Todd S. Sherman

Estrogen Upregulates Endothelial Nitric Oxide Synthase Gene Expression in Fetal Pulmonary Artery Endothelium

NO, produced by endothelial NO synthase (eNOS), is a key mediator of pulmonary vasodilation during cardiopulmonary transition at birth. The capacity for NO production is maximal at term because pulmonary eNOS expression increases during late gestation. Since fetal estrogen levels rise markedly during late gestation and there is indirect evidence that the hormone enhances nonpulmonary NO production in adults, estrogen may upregulate eNOS in fetal pulmonary artery endothelium. Therefore, we studied the direct effects of estrogen on eNOS expression in ovine fetal pulmonary artery endothelial cells (PAECs). Estradiol-17beta caused a 2.5-fold increase in NOS enzymatic activity in PAEC lysates. This effect was evident after 48 hours, and it occurred in response to physiological concentrations of the hormone (10(-10) to 10(-6) mol/L). The increase in NOS activity was related to an upregulation in eNOS protein expression, and eNOS mRNA abundance was also enhanced. Estrogen receptor antagonism with ICI 182,780 completely inhibited estrogen-mediated eNOS upregulation, indicating that estrogen receptor activation is necessary for this response. In addition, immunocytochemistry revealed that fetal PAECs express estrogen receptor protein. Furthermore, transient transfection assays with a specific estrogen-responsive reporter system have demonstrated that the endothelial estrogen receptor is capable of estrogen-induced transcriptional transactivation. Thus, estrogen upregulates eNOS gene expression in fetal PAECs through the activation of PAEC estrogen receptors. This mechanism may be responsible for pulmonary eNOS upregulation during late gestation, thereby optimizing the capacity for NO-mediated pulmonary vasodilation at birth.

Nitric oxide synthase isoform expression in the developing lung epithelium

Nitric oxide (NO), generated by NO synthase (NOS), is an important mediator of physiological processes in the airway and lung parenchyma, and there is evidence that the pulmonary expression of the endothelial isoform of NOS (eNOS) is developmentally regulated. The purpose of the present study was to delineate the cellular distribution of expression of eNOS in the developing respiratory epithelium and to compare it with inducible (iNOS) and neuronal (nNOS) NOS. Immunohistochemistry was performed on fetal (125-135 days gestation, term 144 days), newborn (2-4 wk), and maternal sheep lungs. In fetal lung, eNOS expression was evident in bronchial and proximal bronchiolar epithelia but was absent in terminal and respiratory bronchioles and alveolar epithelium. Similar to eNOS, iNOS was detected in bronchial and proximal bronchiolar epithelia but not in alveolar epithelium. However, iNOS was also detected in terminal and respiratory bronchioles. nNOS was found in epithelium at all levels including the alveolar wall. iNOS and nNOS were also detected in airway and vascular smooth muscle. The cellular distribution of all three isoforms was similar in fetal, newborn, and adult lungs. Findings in the epithelium were confirmed by isoform-specific reverse transcription-polymerase chain reaction assays and NADPH diaphorase histochemistry. Thus the three NOS isoforms are commonly expressed in proximal lung epithelium and are differentially expressed in distal lung epithelium. All three isoforms may be important sources of epithelium-derived NO throughout lung development.

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.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.

Nitric Oxide Synthase Isoforms are Differentially Expressed in Developing Lung Epithelium • 1969

Nitric Oxide Synthase Isoforms are Differentially Expressed in Developing Lung Epithelium • 1969

Estrogen Acutely Stimulates Nitric Oxide Synthase in Fetal Pulmonary Artery Endothelium via Activation of Endothelial Estrogen Receptors and Calcium-dependent Potassium Channels |[bull]| 1531

Estrogen (E) has nitric oxide (NO)-mediated vasodilatory effects in specific vascular beds including the pulmonary circulation. Since fetal E levels rise markedly with parturition, E may play a role in the regulation of NO-mediated pulmonary vasodilation at birth. We therefore tested the hypothesis that E stimulates endothelial NO synthase (eNOS) in fetal pulmonary artery endothelial cells (PAEC). To examine the direct effects of the hormone, NOS activity was assessed in cultured ovine fetal PAEC by measuring3 H-L-arginine conversion to 3H-L-citrulline in intact cells over 15 min. NOS activity in the presence of 10-14 to 10 -6M estradiol-17β (E2β) rose in a dose-dependent manner, with a threshold concentration of 10-10M and a maximal increase of 294% at 10-8M E2β. This effect was also detectable within 5 min of E2β exposure (248% increase), and it was comparable to that obtained with acetylcholine (250% increase) which had a threshold concentration of 10-8M. NOS activity in cell lysates was similar in control and E2β-treated cells, indicating that there is not upregulation of NOS expression. The estrogen receptor (ER) antagonist ICI 182,780 fully inhibited E2β-stimulated NOS activity in the intact cells. In addition, ER mRNA expression was evident in PAEC in reverse transcription-PCR assays, and ER protein expression was demonstrated by immunocytochemistry. E2β-stimulated NOS activity was also fully inhibited by removal of extracellular calcium (Ca++), and by LaCl3 (10-4M) and NiCl2 (10-3M), which are inhibitors of Ca++ influx. In contrast, intracellular Ca++ store depletion with thapsigargin (10-6M) had no effect on E2β-stimulated NOS activity. Furthermore, inactivation of Ca++-dependent potassium channels with tetraethylammonium ions (5× 10-4M) fully inhibited E2β-stimulated NOS activity. Thus, physiologic concentrations of E acutely stimulate eNOS activity in fetal PAEC by a mechanism that involves the activation of endothelial ER and Ca++-dependent potassium channels, leading to Ca++ influx. This mechanism may play a role in NO-mediated pulmonary vasodilation at birth, and it may underly E responsiveness in other vascular beds.

Cell Growth Modulates Nitric Oxide Synthase Expression in Fetal Pulmonary Artery Endothelium |[bull]| 1833

Nitric oxide (NO), produced by the endothelial isoform of nitric oxide synthase (eNOS), is a critical mediator of pulmonary vascular structure and function in the developing lung, causing vasodilation and the inhibition of vascular smooth muscle growth. We have previously shown that pulmonary eNOS gene expression is developmentally regulated during late fetal and early postnatal life, suggesting the eNOS gene expression may be modulated by the state of pulmonary artery endothelial cell (PAEC) growth. We therefore studied the specific effects of cell growth on eNOS expression in cultured ovine fetal PAEC. To examine the effects of cell growth modified by two independent approaches, experiments were performed in cells at varying confluence, and in control cells versus cells stimulated with 20% ovine serum for 24-48 h. Cell proliferation was assessed by examining the incorporation of the thymidine analogue 5-bromo-2′-deoxyuridine (BrdU) into replicating DNA during thymidilate synthetase inhibition with 5-flouro-2′-deoxyuridine. BrdU-positive nuclei were identified by immunocytochemistry with a specific monoclonal antibody. eNOS expression was evaluated by determinations of cell lysate NOS enzymatic activity, which is a highly sensitive measure of changes in enzyme abundance. Compared to 100% confluent cells, cell proliferation was greater at 50% confluence (3.5±0.6% vs. 9.4±0.8% BrdU-positive nuclei, respectively). Greater cell proliferation was associated with attenuated NOS activity, which was 11.9±2.4 vs. 2.3±0.1 pmol/mg prot/min at 100% vs. 50% confluence, respectively. With serum stimulation, cell proliferation increased from 2.8±0.3% to 5.9±0.4% BrdU-positive nuclei. Paralleling the findings with varying confluence, NOS activity was attenuated in the more rapidly-proliferating cells, falling from 2.0±0.3 to 0.5±0.1 pmol/mg prot/min with serum stimulation. These findings contrast with those in systemic endothelium, which exhibit enhanced eNOS expression with greater cell growth. Thus, eNOS expression is markedly attenuated with the activation of cell growth in fetal PAEC. We postulate that eNOS downregulation in rapidly-growing PAEC may facilitate concomitant smooth muscle replication, thereby coordinating the growth of the two cell types during vascularization of the developing lung.

ESTROGEN UPREGULATES CYCLOOXYGENASE GENE EXPRESSION IN FETAL PULMONARY ARTERY ENDOTHELIAL CELLS. • 164

ESTROGEN UPREGULATES CYCLOOXYGENASE GENE EXPRESSION IN FETAL PULMONARY ARTERY ENDOTHELIAL CELLS. • 164