Age-dependent endothelial nitric oxide synthase uncoupling in pulmonary arteries of endoglin heterozygous mice.

Endoglin is a TGF-beta superfamily receptor critical for endothelial cell function. Mutations in this gene are associated with hereditary hemorrhagic telangiectasia type I (HHT1), and clinical signs of disease are generally more evident later in life. We previously showed that systemic vessels of adult Eng heterozygous (Eng(+/-)) mice exhibit increased vasorelaxation due to uncoupling of endothelial nitric oxide synthase (eNOS). We postulated that these changes may develop with age and evaluated pulmonary arteries from newborn and adult Eng(+/-) mice for eNOS-dependent, acetylcholine (ACh-induced) vasorelaxation, compared with that of age-matched littermate controls. While ACh-induced vasorelaxation was similar in all newborn mice, it was significantly increased in the adult Eng(+/-) vs. control vessels. The vasodilatory responses were inhibited by l-NAME suggesting eNOS dependence. eNOS uncoupling was observed in lung tissues of adult, but not newborn, heterozygous mice and was associated with increased production of reactive O(2) species (ROS) in adult Eng(+/-) vs. control lungs. Interestingly, ROS generation was higher in adult than newborn mice and so were the levels of NADPH oxidase 4 and SOD 1, 2, 3 isoforms. However, enzyme protein levels and NADPH activity were normal in adult Eng(+/-) lungs indicating that the developmental maturation of ROS generation and scavenging cannot account for the increased vasodilatation observed in adult Eng(+/-) mice. Our data suggest that eNOS-dependent H(2)O(2) generation in Eng(+/-) lungs accounts for the heightened pulmonary vasorelaxation. To the extent that these mice mimic human HHT1, age-associated pulmonary vascular eNOS uncoupling may explain the late childhood and adult onset of clinical lung manifestations.

Capillary electrophoresis analysis of nitrite and nitrate in sub-microliter quantities of airway surface liquid.

We developed a simple capillary electrophoresis (CE) method to measure nitrite and nitrate concentrations in submicroliter samples of rat airway surface liquid (ASL), a thin (10-30 microm) layer of liquid covering the epithelial cells lining the airways of the lung. The composition of ASL has been poorly defined, in large part because of the small sample volume (approximately 1-3 microl per cm2 of epithelium) and difficulty of harvesting ASL. We have used capillary tubes for ASL sample collection, with microanalysis by CE using a 50 mM phosphate buffer (pH 3), with 0.5 mM spermine as a dynamic flow modifier, and direct UV detection at 214 nm. The limit of detections (LODs), under conditions used, for ASL analysis were 10 microM for nitrate and 30 microM for nitrite (SIN= 3). Nitrate and nitrite were also measured in rat plasma. The concentration of nitrate was 102+/-12 microM in rat ASL and 70+/-1.0 microM in rat plasma, whereas nitrite was 83+/-28 microM in rat ASL and below the LOD in rat plasma. After instilling lipopolysaccharide intratracheally to induce increased NO production, the nitrate concentration in ASL increased to 387+/-16 microM, and to 377+/-88 microM in plasma. The concentration of nitrite increased to 103+/-7.0 microM for ASL and 138+/-17 microM for plasma.

Downregulation of endothelial nitric oxide synthase in rat aorta after prolonged hypoxia in vivo.

The goal of this study was to determine whether hypoxia alters expression of endothelial nitric oxide synthase (eNOS) in the systemic circulation. Rats breathed either air or 10% oxygen for 12 hours, 48 hours, or 7 days. Thoracic aortas were excised and either mounted in organ bath myographs or frozen in liquid nitrogen for later extraction of protein and RNA. eNOS protein (Western blotting) was decreased (20% of normoxic control) after 12 hours, 48 hours, and 7 days of hypoxia. eNOS mRNA (ribonuclease protection assay) was similarly reduced. Acetylcholine (10(-4) mol/L) reversed phenylephrine (10(-5) mol/L) preconstriction by 53.3+/-5.6% in aortic rings from normoxic rats and 26.1+/-4.8% in rings from rats exposed to hypoxia for 48 hours (P<0.05), with comparable impairment of relaxation by the calcium ionophore A23187 (10(-5) mol/L). Responses to diethylamine nitric oxide and 8-bromo-cGMP were unaffected. Aortic cGMP levels after incubation with acetylcholine (10(-6) mol/L) averaged 14.0+/-1.8 fmol/mg in rings from normoxic rats compared with 8.7+/-1.0 fmol/mg in rings from hypoxic rats (P<0. 05). Similarly, nitrate concentration (by capillary electrophoresis) in the media in which the rings were incubated was reduced in the hypoxic group (5.6+/-0.23 micromol/L for hypoxic rats and 7.8+/-0.7 micromol/L for normoxic rats). Impaired endothelial NO release may handicap the vascular responses that defend vital organ function during hypoxia.

Enhancement of aortic contractility by endothelin following prolonged hypoxia in vivo.

The purpose of this study was to determine if endothelin-1 (ET-1) mediates endothelium-dependent enhancement of rat aortic contractility following exposure to hypoxia. Rats breathed room air or 10% oxygen for 12 or 48 h. Thoracic aortas and plasma were analysed for ET-1 content by radioimmunassay. Aortic rings were mounted in organ bath myographs for measurement of isometric tension during activation by phenylephrine (10(-9)-10(-4) M), in the presence and absence of BQ-123. In some rings, the endothelium was removed. Plasma ET-1 levels were 0.79+/-0.09 pg/ml, 2.00+/-0.36 and 1.88+/-0.21 pg/ml, in normoxic rats and rats exposed to hypoxia for 12 or 48 h respectively (P<0.001, 12 or 48 h vs. control). Aortic ET-1 concentrations were 202.3+/-20.8 fg/mg in normoxic rats, compared to 274.9+/-40.6 fg/mg and 292.4+/-24.4 fg/mg in rats exposed to hypoxia for 12 and 48 h, respectively (P<0.01, 12 or 48 h vs. control) and 155.0+/-43.1 fg/mg in de-endothelialized aortas from rats exposed to hypoxia for 48 h (P>0.05 vs. normoxic controls). Maximum tension during phenylephrine-induced contraction was 0.46+/-0.04 mg/g and 0.33+/-0.03 mg/g in endothelialized rings from rats exposed to hypoxia for 48 h in the presence and absence of BQ-123, respectively (P<0.05 for difference), and 0.28+/-0.07 mg/g in rings in which the endothelium had been removed. Local endothelin release is an important mechanism by which the responsiveness of the systemic vasculature to agonists may be preserved during hypoxia.

Hyporeactivity of rat diaphragmatic arterioles after exposure to hypoxia in vivo. Role of the endothelium.

The effect of prior in vivo hypoxia on the in vitro responses to changes in transmural pressure, alpha-adrenoceptor activation, and depolarization with KCl were evaluated in first-order diaphragmatic arterioles. Rats (n = 14 per group) were exposed to normoxia (controls) or to hypoxia (inspired O2 concentration = 10%) for 12 or 48 h. The arteriolar pressure-diameter relationships were recorded over a pressure range from 10 to 200 mm Hg. In separate groups of arterioles (n = 12 per group), the diaphragmatic arteriolar responses to phenylephrine (10(-8) to 10(-5 M) or KCl (10 to 100 mM) were determined after exposure to either room air or hypoxia for 48 h. In half of the arterioles studied, the endothelium was removed. After 12 h of hypoxia, the pressure-diameter relationship was normal in endothelialized arterioles but was shifted upward in de-endothelialized vessels (p < 0.05). After 48 h of hypoxia, the constrictor response to increasing transmural pressure was severely suppressed in all arterioles. The intraluminal diameters during activation with phenylephrine and KCl were larger in arterioles from rats exposed to hypoxia (103 +/- 8 and 81 +/- 7 microns, respectively) than in control arterioles (41 +/- 5 and 54 +/- 6 microns, respectively; p < 0.05 for differences). During maximum phenylephrine- and KCl-induced constriction in de-endothelialized arterioles, diameters averaged 125 +/- 8 and 105 +/- 8 microns, respectively, for arterioles from hypoxic rats and 32 +/- 6 and 40 +/- 5 microns, respectively, for arterioles from control vessels. Exposure to hypoxia results in impairment of diaphragmatic arteriolar smooth muscle reactivity and reversal of the normal inhibitory influence of the endothelium on diaphragmatic arteriolar tone.