Nitric oxide (NO) produced by the enzyme endothelial nitric oxide synthase(eNOS) is critically involved in the cardiopulmonary transition from fetal to neonatal life. We have previously demonstrated that NO-dependent relaxation is attenuated in intrapulmonary arteries from lambs with neonatal pulmonary hypertension (PHT) created by prenatal ligation of the ductus arteriosus. In the present study we determined if pulmonary eNOS expression is altered in the lamb PHT model. Using immunoblot analyses, eNOS and neuronal NOS (nNOS) protein expression were assessed in lungs from near-term PHT lambs and control lambs who underwent ductal ligation or sham surgery, respectively, 10 days earlier. eNOS protein expression in PHT lung was decreased to 51±9% of control*. In contrast, nNOS protein abundance was unchanged (114±60% of control). Factor VIII-associated antigen expression was also unaltered in PHT lung (82±12% of control), indicating that the change in eNOS is not related to differences in endothelial cell density. In association with the decrease in eNOS protein abundance, NOS enzymatic activity was also diminished(60±3 versus 110±7 fmol/mg prot/min*, in PHT and control lung, respectively). To study eNOS mRNA expression, sheep eNOS cDNA was obtained from cultured ovine pulmonary endothelium by degenerate PCR. Sequencing of sheep eNOS cDNA revealed 98% identity, respectively, with bovine and human eNOS at the nucleotide level. Specific primers were then designed to evaulate pulmonary eNOS mRNA abundance in semiquantitative reverse transcription(RT)-PCR assays which included PCR for the house-keeping gene malate dehydrogenase to control for the RT step and for RNA stability. Paralleling the decline in eNOS protein expression and NOS enzymatic activity, eNOS mRNA abundance was also decreased in PHT lung (36±16% of control*). Thus, pulmonary eNOS protein and mRNA expression are attenuated in this lamb model of neonatal PHT. These findings suggest that diminished eNOS gene expression may contribute to certain forms of neonatal PHT. *p<0.05.
