Nitric oxide: a physiologic mediator of penile erection.

Nitric oxide (NO) is a cytotoxic agent of macrophages, a messenger molecule of neurons, and a vasodilator produced by endothelial cells. NO synthase, the synthetic enzyme for NO, was localized to rat penile neurons innervating the corpora cavernosa and to neuronal plexuses in the adventitial layer of penile arteries. Small doses of NO synthase inhibitors abolished electrophysiologically induced penile erections. These results establish NO as a physiologic mediator of erectile function.

Nitric oxide inhibition of coxsackievirus replication in vitro.

Nitric oxide is a radical molecule with antibacterial, -parasitic, and -viral properties. We investigated the mechanism of NO inhibition of Coxsackievirus B3 (CVB3) replication in vitro by determining the effect of NO upon a single replicative cycle of CVB3 grown in HeLa cells. Transfection of inducible NO synthase cDNA into HeLa cells reduces the number of viral particles produced during a single cycle of growth. Similarly, a noncytotoxic concentration of the NO donor S-nitroso-amino-penicillamine reduces the number of viral particles in a dose-dependent manner. To explore the mechanisms by which NO exerts its antiviral effect, we assayed the attachment, replication, and translation steps of the CVB3 life cycle. NO does not affect the attachment of CVB3 to HeLa cells. However, NO inhibits CVB3 RNA synthesis, as shown by a [3H]uridine incorporation assay, reverse transcription-PCR, and Northern analysis. In addition, NO inhibits CVB3 protein synthesis, as shown by [35S]methionine protein labeling and Western blot analysis of infected cells. Thus, NO inhibits CVB3 replication in part by inhibiting viral RNA synthesis by an unknown mechanism.

Nitric oxide inhibits viral replication in murine myocarditis.

Nitric oxide (NO) is a radical molecule that not only serves as a vasodilator and neurotransmitter but also acts as a cytotoxic effector molecule of the immune system. The inducible enzyme making NO, inducible NO synthase (iNOS), is transcriptionally activated by IFN-gamma and TNF-alpha, cytokines which are produced during viral infection. We show that iNOS is induced in mice infected with the Coxsackie B3 virus. Macrophages expressing iNOS are identified in the hearts and spleens of infected animals with an antibody raised against iNOS. Infected mice have increased titers of virus and a higher mortality when fed NOS inhibitors. Thus, viral infection induces iNOS in vivo, and NO inhibits viral replication. NO is a novel, nonspecific immune defense against viruses in vivo.

Intersection of interferon and hypoxia signal transduction pathways in nitric oxide-induced tumor apoptosis.

Activated macrophages play a central role in antitumor immunity. However, the stimuli that activate macrophages to kill tumor cells are not completely understood. Because the center of solid tumors can be hypoxic, we hypothesized that hypoxia may be an important signal in activating macrophages to kill tumor cells. Hypoxia stimulates IFN-primed macrophages to express the inducible nitric oxide synthase (NOS2) and to synthesize nitric oxide (NO). We show that this synergy between IFN and hypoxia is mediated by the direct interaction of the hypoxia inducible factor-1 (HIF-1) and IFN regulatory factor-1 (IRF-1), which are both required for the hypoxic transcription of NOS2. This interaction between HIF-1 and IRF-1 may explain the mechanism by which macrophages infiltrating into tumors are activated to express NOS2 and to produce NO, a mediator of tumor apoptosis.

Inducible nitric oxide synthase inhibition of weibel-palade body release in cardiac transplant rejection.

BACKGROUND: Inducible nitric oxide synthase (iNOS, or NOS2) reduces the severity of accelerated graft arteriosclerosis (AGA) in transplanted organs, although the precise mechanism is unclear. METHODS AND RESULTS: We transplanted wild-type murine hearts into either wild-type or NOS2-null recipient mice; we then measured cardiac allograft survival and analyzed tissue sections by immunohistochemistry. We have confirmed that NOS2 increases cardiac allograft survival. We now show that there is less inflammation of cardiac allografts in wild-type hosts than in NOS2-null hosts. Furthermore, staining for von Willebrand factor reveals that the presence of NOS2 is correlated with the presence of Weibel-Palade bodies inside endothelial cells, whereas the absence of NOS2 is correlated with the release of Weibel-Palade bodies. CONCLUSIONS: Weibel-Palade bodies contain mediators that promote thrombosis and inflammation. Therefore, nitric oxide (NO) may stabilize the vessel wall and prevent endothelial activation in part by inhibiting the release of the contents of Weibel-Palade bodies. Prevention of Weibel-Palade body release might be a mechanism by which NO protects the vessel wall from inflammatory disorders such as atherosclerosis or graft arteriosclerosis.

Interaction of interferon regulatory factor-1 and nuclear factor kappaB during activation of inducible nitric oxide synthase transcription.

We investigated the molecular mechanism for the synergistic induction of inducible nitric oxide synthase transcription by TNF-alpha and IFN-gamma. Since TNF-alpha and IFN-gamma stimulate cells in part by activating NF-kappaB and IRF-1, we hypothesized that these two transcription factors interact with each other. IRF-1 and NF-kappaB co-localize in the nucleus of stimulated macrophages. Co-immunoprecipitation experiments show that IRF-1 and NF-kappaB interact in stimulated but not resting cells. Super-shift experiments show that IRF-1 and NF-kappaB interact while binding to their respective DNA binding sites. These results demonstrate the existence of a physical interaction between IRF-1 and NF-kappaB proteins in vivo. We next suggested that this interaction between IRF-1 and NF-kappaB bends the DNA of the iNOS promoter region. Using a cyclization assay, we demonstrate that nuclear extracts from stimulated cells accelerate the rate of conversion of a linear to circular DNA, compared to extracts from resting cells. However, stimulated nuclear extracts cannot affect the rate of cyclization of a promoter with a mutant IRE or kappaB site. Furthermore, stimulated nuclear extracts depleted of IRF-1 and NF-kappaB cannot induce cyclization. We conclude that IRF-1 and NF-kappaB interact in vivo, and that this interaction physically bends the indicible nitric oxide synthase promoter DNA. This interaction may explain the mechanism by which IFN-gamma synergistically augments inducible nitric oxide synthase transcription.

Post-transcriptional regulation of inducible nitric oxide synthase mRNA in murine macrophages by doxycycline and chemically modified tetracyclines.

Chemically modified tetracyclines [CMT-3 (IC50 approximately 6-13 microM = approximately 2.5-5 microg/ml) and CMT-8 (IC50 approximately 26 microM = 10 microg/ml), but not CMT-1, -2 or -5], which lack anti-microbial activity, inhibited nitrite production in LPS-stimulated macrophages. Unlike competitive inhibitors of L-arginine which inhibited the specific activity of inducible nitric oxide synthase (iNOS) in cell-free extracts, CMTs exerted no such direct effect on the enzyme. CMTs could, however, be shown to inhibit both iNOS mRNA accumulation and protein expression in LPS-stimulated cells. Tetracyclines (doxycycline and CMT-3) unlike hydrocortisone had no significant effect on murine macrophages transfected with iNOS promoter (tagged to a luciferase reporter gene) in the presence of LPS. However, doxycycline and CMT-3 augmented iNOS mRNA degradation, in LPS-stimulated murine macrophages. These studies show a novel mechanism of action of tetracyclines which harbours properties to increase iNOS mRNA degradation and decrease iNOS protein expression and nitric oxide production in macrophages. This property of tetracyclines may have beneficial effects in the treatment of various diseases where excess nitric oxide has been implicated in the pathophysiology of these diseases.

Hydrogen peroxide regulation of bovine endothelin-converting enzyme-1.

Vascular injury leads to the production of reactive oxygen species (ROS), but the mechanisms by which ROS contribute to vascular pathology are not completely understood. We hypothesized that ROS increase endothelin converting enzyme (ECE-1) expression. We found that glucose oxidase (GO) increases ECE-1 mRNA, protein, and activity in bovine aortic endothelial cells. Catalase abolishes this effect. Glucose oxidase treatment of endothelial cells transactivates the ECE-1 promoter. The ECE-1 promoter element that mediates this response to GO is located between -444 and -216 bp. This region contains a STAT response element, and GO activates STAT-3 binding to this STAT response element. Our data suggest that STAT3 mediates hydrogen peroxide induction of ECE-1 expression.

Monosialoganglioside GM1 inhibits neurotoxicity after hypothermic circulatory arrest.

BACKGROUND: Prolonged hypothermic circulatory arrest (HCA) causes clinical neurologic injury. This injury involves neuronal apoptosis, or programmed cell death. We have previously demonstrated that HCA causes glutamate excitotoxicity, increased nitric oxide (NO) production, and NO-mediated apoptosis. We hypothesized that monosialoganglioside GM1 inhibits NO synthase. The purpose of this study was to determine whether GM1 inhibits NO production and neuronal apoptosis after HCA. METHODS: Fourteen dogs underwent intracerebral microdialysis to measure excitatory amino acids, glutamate, aspartate, and citrulline, an equal coproduct of NO. They underwent 2 hours of HCA at 18 degrees C and were sacrificed 8 hours after HCA. Group 1 (n = 6) was pretreated with GM1, 30 mg/kg intravenously every day for 3 days, as well as before and after HCA. Group 2 control dogs (n = 8) received vehicle only. Apoptosis was scored from 0 (normal) to 100 (severe injury). RESULTS: Excitatory amino acids, aspartate and glutamate, coagonist glycine, and citrulline levels increased significantly over baseline during HCA and after HCA. GM1 pretreatment did not appreciably alter levels of glutamate, aspartate, and glycine; however, it substantially decreased citrulline and therefore NO production throughout the experiment. GM1 significantly inhibited apoptosis (group 1 vs group 2: 15.56 +/- 13.60 vs 62.92 +/- 6.17; P < .001). CONCLUSIONS: Our results provide the first direct evidence that GM1 inhibits NO synthase to reduce NO production and HCA-induced neuronal apoptosis. GM1 did not affect excitatory glutamate or aspartate levels. GM1 has been used in clinical trials of spinal cord injury and may be efficacious in reducing neurologic injury after HCA.

Induction of neuronal nitric oxide after hypothermic circulatory arrest.

BACKGROUND: Although hypothermic circulatory arrest (HCA) has become routine practice in cardiac surgery, it is associated with substantial neurotoxicity. We tested the hypothesis that increased nitric oxide production during HCA participates in neuronal death. We previously described a canine survival model of HCA that produces a consistent neurologic deficit and histopathologic pattern of selective neuronal death. METHODS: Adult male hound dogs (n = 17) were subjected to 2 hours of HCA at a brain temperature of 18 degrees C and reperfused to normothermia; they were sacrificed at various intervals up to 74 hours. Using in vivo cerebral microdialysis, dogs (n = 5) were given a simultaneous infusion of artificial cerebrospinal fluid containing L-[14C]arginine or L-[14C]arginine and L-nitroarginine methyl ester (a nitric oxide synthase inhibitor) in contralateral hemispheres while undergoing 2 hours of HCA and reperfusion to normothermia. RESULTS: L-[14C]citrulline recovery, a coproduct of nitric oxide, significantly increased during HCA in the hemisphere without the inhibitor (at 300 minutes: control, 236 +/- 94 fmol/min versus L-nitroarginine methyl ester, 6 +/- 6 fmol/min; p < 0.05). Citrulline production in vitro from canine cortical homogenates in the presence of calcium (n = 12) was significantly greater 8 and 20 hours after reperfusion (5.11 +/- 0.54 x 10(-7) mmol.mg-1.min-1 and 7.52 +/- 0.59 x 10(-7) mmol.mg-1.min-1, respectively) than before HCA (1.51 +/- 0.09 x 10(-7) mmol.mg-1.min-1; p < 0.05). Nitric oxide metabolites in the serum were also increased significantly early after reperfusion (baseline, 6.72 +/- 0.95 mmol/L; at 4 hours, 17.58 +/- 1.46 mmol/L; p < 0.05). Immunocytochemical staining of the cortex with neuronal nitric oxide synthase-specific monoclonal antibodies (Transduction Labs) revealed increased neuronal nitric oxide synthase expression 6 to 18 hours after HCA. Darkfield analysis demonstrated neuronal nitric oxide synthase localization to neuronal processes with widespread formation of dense plexi of nitric oxide synthase fibers. CONCLUSIONS: We conclude that neurotoxicity after HCA involves a significant, early induction in neuronal nitric oxide synthase expression in neuronal processes leading to widespread augmented nitric oxide production in the brain.

Nitric oxide mediates neurologic injury after hypothermic circulatory arrest.

BACKGROUND: Prolonged hypothermic circulatory arrest (HCA) causes neurologic injury. However, the mechanism of this injury is unknown. We hypothesized that HCA causes nitric oxide production to result in neuronal necrosis. This study was undertaken to determine whether the neuronal nitric oxide synthase inhibitor 17477AR reduces necrosis after HCA. METHODS: Thirty-two dogs underwent 2 hours of HCA at 18 degrees C. Nitric oxide synthase catalytic assay and intracerebral microdialysis for nitric oxide production were performed in acute nonsurvival experiments (n = 16). Sixteen animals survived for 72 hours after HCA: Group 1 (n = 9) was treated with 17477AR (Astra Arcus), and group 2 (n = 7) received vehicle only. Animals were scored from 0 (normal) to 500 (coma) for neurologic function and from 0 (normal) to 100 (severe) for neuronal necrosis. RESULTS: Administration of 17477AR reduced nitric oxide production in the striatum by 94% (HCA alone), 3.65+/-2.42 micromol/L; HCA and 17477AR, 0.20+/-0.14 micromol/L citrulline). Dogs treated with 17477AR after HCA had superior neurologic function (62.22+/-29.82 for group 1 versus 141.86+/-61.53 for group 2, p = 0.019) and significantly reduced neuronal necrosis (9.33+/-4.67 for group 1 versus 38.14+/-2.23 for group 2, p<0.00001) compared with untreated HCA dogs. CONCLUSIONS: Our results provide evidence that neuronal nitric oxide synthase mediates neuronal necrosis after HCA and plays a significant role in HCA-induced neurotoxicity. Pharmacologic strategies to inhibit neuronal nitric oxide synthase after the ischemic period of HCA may be clinically beneficial.

Neuronal nitric oxide synthase inhibition reduces neuronal apoptosis after hypothermic circulatory arrest.

BACKGROUND: Neurologic injury, including choreoathetosis and learning and memory deficits, occurs after prolonged hypothermic circulatory arrest (HCA). Apoptosis, or programmed cell death, is a possible cause of the neurologic injury seen after HCA. However, the mechanism of apoptosis is unknown. Hypothermic circulatory arrest causes glutamate excitotoxicity, resulting in increased nitric oxide production. We therefore hypothesized that nitric oxide mediates apoptosis. The purpose of this study was to determine if neuronal nitric oxide synthase inhibition reduces neuronal apoptosis in an established canine model of HCA. METHODS: Fourteen male hound dogs (weight, 20 to 27 kg) were placed on closed-chest cardiopulmonary bypass, subjected to 2 hours of HCA at 18 degrees C, rewarmed to normothermia, and sacrificed 8 hours after HCA. Group 1 (n = 7) dogs were treated with the neuronal nitric oxide inhibitor 7-nitroindazole, 25 mg/kg intraperitoneally, before arrest and every 2 hours until sacrifice. Group 2 (n = 7) dogs received vehicle only. The brains were analyzed histopathologically. Apoptosis, identified by hematoxylin-eosin staining, was confirmed by DNA terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end-labeling assay and electron microscopy. Apoptosis was scored by a blinded neuropathologist from 0 (normal) to 100 (severe injury). RESULTS: Apoptosis occurred early after HCA in select neuronal populations, including the hippocampus, stria terminalis, neocortex, and entorhinal cortex. Apoptotic neurons showed a characteristic shrunken cytoplasm and nuclear chromatin condensation. 7-Nitroindazole significantly inhibited apoptosis (group 1 versus 2: 19.17 +/- 14.39 versus 61.11 +/- 5.41; p < .001). CONCLUSIONS: Our results provide evidence that apoptosis is associated with the neurologic injury that occurs after HCA and that nitric oxide mediates the apoptosis that occurs after HCA. Strategies for cerebral protection during HCA may include the inhibition of neuronal nitric oxide synthase.

First place winner of the Conrad Jobst Award in the gold medal paper competition. Nitric oxide synthase inhibitors N-monomethylarginine and aminoguanidine prevent the progressive and severe hypotension associated with a rat model of pancreatitis.

The objective of this study was to determine whether the observed vascular collapse and other pathologic features of severe pancreatitis may be related to the induction of nitric oxide synthase (NOS). The rat model of pancreatitis reported by Schmidt et al. was employed. Rats in the experimental groups received pretreatment with known NOS inhibitors, N-Monomethylarginine (NMMA) or Aminoguanidine (AG). Controls included sham-operated rats without pancreatic insult and a diseased control group which received pretreatment with normal saline (NS). Arterial blood pressure was continuously recorded with a femoral arterial catheter connected to a transducer and monitor. Fluid resuscitation for hypotension followed a strict protocol with the administration of 5.0 cc NS for sustained decreases in systolic blood pressure (SBP) below 90 mm Hg at 5-minute intervals. Laboratory parameters and histopathology confirmed the induction of pancreatitis, with 6 to 15-fold increases in serum amylase levels and an average of approximately 20% decrease in serum ionized Ca++ levels. Immunohistochemical studies of the pancreas revealed that pancreatic insult resulted in the induction of NOS. Rats in the saline control group (n = 5) became hypotensive (SBP less than 90 mm Hg) between 3 and 4 hours post pancreatic insult and required an average of 110.0 cc (3-4 x blood volume) of NS fluid resuscitation. Rats which were not resuscitated (n = 5) did not survive.(ABSTRACT TRUNCATED AT 250 WORDS)

Nitric oxide: a physiologic messenger.

PURPOSE: To review the physiologic role of nitric oxide, an unusual messenger molecule that mediates blood vessel relaxation, neurotransmission, and pathogen suppression. DATA SOURCES: A MEDLINE search of articles published from 1987 to 1993 that addressed nitric oxide and the enzyme that synthesizes it, nitric oxide synthase. STUDY SELECTION: Animal and human studies were selected from 3044 articles to analyze the clinical importance of nitric oxide. Descriptions of the structure and function of nitric oxide synthase were selected to show how nitric oxide acts as a biological messenger molecule. DATA EXTRACTION: Biochemical and physiologic studies were analyzed if the same results were found by three or more independent observers. DATA SYNTHESIS: Two major classes of nitric oxide synthase enzymes produce nitric oxide. The constitutive isoforms found in endothelial cells and neurons release small amounts of nitric oxide for brief periods to signal adjacent cells, whereas the inducible isoform found in macrophages releases large amounts of nitric oxide continuously to eliminate bacteria and parasites. By diffusing into adjacent cells and binding to enzymes that contain iron, nitric oxide plays many important physiologic roles. It regulates blood pressure, transmits signals between neurons, and suppresses pathogens. Excess amounts, however, can damage host cells, causing neurotoxicity during strokes and causing the hypotension associated with sepsis. CONCLUSIONS: Nitric oxide is a simple molecule with many physiologic roles in the cardiovascular, neurologic, and immune systems. Although the general principles of nitric oxide synthesis are known, further research is necessary to determine what role it plays in causing disease.

Effect of azithromycin on murine arteriosclerosis exacerbated by Chlamydia pneumoniae.

Chlamydia pneumoniae infection can exacerbate atherosclerosis in animals. To test the hypothesis that antibiotic therapy inhibits the atherogenic effects of C. pneumoniae infection, 10-week-old apolipoprotein E (ApoE) null mice were infected with C. pneumoniae or placebo, were treated for 2 weeks after infection with azithromycin or placebo, and were killed at 20 weeks of age. Infection did not affect the size of the aortic lesion, and antibiotic treatment had no effect. Another group of mice, 12-week-old ApoE mice, were infected with C. pneumoniae or placebo, were treated for 2 weeks after infection with azithromycin or placebo, and were killed at 26 weeks of age. C. pneumoniae infection increased the size of the lesion in infected mice, but azithromycin did not reduce the size of the aortic lesion in infected mice. Therefore, immediate therapy of acute infection may be necessary to prevent the proatherogenic effects of C. pneumoniae infection.

Cloned and expressed macrophage nitric oxide synthase contrasts with the brain enzyme.

Nitric oxide (NO) is a messenger molecule of macrophages, endothelial cells in blood vessels, and neurons. A neuronal form of NO synthase (NOS) has been previously cloned. We now report the molecular cloning of macrophage NOS. The macrophage enzyme displays 50% sequence identity to the neuronal enzyme. Like neuronal NOS, macrophage NOS has recognition sites for FAD, FMN, and NADPH and also has a consensus calmodulin binding site. Macrophage NOS mRNA is strikingly inducible; it is absent in quiescent macrophages or spleen but is prominent 2-6 hr after endotoxin treatment.

Midkine induces tumor cell proliferation and binds to a high affinity signaling receptor associated with JAK tyrosine kinases.

The G401 cell line derived from a rhabdoid tumor of the kidney secretes the heparin-binding growth factors midkine and pleiotrophin. Both proteins act as mitogens for diverse cells, but only midkine serves as an autocrine mitogen for G401 tumor cells. We show that midkine specifically binds a protein or complex of molecular mass greater than 200 kDa with high affinity (Kd = 0.07 +/- 0.01 nM). Midkine, but not pleiotrophin, stimulates tyrosine phosphorylation of several cellular proteins with molecular mass of 100, 130, and 200+ kDa. Upon midkine binding, the midkine-receptor complex associates with the Janus tyrosine kinases, JAK1 and JAK2. MK stimulates tyrosine phosphorylation of JAK1, JAK2, and STAT1alpha. Our initial characterization of the midkine receptor suggests that midkine autocrine stimulation of tumor cell proliferation is mediated by a cell-surface receptor which in turn might activate the JAK/STAT pathway.