Estrogen receptor alpha and endothelial nitric oxide synthase are organized into a functional signaling module in caveolae.

Estrogen causes nitric oxide (NO)-dependent vasodilation due to estrogen receptor (ER) alpha-mediated, nongenomic activation of endothelial NO synthase (eNOS). The subcellular site of interaction between ERalpha and eNOS was determined in studies of isolated endothelial cell plasma membranes. Estradiol (E(2), 10(-8) mol/L) caused an increase in eNOS activity in plasma membranes in the absence of added calcium, calmodulin, or eNOS cofactors, which was blocked by ICI 182,780 and ERalpha antibody. Immunoidentification studies detected the same 67-kDa protein in endothelial cell nucleus, cytosol, and plasma membrane. Plasma membranes from COS-7 cells expressing eNOS and ERalpha displayed ER-mediated eNOS stimulation, whereas membranes from cells expressing eNOS alone or ERalpha plus a myristoylation-deficient mutant eNOS were insensitive. Fractionation of endothelial cell plasma membranes revealed ERalpha protein in caveolae, and E(2) caused stimulation of eNOS in isolated caveolae that was ER-dependent; noncaveolae membranes were insensitive. Acetylcholine and bradykinin also activated eNOS in isolated caveolae. Furthermore, the effect of E(2) on eNOS in caveolae was prevented by calcium chelation. Thus, a subpopulation of ERalpha is localized to endothelial cell caveolae where they are coupled to eNOS in a functional signaling module that may regulate the local calcium environment. The full text of this article is available at http://www.circresaha.org.

Cell growth modulates nitric oxide synthase expression in fetal pulmonary artery endothelial cells.

Nitric oxide (NO), produced by endothelial (e) nitric oxide synthase (NOS), is a critical mediator of vascular function and growth in the developing lung. Pulmonary eNOS expression is diminished in conditions associated with altered pulmonary vascular development, suggesting that eNOS may be modulated by changes in pulmonary artery endothelial cell (PAEC) growth. We determined the effects of cell growth on eNOS expression in cultured ovine fetal PAEC studied at varying levels of confluence. NOS enzymatic activity was sixfold greater in quiescent PAEC at 100% confluence compared with more rapidly replicating cells at 50% confluence. To determine if there is a reciprocal effect of NO on PAEC growth, studies of NOS inhibition or the provision of exogenous NO from spermine NONOate were performed. Neither intervention had a discernable effect on PAEC growth. The influence of cell growth on NOS activity was unique to pulmonary endothelium, because varying confluence did not alter NOS activity in fetal systemic endothelial cells. The effects of cell growth induced by serum stimulation were also evaluated, and NOS enzymatic activity was threefold greater in quiescent, serum-deprived cells compared with that in serum-stimulated cells. The increase in NOS activity observed at full confluence was accompanied by parallel increases in eNOS protein and mRNA expression. These findings indicate that eNOS gene expression in fetal PAEC is upregulated during cell quiescence and downregulated during rapid cell growth. Furthermore, the interaction between cell growth and NO in the PAEC is unidirectional.

Estrogen acutely stimulates endothelial nitric oxide synthase in H441 human airway epithelial cells.

Nitric oxide (NO) is an important mediator of physiologic processes in the airway. Levels of exhaled NO are greatest and asthma symptoms are least in menstruating women during midcycle, when estrogen levels are highest. To better understand the role of estrogen in airway function, we tested the hypothesis that estrogen stimulates endothelial NO synthase (eNOS) in NCI-H441 human bronchiolar epithelial cells. eNOS activation was assessed by measuring conversion of [3H]L-arginine to [3H]L-citrulline in intact cells. eNOS activity rose in the presence of estradiol-17beta (E2beta), with a maximum stimulation of 243% at 10(-8) M E2beta. This response was comparable to the 201% increase elicited by the calcium (Ca2+) ionophore A23187 (10(-5) M), and was evident as early as 5 min after such treatment. Actinomycin D had no effect on the response to E2beta, and eNOS abundance was similar in control and E2beta-treated cells. E2beta-stimulated eNOS activity was dependent on the influx of extracellular Ca2+, and was completely inhibited by the estrogen receptor (ER) antagonist ICI182,780. Messenger RNA and protein for the alpha isoform of ER (ERalpha) were evident in the H441 cells, and freshly isolated ovine airway epithelial cells also coexpressed eNOS and ERalpha. These findings indicate that estrogen acutely activates existing eNOS in H441 airway epithelial cells, through a process that involves the stimulation of epithelial ER and Ca2+ influx. This process may play a role in the hormonal modulation of airway function.

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.

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.