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Reactive species of oxygen and nitrogen have been collectively implicated in pulmonary oxygen toxicity, but the contributions of specific molecules are unknown. Therefore, we assessed the roles of several reactive species, particularly nitric oxide, in pulmonary injury by exposing wild-type mice and seven groups of genetically altered mice to >98% 02 at 1, 3, or 4 atmospheres absolute. Genetically altered animals included knockouts lacking either neuronal nitric oxide synthase ([nNOS.sup.-/-]), endothelial nitric oxide synthase ([eNOS.sup.-/-] ), inducible nitric oxide synthase ([iNOS.sup.-/-]), extracellular superoxide dismutase (SOD3 /-), or glutathione peroxidase 1 ([GPxl.sup.-/-]), as well as two transgenic variants (S1179A and S1179D) having altered eNOS activities. We confirmed our earlier finding that normobaric hyperoxia (NB[O.sub.2]) and hyperbaric hyperoxia (HB[O.sub.2]) result in at least two distinct but overlapping patterns of pulmonary injury. Our new findings are that the role of nitric oxide in the pulmonary pathophysiology of hyperoxia depends both on the specific NOS isozyme that is its source and on the level of hyperoxia. Thus, iNOS predominates in the etiology of lung injury in NB[O.sub.2], and SOD3 provides an important defense. But in HB[O.sub.2], nNOS is a major contributor to pulmonary injury, whereas eNOS is protective. In addition, we demonstrated that nitric oxide derived from nNOS is involved in a neurogenic mechanism of HB[O.sub.2]-induced lung injury that is linked to central nervous system oxygen toxicity through adrenergic/cholinergic pathways. normobaric oxygen toxicity; hyperbaric oxygen toxicity; superoxide dismutase; glutathione peroxidase 1 ; neurogenic pulmonary oxygen toxicity

The objective of this study was to determine whether absence of endothelial nitric oxide synthase (eNOS) affects the expression of cell surface adhesion molecules in endothelial cells. Murine lung endothelial cells (MLECs) were prepared by immunomagnetic bead selection from wild-type and eNOS knockout mice. Wild-type cells expressed eNOS, but eNOS knockout cells did not. Expression of neuronal NOS and inducible NOS was not detectable in cells of either genotype. Upon stimulation, confluent wild-type MLECs produced significant amounts of NO compared with [N.sup.[omega]]-monomethyl-L-arginine-treated wild-type cells. eNOS knockout and wild-type cells showed no difference in the expression of E-selectin, P-selectin, intracellular adhesion molecule-1, and vascular cell adhesion molecule-1 as measured by flow cytometry on the surface of platelet endothelial cell adhesion molecule-1 (PECAM- 1/CD31)-positive cells. Both eNOS knockout and wild-type cells displayed the characteristics of resting endothelium. Adhesion studies in a parallel plate laminar flow chamber showed no difference in leukocyte-endothelial cell interactions between the two genotypes. Cytokine treatment induced endothelial cell adhesion molecule expression and increased leukocyte-endothelial cell interactions in both genotypes. We conclude that in resting murine endothelial cells, absence of endothelial production of NO by itself does not initiate endothelial cell activation or promote leukocyte-endothelial cell interactions. We propose that eNOS derived NO does not chronically suppress endothelial cell activation in an autocrine fashion but serves to counterbalance signals that mediate activation. vascular biology; atherosclerosis; mouse models

Meck, Janice V., Wendy W. Waters, Michael G. Ziegler, Heidi F. deBlock, Paul J. Mills, David Robertson, and Paul L. Huang. Mechanisms of postspaceflight orthostatic hypotension: low [[alpha].sub.1]-adrenergic receptor responses before flight and central autonomic dysregulation postflight. Am J Physiol Heart Circ Physiol 286: H1486--H1495, 2004. First published December 11, 2003; 10.1152/ ajpheart.00740.2003.--Although all astronauts experience symptoms of orthostatic intolerance after short-duration spaceflight, only ~20% actually experience presyncope during upright posture on landing day. The presyncopal group is characterized by low vascular resistance before and after flight and low norepinephrine release during orthostatic stress on landing day. Our purpose was to determine the mechanisms of the differences between presyncopal and nonpresyncopal groups. We studied 23 astronauts 10 days before launch, on landing day, and 3 days after landing. We measured pressor responses to phenylephrine injections; norepinephrine release with tyramine injections; plasma volumes; resting plasma levels of chromogranin A (a marker of sympathetic nerve terminal release), endothelin, dihydroxyphenylglycol (DHPG, an intracellular metabolite of norepinephrine); and lymphocyte [[beta].sub.2]-adrenergic receptors. We then measured hemodynamic and neurohumoral responses to upright tilt. Astronauts were separated into two groups according to their ability to complete 10 min of upright tilt on landing day. Compared with astronauts who were not presyncopal on landing day, presyncopal astronauts had 1) significantly smaller pressor responses to phenylephrine both before and after flight; 2) significantly smaller baseline norepinephrine, but significantly greater DHPG levels, on landing day; 3) significantly greater norepinephrine release with tyramine on landing day; and 4) significantly smaller norepinephrine release, but significantly greater epinephrine and arginine vasopressin release, with upright tilt on landing day. These data suggest that the etiology of orthostatic hypotension and presyncope after spaceflight includes low [[alpha].sub.l]-adrenergic receptor responsiveness before flight and a remodeling of the central nervous system during spaceflight such that sympathetic responses to baroreceptor input become impaired. tyramine; phenylephrine; dihydroxyphenylglycol; tilt tests; arginine vasopressin

Research was conducted to examine the leukocyte-endothelial cell interactions in nitric oxide synthase-deficient mice. The objective was to investigate the effects of endothelial cell nitric oxide synthase (ecNOS), neuronal NOS or induced NOS deficiency on leukocyte-endothelial cell interactions in the microcirculation of the mouse mesentery. Results suggest that the NO obtained from the ecNOS and nNOS was required in the regulation of leukocyte-endothelial cell interactions.

Wild-type and endothelial cell nitric oxide synthase (ecNOS)-deficient mice are subjected to 20 min of coronary artery occlusion and 120 min of reperfusion to investigate the role of ecNOS in myocardial ischemia/reperfusion (I/R) injury. A radiolabeled monoclonal anti-body is used to measure the P-selectin, which is thought to be involved with the pathogenesis of neutrophil-mediated I/R injury. Results suggest that the ecNOS derived nitric oxide plays a cardioprotective role in the ischemic-reperfused mouse heart.

The mechanism of acetyl-choline induced arterial dilatation is investigated in the pial arteries of mice in vitro. Inhibiting the neuronal nitric oxide synthase (NOS) in mutant mice without the endothelial isozyme of NOS inhibited the arteriolar dilatation mediated by acetyl choline administration. This indicates that neuronal NOS compensate for endothelial NOS in this strain of mutant mice.

A study was conducted on the source of expired nitric oxide in mice tracheal cannula mechanical ventilation. Results show that mice with -/-nnitric oxide synthase exhibited marked reduction in methacholine airway responsiveness compared to wild-type mice. Results also suggest that baseline source of expired nitric oxide and airway hyperresponsiveness is influenced by nitric oxide synthase.

The interaction between infarct volume reduction and NO-dependent augmentation of regional cerebral blood flow (rCBF) was analyzed in endothelial NO synthase (NOS) mutant mice. Intravenous administration of basic fibroblast growth factor (bFGF) in genetically engineered eNOS mutant mice reduced infarct volume via a mechanism that was independent of changes in rCBF. Furthermore, the lack of increased rCBF in the ischemic penumbra in eNOS-deficient mice receiving bFGF indicated the role of eNOS in promoting the vasodilatory effects of the polypeptide.

The effect of acetylcholine in endothelial nitric oxide synthase (eNOS) mutants was determined in mice with deletions in eNOS by intravital microscopy. Measurements of vessel diameter and NOS activity assays reveal that eNOS mice respond to ACh by dilation of the pial arterioles. The mechanism of this response was dependent on the activity of the neuronal NOS gene.

Regional cerebral blood flow within the brain has been associated with the activation of local neuronal activity by vasodilators such as the neurotransmitter nitric oxide (NO), which is synthesized by the enzyme NO synthase. Herein, it was shown that rCBF response to vibrissal stimulation in mice lacking expression of neuronal NOS is not mediated by endothelial NO production.

The effects of type III nitric oxide synthase (NOS) inhibition were analyzed in mutant mice that express type I NOS with a 96% reduction in type III NOS within their aortic homogenates. Decreased regional cerebral blood flow (rCBF) was detected in the mutant mice after the inhibition of type I NOS activity during moderate hypercapnia. Furthermore, rCBF was not inhibited by NG-nitro-L-arginine in type III NOS mutant mice but a decrease in baseline rCBF was detected in type I NOS mutant mice.

Regulation of murine airway responsiveness by endothelial nitric oxide synthase. Am J Physiol Lung Cell Mol Physiol 281: L258-L267, 2001.--Nitric oxide (NO) is a potent vasodilator, but it can also modulate contractile responses of the airway smooth muscle. Whether or not endothelial (e) NO synthase (NOS) contributes to the regulation of bronchial tone is unknown at present. Experiments were designed to investigate the isoforms of NOS that are expressed in murine airways and to determine whether or not the endogenous release of NO modulates bronchial tone in wild-type mice and in mice with targeted deletion of eNOS [eNOS(-/-)]. The presence of neuronal NOS (nNOS), inducible NOS (iNOS), and eNOS in murine trachea and lung parenchyma was assessed by RT-PCR, immunoblotting, and immunohistochemistry. Airway resistance was measured in conscious unrestrained mice by means of a whole body plethysmography chamber. The three isoforms of NOS were constitutively present in lungs of wild-type mice, whereas only iNOS and nNOS were present in eNOS(-/-) mice. Labeling of nNOS was localized in submucosal airway nerves but was not consistently detected, and iNOS immunoreactivity was observed in tracheal and bronchiolar epithelial cells, whereas eNOS was expressed in endothelial cells. In wild-type mice, treatment with N-nitro-L-arginine methyl ester, but not with aminoguanidine, potentiated the increase in airway resistance produced by inhalation of methacholine, eNOS(-/-) mice were hyperresponsive to inhaled methacholine and markedly less sensitive to N-nitro-L-arginine methyl ester. These results demonstrate that the three NOS isoforms are expressed constitutively in murine lung and that NO derived from eNOS plays a physiological role in controlling bronchial airway reactivity. epithelium; immunohistochemistry; methacholine; endothelial nitric oxide synthase knockout mice; N-nitro-L-arginine methyl ester; airway reactivity; reverse transcription-polymerase chain reaction; Western blot

Gyurko, Robert, Peter Kuhlencordt, Mark C. Fishman, and Paul L. Huang. Modulation of mouse cardiac function in vivo by eNOS and ANP. Am. J. Physiol. Heart Circ. Physiol. 278: H971-H981, 2000. To study the role of endothelial nitric oxide synthase (eNOS) in cardiac function, we compared eNOS expression, contractility, and relaxation in the left ventricles of wild-type and eNOS-deficient mice. eNOS immunostaining is localized to the macro- and microvascular endothelium throughout the myocardium in wild-type mice and is absent in eNOS-/- mice. Whereas blood pressure is elevated in eNOS-/- mice, baseline cardiac contractility (dP/[dt.sub.max]) is similar in wild-type and eNOS-/- mice (9,673 [+ or -] 2,447 and 9,928 [+ or -] 1,566 mmHg/s, respectively). The [Beta]-adrenergic agonist isoproterenol (Iso) at doses of [is greater than or equal to] 1 ng causes enhanced increases in dP/[dt.sub.max] in eNOS-/- mice compared with wild-type controls in vivo (P [is less than] 0.01) as well as in Langendorff isolated heart preparations (P [is less than] 0.02). [Beta]-Adrenergic receptor binding ([B.sub.max]) is not significantly different in the two groups of animals ([B.sub.max] = 41.4 [+ or -] 9.4 and 36.1 [+ or -] 5.1 fmol/mg for wild-type and eNOS-/-). Iso-stimulated ventricular relaxation is also enhanced in the eNOS-/- mice, as measured by dP/[dt.sub.min] in the isolated heart. However, baseline ventricular relaxation is normal in eNOS-/- mice ([Tau] = 5.2 [+ or -] 1.0 and 5.6 [+ or -] 1.5 ms for wild-type and eNOS-/-, respectively), whereas it is impaired in wild-type mice after NOS inhibition ([Tau] = 8.3 [+ or -] 2.4 ms). cGMP levels in the left ventricle are unaffected by eNOS gene deletion (wild-type: 3.1 [+ or -] 0.8 pmol/mg, eNOS-/-: 3.1 [+ or -] 0.6 pmol/mg), leading us to examine the level of another physiological regulator of cGMP. Atrial natriuretic peptide (ANP) expression is markedly upregulated in the eNOS-/- mice, and exogenous ANP restores ventricular relaxation in wild-type mice treated with NOS inhibitors. These results suggest that eNOS attenuates both inotropic and lusitropic responses to [Beta]-adrenergic stimulation, and it also appears to regulate baseline ventricular relaxation in conjunction with ANP. left ventricle; contractility; diastolic relaxation

Fagan, Karen A., Robert C. Tyler, Koichi Sato, Brian W. Fouty, Kenneth G. Morris, Jr., Paul L. Huang, Ivan F. McMurtry, and David M. Rodman. Relative contributions of endothelial, inducible, and neuronal NOS to tone in the murine pulmonary circulation. Am. J. Physiol. 277 (Lung Cell. Mol. Physiol. 21): L472-L478, 1999.--Nitric oxide plays an important role in modulating pulmonary vascular tone. All three isoforms of nitric oxide synthase (NOS), neuronal (nNOS, NOS I), inducible (iNOS, NOS II), and endothelial (eNOS, NOS III), are expressed in the lung. Recent reports have suggested an important role for eNOS in the modulation of pulmonary vascular tone chronically; however, the relative contribution of the three isoforms to acute modulation of pulmonary vascular tone is uncertain. We therefore tested the effect of targeted disruption of each isoform on pulmonary vascular reactivity in transgenic mice. Isolated perfused mouse lungs were used to evaluate the effect of selective loss of pulmonary nNOS, iNOS, and eNOS with respect to hypoxic pulmonary vasoconstriction (HPV) and endothelium-dependent and -independent vasodilation, eNOS null mice had augmented HPV (225 [+ or -] 65% control, P [is less than] 0.02, mean [+ or -] SE) and absent endothelium-dependent vasodilation, whereas endothelium-independent vasodilation was preserved. HPV was minimally elevated in iNOS null mice and normal in nNOS null mice. Both nNOS and iNOS null mice had normal endothelium-dependent vasodilation. In wild-type lungs, nonselective NOS inhibition doubled HPV, whereas selective iNOS inhibition had no detectable effect. In intact, lightly sedated mice, right ventricular systolic pressure was elevated in eNOS-deficient (42.3 [+ or -] 1.2 mmHg, P [is less than] 0.001) and, to a lesser extent, in iNOS-deficient (37.2 [+ or -] 0.8 mmHg, P [is less than] 0.001) mice, whereas it was normal in nNOS-deficient mice (30.9 [+ or -] 0.7 mmHg, P = not significant) compared with wild-type controls (31.3 [+ or -] 0.7 mmHg). We conclude that in the normal murine pulmonary circulation 1) nNOS does not modulate tone, 2) eNOS-derived nitric oxide is the principle mediator of endothelium-dependent vasodilation in the pulmonary circulation, and 3) both eNOS and iNOS play a role in modulating basal tone chronically. nitric oxide; pulmonary hypertension; vasoconstriction

The adhesion of uropathogenic Escherichia coli to the urothelial surface of the bladder is a prerequisite for the establishment of bladder infections. This adhesion process relies on E. coli adhesins and their cognate urothelial receptors, and it also is influenced by an intricate array of defense mechanisms of the urinary system. In this study, we examined the in vivo role of Tamm-Horsfall protein (THP), the most abundant urinary protein, in innate urinary defense. We genetically ablated the mouse THP gene and found that THP deficiency predisposes mice to bladder infections by type 1-timbriated E. coli. Inoculation of too few type 1-fimbriated E. coli to colonize wild-type mice caused significant bladder colonization in THP-knockout mice. In contrast, THP deficiency did not enhance the ability of P-fimbriated E. coli to colonize the bladder. Our results provide the first in vivo evidence indicating that under physiological conditions, the mannosylated THP can serve as an effective soluble "receptor," binding to the type 1-fimbriated E. coli and competitively inhibiting them from adhering to the uroplakin Ia receptors present on the urothelial surface. These results suggest that potential THP defects, either quantitative or qualitative, could predispose the urinary bladder to bacterial infections. The generation of THP-deficient mice established the role of THP as a first line of urinary defense and should help elucidate other potential functions of this major protein in urinary tract physiology and diseases. [ABSTRACT FROM AUTHOR]

Although all astronauts experience symptoms of orthostatic intolerance after short-duration spaceflight, only ∼20% actually experience presyncope during upright posture on landing day. The presyncopal group is characterized by low vascular resistance before and after flight and low norepinephrine release during orthostatic stress on landing day. Our purpose was to determine the mechanisms of the differences between presyncopal and nonpresyncopal groups. We studied 23 astronauts 10 days before launch, on landing day, and 3 days after landing. We measured pressor responses to phenylephrine injections; norepinephrine release with tyramine injections; plasma volumes; resting plasma levels of chromogranin A (a marker of sympathetic nerve terminal release), endothelin, dihydroxyphenylglycol (DHPG, an intracellular metabolite of norepinephfine); and lymphocyte β2-adrenergic receptors. We then measured hemodynamic and neurohumoral responses to upright tilt. Astronauts were separated into two groups according to their ability to complete 10 min of upright tilt on landing day. Compared with astronauts who were not presyncopal on landing day, presyncopal astronauts had 1) significantly smaller pressor responses to phenylephrine both before and after flight; 2) significantly smaller baseline norepinephrine, but significantly greater DHPG levels, on landing day; 3) significantly greater norepinephrine release with tyramine on landing day; and 4) significantly smaller norepinephrine release, but significantly greater epinephrine and arginine vasopressin release, with upright tilt on landing day. These data suggest that the etiology of orthostatic hypotension and presyncope after spaceflight includes low α1-adrenergic receptor responsiveness before flight and a remodeling of the central nervous system during spaceflight such that sympathetic responses to baroreceptor input become impaired. [ABSTRACT FROM AUTHOR]

Evaluates neuronal nitric oxide synthase (NOS)-guanosine 3,5-cyclic monophosphate-dependent acetylcholine (ACh)-induced relaxation in pial arterioles of endothelial NOS knockout mice. Function of ACh; In vitro muscarinic ACh receptor autoradiography; Measurement of carbon dioxide in arterial blood.

Insulin resistance (IR) and systemic hypertension are independently associated with heart failure. We reported previously that nitric oxide synthase 3 (NOS3) has a beneficial effect on left ventricular (LV) remodeling and function after pressure-overload in mice. The aim of our study was to investigate the interaction of IR and NOS3 in pressure-overload-induced LV remodeling and dysfunction. Wild-type (WT) and NOS3-deficient (NOS3(-/-)) mice were fed either a standard diet (SD) or a high-fat diet (HFD) to induce IR. After 9 days of diet, mice underwent transverse aortic constriction (TAC). LV structure and function were assessed serially using echocardiography. Cardiomyocytes were isolated, and levels of oxidative stress were evaluated using 2',7'-dichlorodihydrofluorescein diacetate. Cardiac mitochondria were isolated, and mitochondrial respiration and ATP production were measured. TAC induced LV remodeling and dysfunction in all mice. The TAC-induced decrease in LV function was greater in SD-fed NOS3(-/-) mice than in SD-fed WT mice. In contrast, HFD-fed NOS3(-/-) developed less LV remodeling and dysfunction and had better survival than did HFD-fed WT mice. Seven days after TAC, oxidative stress levels were lower in cardiomyocytes from HFD-fed NOS3(-/-) than in those from HFD-fed WT. N(ω)-nitro-L-arginine methyl ester and mitochondrial inhibitors (rotenone and 2-thenoyltrifluoroacetone) decreased oxidative stress levels in cardiomyocytes from HFD-fed WT mice. Mitochondrial respiration was altered in NOS3(-/-) mice but did not worsen after HFD and TAC. In contrast with its protective role in SD, NOS3 increases LV adverse remodeling after pressure overload in HFD-fed, insulin resistant mice. Interactions between NOS3 and mitochondria may be responsible for increased oxidative stress levels in HFD-fed WT mice hearts.