Functional differences between the arteries perfusing gas exchange and nutritional membranes in the late chicken embryo.

The chicken extraembryonic arterial system comprises the allantoic arteries, which irrigate the gas exchange organ (the chorioallantoic membrane, CAM) and the yolk sac (YS) artery, which irrigates the nutritional organ (the YS membrane). We compared, using wire myography, the reactivity of allantoic and YS arteries from 19-day chicken embryos (total incubation 21 days). The contractions induced by KCl, the adrenergic agonists norepinephrine (NE, nonselective), phenylephrine (α1), and oxymetazoline (α2), electric field stimulation (EFS), serotonin, U46619 (TP receptor agonist), and endothelin (ET)-1 and the relaxations induced by acetylcholine (ACh), sodium nitroprusside (SNP, NO donor), forskolin (adenylate cyclase activator), and isoproterenol (ß-adrenergic agonist) were investigated. Extraembryonic allantoic arteries did not show α-adrenergic-mediated contraction (either elicited by exogenous agonists or EFS) or ACh-induced (endothelium-dependent) relaxation, whereas these responses were present in YS arteries. Interestingly, the intraembryonic segment of the allantoic artery showed EFS- and α-adrenergic-induced contraction and ACh-mediated relaxation. Moreover, glyoxylic acid staining showed the presence of catecholamine-containing nerves in the YS and the intraembryonic allantoic artery, but not in the extraembryonic allantoic artery. Isoproterenol- and forskolin-induced relaxation and ET-1-induced contraction were higher in YS than in allantoic arteries, whereas serotonin- and U46619-induced contraction and SNP-induced relaxation did not significantly differ between the two arteries. In conclusion, our study demonstrates a different pattern of reactivity in the arteries perfusing the gas exchange and the nutritional membranes of the chicken embryo.

Pulmonary vascular endothelial growth factor expression and disaturated phospholipid content in a chicken model of hypoxia-induced fetal growth restriction.

BACKGROUND: Prenatal hypoxia is an important cause of intrauterine growth retardation that affects fetal lung maturation, although previous studies have rendered conflicting results. The fetal chicken model allows the study of the isolated effects of hypoxia during development. OBJECTIVES: We hypothesized that prenatal hypoxia would differentially affect surfactant synthesis, depending on timing and duration of hypoxia. Pulmonary vascular endothelial growth factor (VEGF) expression was analyzed as a possible link between oxygen sensing and surfactant production. METHODS: Fertilized White Leghorn eggs were incubated in normoxia, hyperoxia (60% O(2)) from day 15 or hypoxia (15% O(2)) from either day 6 or day 15 of incubation. Whole lung disaturated phospholipids (DSPL) and mRNA expression of VEGF isoforms were quantified at day 16 and 19. RESULTS: Lung DSPL content increased approximately threefold between day 16 and 19 in control animals. Both hypoxia and hyperoxia from day 15 significantly increased DSPL content at day 19 versus control (103 +/- 22 and 116 +/- 18 vs. 81 +/- 15 microg/mg protein, p < 0.01 and p < 0.001, respectively), while long-term hypoxia tended to decrease DSPL content (65 +/- 17 microg/mg protein, p = 0.056). No differences in DSPL content were observed at day 16. Short-term hypoxia transiently up-regulated VEGF146 1.5-fold at day 16 (p < 0.05). A similar trend was observed for VEGF122 (p = 0.058) and VEGF190 (p = 0.08), while no differences were present at day 19. CONCLUSIONS: Both prenatal hypoxia and hyperoxia induced during critical windows of lung development differentially modulate surfactant synthesis. Our data support the concept that fetal oxygen tension is a key signal in the regulation of the surfactant system.

Ontogeny of chicken ductus arteriosus response to oxygen and vasoconstrictors.

The present study aimed to characterize the contractile reactivity of the chicken ductus arteriosus (DA) from the last stage of prenatal development and throughout the perinatal period. Isolated DA rings from 15-day, noninternally-pipped 19-day, and externally-pipped 21-day embryos were studied using myograph techniques. On embryonic day 15, the chicken DA did not respond to O(2) (0 to 21%), norepinephrine (NE), or phenylephrine (Phe) but contracted in response to high-K(+) solution, the inhibitor of voltage-gated channels 4-aminopyridine, U-46619, and endothelin (ET)-1. These responses increased with advancing incubation age. Contractile responses to O(2), NE, and Phe were present in the 19- and 21-day embryo. Oxygen-induced contraction was restricted to the pulmonary side of the DA and was augmented by the nitric oxide synthase inhibitor N(omega)-nitro-l-arginine methyl ester and the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one and reduced by the peptidic ET(A) and ET(B)-receptor antagonist PD-142,893. Transmural electrical stimulation of nerves, the nonselective cyclooxygenase (COX) inhibitor indomethacin, the COX-1 inhibitor valeryl salicylate, the COX-2 inhibitor nimesulide, the inhibitor of ATP-sensitive K(+) channels glibenclamide, and the inhibitor of Ca(2+)-activated K(+) channels tetraethylammonium did not cause contraction of the DA rings at any age. We conclude that transition to ex ovo life is accompanied by dramatic changes in chicken DA reactivity. At 0.7 incubation, excitation-contraction and pharmacomechanical coupling for several contractile agonists are already present, whereas the constrictor effects of O(2) and cathecolamines appear later in development and are located in the pulmonary side of the DA.

Cardiopulmonary effects of chronic administration of the NO synthase inhibitor L-NAME in the chick embryo.

BACKGROUND: Experimental observations in mammalian models suggest that endothelial nitric oxide (NO) synthase (NOS) content and activity are decreased in persistent pulmonary hypertension of the newborn. OBJECTIVES: To test the hypothesis that disruption of NO signaling in the developing chick embryo lung may contribute to pulmonary hypertension. METHODS: We analyzed pulmonary arterial reactivity and structure and heart morphology of 19-day chick embryos (incubation time 21 days) that received a daily injection of the NOS inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME, 20 mug per gram egg) or vehicle from day 12 until day 18. RESULTS: Exposure to L-NAME did not affect embryonic survival or body mass of the embryos. The contractile responses to KCl, endothelin-1, the thromboxane A2 mimetic U46619, noradrenaline, and electrical-field stimulation were not affected by exposure to L-NAME. In contrast, in ovo L-NAME exposure reduced the sensitivity of pulmonary arteries to acetylcholine (pD2: 6.53 +/- 0.14 vs. 6.96 +/- 0.13; p < 0.05) and this effect was reversed by the NOS substrate L-arginine. Relaxations induced by sodium nitroprusside or forskolin were not altered by chronic L-NAME. Pulmonary vessel density was not different, but the percentage medial wall area of small pulmonary arteries (external diameter 10-50 microm) was slightly but significantly increased in the embryos exposed to L-NAME. In addition, hearts of L-NAME-exposed embryos showed an increase in right and left ventricular wall area. CONCLUSIONS: Chronic inhibition of NOS produced, in the chick embryo, impairment of endothelium-dependent relaxation, structural remodeling of the pulmonary vascular bed and biventricular cardiac enlargement.

Chronic in ovo hypoxia decreases pulmonary arterial contractile reactivity and induces biventricular cardiac enlargement in the chicken embryo.

Although chronic prenatal hypoxia is considered a major cause of persistent pulmonary hypertension of the newborn, experimental studies have failed to consistently find pulmonary hypertensive changes after chronic intrauterine hypoxia. We hypothesized that chronic prenatal hypoxia induces changes in the pulmonary vasculature of the chicken embryo. We analyzed pulmonary arterial reactivity and structure and heart morphology of chicken embryos maintained from days 6 to 19 of the 21-day incubation period under normoxic (21% O(2)) or hypoxic (15% O(2)) conditions. Hypoxia increased mortality (0.46 vs. 0.14; P < 0.01) and reduced the body mass of the surviving 19-day embryos (22.4 +/- 0.5 vs. 26.6 +/- 0.7 g; P < 0.01). A decrease in the response of the pulmonary artery to KCl was observed in the 19-day hypoxic embryos. The contractile responses to endothelin-1, the thromboxane A(2) mimetic U-46619, norepinephrine, and electrical-field stimulation were also reduced in a proportion similar to that observed for KCl-induced contractions. In contrast, no hypoxia-induced decrease of response to vasoconstrictors was observed in externally pipped 21-day embryos (incubated under normoxia for the last 2 days). Relaxations induced by ACh, sodium nitroprusside, or forskolin were unaffected by chronic hypoxia in the pulmonary artery, but femoral artery segments of 19-day hypoxic embryos were significantly less sensitive to ACh than arteries of control embryos [pD(2) (= -log EC(50)): 6.51 +/- 0.1 vs. 7.05 +/- 0.1, P < 0.01]. Pulmonary vessel density, percent wall area, and periarterial sympathetic nerve density were not different between control and hypoxic embryos. In contrast, hypoxic hearts showed an increase in right and left ventricular wall area and thickness. We conclude that, in the chicken embryo, chronic moderate hypoxia during incubation transiently reduced pulmonary arterial contractile reactivity, impaired endothelium-dependent relaxation of femoral but not pulmonary arteries, and induced biventricular cardiac hypertrophy.

Role of superoxide anion on basal and stimulated nitric oxide activity in neonatal piglet pulmonary vessels.

The superoxide anion (O2*-) appears to be an important modulator of nitric oxide bioavailability. Enzymatic scavenging of O2*- is carried out by superoxide dismutase (SOD). The present study was designed to characterize the developmental changes on pulmonary vascular reactivity induced by 1) exogenous Cu/Zn SOD, 2) several putative SOD mimetics, and 3) endogenous SOD inhibition. We also analyzed age-related changes on pulmonary SOD activity and vascular O2*- levels. SOD (1-300 U/mL) produced endothelium-dependent relaxation of U46619-contracted intrapulmonary arteries (fourth branch) and veins from 12- to 24-h-old and 2-wk-old piglets. SOD-induced relaxation was greater in pulmonary arteries and was abolished by the nitric oxide synthase inhibitor N omega-nitro-L-arginine methyl ester. SOD induced a greater pulmonary artery relaxation in the 2-wk-old than in the 12- to 24-h-old piglet. SOD (100 U/mL) did not modify acetylcholine-induced relaxation in pulmonary arteries. In contrast, endogenous SOD inhibition by diethyldithiocarbamate (3 mM) impaired acetylcholine-induced relaxation in pulmonary arteries from newborn but not from 2-wk-old piglets. Total SOD activity in lung tissue did not change with postnatal age. With the use of dihydroethidium, an oxidant-sensitive fluorescent probe, we did not find significant age- or vessel-related differences in O2*- presence. From the putative SOD mimetics tested, only the metal salts MnCl2 and CuSO4 reproduced the vascular effects of SOD. In summary, SOD produces endothelium-dependent pulmonary vascular relaxation by protecting nitric oxide from destruction by O2*-. This effect was less marked in newborns than in 2-wk-old piglets. In contrast, pulmonary arteries from newborn piglets are more sensitive to the inhibition of endogenous SOD.