Overexpression of endothelial nitric oxide synthase in endothelial cells is protective against ischemia-reperfusion injury in mouse skeletal muscle

Microvascular injury has been proposed to be a main cause of ischemia-reperfusion (I/R) injury. The roles of endothelial nitric oxide synthase (eNOS)-derived NO, a key regulator of vascular function, in I/R injury are incompletely understood. We used transgenic mice overexpressing eNOS in endothelial cells (eNOS-Tg) and their littermates wild-type mice (WT) to investigate the roles of eNOS in I/R injury in skeletal muscle. Superoxide levels in the affected muscles were reduced by approximately 50% in eNOS-Tg compared with WT during reperfusion. In WT, the disassembly of endothelial junctional proteins seen in the early period of reperfusion was recovered in the later phase. These findings were correlated with the increased vascular permeability in vivo. In contrast, eNOS-Tg maintained the endothelial junction assembly as well as vascular permeability during reperfusion. Leukocyte extravasation into tissue and up-regulated expression of adhesion molecules in the reperfused vessels were significantly inhibited in eNOS-Tg. Tissue viability of the affected muscle was decreased in WT time-dependently after reperfusion, whereas eNOS-Tg showed no significant reduction. NOS inhibition completely reversed these protective effects of eNOS overexpression in I/R injury. Thus, eNOS overexpression appears to prevent the I/R injury in skeletal muscle by maintaining vascular integrity.     

Regulatory effects of iNOS on acute lung inflammatory responses in mice

The role of endogenous NO in the regulation of acute lung injury is not well defined. We investigated the effects of inducible nitric oxide synthase (iNOS) and endothelial NOS (eNOS) on the acute inflammatory response in mouse lungs. Acute lung injury was induced by intratracheal instillation of bacterial lipopolysaccharide (LPS) into wild-type (WT) mice and mice deficient in iNOS (iNOS(-/-)) or eNOS (eNOS(-/-)). Endpoints of inflammatory injury were myeloperoxidase (MPO) content and leak of albumin into lung. Inflammatory injury was similar in WT and eNOS(-/-) mice but was substantially increased in iNOS(-/-) mice. Bronchoalveolar lavage (BAL) fluids of iNOS(-/-) and WT mice showed similar levels of CXC chemokines (MIP-2, KC) but enhanced levels of CC chemokines (MCP-1, MCP-3). Increased lung content of MPO in iNOS(-/-) mice was reduced by anti-MCP-1 to values found in WT mice. In vitro stimulation of microvascular endothelial cells with LPS and IFN gamma revealed elevated production of CXC and CC chemokines in cells from iNOS(-/-) mice when compared to endothelial cells from iNOS(+/+) mice. Peritoneal macrophages from iNOS(-/-) donors also revealed increased production of CC chemokines after stimulation with LPS and interferon (IFN gamma). These data indicate that absence of iNOS causes enhanced lung inflammatory responses in mice which may be related to enhanced production of MCP-1 by endothelial cells and macrophages. It appears that iNOS affects the lung inflammatory response by regulating chemokine production.     

Endothelial NOS is required for SDF-1alpha/CXCR4-mediated peripheral endothelial adhesion of c-kit+ bone marrow stem cells

In the era of intravascular approaches for regenerative cell therapy, the underlying mechanisms of stem cell migration to non-marrow tissue have not been clarified. We hypothesized that next to a local inflammatory response implying adhesion molecule expression, endothelial nitric oxide synthase (eNOS)-dependent signaling is required for stromal- cell-derived factor-1 alpha (SDF-1alpha)-induced adhesion of c-kit+ cells to the vascular endothelium. SDF-1alpha/tumor necrosis factor-alpha (TNF-alpha)-induced c-kit+-cell shape change and migration capacity was studied in vitro using immunohistochemistry and Boyden chamber assays. In vivo interaction of c-kit+ cells from bone marrow with the endothelium in response to SDF-1alpha/TNF-alpha stimulation was visualized in the cremaster muscle microcirculation of wild-type (WT) and eNOS (-/-) mice using intravital fluorescence microscopy. In addition, NOS activity was inhibited with N-nitro-L-arginine-methylester-hydrochloride in WT mice. To reveal c-kit+-specific adhesion behavior, endogenous leukocytes (EL) and c-kit+ cells from peripheral blood served as control. Moreover, intercellular adhesion molecule-1 (ICAM-1) and CXCR4 were blocked systemically to determine their role in inflammation-related c-kit+-cell adhesion. In vitro, SDF-1alpha enhanced c-kit+-cell migration. In vivo, SDF-1alpha alone triggered endothelial rolling-not firm adherence-of c-kit+ cells in WT mice. While TNF-alpha alone had little effect on adhesion of c-kit+ cells, it induced maximum endothelial EL adherence. However, after combined treatment with SDF-1alpha+TNF-alpha, endothelial adhesion of c-kit+ cells increased independent of their origin, while EL adhesion was not further incremented. Systemic treatment with anti-ICAM-1 and anti-CXCR4-monoclonal antibody completely abolished endothelial c-kit+-cell adhesion. In N-nitro-L-arginine-methylester-hydrochloride-treated WT mice as well as in eNOS (-/-) mice, firm endothelial adhesion of c-kit+ cells was entirely abrogated, while EL adhesion was significantly increased. The chemokine SDF-1alpha mediates firm adhesion c-kit+ cells only in the presence of TNF-alpha stimulation via an ICAM-1- and CXCR4-dependent mechanism. The presence of eNOS appears to be a crucial and specific factor for firm c-kit+-cell adhesion to the vascular endothelium.     

Endothelial nitric oxide synthase mediates protective effects of hypoxic preconditioning in lungs

To elucidate the protective mechanism of whole-body hypoxic preconditioning (WHPC) on pulmonary ischemia-reperfusion injury focussing on nitric oxide synthases (NOS), mice were placed in a hypoxic chamber (FIO(2)=0.1) for 4h followed by 12h of normoxia. Then, pulmonary ischemia for 1h followed by 5h of reperfusion was performed by clamping the left hilum in vivo (I/R). WHPC protected WT mice from pulmonary leukocyte infiltration as assessed by myeloperoxidase (MPO) activity, associated with a mild further increase in endothelial permeability (Evans Blue extravasation). When all NOS isoforms were inhibited during WHPC by L-NAME, mortality and MPO activity after I/R markedly increased. To determine the responsible NOS isoform, quantitative RT-PCR was performed for eNOS and iNOS mRNA, showing that only eNOS was upregulated in response to WHPC. While eNOS total protein expression remained unchanged, the amount of phosphorylated eNOS also increased. The WHPC/IR experiments were then repeated with eNOS knockout mice. Here, we found that the protective effect of WHPC on pulmonary leukocyte sequestration was abrogated, and endothelial leakage was further exacerbated. We conclude that WHPC limits neutrophil sequestration via an eNOS-dependent mechanism, and that eNOS helps preserve endothelial permeability during hypoxia and I/R.     

Nitric oxide synthases modulate progenitor and resident endothelial cell behavior in galactosemia

We used knockout animals of either inducible nitric oxide synthase (iNOS(/)) or endothelial NOS (eNOS(/)) to characterize the role of NOS in galactosemia, a model of diabetic retinopathy. NADH oxidase and nitrotyrosine were used as biomarkers of oxidative stress and vascular dysfunction. These animals were engrafted with hematopoietic stem cells (HSC) expressing green fluorescence protein (gfp(+)) to characterize the contribution of HSC and endothelial progenitor cells to neovascularization. Increased NADH oxidase activity and superoxide generation occurred in all galactose-fed mice. eNOS(/) mice demonstrated increased iNOS immunoreactivity in their retinal vasculature. Nitrotyrosine levels were low at baseline in the wild-type (WT) mice, eNOS(/) and iNOS(/) mice, and the galactose-fed iNOS mice and increased following galactose feeding in eNOS(/) and WT. Galactose-fed WT.gfp and iNOS(/).gfp chimeric animals had areas of perfused new vessels composed of gfp(+) cells. In contrast, galactose-fed eNOS(/).gfp mice produced copious, unbranched, nonperfused tubes. Thus, nitric oxide modulates HSC behavior and vascular phenotype in the retina. Although there is increased NADH oxidase and superoxide in galactosemic mice of all isoforms, iNOS is the source of nitric oxide responsible for peroxynitrite and nitrotyrosine formation that leads to the pathology observed in galactosemic mice.     

腺苷对肺缺血再灌注大鼠NOS活性的影响

目的:观察腺苷(Adenosine)对大鼠肺缺血再灌注损伤(I/R)的保护作用及一氧化碳合酶(NOS)活性的影响。方法:将60只清洁级SD大鼠随机分为假手术组(Sham组,开胸但不阻断肺门)、I/R组(开胸后左肺门阻断60min+开放再灌注120min)、Adenosine预处理组,每组20只。A-denosine预处理组术前30min开始由股静脉持续静脉滴入Adenosine(0.1mg/kg/min)。实验结束前测定各组平均肺动脉压(mPAP)、动脉血氧分压(PaO2)、肺湿/干重比(W/D)及血清和肺组织匀浆中内皮型一氧化碳合酶(eNOS)、诱导型一氧化碳合酶(iNOS)活性。结果:I/R组mPAP、W/D均高于Sham组(P<0.01),PaO2低于Sham组(P<0.01)。与I/R组比较,Adenosine预处理组mPAP、W/D明显降低,PaO2显著增高。与Sham组比较,I/R组血清及肺组织匀浆中eNOS活性明显降低,iNOS活性显著增加(P<0.01);Adenosine预处理能明显升高I/R大鼠eNOS活性,降低iNOS活性(与I/R组比较,P<0.01)。结论:Adenosine可通过减轻再灌注后肺水肿、降低肺动脉压及改善肺组织氧合功能,从而有效实现对I/R肺组织的保护,其保护作用可能与NOS不同亚型的表达变化有关。     

Protective role of endothelial nitric oxide synthase

Nitric oxide is a versatile molecule, with its actions ranging from haemodynamic regulation to anti-proliferative effects on vascular smooth muscle cells. Nitric oxide is produced by the nitric oxide synthases, endothelial NOS (eNOS), neural NOS (nNOS), and inducible NOS (iNOS). Constitutively expressed eNOS produces low concentrations of NO, which is necessary for a good endothelial function and integrity. Endothelial derived NO is often seen as a protective agent in a variety of diseases.This review will focus on the potential protective role of eNOS. We will discuss recent data derived from studies in eNOS knockout mice and other experimental models. Furthermore, the role of eNOS in human diseases is described and possible therapeutic intervention strategies will be discussed.     

Arrest of B16 melanoma cells in the mouse pulmonary microcirculation induces endothelial nitric oxide synthase-dependent nitric oxide release that is cytotoxic to the tumor cells

Metastatic cancer cells seed the lung via blood vessels. Because endothelial cells generate nitric oxide (NO) in response to shear stress, we postulated that the arrest of cancer cells in the pulmonary microcirculation causes the release of NO in the lung. After intravenous injection of B16F1 melanoma cells, pulmonary NO increased sevenfold throughout 20 minutes and approached basal levels by 4 hours. NO induction was blocked by N(G)-nitro-L-arginine methyl ester (L-NAME) and was not observed in endothelial nitric oxide synthase (eNOS)-deficient mice. NO production, visualized ex vivo with the fluorescent NO probe diaminofluorescein diacetate, increased rapidly at the site of tumor cell arrest, and continued to increase throughout 20 minutes. Arrested tumor cells underwent apoptosis with apoptotic counts more than threefold over baseline at 8 and 48 hours. Neither the NO signals nor increased apoptosis were seen in eNOS knockout mice or mice pretreated with L-NAME. At 48 hours, 83% of the arrested cells had cleared from the lungs of wild-type mice but only approximately 55% of the cells cleared from eNOS-deficient or L-NAME pretreated mice. eNOS knockout and L-NAME-treated mice had twofold to fivefold more metastases than wild-type mice, measured by the number of surface nodules or by histomorphometry. We conclude that tumor cell arrest in the pulmonary microcirculation induces eNOS-dependent NO release by the endothelium adjacent to the arrested tumor cells and that NO is one factor that causes tumor cell apoptosis, clearance from the lung, and inhibition of metastasis.     

Cell-specific nitric oxide synthase-isoenzyme expression and regulation in response to endotoxin in intact rat lungs

Nitric oxide (NO) produced by NO synthase (NOS) serves as a ubiquitous mediator molecule involved in many physiologic lung functions, including regulation of vascular and bronchial tone, immunocompetence, and neuronal signaling. On the other hand, excessive and inappropriate NO synthesis in inflammation and sepsis has been implicated in vascular abnormalities and cell injury. At least three different NOS isoforms (neuronal/brain [bNOS], inducible [iNOS], and endothelial [eNOS]) have been described, which are all expressed in normal lung tissue. We investigated the cell-specific expression of bNOS, iNOS, and eNOS in perfused control rat lungs and lungs undergoing stimulation with endotoxin in the presence and absence of plasma constituents. Lung immunohistochemistry and quantitative evaluation of staining intensity showed endotoxin-induced increase in iNOS expression in particular in bronchial epithelial cells, cells of the bronchus-associated lymphoid tissue (BALT), alveolar macrophages, and vascular smooth muscle cells in a time- and dose-dependent fashion. In endothelial cells, which did not express iNOS at baseline, newly induced iNOS was found in response to endotoxin. In contrast, expression of eNOS was markedly suppressed under endotoxin challenge, particularly in bronchial epithelium, BALT, and alveolar macrophages but also in vascular smooth muscle cells and endothelial cells. eNOS expression in bronchial smooth muscle cells was not altered. In contrast to iNOS and eNOS, cellular expression of bNOS in epithelial cells, nerve fibers, BALT, and endothelial cells did not change in response to endotoxin. All changes in NOS regulation were found to be independent of plasma constituents. We conclude that endotoxin exerts a profound impact on the cell-specific NOS regulation in a large number of lung cell types. Prominent features include de novo synthesis or up-regulation of iNOS, in contrast to down-regulation of eNOS, which may well contribute to vascular abnormalities, inflammatory sequelae, and loss of physiologic functions in septic lung failure.     

Effect of adenovirus-mediated gene transfer of nitric oxide synthase on vascular reactivity of rat isolated pulmonary arteries

Aerosol gene transfer of endothelial nitric oxide synthase (eNOS) and inducible NOS (iNOS) to rat lungs increased NOS expression and activity, and prevented hypoxic pulmonary vasoconstriction (HPV) in vivo. Hereby, we examined the effect of eNOS and iNOS aerosol gene transfer on the endothelium-dependent relaxation (EDR) and on acute HPV in isolated rat pulmonary arteries. Changes in isometric forces were recorded in organ baths for large conduit arteries (diameter 1.8±0.1 mm) and in a wire myograph for small resistance arteries (258±35 μm). Male Wistar rats were randomly aerosolized with adenovirus (Ad) encoding β-galactosidase (control), eNOS, or iNOS. Four days later, exhaled nitric oxide was measured, NOS expression within rat lungs was evaluated by quantitative real-time polymerase chain reaction and immunohistochemistry, vasoconstricting agonist and acetylcholine concentration response curves were generated, and the time course of HPV was recorded. Human eNOS and murine iNOS were expressed within rat lung tissue mostly in parenchyma and endothelial cells. Large arteries isolated from Ad-i, eNOS-aerosolized rats developed lower agonist-induced tension than those of control rats. The first and second contractions of the HPV were smaller in the Ad-i, eNOS-aerosolized rats. Contractions were modestly, but significantly and inversely, related to exhaled NO. Agonist- and hypoxia-induced contractions were even more reduced after eNOS aerosolization. There was no significant effect on EDR and no notable difference between small and large vessels. We conclude that adenovirus (Ad)-mediated NOS gene transfer can counteract both pharmacologically and hypoxia-induced increases in pulmonary vascular tone in isolated rat pulmonary arteries. eNOS seems as efficient as iNOS in regulating pulmonary vascular tone.     

Inducible and endothelial nitric oxide synthase expression during development of transplant arteriosclerosis in rat aortic grafts.

In the vascular system, distinct isoforms of nitric oxide synthase (NOS) generate nitric oxide (NO), which acts as a biological messenger. Its role in the development of transplant arteriosclerosis (TA) is still unclear. To investigate whether NO is involved in TA, we studied the expression of NOS isoforms, inducible NOS (iNOS) and endothelial NOS (eNOS), by immunohistochemistry and in situ hybridization during the first two post-transplantation months and their relation with cold ischemia (1 to 24 hours) and reperfusion injury using an aortic transplantation model in the rat. We found an increased iNOS expression in the intima and adventitia and a decreased expression in the media, whereas eNOS expression was not significantly altered during the development of TA. Co-localization studies suggested that iNOS-positive cells were vascular smooth muscle cells, monocyte-derived macrophages, and endothelial cells. Prolonged ischemic storage time resulted in an increase in eNOS expression in the neointima. In situ hybridization showed iNOS mRNA expression by vascular cells in the neointima and media. NO produced by iNOS and eNOS may be involved, at least in part, in the pathogenesis of TA in aortic grafts. Additional studies are needed to confirm the modulatory mechanism of NO during the development of TA.     

Neuronal nitric oxide synthase is the dominant nitric oxide supplier for the survival of dorsal root ganglia after peripheral nerve axotomy

This study was designed to determine whether nitric oxide supply may be a major factor in the survival of dorsal root ganglia in a sciatic nerve injury model. Wild-type (WT) mice were compared with knockout (KO) mice lacking neuronal nitric oxide synthase (nNOS) or endothelial (eNOS). The NO-generating capacities were analysed by NOS immunohistochemistry and NADPH-diaphorase staining 1, 2, 6, and 12 weeks after nerve transection. The occurrence and morphological type of neuronal death were determined by TUNEL reaction and ultrastructural examination. Cell loss following nerve section, whist dependent on the availability of NO, as shown by its marked elevation in nNOS KO mice, did not correlate well with nNOS expression in WT animals. Whereas a lack of eNOS was tolerated, deficiency of nNOS led to an enhanced cell loss. The results suggest a crucial role of NO supply after transection of peripheral nerves with a particular significance of the nNOS isoform.     

Interplay of endothelial and inducible nitric oxide synthases modulates the vascular response to ischaemia-reperfusion in the rabbit lung

Aim: Lung ischaemia–reperfusion induces nitric oxide synthesis and reactive nitrogen species, decreasing nitric oxide bioavailability. We hypothesized that in the ventilated lung, this process begins during ischaemia and intensifies with reperfusion, contributing to ischaemia–reperfusion‐induced pulmonary vasoconstriction. The aim was to determine whether ischaemia–reperfusion alters inducible and endothelial nitric oxide synthase expression/activity, reactive nitrogen species generation, and nitric oxide bioavailability, potentially affecting pulmonary perfusion. Methods: Ischaemia–reperfusion was induced for various times in anesthetized rabbits with ventilated lungs by reversibly occluding the right pulmonary artery and initiating reperfusion. Nitric oxide synthase activity/expression and phosphorylation, reactive nitrogen species generation and total nitrate/nitrite were determined in lung tissue. Results: Inducible nitric oxide synthase expression and activity, and reactive nitrogen species formation coincided with increased pulmonary vascular resistance during reperfusion and increased with ischaemia duration, further increasing after 2‐h reperfusion. Total nitrate/nitrite also increased with ischaemia but decreased after 2‐h reperfusion. Pre‐treatment with an inducible nitric oxide synthase inhibitor (1400W; Cayman Chemical Company, Ann Arbor, MI, USA) attenuated inducible nitric oxide synthase activity, reactive nitrogen species generation and pulmonary vascular resistance, but did not affect total nitrate/nitrite. Endothelial nitric oxide synthase expression was unchanged by ischaemia–reperfusion; however, its phosphorylation on serine 1177 and dephosphorylation on threonine 495 was uncoupled, suggesting decreased endothelial nitric oxide synthase activity. 1400W prevented uncoupling of endothelial nitric oxide synthase phosphorylation, maintaining its activity during reperfusion. Conclusion: Ischaemia–reperfusion up‐regulates inducible nitric oxide synthesis and/activity, which coincides with reduced endothelial nitric oxide synthase activity as suggested by its uncoupling and may contribute to ischaemia–reperfusion‐induced pulmonary vasoconstriction.     

Hypothermic preservation of lung allograft inhibits cytokine-induced chemoattractant-1, endothelial leucocyte adhesion molecule, vascular cell adhesion molecule-1 and intracellular adhesion molecule-1 expression

Organ dysfunction is a major clinical problem after lung transplantation. Prolonged cold ischaemia and reperfusion injury are believed to play a central role in this complication. The influence of cold preservation on subsequent warm reperfusion was studied in an isolated, ventilated and perfused rat lung. Rat lungs were flushed with cold Perfadex-solution and stored at 4 degrees C for different time periods. Thereafter lungs were perfused and ventilated for up to 3 h. Physiological parameters, production of inflammatory mediators and leucocyte infiltration were measured before and after perfusion. Lungs subjected to a cold ischaemia time of up to 6 h showed stable physiological conditions when perfused for 3 h. However, cold-ischaemia time beyond 6 h resulted in profound tissue oedema, thereby impairing ventilation and perfusion. Warm reperfusion and ventilation per se induced a strong inflammatory response, as demonstrated by a significant up-regulation of chemokines and adhesion molecules (cytokine-induced chemoattractant-1, intracellular adhesion molecule and endothelial leucocyte adhesion molecule), accompanied by enhanced leucocyte infiltration. Although the up-regulation of inflammatory mediators was blunted in lungs that were subjected to cold ischaemia, this did not influence leucocyte infiltration. In fact, cold ischaemia time correlated with leucocyte sequestration. Although cold preservation inhibits the expression of inflammatory mediators it does not affect leucocyte sequestration during warm reperfusion. Cold preservation might cause impairment of the endothelial barrier function, as evidenced by tissue oedema and profound leucocyte infiltration.     

Renal expression of endothelial and inducible nitric oxide synthase, and formation of peroxynitrite-modified proteins and reactive oxygen species in Wegener's granulomatosis

To investigate the role of nitric oxide (NO) in glomerular inflammation, the expression of endothelial NO synthase (eNOS) and inducible NOS (iNOS) was studied in conjunction with inflammatory cell influx, H2O2 production, and the formation of nitrotyrosines in renal biopsies from patients with Wegener's granulomatosis (WG). Renal cryostat sections from patients with WG (n=15) were stained by immunohistochemistry for eNOS, iNOS, endothelial cells (CD31), nitrotyrosines, polymorphonuclear cells (PMNs, CD15), and monocytes/macrophages (CD14, CD68). Production of H2O2 was identified by enzyme cytochemistry using diaminobenzidine. In control tissues, strong staining for eNOS was found in glomerular and interstitial tubular capillaries and cortical vessels. A significant reduction in eNOS expression was found in WG biopsies, which was associated with a reduction in CD31 expression. Expression of iNOS was found in infiltrating inflammatory cells, mainly located in the interstitium. H2O2-producing cells were detected in glomeruli and were abundantly present in the interstitium. Nitrotyrosine-positive cells, however, were almost exclusively found in the interstitium. It is concluded that renal inflammation in WG is associated with the induction of iNOS in inflammatory cells and the formation of nitrotyrosines. Expression of eNOS in glomerular capillaries is lost, most likely due to endothelial cell damage. These results suggest that decreased NO production by endothelial cells, in conjunction with increased NO production by iNOS-positive inflammatory cells, is involved in renal tissue injury in WG.     

Differential roles of neuronal and endothelial nitric oxide synthases during carrageenan-induced inflammatory hyperalgesia

The present study investigated the role of neuronal nitric oxide synthase (nNOS) in carrageenan-induced inflammatory pain by combining genomic and pharmacological strategies. Intrathecal injection of the nNOS inhibitor 7-nitroindazole dose-dependently inhibited carrageenan-induced thermal hyperalgesia in both early and late phases in wild-type mice. However in nNOS knockout mice, carrageenan-induced thermal hyperalgesia remained intact in the early phase but was reduced in the late phase. Spinal Ca2+ -dependent nitric oxide synthase (NOS) activity in nNOS knockout mice was significantly lower than that in wild-type mice. Following carrageenan injection, although the spinal Ca2+ -dependent NOS activity in both wild-type and knockout mice increased, the enzyme activity in nNOS knockout mice reached a level similar to that in wild-type mice. On the other hand, no significant difference in spinal Ca2+ -independent NOS activity was noted between wild-type and nNOS knockout mice before and after carrageenan injection. Furthermore, intrathecal administration of the endothelial NOS (eNOS) inhibitor L-N5-(1-iminoethyl)-ornithinein nNOS knockout mice inhibited the thermal hyperalgesia in both early and late phases, though this inhibitor had no effect in wild-type mice. Meanwhile, Western blot showed that eNOS expression in the spinal cord of nNOS knockout mice was up-regulated compared with wild-type mice; immunohistochemical staining showed that the spinal eNOS was mainly distributed in superficial laminae of the dorsal horn. Finally, double staining with confocal analysis showed that the enhanced spinal eNOS was expressed in astrocytes, but not in neurons. Our current results indicate that nNOS plays different roles in the two phases of carrageenan-induced inflammatory pain. In this model, enhanced spinal eNOS appears to compensate for the role of nNOS in nNOS knockout mice.     

Carbon monoxide inhalation protects rat intestinal grafts from ischemia/reperfusion injury

Carbon monoxide (CO), a byproduct of heme catalysis by heme oxygenases, has been shown to exert anti-inflammatory effects. This study examines the cytoprotective efficacy of inhaled CO during intestinal cold ischemia/reperfusion injury associated with small intestinal transplantation. Orthotopic syngenic intestinal transplantation was performed in Lewis rats after 6 hours of cold preservation in University of Wisconsin solution. Three groups were examined: normal untreated controls, control intestinal transplant recipients kept in room air, and recipients exposed to CO (250 ppm) for 1 hour before and 24 hours after surgery. In air grafts, mRNA levels for interleukin-6, cyclooxygenase-2, intracellular adhesion molecule (ICAM-1), and inducible nitric oxide synthase rapidly increased after intestinal transplant. Histopathological analysis revealed severe mucosal erosion, villous congestion, and inflammatory infiltrates. CO effectively blocked an early up-regulation of these mediators, showed less severe histopathological changes, and resulted in significantly improved animal survival of 92% from 58% in air-treated controls. CO also significantly reduced mRNA for proapoptotic Bax, while it up-regulated anti-apoptotic Bcl-2. These changes in CO-treated grafts correlated with well-preserved CD31(+) vascular endothelial cells, less frequent apoptosis/necrosis in intestinal epithelial and capillary endothelial cells, and improved graft tissue blood circulation. Protective effects of CO in this study were mediated via soluble guanylyl cyclase, because 1H-(1,2,4)oxadiazole (4,3-alpha) quinoxaline-1-one (soluble guanylyl cyclase inhibitor) completely reversed the beneficial effect conferred by CO. Perioperative CO inhalation at a low concentration resulted in protection against ischemia/reperfusion injury to intestinal grafts with prolonged cold preservation.     

Overexpression of P104L mutant caveolin-3 in mice develops hypertrophic cardiomyopathy with enhanced contractility in association with increased endothelial nitric oxide synthase activity

The effect of endogenous nitric oxide synthase (NOS) on cardiac contractility and architecture has been a matter of debate. A role for NOS in cardiac hypertrophy has recently been demonstrated by studies which have shown hypertrophic cardiomyopathy (HCM) with altered contractility in constitutive NOS (cNOS) knockout mice. Caveolin-3, a strong inhibitor of all NOS isoforms, is expressed in sarcolemmal caveolae microdomains and binds to cNOS in vivo: endothelial nitric oxide synthase (eNOS) in cardiac myocytes and neuronal nitric oxide synthase (nNOS) in skeletal myocytes. The current study characterized the biochemical and cardiac parameters of P104L mutant caveolin-3 transgenic mice, a model of an autosomal dominant limb-girdle muscular dystrophy (LGMD1C). Transgenic mouse hearts demonstrated HCM, enhanced basal contractility, decreased left ventricular end diastolic diameter, and loss and cytoplasmic mislocalization of caveolin-3 protein. Surprisingly, cardiac muscle showed activation of eNOS catalytic activity without increased expression of all NOS isoforms. These data suggest that a moderate increase in eNOS activity associated with loss of caveolin-3 results in HCM.     

Ejaculatory abnormalities in mice lacking the gene for endothelial nitric oxide synthase (eNOS-/-).

Nitric oxide (NO) has been established as a neurotransmitter in both the central and peripheral nervous systems. Three isoforms of its synthetic enzyme, NO synthase (NOS), have been identified: 1) in the endothelial lining of blood vessels (eNOS), 2) an inducible form found in macrophages (iNOS), and 3) in neurons (nNOS). Previous studies using pharmacological agents that block all three isoforms of NOS have revealed that NO mediates several aspects of reproductive physiology and behavior, including anomalies in male sexual behavior and erectile function. To determine the specific contribution of the endothelial isoform of NOS in male reproductive behavior, we studied mice missing the gene for only eNOS (eNOS-/-). Wild-type (WT) and eNOS-/- animals were placed with an estrous WT female and observed for 45 min. Both WT and eNOS-/- mice displayed equivalent motivation to mount the stimulus female. However, eNOS-/- mice exhibited striking anomalies in ejaculatory function. A higher percentage of eNOS-/- than WT mice ejaculated during the testing period (p < 0.001). This increased propensity to ejaculate was apparently due to reduced stimulation required to elicit ejaculation; eNOS-/- mice required significantly fewer mounts (p < 0.003) and intromissions (p < 0.001) to ejaculate compared to WT mice. Taken together, these results suggest that NO synthesized by eNOS may be involved in ejaculatory physiology, but not sexual motivation.     

Localization of inducible nitric oxide synthase to mast cells during ischemia/reperfusion injury of skeletal muscle

Nitric oxide contributes to tissue necrosis after ischemia-reperfusion (IR). A biochemical and immunohistochemical study was made of the amounts and localization of both Ca++-independent nitric oxide synthase (NOS) II and Ca++-dependent (NOS I and NOS III) in rat skeletal muscle after ischemia and 0.5, 2, 8, 16, and 24 hours reperfusion. NOS II was not detectable in control muscle or during ischemia, was first detected after 2 hours reperfusion, increased further by 8 hours, and remained elevated at 24 hours. Both NOS II and nitrotyrosine, a marker of peroxynitrite formation, were localized exclusively to mast cells except after 24 hours reperfusion when some macrophages and neutrophils also showed positive immunoreactivity. Mast cells underwent extensive degranulation during reperfusion. NOS I was not detected in injured or control muscle. The level of NOS III, which was localized to the endothelium of blood vessels of all sizes in control muscle, decreased progressively during ischemia and reperfusion to reach undetectable levels after 16 hours reperfusion. These findings indicate that most of the nitric oxide formed during IR injury is generated by NOS II located almost exclusively in mast cells.