Nitric oxide exposure inhibits endothelial NOS activity but not gene expression: a role for superoxide.

Recent studies have characterized a rebound pulmonary vasoconstriction with abrupt withdrawal of inhaled nitric oxide (NO) during therapy for pulmonary hypertension, suggesting that inhaled NO may downregulate basal NO production. However, the exact mechanism of this rebound pulmonary hypertension remains unclear. The objectives of these studies were to determine the effect of NO exposure on endothelial NO synthase (eNOS) gene expression, enzyme activity, and posttranslational modification in cultured pulmonary arterial endothelial cells. Sodium nitroprusside (SNP) treatment had no effect on eNOS mRNA or protein levels but did produce a significant decrease in enzyme activity. Furthermore, although SNP treatment induced protein kinase C (PKC)-dependent eNOS phosphorylation, blockade of PKC activity did not protect against the effects of SNP. When the xanthine oxidase inhibitor allopurinol or the superoxide scavenger 4,5-dihydroxy-1-benzene-disulfonic acid were co-incubated with SNP, the inhibitory effects on eNOS activity could be partially alleviated. Also, the levels of superoxide were found to be elevated 4.5-fold when cultured pulmonary arterial endothelial cells were exposed to the NO donor spermine/NO. This suggests that NO can stimulate xanthine oxidase to cause an increase in cellular superoxide generation. A reaction between NO and superoxide would produce peroxynitrite, which could then react with the eNOS protein, resulting in enzyme inactivation. This mechanism may explain, at least in part, how NO produces NOS inhibition in vivo and may delineate, in part, the mechanism of rebound pulmonary hypertension after withdrawal of inhaled NO.

Nitric oxide is an important determinant of coronary flow at rest and during hypoxemic stress in fetal lambs.

Fourteen fetal lambs were instrumented with atrial, coronary sinus, and arterial catheters and a proximal left circumflex coronary artery Doppler probe and were studied at a mean gestational age of 130 +/- 3 (SD) days, 7 +/- 2 days after surgery. Myocardial blood flow was assessed using 15-microns microspheres and Doppler flow velocities. In 11 fetuses, the maximal myocardial flow response to left atrial adenosine infusion was 802 +/- 215 ml.min-1 x 100 g-1, 3.5-fold greater than baseline flow. Acute fetal hypoxemia in six fetuses to an arterial PO2 of 8.8 +/- 0.8 mmHg and an arterial O2 content (CaO2) of 1.7 +/- 0.2 ml/dl was not associated with significant change in coronary perfusion pressure; yet left ventricular myocardial flow increased to 1,020 +/- 198 ml.min-1 x 100 g-1, a value significantly greater than that seen with adenosine (P < 0.05). Left atrial N omega-nitro-L-arginine (L-NNA), a competitive inhibitor of nitric oxide synthase (NOS), was infused at a dosage of approximately 1 mg.kg-1.min-1 for 60 min in 10 fetuses. Although L-NNA was associated with a significant increase in arterial pressure, left ventricular myocardial flow decreased (162 +/- 79 ml.min-1 x 100 g-1) as did myocardial O2 consumption (P < 0.05). Acute hypoxemia in five fetuses that received L-NNA was associated with significant further increases in systemic arterial pressure; however, left ventricular myocardial flow was only 771 +/- 237 ml.min-1 x 100 g-1, a value similar to that seen with adenosine and approximately 75% of that seen with acute hypoxemia alone. We conclude that nitric oxide plays an important role in the regulation of fetal myocardial flow during basal conditions as well as in the exuberant vasodilatory response associated with acute hypoxemic stress.