Abstract-Estrogen causes nitric oxide (NO)-dependent vasodilation due to estrogen receptor (ER) ␣-mediated, nongenomic activation of endothelial NO synthase (eNOS). The subcellular site of interaction between ER␣ 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 ER␣ 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 ER␣ displayed ER-mediated eNOS stimulation, whereas membranes from cells expressing eNOS alone or ER␣ plus a myristoylation-deficient mutant eNOS were insensitive. Fractionation of endothelial cell plasma membranes revealed ER␣ 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 ER␣ 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. (Circ Res. 2000;87:e44-e52.) Key Words: acetylcholine Ⅲ bradykinin Ⅲ caveolin Ⅲ cell membrane Ⅲ endothelium Ⅲ estrogens T he hormone estrogen classically exerts its effects by modifying gene expression through the activation of estrogen receptors (ERs), which serve as transcription factors. 1-3 However, there are also rapid, presumably nongenomic effects of estrogen in a variety of tissues including the vasculature. 4 -6 Estrogen has important atheroprotective properties that are at least partially related to its capacity to enhance the bioavailability of nitric oxide (NO). [5][6][7] NO is a potent regulator of blood pressure, platelet aggregation, leukocyte adhesion, and vascular smooth muscle mitogenesis that is produced in the vascular wall primarily by the endothelial isoform of NO synthase (eNOS) on the conversion of the substrate L-arginine to L-citrulline. 8 The function of the L-arginine/eNOS system is altered in a variety of vascular disorders. 9 We have previously shown that estrogen rapidly stimulates eNOS activity in endothelial cells, that the response is attenuated by ER antagonism but not by inhibiting gene transcription, and that ER␣ is expressed in endothelium. 10,11 We have also shown that the overexpression of ER␣ in endothelial cells causes enhancement of the acute response to estradiol (E 2 ) that is blocked by ER antagonism, specific to E 2 versus other agonists, and dependent on the ER␣ hormone binding domain. In addition, the acute stimulation of eNOS by E 2 can be reconstituted in COS-7 cells cotransfected with wild-type ER␣ and eNOS. 11 Thus, the short-term ef...

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 (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.

Nitric oxide (NO), produced by NO synthase (NOS), serves multiple functions in the perinatal lung. In fetal baboons, neuronal (nNOS), endothelial (eNOS), and inducible NOS (iNOS) are all primarily expressed in proximal respiratory epithelium. In the present study, NOS expression and activity in proximal lung and minute ventilation of NO standard temperature and pressure (VeNO(STP)) were evaluated in a model of chronic lung disease (CLD) in baboons delivered at 125 days (d) of gestation (term = 185 d) and ventilated for 14 d, obtaining control lung samples from fetuses at 125 or 140 d of gestation. In contrast to the normal 73% increase in total NOS activity from 125 to 140 d of gestation, there was an 83% decline with CLD. This was related to marked diminutions in both nNOS and eNOS expression and enzymatic activity. nNOS accounted for the vast majority of enzymatic activity in all groups. The normal 3.3-fold maturational rise in iNOS protein expression was blunted in CLD, yet iNOS activity was elevated in CLD compared with at birth. The contribution of iNOS to total NOS activity was minimal in all groups. VeNO(STP) remained stable in the range of 0.5-1.0 nl x kg(-1) x min(-1) from birth to day 7 of life, and it then rose by 2.5-fold. Thus the baboon model of CLD is characterized by deficiency of the principal pulmonary isoforms, nNOS and eNOS, and enhanced iNOS activity over the first 2 wk of postnatal life. It is postulated that these alterations in NOS expression and activity may contribute to the pathogenesis of CLD.

. Developmental changes in nitric oxide synthase isoform expression and nitric oxide production in fetal baboon lung. Am J Physiol Lung Cell Mol Physiol 283: L1192-L1199, 2002. First published July 3, 2002 10.1152/ajplung.00112.2002, produced by NO synthase (NOS), plays a critical role in multiple processes in the lung during the perinatal period. To better understand the regulation of pulmonary NO production in the developing primate, we determined the cell specificity and developmental changes in NOS isoform expression and action in the lungs of third-trimester fetal baboons. Immunohistochemistry in lungs obtained at 175 days (d) of gestation (term ϭ 185 d) revealed that all three NOS isoforms, neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS), are primarily expressed in proximal airway epithelium. In proximal lung, there was a marked increase in total NOS enzymatic activity from 125 to 140 d gestation due to elevations in nNOS and eNOS, whereas iNOS expression and activity were minimal. Total NOS activity was constant from 140 to 175 d gestation, and during the latter stage (160-175 d gestation), a dramatic fall in nNOS and eNOS was replaced by a rise in iNOS. Studies done within 1 h of delivery at 125 or 140 d gestation revealed that the principal increase in NOS during the third trimester is associated with an elevation in exhaled NO levels, a decline in expiratory resistance, and greater pulmonary compliance. Thus, there are developmental increases in pulmonary NOS expression and NO production during the early third trimester in the primate that may enhance airway and parenchymal function in the immediate postnatal period. airway epithelium; compliance; expiratory resistance; primate THE SIGNALING MOLECULE nitric oxide (NO), produced by nitric oxide synthase (NOS), plays a critical role in physiological processes in the pulmonary epithelium (1,10,17). NO is detectable in expired gas (9), and studies in both animals and humans suggest that the principle source of expired NO is the lung epithelium rather than the pulmonary vasculature (7, 12). The functions of NO in the mature airway include neurotransmission, smooth muscle relaxation, and bacteriostasis, and also the modulation of mucin secretion, ciliary motility, and plasma exudation (1, 10).There is mounting evidence that NO is of great importance to lung epithelial function in the perinatal period. The stimulation of NO synthesis by acetylcholine or bradykinin causes marked decreases in lung liquid production in late-gestation fetal lambs (5), and the instillation of NO or cGMP, the second messenger for NO, into the fetal lung liquid has the same effect (4, 6). The decrease in lung liquid production by the respiratory epithelium at the time of birth is an essential component of the transition of the fetus from a liquidbreathing to an air-breathing status. Epithelium-derived NO is also critical to the regulation of bronchomotor tone in the early newborn period, playing an important role in the opposition of airway contraction (14). ...