Clinically significant increases in pulmonary vascular resistance have been noted on acute withdrawal of inhaled nitric oxide (NO). Endothelin (ET)-1 is a vasoactive peptide produced by the vascular endothelium that may participate in the pathophysiology of pulmonary hypertension. The objectives of this study were to determine the effects of inhaled NO on endogenous ET-1 production in vivo in the intact lamb and to determine the potential role of ET-1 in the rebound pulmonary hypertension associated with the withdrawal of inhaled NO. Seven 1-mo-old vehicle-treated control lambs and six PD-156707 (an ET(A) receptor antagonist)-treated lambs were mechanically ventilated. Inhaled NO (40 parts per million) was administered for 24 h and then acutely withdrawn. After 24 h of inhaled NO, plasma ET-1 levels increased by 119.5 +/- 42.2% (P < 0.05). Western blot analysis revealed that protein levels of preproET-1, endothelin-converting enzyme-1alpha, and ET(A) and ET(B) receptors were unchanged. On acute withdrawal of NO, pulmonary vascular resistance (PVR) increased by 77.8% (P < 0.05) in control lambs but was unchanged (-5.5%) in PD-156707-treated lambs. Inhaled NO increased plasma ET-1 concentrations but not gene expression in the intact lamb, and ET(A) receptor blockade prevented the increase in PVR after NO withdrawal. These data suggest a role for ET-1 in the rebound pulmonary hypertension noted on acute withdrawal of inhaled NO.

Life-threatening increases in pulmonary vascular resistance have been noted on acute withdrawal of inhaled nitric oxide (NO), although the mechanisms remain unknown. In vitro data suggest that exogenous NO exposure inhibits endothelial NO synthase (NOS) activity. Thus the objectives of this study were to determine the effects of inhaled NO therapy and its acute withdrawal on endogenous NOS activity and gene expression in vivo in the intact lamb. Six 1-mo-old lambs were mechanically ventilated and instrumented to measure vascular pressures and left pulmonary blood flow. Inhaled NO (40 ppm) acutely decreased left pulmonary vascular resistance by 27. 5 +/- 4.7% (P < 0.05). This was associated with a 207% increase in plasma cGMP concentrations (P < 0.05). After 6 h of inhaled NO, NOS activity was reduced to 44.3 +/- 5.9% of pre-NO values (P < 0.05). After acute withdrawal of NO, pulmonary vascular resistance increased by 52.1 +/- 11.6% (P < 0.05) and cGMP concentrations decreased. Both returned to pre-NO values within 60 min. One hour after NO withdrawal, NOS activity increased by 48.4 +/- 19.1% to 70% of pre-NO values (P < 0.05). Western blot analysis revealed that endothelial NOS protein levels remained unchanged throughout the study period. These data suggest a role for decreased endogenous NOS activity in the rebound pulmonary hypertension noted after acute withdrawal of inhaled NO.

Ligation of the ductus arteriosus in utero produces fetal and neonatal pulmonary hypertension and alterations in the hemodynamic responses to nitric oxide and endothelin-1 in fetal and newborn lambs. To determine whether fetal pulmonary hypertension alters the expression of the genes of the nitric oxide and endothelin-1 pathways, seven fetal lambs (123-126-d gestation) underwent ligation of the ductus arteriosus. Near-term (138-139-d gestation), total lung RNA, and protein were prepared from control and ductal ligation fetal lambs for RNase protection assays and Western blotting. Ligation of the ductus arteriosus was associated with decreased expression of endothelial nitric oxide synthase mRNA and protein, and the alpha1 and the beta1 subunits of soluble guanylate cyclase protein; and with increased expression of phosphodiesterase V mRNA. Ligation of the ductus arteriosus was also associated with increased expression of preproendothelin-1 mRNA and with decreased expression of endothelin B receptor (ET(B)) mRNA. These results suggest that there is coordinated regulation of genes of the nitric oxide pathway, which would decrease nitric oxide and cGMP concentration, thereby decreasing pulmonary vasodilator activity. There is also coordinated regulation of genes of the endothelin-1 pathway, which would increase endothelin-1 concentration and limit ET(B) receptor activation, thereby increasing pulmonary vasoconstrictor activity. These alterations in gene expression would increase fetal pulmonary vascular resistance, contributing to the development of pulmonary hypertension after birth.

At birth, ventilation and oxygenation immediately decrease pulmonary vascular resistance (PVR) and increase pulmonary blood flow (PBF); more gradual changes occur over the next several hours. Nitric oxide, produced by endothelial nitric oxide synthase (eNOS), mediates these gradual changes. To determine how ventilation and oxygenation affect eNOS gene expression, 12 fetal lambs were ventilated for 8 h without changing fetal descending aortic blood gases or pH (rhythmic distension) or with 100% oxygen (O 2 ventilation). Vascular pressures and PBF were measured. Total RNA, protein, and tissue sections were prepared from lung tissue for RNase protection assays, Western blotting, and in situ hybridization. O 2 ventilation increased PBF and decreased PVR more than rhythmic distension ( P Ͻ 0.05). Rhythmic distension increased eNOS mRNA expression; O 2 ventilation increased eNOS mRNA expression more and increased eNOS protein expression ( P Ͻ 0.05). To define the mechanisms responsible for these changes, ovine fetal pulmonary arterial endothelial cells were exposed to 1, 21, or 95% O 2 or to shear stress. 95% O 2 increased eNOS mRNA and protein expression ( P Ͻ 0.05). Shear stress increased eNOS mRNA and protein expression ( P Ͻ 0.05). Increased oxygenation but more importantly increased PBF with increased shear stress induce eNOS gene expression and contribute to pulmonary vasodilation after birth. (

Plasma concentrations of endothelin-1 (ET-1) are increased in children with congenital heart disease associated with increased pulmonary blood flow. However, the role of ET-1 in the pathophysiology of pulmonary hypertension remains unclear. Preproendothelin-1 gene expression is increased in adults with advanced pulmonary hypertension. To characterize potential early molecular alterations in the ET-1 cascade induced by increased pulmonary blood flow and pulmonary hypertension, fetal lambs underwent in utero placement of an aortopulmonary vascular graft (shunt). RNase protection assays and Western blot analysis were performed on lung tissue prepared from 4-wk-old shunt lambs and age-matched controls. Endothelin-converting enzyme-1 [the enzyme responsible for the production of active ET-1 from big ET-1, mRNA (411%, p<0.05)] and protein (170%, p<0.05) were increased in lung tissue prepared from shunt lambs, compared with age-matched controls. Endothelin type A receptor (the receptor that mediates vasoconstriction), mRNA (246%, p<0.05), and protein (176%, p<0.05) also were increased in lung tissue prepared from shunt lambs compared with age-matched controls. Conversely, endothelin type B receptor (the receptor that mediates vasodilation), mRNA (46%, p<0.05), and protein (65%, p<0.05) were decreased in shunt lambs. Both the mRNA and protein levels for preproendothelin-were unchanged. Thus we conclude that increased pulmonary blood flow and pulmonary hypertension induce early alterations in the ET-1 cascade that result in increased ET-1 production, increased ET-1-mediated vasoconstriction, and decreased vasodilation. These early alterations in gene expression may contribute to the development of pulmonary hypertension and its associated enhanced pulmonary vascular reactivity.

SM. Progressive dysfunction of nitric oxide synthase in a lamb model of chronically increased pulmonary blood flow: a role for oxidative stress. Am J Physiol Lung Cell Mol Physiol 295: L756 -L766, 2008. First published August 29, 2008 doi:10.1152/ajplung.00146.2007.-Cardiac defects associated with increased pulmonary blood flow result in pulmonary vascular dysfunction that may relate to a decrease in bioavailable nitric oxide (NO). An 8-mm graft (shunt) was placed between the aorta and pulmonary artery in 30 late gestation fetal lambs; 27 fetal lambs underwent a sham procedure. Hemodynamic responses to ACh (1 g/kg) and inhaled NO (40 ppm) were assessed at 2, 4, and 8 wk of age. Lung tissue nitric oxide synthase (NOS) activity, endothelial NOS (eNOS), neuronal NOS (nNOS), inducible NOS (iNOS), and heat shock protein 90 (HSP90), lung tissue and plasma nitrate and nitrite (NO x), and lung tissue superoxide anion and nitrated eNOS levels were determined. In shunted lambs, ACh decreased pulmonary artery pressure at 2 wk (P Ͻ 0.05) but not at 4 and 8 wk. Inhaled NO decreased pulmonary artery pressure at each age (P Ͻ 0.05). In control lambs, ACh and inhaled NO decreased pulmonary artery pressure at each age (P Ͻ 0.05). Total NOS activity did not change from 2 to 8 wk in control lambs but increased in shunted lambs (ANOVA, P Ͻ 0.05). Conversely, NO x levels relative to NOS activity were lower in shunted lambs than controls at 4 and 8 wk (P Ͻ 0.05). eNOS protein levels were greater in shunted lambs than controls at 4 wk of age (P Ͻ 0.05). Superoxide levels increased from 2 to 8 wk in control and shunted lambs (ANOVA, P Ͻ 0.05) and were greater in shunted lambs than controls at all ages (P Ͻ 0.05). Nitrated eNOS levels were greater in shunted lambs than controls at each age (P Ͻ 0.05). We conclude that increased pulmonary blood flow results in progressive impairment of basal and agonist-induced NOS function, in part secondary to oxidative stress that decreases bioavailable NO. pulmonary circulation; oxidant stress; congenital heart disease; reactive oxygen species INFANTS AND CHILDREN with congenital cardiac defects that cause significantly increased pulmonary blood flow suffer morbidity due to early aberrations in pulmonary vascular function (17). In fact, this early pulmonary vascular dysfunction is often exacerbated in the immediate postoperative period, manifesting as increased vascular reactivity that may produce severe hypoxemia, acidosis, low cardiac output, and death if not treated immediately (7,11,31). A complete understanding of the mechanisms responsible for this pulmonary vascular dysfunction is lacking, but evidence suggests that aberrant nitric oxide (NO)-cGMP signaling and oxidative stress may participate (1,3,6,11,15,33,39,41).Basal NO production by the vascular endothelium is integral to the maintenance of the normal low resistance state of the pulmonary vasculature, and dynamic alterations in NO production modulate vascular relaxation and constriction in response to various stimuli. NO is produced in ...

To investigate early endothelial function associated with increased pulmonary blood flow, vascular shunts were placed between the ascending aorta and main pulmonary artery in 18 late-gestation fetal sheep. Four weeks after delivery, the lambs were instrumented to measure vascular pressures and blood flows, and blood was collected to measure plasma concentrations of guanosine 3',5'-cyclic monophosphate [cGMP, the second messenger to nitric oxide (NO)-mediated vasodilation] and L-arginine (the precursor for NO synthesis). The responses to the endothelium-dependent vasodilators acetylcholine (ACh, 1.0 microgram/kg) and ATP (0.1 mg.kg-1.min-1), the endothelium-independent vasodilators M & B-22948 (a cGMP-specific phosphodiesterase inhibitor, 2.5 mg/kg) and inhaled NO (40 ppm), and N omega-nitro-L-arginine (an inhibitor of NO synthase, 5 mg/kg) were then compared with responses in 12 age-matched controls. Vasodilator responses in control lambs were determined during pulmonary hypertension induced by U-46619 (a thromboxane A2 mimic). Shunted lambs displayed a selective impairment of endothelium-dependent pulmonary vasodilation, an augmented pulmonary vasoconstricting response to NO synthase inhibition, increased plasma cGMP concentrations, and decreased L-arginine concentrations. Taken together, these data suggest that lambs with pulmonary hypertension and increased pulmonary blood flow have early aberrations in endothelial function, as manifested by increased basal NO activity, that cannot be further increased by agonist-induced endothelium-dependent vasodilators.

Background-Endothelin-1 (ET-1) has been implicated in the pathophysiology of pulmonary hypertension. In 1-monthold lambs with increased pulmonary blood flow, we have demonstrated early alterations in the ET-1 cascade. The objective of this study was to investigate the role of potential later alterations of the ET cascade in the pathophysiology of pulmonary hypertension secondary to increased pulmonary blood flow. Methods and Results-Eighteen fetal lambs underwent in utero placement of an aortopulmonary vascular graft (shunt) and were studied 8 weeks after spontaneous delivery. Compared with age-matched control lambs, lung tissue ET-1 levels were increased in shunt lambs (317.2Ϯ113.8 versus 209.8Ϯ61.8 pg/g, PϽ0.05). In shunt lambs (nϭ9), exogenous ET-1 induced potent pulmonary vasoconstriction, which was blocked by the ET A receptor antagonist PD 156707 (nϭ3). This pulmonary vasoconstriction was mimicked by exogenous Ala 1,3,11,15 ET-1 (4 Ala ET-1), the ET B receptor agonist, and was blocked by the ET B receptor antagonist BQ 788 (nϭ3). However, in control lambs (nϭ7), ET-1 and 4 Ala ET-1 did not change pulmonary vascular tone. In contrast to 4-week-old shunt lambs, immunohistochemistry revealed the emergence of ET B receptors on smooth muscle cells in the vasculature of 8-week-old shunt lambs. Conclusions-Over