Developmental changes in prostacyclin synthesis are conserved in cultured pulmonary endothelium and vascular smooth muscle.

Prostacyclin (PGI2) is a key mediator of pulmonary vascular and parenchymal function during late fetal and early postnatal life, and its synthesis in intrapulmonary arteries increases markedly during that period. The rate-limiting enzyme in PGI2 synthesis in the developing lung is cyclooxygenase (COX). To understand better the mechanisms underlying the developmental increase in PGI2 synthesis, we evaluated PGI2 production in early-passage, cultured pulmonary artery endothelial cells (PAEC) and pulmonary vascular smooth-muscle cells (VSM) from fetal and newborn lambs. In arterial segments, PGI2 synthesis was sevenfold greater in intact arteries from newborn than from fetal lambs, and it was 12-fold greater in endothelium-denuded newborn than in fetal arteries, indicating that the developmental increase occurs in both the endothelium and medial layer. Similarly, basal PGI2 production was three-fold greater in newborn than in fetal PAEC, and 2.5-fold greater in newborn than in fetal pulmonary VSM cells. Calcium ionophore (A23187)-stimulated and arachidonic acid-stimulated PGI2 synthesis were also greater in newborn than in fetal PAEC and VSM, revealing a developmental upregulation in COX enzymatic activity in both cell types. Immunoblot analysis showed that this is due to greater COX-1 protein expression in newborn than in fetal vascular cells; COX-2 protein expression was not detected. In addition, COX-1 messenger RNA (mRNA) abundance was greater in newborn than in fetal PAEC, and this was not due to a difference in COX-1 mRNA stability. Thus, the developmental upregulation of PGI2 synthesis is conserved in early-passage PAEC and pulmonary VSM, and is related to a maturational increase in COX-1 gene expression. Further studies with the cultured cell model will enable determination of the factors that directly regulate COX-1 expression in the developing pulmonary vasculature.

Ontogeny of cyclooxygenase-1 and cyclooxygenase-2 gene expression in ovine lung.

Prostacyclin is a key mediator of pulmonary vascular and parenchymal function during late fetal and early postnatal life, and its synthesis in whole lung increases during that period. The rate-limiting enzyme in prostacyclin synthesis in the developing lung is cyclooxygenase (COX). We investigated the ontogeny and cellular localization of COX-1 (constitutive) and COX-2 (inducible) gene expression in lungs from late-gestation fetal lambs, 1-wk-old newborn lambs (NB1), and 1- to 4-mo-old newborn lambs (NB2). COX-1 mRNA abundance rose progressively from fetal to NB1 to NB2, increasing 12-fold overall. In parallel, immunoblot analysis revealed a progressive increase in COX-1 protein, rising fourfold from fetal lambs to NB2. COX-2 mRNA levels increased fivefold from fetal to NB1 but were similar in NB1 and NB2. However, COX-2 protein was not detectable by immunoblot analysis in any age group. Immunohistochemistry for COX-1 showed intense immunostaining in endothelial cells at all ages. COX-1 was also expressed in airway epithelium at all ages, with a greater number of epithelial cells staining positively in NB2 compared with fetal and NB1 groups. In addition, COX-1 was expressed in airway smooth muscle from NB1. COX-2 immunostaining was absent in all age groups. These findings indicate that there is differential expression of COX-1 and COX-2 in the developing lung and that the enzymes are expressed in a cell-specific manner. The developmental upregulation in COX-1 may optimize the capacity for prostaglandin-mediated vasodilation, bronchodilation, and surfactant synthesis in the newborn lung.

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.

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.

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.

Milrinone effects in the isolated immature rabbit heart.

Milrinone, a new positive inotropic agent, was evaluated and compared to isoproterenol in an immature isolated isovolumic rabbit heart model. Three age groups were studied: newborns (0-6 days), juveniles (4-6 weeks old) and adults (5-7 months old). Heart rate did not change significantly with milrinone or isoproterenol in adults or juveniles, but increased in newborns from 144 +/- 1 to 162 +/- (SEM) 6 beats/min at peak milrinone effect. Milrinone had a greater effect on the contractility (maximum positive dP/dt) of the mature hearts, with newborns increasing to 134 +/- 6% of baseline, juveniles to 154 +/- 8% and adults to 216 +/- 15%. Results were similar for isoproterenol, although the positive inotropic effect occurred over a wider dosage range for this drug. No additive effects of the two drugs were noted. We conclude, that although milrinone is a positive inotropic drug in all age groups studied, the response of the newborn heart is quantitatively much weaker than that of the adult.