Altered pulmonary vascular reactivity secondary to increased pulmonary blood flow with pulmonary hypertension is a source of morbidity and mortality for children with congenital heart defects. The mechanisms responsible for this are not understood. Injury to the pulmonary vascular endothelium may be important, since endothelial cells produce substances which mediate pulmonary vascular tone. We have established a model of increased pulmonary blood flow with pulmonary hypertension in the lamb, following in utero placement of an aortopulmonary vascular graft. By 4 weeks of age, these lambs(Shunt) have endothelial dysfunction evidenced by: (1) decreased endothelial nitric oxide (NO)-mediated pulmonary vasodilation; (2) augmented pulmonary vasoconstriction in response to NO synthesis inhibition; (3) increased plasma cGMP concentrations; (4) loss of endothelin-1 (ET-1)- and endothelin b (ETb) receptor agonist-mediated pulmonary vasodilation; and (5) increased plasma ET-1 concentrations. To understand the molecular events underlying these physiological responses, we cloned ovine cDNA fragments for endothelial nitric oxide synthase (eNOS, the enzyme responsible for NO synthesis), big ET-1 (the precursor of ET-1), endothelin converting enzyme -1 (ECE-1, the enzyme responsible for the conversion to ET-1), and the two endothelin receptors, ETa and ETb. RNase protection assays and in situ hybridization were performed on lung tissue prepared from Shunt lambs and from 4-week-old lambs with normal pulmonary blood flow. In Shunt lambs, gene expression was altered. eNOS gene expression was increased 5-fold, big ET-1 2-fold, and ECE-1 5-10-fold. ETa receptor gene expression was decreased 2-fold and ETb 10-fold. These changes in gene expression explain, in part, the altered pulmonary vascular reactivity in Shunt lambs. Increased pulmonary blood flow with pulmonary hypertension alters the molecular regulation of pulmonary vascular tone.