8-bromocyclic-gmp and sapropterin

Nitric oxide (NO) regulates the biological activity of many enzymes and other functional proteins as well as gene expression. In this study, we tested whether pretreatment with NO regulates NO production in response to cytokines in cultured rat hepatocytes. Hepatocytes were recovered in fresh medium for 24 h following pretreatment with the NO donor S-nitroso-N-acetyl-d,l-penicillamine (SNAP) and stimulated to express the inducible NO synthase (iNOS) with interleukin-1beta and interferon-gamma or transfected with the human iNOS gene. NO pretreatment resulted in a significant increase in NO production without changing iNOS expression for both conditions. This effect, which did not occur in macrophages and smooth muscle cells, was inhibited when NO was scavenged using red blood cells. Pretreatment with oxidized SNAP, 8-Br-cGMP, NO(2)(-), or NO(3)(-) did not increase the cytokine-induced NO production. SNAP pretreatment increased cytosolic iNOS activity measured only in the absence of exogenous tetrahydrobiopterin (BH(4)). SNAP pretreatment suppressed the level of GTP cyclohydrolase I (GTPCHI) feedback regulatory protein (GFRP) and increased GTPCHI activity without changing GTPCHI protein level. SNAP pretreatment also increased total cellular levels of biopterin and active iNOS dimer. These results suggest that SNAP pretreatment increased NO production from iNOS by elevating cellular BH(4) levels and promoting iNOS subunit dimerization through the suppression of GFRP levels and subsequent activation of GTPCHI.

Topics: Animals; Biopterins; Blotting, Northern; Blotting, Western; Cells, Cultured; Cyclic GMP; Cytokines; Dimerization; Dose-Response Relationship, Drug; Enzyme Activation; GTP Cyclohydrolase; Hepatocytes; Humans; Male; Muscle, Smooth; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitrites; Penicillamine; Protein Binding; Rats; Rats, Sprague-Dawley; Time Factors; Transfection

Substitution of the nitric oxide- (NO-) pathway improves early graft function following lung transplantation. We previously demonstrated that 8-Br-cGMP (second messenger of NO) to the flush solution and tetrahydrobiopterin (BH4, coenzyme of NO synthase) given as additive during reperfusion improve post-transplant graft function. In the present study, the combined treatment with 8-Br-cGMP and BH4 was evaluated.. Unilateral left lung transplantation was performed in weight matched outbred pigs (24-31 kg). In group I, grafts were preserved for 30 h (n=5). 8-Br-cGMP (1mg/kg) was added to the flush solution (Perfadex, 1.5l, 1 degrees C) and BH4 (10mg/kg/h) was given to the recipient for 5h after reperfusion. In group II, lungs were transplanted after a preservation time of 30 h (n=3) and prostaglandin E(1) (250 g) was given into the pulmonary artery (PA) prior to flush. In all recipients 1h after reperfusion the contralateral right PA and bronchus were ligated to assess graft function only. Survival time after reperfusion, extravascular lung water index (EVLWI), hemodynamic variables, and gas exchange (PaO(2)) were assessed during a 12h observation period.. All recipients in group I survived the 12h assessment, whereas none of the group II animals survived more than 4h after reperfusion with a rapid increase of EVLWI up to 24.8+/-6.7 ml/kg. In contrast, in group I EVLWI reached up to 8.9+/-1.5 ml/kg and returned to nearly normal levels at 12h (6.1+/-0.8 ml/kg). In two animals of group I the gas exchange deteriorated slightly. The other three animals showed normal arterial oxygenation over the entire observation time.. Our data indicate that the combined substitution of the NO pathway during preservation and reperfusion reduces ischemia/reperfusion injury substantially and that this treatment even allows lung transplantation after 30 h preservation in this model.

Topics: Animals; Biopterins; Cell Movement; Coenzymes; Cyclic GMP; Extravascular Lung Water; Graft Survival; Hemodynamics; Infusions, Intravenous; Lipid Peroxidation; Lung; Lung Transplantation; Neutrophils; Nitric Oxide Synthase; Organ Preservation; Organ Preservation Solutions; Peroxidase; Pulmonary Gas Exchange; Reperfusion Injury; Swine; Thiobarbituric Acid Reactive Substances; Time Factors

We assessed the role of .NO in recombinant adenovirus-mediated gene transfer both in vitro and in vivo. NIH3T3 fibroblasts, stably transfected with the human inducible nitric oxide synthase, but lacking tetrahydrobiopterin (NIH3T3/iNOS [inducibile nitric oxide synthase]), were infected with replication-deficient adenovirus (E1-deleted), containing either the luciferase or the Lac Z reporter genes (AdCMV-Luc and AdCMV-Lac Z; 1-10 plaque forming units [pfu]/cell). Incubation of infected cells with sepiapterin (50 microM), a precursor of tetrahydrobiopterin, progressively increased nitrate/nitrite levels in the medium and decreased both luciferase and beta-galactosidase protein expression to approximately 60% of their corresponding control values, 24 h later. NIH3T3/iNOS cells had normal ATP (adenosine 5'-triphosphate) levels and did not release LDH(lactic dehydrogenase) into the medium. Pretreatment of these cells with N(G)-monomethyl-L-arginine (L-NMMA; 1 mM), an inhibitor of iNOS, prevented the sepiapterin-mediated induction of .NO and restored gene transfer to baseline values. Incubation of NIH3T3/iNOS with 8-bromo-cGMP (400 microM) in the absence of sepiapterin, or exposure of AdCMV-Luc to large concentrations of .NO, did not alter the efficacy of gene transfer. .NO produced by NIH3T3/iNOS cells also suppressed beta-galactosidase expression in NIH3T3 cocultured cells stably transfected with beta-galactosidase gene, suggesting .NO inhibited gene expression at either the transriptional or posttranscriptional levels. To investigate the effects of inhaled .NO on gene transfer in vivo, CD1 mice received an intratracheal instillation of AdCMV-Luc (4 x 10(9) pfu in 80 microl of saline) and exposed to .NO (25 ppm in room air) for 72 h. At that time, no significant degree of lung inflammation was detected by histological examination. However, lung luciferase activity decreased by 53% as compared with air breathing controls (P < 0.05; n > or = 8). We concluded that overproduction of .NO decreases the efficiency of adenovirus-mediated gene transfer in lung cells in the absence of cytotoxicity or inflammation.

Topics: 3T3 Cells; Adenoviruses, Human; Animals; Antioxidants; beta-Galactosidase; Biopterins; Cell Survival; Cyclic GMP; Enzyme Inhibitors; Gene Transfer Techniques; Genes, Reporter; Genetic Vectors; Humans; Luciferases; Lung; Mice; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitrites; omega-N-Methylarginine; Pteridines; Pterins; Transfection