Nitric oxide (NO) upregulates NO synthase expression in fetal intrapulmonary artery endothelial cells.

Endothelium-derived nitric oxide (NO) generated by endothelial NO synthase (eNOS) is critically involved in pulmonary vasodilation during cardiopulmonary transition at birth. Inhaled NO therapy has recently been considered for patients with persistent pulmonary hypertension of the newborn (PPHN). To better understand the mechanisms regulating NO synthesis in the developing pulmonary circulation and the possible ramifications of NO therapy, studies were performed with early passage ovine fetal intrapulmonary artery endothelial cells (PAEC) to determine whether NO directly modulates eNOS expression. To examine the effects of exogenous NO, PAEC were treated with the NO donor spermine NONOate or the parent compound spermine. Exogenous NO caused increases in eNOS protein expression and NOS enzymatic activity that were detectable within 16 h of exposure. In contrast, the inhibition of endogenous NO production with nitro-L-arginine-methyl ester (L-NAME) caused a reduction in eNOS protein expression that was evident within 8 h. Paralleling the changes in eNOS protein, eNOS messenger RNA (mRNA) abundance was upregulated by exogenous NO and downregulated by L-NAME, suggesting that NO modulation of eNOS expression involves processes at the level of gene transcription or mRNA stability. Thus, in fetal PAEC there is positive-feedback regulation of eNOS expression by both exogenous and endogenous NO. These findings suggest that difficulties with transient effectiveness or prolonged requirements for NO therapy in certain PPHN patients are not due to declines in eNOS expression. Further, conditions such as fetal hypoxemia that impair PAEC NO production may attenuate eNOS expression through this mechanism, thereby contributing to the pathogenesis of PPHN.

Oxygen upregulates nitric oxide synthase gene expression in ovine fetal pulmonary artery endothelial cells.

Nitric oxide (NO) is critically involved in oxygen-mediated pulmonary vasodilatation in the fetus and newborn. We determined the effects of prolonged alterations in oxygenation on endothelial NO synthase (eNOS) gene expression in early passage ovine fetal intrapulmonary artery endothelial cells (PAEC). PAEC were exposed to PO2 = 50 or 150 mmHg for 48 h, and eNOS protein expression was evaluated by immunoblot analysis. eNOS protein expression was 2.7-fold greater at higher oxygen tension; eNOS upregulation was also evident after 24 h. Inducible NOS protein was not detectable by immunoblot at either level of oxygenation. In the lung, the effect of oxygen on eNOS expression may be specific to the endothelium, as eNOS expression in bronchiolar epithelial cells of Clara cell lineage was not altered by varying oxygen tension. The oxygen-related increase in eNOS protein in the fetal PAEC was associated with 2.5-fold greater NOS enzymatic activity. In parallel, there was a 2.8-fold rise in eNOS mRNA abundance. Thus eNOS gene expression in ovine fetal PAEC is upregulated by oxygen, and this is mediated at the level of gene transcription or mRNA stability. This process may play an important role in oxygen modulation of pulmonary vasomotor tone in the fetus and newborn.

Pulmonary endothelial nitric oxide synthase gene expression is decreased in a rat model of congenital diaphragmatic hernia.

Nitric oxide (NO) produced by the enzyme nitric oxide synthase (NOS) is critically involved in the cardiopulmonary transition from fetal to neonatal life. In congenital diaphragmatic hernia (CDH) this transition often does not occur normally, resulting in persistent pulmonary hypertension of the newborn (PPHN). We sought to determine if pulmonary NOS expression is altered in a rat model of CDH induced by maternal ingestion of the herbicide 2,4-dichlorophenyl-p-nitrophenyl ether (Nitrofen) on day 9 of gestation (term = 22 days). Sixty-three percent of Nitrofen-exposed fetuses developed CDH. Endothelial NOS (eNOS) and neuronal NOS (nNOS) protein expression were assessed in ipsilateral CDH lungs and in control lungs (Nitrofen-treated, no hernia) at 20 d gestation using immunoblot analyses. eNOS and nNOS have been immunohistochemically localized to rat pulmonary endothelium and bronchiolar epithelium, respectively, and we have previously demonstrated that their expression normally increases during late gestation to be maximal near term. eNOS protein expression was decreased in CDH versus control lung (58 +/- 6 versus 100 +/- 6% of control, n = 5). In contrast, nNOS protein abundance was similar. Factor VIII-associated antigen expression was comparable in CDH and control lung, indicating that the change in eNOS is not related to differences in endothelial cell density. eNOS mRNA abundance was evaluated in semiquantitative reverse transcription-polymerase chain reaction assays. Paralleling the decline in eNOS protein expression, eNOS mRNA was decreased in CDH versus control lung (22 +/- 8 versus 100 +/- 31% of control, n = 4).(ABSTRACT TRUNCATED AT 250 WORDS)

Prolonged in vivo hypoxia enhances nitric oxide synthase type I and type III gene expression in adult rat lung.

Prolonged hypoxia in the adult rat causes a decline in endothelium-derived nitric oxide (NO) production in the pulmonary circulation. To evaluate whether this is related to a decrease in endothelial NO synthase (NOS-III) expression, we determined the effects of hypobaric hypoxia (7 or 21 days) on NOS-III gene expression in adult rat lung. Neuronal NOS (NOS-I) expression was also examined; NOS-I has been immunohistochemically localized to rat bronchiolar epithelium. NOS-III and NOS-I mRNA abundance were assessed in reverse transcription-polymerase chain reaction assays and the proteins were evaluated by immunoblot analysis. After 7 and 21 days of hypoxia, there were increases in the steady-state levels of both NOS-III and NOS-I mRNA, rising 2.7- to 3.0-fold and 2.5- to 2.8-fold, respectively. These findings were confirmed by Northern analyses. In parallel, NOS-III and NOS-I protein abundance were also increased with hypoxia by 3.0- to 3.5-fold and 2.4- to 3.0-fold, respectively. NOS activity detected by [3H]arginine to [3H]citrulline conversion rose 109%. Thus, prolonged in vivo hypoxia causes enhancement of NOS-III and NOS-I gene expression in adult rat lung, indicating that the pulmonary expression of these genes is modulated in vivo. The increase in NOS-III expression does not explain the declines in pulmonary endothelial NO production previously observed following prolonged hypoxia in this model. Alternatively, the fall in NO production may be related to diminished NOS co-factor availability.

Endothelial nitric oxide synthase is expressed in cultured human bronchiolar epithelium.

Nitric oxide (NO) is an important mediator of physiologic and inflammatory processes in the lung. To better understand the role of NO in the airway, we examined constitutive NO synthase (NOS) gene expression and function in NCI-H441 human bronchiolar epithelial cells, which are believed to be of Clara cell lineage. NOS activity was detected by [3H]arginine to [3H]citrulline conversion (1,070 +/- 260 fmol/mg protein per minute); enzyme activity was inhibited 91% by EGTA, consistent with the expression of a calcium-dependent NOS isoform. Immunoblot analyses with antisera directed against neuronal, inducible, or endothelial NOS revealed expression solely of endothelial NOS protein. Immunocytochemistry for endothelial NOS revealed staining predominantly in the cell periphery, consistent with the association of this isoform with the cellular membrane. To definitively identify the NOS isoform expressed in H441 cells, NOS cDNA was obtained by degenerate PCR. Sequencing of the H441 NOS cDNA revealed 100% identity with human endothelial NOS at the amino acid level. Furthermore, the H441 NOS cDNA hybridized to a single 4.7-kb mRNA species in poly(A)+ RNA isolated from H441 cells, from rat, sheep, and pig lung, and from ovine endothelial cells, coinciding with the predicted size of 4.7 kb for endothelial NOS mRNA. Guanylyl cyclase activity in H441 cells, assessed by measuring cGMP accumulation, rose 6.6- and 5.4-fold with calcium-mediated activation of NOS by thapsigargin and A23187, respectively. These findings indicate that endothelial NOS is expressed in select bronchiolar epithelial cells, where it may have autocrine effects through activation of guanylyl cyclase. Based on these observations and the previous identification of endothelial NOS in a kidney epithelial cell line, it is postulated that endothelial NOS may be expressed in unique subsets of epithelial cells in a variety of organs, serving to modulate ion flux and/or secretory function.

Oxygen modulates nitric oxide production selectively in fetal pulmonary endothelial cells.

Acute hypoxia causes pulmonary hypertension in the fetus and newborn that is contrasted by systemic hypotension or normotension. To better understand the role of nitric oxide (NO) in this specific pulmonary vascular response, we determined the acute effects of decreased oxygenation on NO production in ovine fetal pulmonary and systemic (mesenteric) endothelial cells. NO was assessed by measuring cGMP accumulation in fetal vascular smooth muscle (VSM) cells during co-culture incubations of endothelium and VSM (40 s) in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine. Changes in cGMP were dependent on the endothelium and on NO synthase and guanylate cyclase activity. At high O2 (680 mm Hg), basal NO was detectable and NO increased 6- to 10-fold with bradykinin or A23187. In pulmonary endothelium, basal NO fell 58% at pO2 = 150 mm Hg and 51% at 40 mm Hg versus 680 mm Hg, while NO with bradykinin fell 56% and 63%, respectively. NO with A23187, however, was unchanged at 150 mm Hg, but it fell 56% at 40 mm Hg. In contrast, in systemic endothelium basal and stimulated NO production were not altered at lower O2. Findings were similar using pulmonary or systemic detector VSM cells, and exogenous L-arginine had no effect. Thus, decreased O2 acutely attenuates NO production specifically in fetal pulmonary endothelial cells. This process is not related to changes in O2 or L-arginine availability as substrates for NO synthase; alternatively, it may be partially mediated by specific effects of O2 on pulmonary endothelial cell calcium homeostasis.

Hypoxia stimulates prostacyclin synthesis in newborn pulmonary artery endothelium by increasing cyclooxygenase-1 protein.

In newborn lambs, pulmonary prostacyclin (PGI2) production increases acutely in response to low oxygen. We tested the hypothesis that decreased oxygenation directly stimulates PGI2 synthesis in arterial segments and cultured endothelial cells from newborn lamb intrapulmonary arteries. In segments studied at PO2 of 680 mm Hg, the synthesis of PGI2 exceeded prostaglandin E2 (PGE2) by 73%. Endothelium removal lowered PGI2 by 77% and PGE2 by 66%. At low oxygen tension (PO2, 40 mm Hg), PGI2 and PGE2 synthesis rose by 96% and 102%, respectively. Similarly, in endothelial cells studied at PO2 of 680 mm Hg, the synthesis of PGI2 exceeded PGE2 by 50%, and at low oxygen tension both PGI2 and PGE2 increased (89% and 64%, respectively). Endothelial cell PGI2 synthesis maximally stimulated by bradykinin, A23187, or arachidonic acid was also increased at low PO2 by 50%, 66%, and 48%, respectively. PGE2 synthesis was similarly altered, increasing by 33%, 37%, and 41%, respectively. In contrast, lowering oxygen had minimal effect on PGI2 and PGE2 synthesis with exogenous PGH2, which is the product of cyclooxygenase. Immunoblot analyses revealed that there was a 2.6-fold greater abundance of cyclooxygenase-1 protein at PO2 of 40 versus 680 mm Hg, and the increase at lower oxygen tension was inhibited by cycloheximide. The cyclooxygenase-2 isoform was not detected. Thus, attenuated oxygenation directly stimulates PGI2 and PGE2 synthesis in intrapulmonary arterial segments and endothelial cells from newborn lambs. This process is due to enhanced cyclooxygenase activity related to increased abundance of the cyclooxygenase-1 protein, and this effect may be due to increased synthesis of the enzyme protein.

Nitric oxide synthase type I and type III gene expression are developmentally regulated in rat lung.

The successful transition from fetal to neonatal life involves a marked decline in pulmonary vascular resistance which is modulated in part by endothelium-derived nitric oxide. To define the molecular processes which prepare the pulmonary circulation for nitric oxide mediation of vasodilatation at the time of birth, we determined the ontogeny of endothelial nitric oxide synthase (NOS-III) gene expression in lungs from fetal and newborn rats. Maturational changes in lung neuronal NOS (NOS-I) expression were also investigated; the latter isoform has been localized to rat bronchiolar epithelium. NOS proteins were examined by immunoblot analysis, and mRNA abundance was assessed in reverse transcription-polymerase chain reaction assays. Both NOS-III and NOS-I protein were detectable in 16-day fetal lung, they increased 3.8- and 3.1-fold, respectively, to maximal levels at 20 days of gestation (term = 22 day), and they fell postnatally (1-5 days). In parallel with the findings for NOS-III protein, NOS-III mRNA increased from 16 to 20 days gestation and fell after birth. In contrast, NOS-I mRNA abundance declined during late fetal life and rose postnatally. These findings were confirmed by Northern analyses. Thus NOS-III and NOS-I gene expression are developmentally regulated in rat lung, with maximal NOS-III and NOS-I protein present near term. The regulation of pulmonary NOS-III may primarily involve alterations in transcription or mRNA stability, whereas NOS-I expression in the maturing lung may also be mediated by additional posttranscriptional processes.

Prostacyclin synthesis in ovine pulmonary artery is developmentally regulated by changes in cyclooxygenase-1 gene expression.

Prostacyclin (PGI2) is a key mediator of pulmonary vasomotor tone during late gestation and in the newborn, and its production in whole lung increases during that period. We investigated the developmental regulation of PGI2 synthesis in ovine intrapulmonary artery (PA) segments from 110 to 115 d (F1) and 125 to 135 d gestation fetal lambs (F2, term = 144 d) and 1- and 4-wk-old newborn lambs (NB1 and NB2). Basal PGI2 rose fourfold from F1 to F2, fourfold from F2 to NB1, and twofold from NB1 to NB2. In all age groups 66-72% of PGI2 was derived from the endothelium. Similar fold increases in PGI2 were observed with maturation in intact and endothelium-denuded segments. In intact PA from F2, NB1, and NB2, basal PGI2 synthesis and synthesis maximally stimulated by bradykinin, A23187, or arachidonic acid rose with development in a comparable manner. In contrast, PGI2 synthesis stimulated by exogenous PGH2, the product of cyclooxygenase, was similar at all ages. Immunoblot analyses of PA from F2, NB1, and NB2 revealed that there is a sixfold maturational increase in cyclooxygenase-1 protein; the cyclooxygenase-2 isoform was not detectable. Cyclooxygenase-1 mRNA abundance in whole lung also rose with development. Thus, PGI2 synthesis in ovine PA endothelium and vascular smooth muscle increases markedly during late fetal and early newborn life; the increase is due to a rise in cyclooxygenase activity related to enhanced expression of cyclooxygenase-1. We conclude that there is developmental regulation of PA cyclooxygenase-1 gene expression, and that this may be critical to successful cardiopulmonary transition and function in the newborn.

Acute and prolonged hypoxia attenuate endothelial nitric oxide production in rat pulmonary arteries by different mechanisms.

Hypoxic pulmonary hypertension complicates many primary respiratory and cardiac conditions. To define the potential role of endothelial nitric oxide (NO) further in both the acute and chronic forms of this disorder, we determined the effects of acute changes in O2 in vitro and prolonged variations in O2 in vivo on endothelial NO production in rat main pulmonary arteries. NO production was assessed by measuring segment cyclic GMP synthesis, which was dependent on the presence of the endothelium and on NO synthase and soluble guanylate cyclase activity. With an acute decrease in pO2 in vitro from 150 to 40 mm Hg, basal endothelial NO production was attenuated by 52%. NO production stimulated by acetylcholine (ACh) or A23187, however, was not altered, suggesting that the underlying mechanism involves acute changes in endothelial intracellular calcium homeostasis or in the production or action of a local activator of endothelial NO synthase. Although prolonged hypoxia in vivo (7 days) also caused a 52% decrease in basal endothelial NO production, ACh- and A23187-stimulated production were diminished as well, by 69 and 73%, respectively; the attenuation in NO production was evident when tested at high pO2 in vitro, was not altered by exogenous L-arginine, and was reversed by 3 days of normoxic recovery, indicating that the chronic process may involve diminished availability of cofactor(s) required for NO synthase activity. Parallel studies of aortic segments showed that these effects are specific to the pulmonary endothelium. Thus, both acute and prolonged hypoxia selectively attenuate pulmonary endothelial NO production by different mechanisms.