IntroductionNitric oxide (NO) is an important mediator of physiologic and inflammatory processes in the lung. (2), and endothelium-derived NO relaxes tracheal smooth muscle in vitro (3). In addition, endogenous NO gas is present in the exhaled air of animals and humans (4), and endogenous nitrogen oxides and bronchodilator S-nitrosothiols have been demonstrated in human airways (5). Furthermore, air or helium embolization of the rabbit pulmonary artery does not alter exhaled NO concentrations, suggesting that the exhaled NO may be derived from the bronchial tree and not the pulmonary vasculature (4).There is indirect evidence that the epithelium may be a source of NO production in the airway. In the canine bronchial tree, the epithelium releases a relaxing factor which reduces contractile responses in larger airways and enhances relaxation responses in smaller airways (6). Immunohistochemical studies in the rat using NADPH diaphorase staining indicative of NOS activity and antisera which recognize neuronal and endothelial NOS localize NOS to the airway epithelium (7,8). In human lung, it has been reported that NADPH diaphorase staining is evident throughout the airway epithelium but staining for constitutive NOS isoforms is negative, whereas large airway epithelium stains positive for inducible NOS (7). There are preliminary reports of NOS expression and activity in cultured human airway epithelium (9, 10), but the isoform(s) constitutively expressed in those cells has not been fully characterized.The purpose of this investigation was to examine constitutive NOS gene expression and function in NCI-H441 human bronchiolar epithelial cells, which originated from a papillary adenocarcinoma (11,12). Experiments were performed to address the following questions: (a) Is NOS activity present in human bronchiolar epithelial cells?; (b) Which NOS isoform(s) is expressed?; and (c) Does NO have autocrine effects in bronchiolar epithelium?1. Abbreviations used in this paper: FMN, flavin mononucleotide; IBMX, isobutylmethylxanthine; L-NAME, nitro-L-arginine methyl ester; NO, nitric oxide; NOS, nitric oxide synthase; PAEC, pulmonary artery endothelial cells.Nitric Oxide Synthase in Airway Epithelium 2231 J. Clin. Invest.
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.
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 (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 (Fl) 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. (J. Clin. Invest. 1994.93:2230-2235
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