Biomedical subjects

P L Huang

Publications and source records attributed to P L Huang.

At least 19 recordsLinked to original sources

PubMed2004

Mouse model of microembolic stroke and reperfusion.

BACKGROUND AND PURPOSE: To test the role of fibrinolysis in stroke, we used a mouse model in which preformed 2.5- to 3-microm-diameter fibrin microemboli are injected into the cerebral circulation. The microemboli lodge in the downstream precapillary vasculature and are susceptible to fibrinolysis. METHODS: We injected various doses of microemboli into the internal carotid artery in mice and characterized their distribution, effects on cerebral blood flow, neurological deficit, infarct area, and spontaneous dissolution. By comparing wild-type and tissue plasminogen activator (tPA) knockout (tPA-/-) mice, we analyzed the role of endogenous tPA in acute thrombotic stroke. RESULTS: Microemboli cause dose-dependent brain injury. Although moderate doses of microemboli are followed by spontaneous reperfusion, they result in reproducible injury. Gene knockout of tPA markedly delays dissolution of cerebral emboli and restoration of blood flow and aggravates ischemic thrombotic infarction in the brain. CONCLUSIONS: We describe a microembolic model of stroke, in which degree of injury can be controlled by the dose of microemboli injected. Unlike vessel occlusion models, this model can be modulated to allow spontaneous fibrinolysis. Application to tPA-/- mice supports a key role of endogenous tPA in restoring cerebral blood flow and limiting infarct size after thrombosis.

Animals↗

PubMed2003

Contributions of endothelial and neuronal nitric oxide synthases to cerebrovascular responses to hyperoxia.

Hyperoxia causes a transient decrease in CBF, followed by a later rise. The mediators of these effects are not known. We used mice lacking endothelial or neuronal nitric oxide synthase (NOS) isoforms (eNOS-/- and nNOS-/- mice) to study the roles of the NOS isoforms in mediating changes in cerebral vascular tone in response to hyperoxia. Resting regional cerebral blood flow (rCBF) did not differ between wild type (WT), eNOS-/- mice, and nNOS-/- mice. eNOS-/- mice showed decreased cerebrovascular reactivities to NG-nitro-L-arginine methyl ester (L-NAME), PAPA NONOate, acetylcholine (Ach), and SOD1. In response to hyperbaric oxygen (HBO2) at 5 ATA, WT and nNOS-/- mice showed decreases in rCBF over 30 minutes, but eNOS-/- mice did not. After 60 minutes HBO2, rCBF increased more in WT mice than in eNOS-/- or nNOS-/- mice. Brain NO-metabolites (NOx) decreased in WT and eNOS-/- mice within 30 minutes of HBO2, but after 45 minutes, NOx rose above control levels, whereas they did not change in nNOS-/- mice. Brain 3NT increased during HBO2 in WT and eNOS-/- but did not change in nNOS-/- mice. These results suggest that modulation of eNOS-derived NO by HBO2 is responsible for the early vasoconstriction responses, whereas late HBO2-induced vasodilation depends upon both eNOS and nNOS.

Animals↗

PubMed2001

Hypertension does not account for the accelerated atherosclerosis and development of aneurysms in male apolipoprotein e/endothelial nitric oxide synthase double knockout mice.

BACKGROUND: Apolipoprotein E (apoE)/endothelial nitric oxide synthase (eNOS) double knockout (DKO) mice demonstrate accelerated atherosclerosis and develop abdominal aortic aneurysms and aortic dissection, suggesting a role for eNOS in suppressing atherogenesis. To test whether accelerated atherosclerosis and aortic aneurysms were due to hypertension, we administered hydralazine to male apoE/eNOS DKO mice to reduce blood pressure. METHODS AND RESULTS: Male apoE/eNOS DKO mice were treated with hydralazine in their drinking water (250 mg/L) using a dose that lowers the blood pressure to levels seen in apoE KO mice. The mice were fed a Western-type diet for 16 weeks, and lesion formation was assessed by inspection of the vessel and staining with Sudan IV. Hydralazine-treated, normotensive male apoE/eNOS DKO mice developed increased aortic lesion areas (30.0+/-2.8%, n=11) compared with male apoE KO mice (14.6+/-0.8%, n=7). The extent of lesion formation was not significantly different from male apoE/eNOS DKO mice that were not given hydralazine (28.3+/-3.1%, n=9). Four of 11 hydralazine-treated male apoE/eNOS DKO mice developed abdominal aortic aneurysms. CONCLUSIONS: Hypertension is not required for the accelerated atherosclerosis seen in apoE/eNOS DKO animals, and control of hypertension during a 16-week period does not prevent aortic aneurysm formation.

Animals↗

PubMed2001

Anti-HIV agent MAP30 modulates the expression profile of viral and cellular genes for proliferation and apoptosis in AIDS-related lymphoma cells infected with Kaposi's sarcoma-associated virus.

The anti-HIV agent MAP30 (Momordica anti-HIV protein, 30 kDa) inhibits the proliferation of BC-2, an AIDS-related primary effusion lymphoma (PEL) cell line derived from an AIDS patient. BC-2 cells are latently infected with Kaposi's sarcoma-associated herpes virus (KSHV), also known as human herpes virus 8 (HHV8). We examined the effect of MAP30 on the expression of viral and cellular genes in BC-2 during latent and lytic states of the viral life cycle. By Northern analysis and RT-PCR, we found that MAP30 downregulates the expression of viral cyclin D (vCD), viral interleukin-6 (vIL-6), and viral FLIP (vFLIP), genes involved in cell cycle regulation, viral pathogenesis, and apoptosis. By pathway-specific cDNA microarray analysis, we found that BC-2 cells express high levels of egr-1, ATF-2, hsp27, hsp90, IkappaB, mdm2, skp1, and IL-2, cellular genes involved in mitogenesis, tumorigenesis, and inhibition of apoptosis in NFkappaB and p53 signaling pathways. These results define for the first time the specific cellular pathways involved in AIDS-related tumorigenesis and suggest specific novel targets for the treatment. Furthermore, we found that MAP30 downregulates the expression of egr-1, ATF-2, hsp27, hsp90, IkappaB, mdm2, and Skp1, while it upregulates the pro-apoptotic-related genes Bax, CRADD, and caspase-3. Thus, MAP30 modulates the expression of both viral and cellular genes involved in KS pathogenesis. These results provide valuable insight into the molecular mechanisms of MAP30 anti-KS action and suggest its utility as a therapeutic agent against AIDS-related tumors.

Anti-HIV Agents↗

PubMed2001

Endothelial nitric oxide synthase limits left ventricular remodeling after myocardial infarction in mice.

Background- To investigate the role of endothelial nitric oxide synthase (NOS3) in left ventricular (LV) remodeling after myocardial infarction (MI), the impact of left anterior descending coronary artery ligation on LV size and function was compared in 2- to 4-month-old wild-type (WT) and NOS3-deficient mice (NOS3(-/-)). Methods and Results- Two days after MI, both strains of mice had a similar LV size, fractional shortening, and ejection fraction by echocardiography. Twenty-eight days after MI, both strains had dilated LVs with decreased fractional shortening and lower ejection fractions. Although the infarcted fraction of the LV was similar in both strains, LV end-diastolic internal diameter, end-diastolic volume, and mass were greater, but fractional shortening, ejection fraction, and the maximum rate of developed LV pressure (dP/dt(max)) were lower in NOS3(-/-) than in WT mice. Impairment of diastolic function, as measured by the time constant of isovolumic relaxation (tau) and the maximum rate of LV pressure decay (dP/dt(min)), was more marked in NOS3(-/-) than in WT mice. Mortality after MI was greater in NOS3(-/-) than in WT mice. Long-term administration of hydralazine normalized blood pressure in NOS3(-/-) mice, but it did not prevent the LV dilatation, impaired systolic and diastolic function, and increased LV mass that followed MI. In WT mice, capillary density and myocyte width in the nonischemic portion of the LV did not differ before and 28 days after MI, whereas in NOS3(-/-) mice, capillary density decreased and myocyte width increased after MI, whether or not hydralazine was administered. Conclusions- These results suggest that the presence of NOS3 limits LV dysfunction and remodeling in a murine model of MI by an afterload-independent mechanism, in part by decreasing myocyte hypertrophy in the remote myocardium.

Animals↗

PubMed2001

Accelerated atherosclerosis, aortic aneurysm formation, and ischemic heart disease in apolipoprotein E/endothelial nitric oxide synthase double-knockout mice.

BACKGROUND: To test whether deficiency in endothelial nitric oxide synthase (eNOS) affects atherosclerosis development, we compared lesion formation in apolipoprotein E (apoE)/eNOS-double knockout (DKO) and apoE-knockout (KO) control animals. METHODS AND RESULTS: After 16 weeks of "Western-type" diet, apoE/eNOS-DKO males and females showed significant increases in lesion area of 93.6% and 59.2% compared with apoE-KO mice. All apoE/eNOS-DKO animals studied developed peripheral coronary arteriosclerosis, associated with perivascular and myocardial fibrosis, whereas none of the apoE-KO mice did. Transthoracic echocardiography showed a significantly increased left ventricular wall thickness and decreased fractional shortening in DKO animals. Mean arterial pressure was increased in DKO mice and was comparable in degree to eNOS-KO animals. Male DKO animals developed atherosclerotic abdominal aneurysms and aortic dissection. CONCLUSIONS: eNOS deficiency increases atherosclerosis in Western-type diet-fed apoE-KO animals and introduces coronary disease and an array of cardiovascular complications, including spontaneous aortic aneurysm and dissection. This phenotype constitutes the first murine model to demonstrate distal coronary arteriosclerosis associated with evidence of myocardial ischemia, infarction, and heart failure. Hypertrophy and reduced left ventricular function cannot be explained by increased blood pressure alone, because eNOS-KO animals do not develop these complications.

Animals↗

PubMed2001

Genetic deficiency of inducible nitric oxide synthase reduces atherosclerosis and lowers plasma lipid peroxides in apolipoprotein E-knockout mice.

BACKGROUND: Inducible nitric oxide synthase (iNOS) is expressed by leukocytes and smooth muscle cells in atherosclerotic lesions. To test whether NO produced by iNOS deficiency affects atherosclerosis, we studied apoE/iNOS-double knockout (dKO) and apoE-knockout (KO) control animals fed a "Western-type" diet. METHODS AND RESULTS: After 16 weeks of Western-type diet, the aortic lesion area in apoE/iNOS-dKO males and females was significantly reduced, by 22% and 21%, respectively, compared with apoE-KO males and females. This effect was more pronounced after 24 weeks of Western-type diet, after which lesion formation in male and female dKO mice was reduced by 38% and 40%, respectively. Plasma levels of lipoperoxides in apoE/iNOS-dKO mice (2.0+/-0.23 micromol/L) were significantly lower than in apoE-KO control animals (3.2+/-0.44 micromol/L; P=0.02). To test whether substrate deficiency plays a role in the proatherogenic actions of iNOS, we administered L-arginine to apoE-KO animals for 16 and 24 weeks. L-Arginine treatment did not affect lesion formation in apoE-KO animals fed a Western-type diet. CONCLUSIONS: Genetic deficiency of iNOS decreases diet-induced atherosclerosis and lowers plasma levels of lipoperoxides, a marker for oxidative stress, in apoE-KO animals. Reduction in iNOS-mediated oxidative stress could partly explain protection from lesion formation in dKO animals. L-Arginine supplementation did not change lesion area in apoE-KO mice, indicating that substrate deficiency is not a likely cause for iNOS-mediated injury in this model of atherosclerosis.

Animals↗

PubMed2001

Simvastatin exerts both anti-inflammatory and cardioprotective effects in apolipoprotein E-deficient mice.

BACKGROUND: Simvastatin attenuates ischemia and reperfusion in normocholesterolemic animals by stabilizing endothelial nitric oxide synthase activity and inhibiting neutrophil-mediated injury. Because endothelial dysfunction is a detrimental effect of hypercholesterolemia, we examined whether short-term treatment with simvastatin could inhibit leukocyte-endothelium interaction and attenuate myocardial ischemia-reperfusion injury in apoE-deficient (apoE(-/-)) mice fed a high-cholesterol diet. METHODS AND RESULTS: We studied leukocyte-endothelium interactions in apoE(-/-) mice fed a normal or a high-cholesterol diet after short-term (ie, 18 hours) simvastatin treatment. We also studied simvastatin treatment in myocardial ischemia-reperfusion injury by subjecting apoE(-/-) mice to 30 minutes of ischemia and 24 hours of reperfusion. ApoE(-/-) mice fed a high-cholesterol diet exhibited higher blood cholesterol levels, which were not affected by short-term simvastatin treatment. However, the increased leukocyte rolling and adherence that occurred in cholesterol-fed apoE(-/-) mice (P<0.001 versus control diet) were significantly attenuated by simvastatin treatment (P<0.01 versus vehicle). Cholesterol-fed apoE(-/-) mice subjected to myocardial ischemia-reperfusion also experienced increased myocardial necrosis (P<0.01 versus control diet), which was significantly attenuated by simvastatin (P<0.01 versus vehicle). Simvastatin therapy also significantly increased vascular nitric oxide production in apoE(-/-) mice. CONCLUSIONS: Simvastatin attenuates leukocyte-endothelial cell interactions and ameliorates ischemic injury in hypercholesterolemic mice independently of lipid-lowering actions.

Animals↗

PubMed2001

Predominant role of endothelial nitric oxide synthase in vascular endothelial growth factor-induced angiogenesis and vascular permeability.

Nitric oxide (NO) plays a critical role in vascular endothelial growth factor (VEGF)-induced angiogenesis and vascular hyperpermeability. However, the relative contribution of different NO synthase (NOS) isoforms to these processes is not known. Here, we evaluated the relative contributions of endothelial and inducible NOS (eNOS and iNOS, respectively) to angiogenesis and permeability of VEGF-induced angiogenic vessels. The contribution of eNOS was assessed by using an eNOS-deficient mouse, and iNOS contribution was assessed by using a selective inhibitor [l-N(6)-(1-iminoethyl) lysine, l-NIL] and an iNOS-deficient mouse. Angiogenesis was induced by VEGF in type I collagen gels placed in the mouse cranial window. Angiogenesis, vessel diameter, blood flow rate, and vascular permeability were proportional to NO levels measured with microelectrodes: Wild-type (WT) > or = WT with l-NIL or iNOS(-/-) > eNOS(-/-) > or = eNOS(-/-) with l-NIL. The role of NOS in VEGF-induced acute vascular permeability increase in quiescent vessels also was determined by using eNOS- and iNOS-deficient mice. VEGF superfusion significantly increased permeability in both WT and iNOS(-/-) mice but not in eNOS(-/-) mice. These findings suggest that eNOS plays a predominant role in VEGF-induced angiogenesis and vascular permeability. Thus, selective modulation of eNOS activity is a promising strategy for altering angiogenesis and vascular permeability in vivo.

Animals↗

PubMed2001

Nitric oxide contributes to vascular smooth muscle relaxation in contracting fast-twitch muscles.

During skeletal muscle contraction, NO derived from neuronal nitric oxide synthase (nNOS) in skeletal muscle fibers or from endothelial cells (eNOS) may relax vascular smooth muscle contributing to functional hyperemia. To examine the relative importance of these pathways, smooth muscle myosin regulatory light chain (smRLC) phosphorylation was assessed as an index of vascular tone in isolated extensor digitorum longus (EDL) muscles from C57, nNOS(-/-), and eNOS(-/-) mice. The smRLC phosphorylation (in mol phosphate per mol smRLC) in C57 resting muscles (0.12 +/- 0.04) was increased 3.7-fold (0.44 +/- 0.03) by phenylephrine (PE). Reversal of this increase with electrical stimulation (to 0.19 +/- 0.03; P < 0.05) was partially blocked by N(omega)-nitro-l-arginine (NLA). In nNOS(-/-) EDL, the PE-induced increase in smRLC phosphorylation (0.10 +/- 0.02 to 0.49 +/- 0.04) was partially decreased by stimulation (0.25 +/- 0.04). In eNOS(-/-) EDL, the control value for smRLC was increased (0.24 +/- 0.04), and PE-induced smRLC phosphorylation (0.36 +/- 0.06) was decreased by stimulation even in the presence of NLA (to 0.20 +/- 0.02; P < 0.05). These results suggest that in addition to NO-independent mechanisms, NO derived from both nNOS and eNOS plays a role in the integrative vascular response of contracting skeletal muscle.

Animals↗

PubMed2001

Effect of isoflurane versus nicardipine on blood flow of lumbar paraspinal muscles during controlled hypotension for spinal surgery.

STUDY DESIGN: This study compared the effects of isoflurane and nicardipine on regional blood flow of the lumbar paraspinal muscles. OBJECTIVES: The purpose of this study was to determine whether treatment with hypotensive agents result in ischemia of the lumbar paraspinal muscles, thereby facilitating surgical procedures. SUMMARY OF BACKGROUND DATA: Despite the general acceptance of controlled hypotension as effective in reducing blood loss during spinal surgery, the changes of blood flow that occur at the lumbar paraspinal muscles when this technique is applied remain unclear. The use of laser Doppler flowmetry allows changes of muscle blood flow to be easily detected in real time with minimal invasion, thereby allowing differences among distinct pharmacological approaches for induction and maintenance of controlled hypotension to be evaluated. METHODS: The prehypotensive and hypotensive (reduction of mean arterial pressure by 20 mm Hg) blood flow of the lumbar paraspinal muscles were assessed with a laser Doppler flowmeter in 40 patients undergoing lumbar spinal surgery. The first half of the patients (n = 20) received isoflurane, whereas the second half received nicardipine to achieve arterial hypotension. RESULTS: Compared with the prehypotensive state, during the hypotensive state, patients in the isoflurane group exhibited a 17% to 46% (mean, 33.7%) decrease in lumbar paraspinal muscle blood flow, whereas patients in the nicardipine group exhibited a 24% to 177% (mean, 82.5%) increase in lumbar paraspinal muscle blood flow. Statistical analysis showed a significant difference in the changes of flux after induced hypotension between the isoflurane and nicardipine group (P < 0.001). CONCLUSIONS: Depending on the pharmacological treatment used to achieve arterial hypotension in spine surgery, there will be either a reduction in paraspinal muscle blood flow (ischemia) or an enhancement of this blood flow (hyperemia).

Adult↗

PubMed2001

Nitric oxide is involved in ischemia-induced apoptosis in brain: a study in neuronal nitric oxide synthase null mice.

Nitric oxide can promote or inhibit apoptosis depending on the cell type and coexisting metabolic or experimental conditions. We examined the impact of nitric oxide on development of apoptosis 6, 24, and 72 h after permanent middle cerebral artery occlusion in mutant mice that lack the ability to generate nitric oxide from neuronal nitric oxide synthase. Adjacent coronal sections passing through the anterior commissure were stained with hematoxylin and eosin or terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). Immunoblotting was used to identify changes in the anti- and proapoptotic proteins Bcl-2 and Bax, respectively. Activation of caspases was assessed by appearance of actin cleavage products using a novel antiserum directed against 32-kDa actin fragment (fractin). In the neuronal nitric oxide synthase mutant mouse, infarct size and TUNEL positive apoptotic neurons were reduced compared to the wild-type controls. At 6 h, Bcl-2 levels in the ischemic hemisphere were increased in mutants but decreased in the wild-type strain. Bax levels did not change significantly. Caspase-mediated actin cleavage appeared in the ischemic hemisphere at this time point, and was significantly less in mutant brains at 72 h compared to the wild-type. The reduction in the number of TUNEL and fractin positive apoptotic cells appears far greater than anticipated based on the smaller lesion size in mutant mice.Hence, from these data we suggest that a deficiency in neuronal nitric oxide production slows the development of apoptotic cell death after ischemic injury and is associated with preserved Bcl-2 levels and delayed activation of effector caspases.

Actins↗

PubMed2001

Neuronal nitric oxide synthase (NOS) regulates leukocyte-endothelial cell interactions in endothelial NOS deficient mice.

1. The present study was designed to examine the possible role of neuronal nitric oxide synthase (nNOS) in regulation of leukocyte - endothelial cell interactions in the absence of endothelial nitric oxide synthase (eNOS), using intravital microscopy of the cremasteric microcirculation of eNOS(-/-) mice. 2. Baseline leukocyte rolling and adhesion revealed no differences between wild-type and eNOS(-/-) mice in either the cremasteric or intestinal microcirculations. 3. Superfusion with L-NAME (100 microM) caused a progressive and significant increase in leukocyte adhesion in both wild-type and eNOS(-/-) mice, without detecting differences between the two strains of mice. 4. Superfusion with 7-nitroindazole (100 microM), a selective inhibitor of nNOS, had no effect on leukocyte adhesion in wild-type animals. However, it increased leukocyte adhesion significantly in eNOS(-/-) mice, which was reversed by systemic L-arginine pre-administration. 5. Stimulation of the microvasculature with H(2)O(2) (100 microM) induced a transient elevation in leukocyte rolling in wild-type mice. Conversely, the effect persisted during the entire 60 min of experimental protocol in eNOS(-/-) mice either with or without 7-nitroindazole. 6. Semi-quantitative analysis by RT - PCR of the mRNA for nNOS levels in eNOS(-/-) and wild-type animals, showed increased expression of nNOS in both brain and skeletal muscle of eNOS(-/-) mice. 7. In conclusion, we have demonstrated that leukocyte-endothelial cell interactions are predominantly modulated by eNOS isoform in postcapillary venules of normal mice, whereas nNOS appears to assume the same role in eNOS(-/-) mice. Interestingly, unlike eNOS there was insufficient NO produced by nNOS to overcome leukocyte recruitment elicited by oxidative stress, suggesting that nNOS cannot completely compensate for eNOS.

Animals↗

PubMed2001

Lack of neuronal NOS has consequences for the expression of POMC and POMC-derived peptides in the mouse pituitary.

The relevance of NO in neuroendocrine signalling has been investigated by analysis of cellular expression of pro-opiomelanocortin (POMC) and the POMC-derived peptides beta-endorphin, alpha-melanocyte stimulating hormone and adrenocorticotropin. Expression patterns were studied in the pituitary gland of 150-day old wild-type and neuronal-NOS (nNOS) knock-out mice by using immunohistochemistry, in situ hybridization and Northern blot analysis. Remaining NO-generating capacities in the knock-out mice were demonstrated by immunohistochemical localization of inducible, endothelial and neuronal NOS isoforms. Quantitative analysis revealed that cellular expression of POMC mRNA was drastically reduced in the pituitary of knock-out mice in comparison to controls. In situ hybridization studies demonstrated that this reduction was most pronounced in the intermediate lobe, while the anterior lobe was much less affected. Immunostaining for the proteolytic fragments of POMC was significantly reduced in the intermediate lobe cells of knock-out mice. A moderate reduction of immunostaining for these peptides was also observed in adenopituitary cells of nNOS knock-out mice. Our data demonstrate that the lack of nNOS substantially affects cellular levels of pituitary opioid peptides, which may have consequences for the response of these animals to stress and pain.

Adrenocorticotropic Hormone↗

PubMed2001

Lack of endothelial nitric oxide synthase aggravates murine pneumococcal meningitis.

Nitric oxide (NO) plays a central role in the pathogenesis of bacterial meningitis. However, the role of NO produced by endothelial NO synthase (eNOS) in meningitis is still unclear. We investigated the influence of eNOS depletion on the inflammatory host response, intracranial complications, and outcome in experimental pneumococcal meningitis. Leukocyte accumulation in the cerebrospinal fluid was more pronounced in infected eNOS-deficient mice than in infected wild type mice. This effect could be attributed to an increased expression of P-selectin, macrophage inflammatory protein-2, keratinocyte-derived cytokine, and interleukin (IL)-1beta in the brain of infected eNOS-deficient mice. However, no differences in the cerebral expression of intercellular adhesion molecule-1, tumor necrosis factor-alpha, and IL-6 as well as of neuronal NOS and inducible NOS could be detected between infected wild type and mutant mice. In addition to enhanced leukocyte infiltration into the CSF, meningitis-associated intracranial complications including blood-brain barrier disruption and the rise in intracranial pressure were significantly augmented in infected eNOS-deficient mice. The aggravation of intracranial complications was paralleled by a worsening of the disease, as evidenced by a more pronounced hypothermia, an enhanced weight reduction, and an increased death rate. The current data indicate that eNOS deficiency is detrimental in bacterial meningitis. This effect seems to be related to an increased expression of (certain) cytokines/chemokines and adhesion molecules; thus leading to increased meningeal inflammation and, subsequently, to aggravated intracranial complications.

Animals↗

PubMed2001

Regulation of murine airway responsiveness by endothelial nitric oxide synthase.

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.

Airway Resistance↗

PubMed2001

Mevastatin, an HMG-CoA reductase inhibitor, reduces stroke damage and upregulates endothelial nitric oxide synthase in mice.

BACKGROUND AND PURPOSE: The 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) lower serum cholesterol and decrease the incidence of stroke and cardiovascular disease. There is growing evidence that statins exert some of their beneficial effects independent of cholesterol lowering. Indeed, we have previously demonstrated that chronic simvastatin administration upregulates endothelial nitric oxide synthase (eNOS), resulting in more functional protein, augmentation of cerebral blood flow, and neuroprotection in a murine model of cerebral ischemia. In this report we examined whether another member of the statin family shared these effects and whether eNOS upregulation is sustained with longer treatment. METHODS: Mevastatin (2 mg/kg or 20 mg/kg per day) was administered to 18- to 22-g male mice for 7, 14, or 28 days before 2-hour middle cerebral artery occlusion with the use of the filament model (n=9 to 12). Neurological deficits and cerebral infarct volumes were assessed at 24 hours. Arterial blood pressure and gases, relative cerebral blood flow, and blood cholesterol levels were monitored in a subset of animals (n=5). Absolute cerebral blood flow was measured by the [(14)C]iodoamphetamine indicator fractionation technique (n=6). eNOS mRNA and protein levels were determined. RESULTS: Mevastatin increased levels of eNOS mRNA and protein, reduced infarct size, and improved neurological deficits in a dose- and time-dependent manner. Greatest protection was seen with 14- and 28-day high-dose treatment (26% and 37% infarct reduction, respectively). Cholesterol levels were reduced only after 28 days of treatment and did not correlate with infarct reduction. Baseline absolute cerebral blood flow was 30% higher after 14-day high-dose treatment. CONCLUSIONS: Chronic prophylactic treatment with mevastatin upregulated eNOS and augmented cerebral blood flow. These changes occurred in the absence of changes in serum cholesterol levels, were sustained for up to 1 month of treatment, and resulted in neuroprotection after middle cerebral artery occlusion.

Animals↗

PubMed2001

Lipopolysaccharide-induced diaphragmatic contractile dysfunction and sarcolemmal injury in mice lacking the neuronal nitric oxide synthase.

In this study we evaluated the role of the neuronal nitric oxide synthase (nNOS) in lipopolysaccharide (LPS)-induced diaphragmatic contractile dysfunction and sarcolemmal injury. Wild-type (WT) mice or mice deficient in the nNOS gene (nNOS(-/-)) were injected with either saline (control) or Escherichia coli LPS (LPS groups) and sacrificed 12 h later. The diaphragm was then examined for NOS expression, NOS activity, and in-vitro contractility. We also assessed sarcolemmal injury in isolated muscle strips under resting condition and after 3 min of artificial stimulations. In WT mice, LPS injection reduced maximum force to about 75% of that of control animals and raised total NOS activity significantly due to the induction of the iNOS isoform. Although muscle fiber injury was minimal under resting condition, the percentage of injured fibers in control and LPS-injected mice approached 27% and 40% of total fibers, respectively, in response to artificial stimulation. By comparison, LPS injection in nNOS(-/-) mice elicited a worsening of muscle contractility (maximum force < 60% of control animals) but elicited degrees of sarcolemmal injury similar to those observed in the WT animals. In addition, muscle NOS activity and iNOS protein level in nNOS(-/-) mice injected with LPS reached about 10% and 60% of that of WT animals, respectively (p < 0.05 compared with WT animals). Protein level of endothelial NOS isoform in the diaphragm was not altered by LPS injection in either WT or nNOS(-/-) animals. We conclude that nNOS plays a protective role in attenuating the negative influence of sepsis on diaphragmatic contractility but is not involved in the pathogenesis of sepsis-induced sarcolemmal injury.

Animals↗