhibit the activity of ENOS.