Nitric Oxide Upregulates Endothelial Nitric Oxide Synthase Expression in Fetal Pulmonary Artery Endothelial Cells • 1783

Abstract

Pulmonary vasodilation during cardiopulmonary transition at birth is mediated by endothelium derived nitric oxide (NO), which is generated by the endothelial isoform of NO synthase (NOS), or eNOS. Inhaled NO therapy is currently often used to treat neonatal pulmonary hypertension. Some patients receiving inhaled NO are difficult to wean from the gas after prolonged treatment, suggesting that long-term NO exposure modifies the function of the NO-cGMP signalling cascade. Although NO is known to acutely and reversibly inhibit eNOS activity, the effects of prolonged NO exposure on eNOS expression are not known. We therefore determined the effects of long-term NO exposure on eNOS expression in early passage ovine fetal pulmonary artery endothelial cells (PAEC). To first examine the effects of endogenous NO, PAEC were treated with 2 mM nitro-L-arginine methyl ester (L-NAME). L-NAME caused a decrease in eNOS protein expression that was evident within 8h and maximal by 16h (67% decrease); the effect persisted for at least 48h. The effects of exogenous NO were examined in PAEC treated with the NO donor spermine NONOate or the control parent compound spermine (10-8M to 10-6M). Exogenous NO caused increases in both eNOS protein and cell lysate NOS activity that were also evident within 8h, maximal at 16h (223% of control), and persistent for at least 48h. However, eNOS mRNA abundance was not altered by changes in the level of endogenous or exogenous NO, indicating that the effect of NO on eNOS expression is mediated at the level of translation. This effect is more rapid than previously demonstrated transcriptional regulation of PAEC eNOS by oxygen(≥ 24h). Thus, both endogenous and exogenous NO cause positive feedback, translational upregulation of eNOS gene expression in fetal PAEC. To our knowledge, this is the first demonstration of eNOS regulation mediated at the level of translation. Furthermore, these findings suggest that difficulties with prolonged requirements for inhaled NO in certain patients are not due to changes in the capacity for endogenous NO production.