cyclic-gmp and Necrosis

Reperfusion injury may cause myocardial cell death and limit the benefit achieved by restoration of coronary artery patency in patients with acute myocardial infarction. The mechanism includes altered Ca(2+) handling with cytosolic and mitochondrial Ca(2+) overload, Ca(2+)- and ATP-dependent hypercontraction, cytoskeletal fragility, mitochondrial permeability transition and gap junction-mediated propagation of cell death, as well as alterations in non-cardiomyocyte cells, in particular platelets and endothelial cells. cGMP modulates favorably all these mechanism, mainly through PKG-mediated actions, but cGMP synthesis is altered in reperfused cardiomyocytes and endothelial cells by mechanisms that are only partially understood. Stimulation of cGMP synthesis during initial reperfusion by means of natriuretic peptides has been found protective in different animal models and in patients. Moreover, increasing evidence indicates that cGMP is an important step in signal transduction of endogenous cardioprotection. Thus, the cGMP pathway appears as a key element in the pathophysiology of myocardial ischaemia-reperfusion and as a promising therapeutic target in patients with acute myocardial infarction.

Topics: Angioplasty, Balloon, Coronary; Animals; Cardiovascular Agents; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Humans; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Necrosis; Second Messenger Systems; Time Factors

Topics: Animals; Autophagy; Cell Communication; Cell Nucleus; Cell Survival; Cyclic AMP; Cyclic GMP; Cytoplasm; Female; Hormones; Hydrolases; Insect Hormones; Macrophages; Male; Metamorphosis, Biological; Morphogenesis; Necrosis; Phagocytosis; Reproduction

The nitric oxide (NO) donor JS-K is specifically activated by glutathione S-transferases (GSTs) in GST-overexpressing cells. We have shown the induction of cell death in glioblastoma multiforme (GBM) cells at high JS-K doses but the mechanism remains unclear. The aim of this study was to determine whether NO-induced cell death is triggered by induction of apoptotic or necrotic pathways. For the first time, we demonstrate that NO induces cell death via mitotic catastrophe (MC) with non-apoptotic mechanisms in GBM cells. Moreover, the level of morphological changes indicating MC correlates with increased necrosis. Therefore, we conclude that MC is the main mechanism by which GBM cells undergo cell death after treatment with JS-K associated with necrosis rather than apoptosis. In addition, we show that PARP1 is not an exclusive marker for late apoptosis but is also involved in MC. Activating an alternative way of cell death can be useful for the multimodal cancer therapy of GBM known for its strong anti-apoptotic mechanisms and drug resistance.

Topics: Adenosine Triphosphate; Apoptosis; Azo Compounds; Blotting, Western; Caspases; Cell Line, Tumor; Cyclic GMP; Enzyme Activation; Flow Cytometry; Glioblastoma; Humans; In Situ Nick-End Labeling; Mitosis; Necrosis; Nitric Oxide; Piperazines; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-akt; Time Factors

Guanylyl cyclase-cyclic guanosine monophosphate signalling plays an important role in endogenous cardioprotective signalling. The aim was to assess the potential of direct pharmacological activation and stimulation of soluble guanylyl cyclase, targeting different redox states of the enzyme, to limit myocardial necrosis during early reperfusion.. Rat isolated hearts were subjected to reversible left coronary artery occlusion (ischaemia-reperfusion) and infarct size was assessed by the tetrazolium staining technique. Administration during early reperfusion of BAY 41-2272, an NO-independent, haem-dependent stimulator of soluble guanylyl cyclase targeting the reduced state, or BAY 60-2770, an NO-independent, haem-independent activator targeting the oxidized state, significantly limited infarct size. Inhibition of NO synthesis did not abrogate this protection, but exogenous perfusion of NO with BAY 41-2272 produced a synergistic effect. The haem site oxidiser, ODQ abrogated the protection afforded by BAY 41-2272 but potentiated the protection afforded by BAY 60-2770. Targeting both the reduced and oxidized forms of sGC together did not afford additive protection.. Targeting either reduced or oxidized forms of sGC during early reperfusion affords cardioprotection, providing support for the concept that direct sGC manipulation at reperfusion has therapeutic potential for the management of acute myocardial infarction.

Topics: Animals; Benzoates; Biphenyl Compounds; Cyclic GMP; Cytoprotection; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Activators; Guanylate Cyclase; Hydrocarbons, Fluorinated; Male; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Necrosis; Nitric Oxide; Oxidation-Reduction; Pyrazoles; Pyridines; Rats; Rats, Sprague-Dawley; Receptors, Cytoplasmic and Nuclear; Second Messenger Systems; Soluble Guanylyl Cyclase; Time Factors

The duration of cell death may allow deducing the underlying degenerative mechanism. To find out how long a photoreceptor takes to die, we used the rd1 mouse model for retinal neurodegeneration, which is characterized by phosphodiesterase-6 (PDE6) dysfunction and photoreceptor death triggered by high cGMP levels. Based on cellular data on the progression of cGMP accumulation, cell death, and survival, we created a mathematical model to simulate the temporal development of the degeneration and the clearance of dead cells. Both cellular data and modelling suggested that at the level of the individual cell, the degenerative process was rather slow, taking around 80 h to complete. Organotypic retinal explant cultures derived from wild-type animals and exposed to the selective PDE6 inhibitor zaprinast, confirmed the surprisingly long duration of an individual photoreceptor cell's death. We briefly discuss the possibility to link different cell death stages and their temporal progression to specific enzymatic activities known to be causally connected to cell death. This in turn opens up new perspectives for the treatment of inherited retinal degeneration, both in terms of therapeutic targets and temporal windows-of-opportunity.

Topics: Animals; Apoptosis; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Cyclic Nucleotide-Gated Cation Channels; Disease Models, Animal; Histone Deacetylases; Mice; Mice, Inbred C3H; Necrosis; Photoreceptor Cells, Vertebrate; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Retinal Degeneration

The nitroderivative 1-nitro-2-phenylethane (NPE) was recently described as a compound possessing heme-dependent soluble guanylyl cyclase (sGC) stimulating properties in vascular smooth muscle cells. In this study, we tested such pharmacological property of NPE in mice pancreatic acinar cells subjected to the bile salt taurocholate, a type of pathological stimulus that simulates pancreatitis. Here, isolated acinar cells were treated with NPE in order to assess the role of sGC on the detrimental effects induced by taurocholate. NPE reduced taurocholate-elicited Ca(2+) overload, production of reactive oxygen species (ROS), apoptosis, necrosis, and exerted a protective effect against mitochondrial membrane potential (ΔΨm) dissipation. These NPE-induced effects were abolished by pretreatment with ODQ and KT 5823, and after the blockade of nitric oxide (NO) synthase with l-NAME, inhibitors of key components of the sGC pathway. Contrarily to cGMP that alone increased ΔΨm collapse and cell damage, the cytoprotective effect of NPE on ΔΨm and cell necrosis was almost reproduced by 8-nitro-cGMP, a second messenger generated by sGC under oxidative stress conditions. In conclusion, putative sGC stimulation with NPE reveals its cytoprotective profile on pancreatic cells subjected to taurocholate. Moreover, ROS and NO conjunctly appear to drive sGC activity in pancreatic acinar cells to implement an adaptive mechanism in response to oxidative and Ca(2+) stress through 8-nitro-cGMPsynthesis.

Topics: Acinar Cells; Animals; Apoptosis; Benzene Derivatives; Calcium Signaling; Cells, Cultured; Cyclic GMP; Male; Mice; Necrosis; Pancreas; Reactive Oxygen Species; Taurocholic Acid

Cinaciguat (BAY 58-2667) is a novel nitric oxide (NO)-independent activator of soluble guanylate cyclase (sGC), which induces cGMP-generation and vasodilation in diseased vessels. We tested the hypothesis that cinaciguat might trigger protection against ischemia/reperfusion (I/R) in the heart and adult cardiomyocytes through cGMP/protein kinase G (PKG)-dependent generation of hydrogen sulfide (H(2)S). Adult New Zealand White rabbits were pretreated with 1 or 10 μg/kg cinaciguat (iv) or 10% DMSO (vehicle) 15 min before I/R or with 10 μg/kg cinaciguat (iv) at reperfusion. Additionally, adult male ICR mice were treated with either cinaciguat (10 μg/kg ip) or vehicle 30 min before I/R or at the onset of reperfusion (10 μg/kg iv). The PKG inhibitor KT5283 (KT; 1 mg/kg ip) or dl-propargylglycine (PAG; 50 mg/kg ip) the inhibitor of the H(2)S-producing enzyme cystathionine-γ-lyase (CSE) were given 10 and 30 min before cinaciguat. Cardiac function and infarct size were assessed by echocardiography and tetrazolium staining, respectively. Primary adult mouse cardiomyocytes were isolated and treated with cinaciguat before simulated ischemia/reoxygenation. Cinaciguat caused 63 and 41% reduction of infarct size when given before I/R and at reperfusion in rabbits, respectively. In mice, cinaciguat pretreatment caused a more robust 80% reduction in infarct size vs. 63% reduction when given at reperfusion and preserved cardiac function following I/R, which were blocked by KT and PAG. Cinaciguat also caused an increase in myocardial PKG activity and CSE expression. In cardiomyocytes, cinaciguat (50 nM) reduced necrosis and apoptosis and increased H(2)S levels, which was abrogated by KT. Cinaciguat is a novel molecule to induce H(2)S generation and a powerful protection against I/R injury in heart.

Topics: Animals; Apoptosis; Benzoates; Cell Survival; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Cystathionine gamma-Lyase; Disease Models, Animal; Enzyme Activation; Enzyme Activators; Enzyme Inhibitors; Guanylate Cyclase; Hydrogen Sulfide; Male; Mice; Mice, Inbred ICR; Myocardial Infarction; Myocardial Reperfusion Injury; Myocytes, Cardiac; Necrosis; Rabbits; Receptors, Cytoplasmic and Nuclear; RNA, Messenger; Soluble Guanylyl Cyclase; Ultrasonography; Up-Regulation; Ventricular Function, Left

Neovascularization is a physiologic repair process that partly depends on nitric oxide. Extracellular superoxide dismutase (EcSOD) is the major scavenger of superoxide. It is an important regulator of nitric oxide bioavailability and thus protects against vascular dysfunction. We hypothesized that overexpression of EcSOD in skeletal muscle would improve recovery from hind-limb ischemia.. Adeno-associated virus serotype 9 (AAV9) vectors expressing EcSOD or luciferase (control) from the cytomegalovirus promoter were cross-packaged into AAV9 capsids and injected intramuscularly into the hind-limb muscles (1 × 10(11) viral genomes/limb) of 12-week-old mice. Ischemia was induced after intramuscular injections. Laser Doppler was used to measure limb perfusion on days 0, 7, and 14 after injection. Values were expressed as a ratio relative to the nonischemic limb. EcSOD expression was measured by Western blotting. Capillary density was documented by immunohistochemical staining for platelet endothelial cell adhesion molecule. Apoptosis was assessed by terminal deoxynucleotide transferase-mediated biotin-deoxy uridine triphosphate nick-end labeling and necrosis was visually evaluated daily.. EcSOD expression was twofold upregulated in EcSOD treated vs control ischemic muscles at day 14. Capillary density (capillaries/fiber) was 1.9-fold higher in treated (1.65 ± 0.02) vs control muscle (0.78 ± 0.17, P < .05). Recovery of perfusion ratio at day 14 after ischemia was 1.5-fold greater in EcSOD vs control mice (P < .05). The percentage of apoptotic nuclei was 1.3% ± 0.4% in EcSOD-treated mice compared with 4.2% ± 0.2% in controls (P < .001). Limb necrosis was also significantly lower in EcSOD vs control mice.. AAV9-mediated overexpression of EcSOD in skeletal muscle significantly improves recovery from hind-limb ischemia in mice, consistent with improved capillary density and perfusion ratios in treated mice.

Topics: Animals; Apoptosis; Blotting, Western; Capillaries; Cyclic GMP; Dependovirus; Disease Models, Animal; Genetic Therapy; Genetic Vectors; Hindlimb; Immunohistochemistry; In Situ Nick-End Labeling; Injections, Intramuscular; Ischemia; Laser-Doppler Flowmetry; Luciferases, Firefly; Male; Mice; Mice, Inbred BALB C; Muscle, Skeletal; Necrosis; Neovascularization, Physiologic; Platelet Endothelial Cell Adhesion Molecule-1; Recombinant Fusion Proteins; Recovery of Function; Regional Blood Flow; Superoxide Dismutase; Time Factors

Nervous tissues express various communication molecules including natriuretic peptides, i.e. Brain Natriuretic Peptide (BNP) and C-type Natriuretic Peptide (CNP). These molecules share structural similarities with cyclic antibacterial peptides. CNP and to a lesser extent BNP can modify the cytotoxicity of the opportunistic pathogen Pseudomonas aeruginosa. The psychrotrophic environmental species Pseudomonas fluorescens also binds to and kills neurons and glial cells, cell types that both produce natriuretic peptides. In the present study, we investigated the sensitivity of Pseudomonas fluorescens to natriuretic peptides and evaluated the distribution and variability of putative natriuretic peptide-dependent sensor systems in the Pseudomonas genus.. Neither BNP nor CNP modified P. fluorescens MF37 growth or cultivability. However, pre-treatment of P. fluorescens MF37 with BNP or CNP provoked a decrease of the apoptotic effect of the bacterium on glial cells and an increase of its necrotic activity. By homology with eukaryotes, where natriuretic peptides act through receptors coupled to cyclases, we observed that cell-permeable stable analogues of cyclic AMP (dbcAMP) and cyclic GMP (8BcGMP) mimicked the effect of BNP and CNP on bacteria. Intra-bacterial concentrations of cAMP and cGMP were measured to study the involvement of bacterial cyclases in the regulation of P. fluorescens cytotoxicity by BNP or CNP. BNP provoked an increase (+49%) of the cAMP concentration in P. fluorescens, and CNP increased the intra-bacterial concentrations of cGMP (+136%). The effect of BNP and CNP on the virulence of P. fluorescens was independent of the potential of the bacteria to bind to glial cells. Conversely, LPS extracted from MF37 pre-treated with dbcAMP showed a higher necrotic activity than the LPS from untreated or 8BcGMP-pre-treated bacteria. Capillary electrophoresis analysis suggests that these different effects of the LPS may be due, at least in part, to variations in the structure of the macromolecule.. These observations support the hypothesis that P. fluorescens responds to natriuretic peptides through a putative sensor system coupled to a cyclase that could interfere with LPS synthesis and thereby modify the overall virulence of the micro-organism.

Topics: Animals; Apoptosis; Cells, Cultured; Cyclic AMP; Cyclic GMP; L-Lactate Dehydrogenase; Lipopolysaccharides; Natriuretic Peptide, Brain; Natriuretic Peptide, C-Type; Necrosis; Neuroglia; Nitric Oxide Synthase; Pseudomonas fluorescens; Rats

Decreased blood flow secondary to peripheral vascular disease underlies a significant number of chronic diseases that account for the majority of morbidity and mortality among the elderly. Blood vessel diameter and blood flow are limited by the matricellular protein thrombospondin-1 (TSP1) through its ability to block responses to the endogenous vasodilator nitric oxide (NO). In this study we investigate the role TSP1 plays in regulating blood flow in the presence of advanced age and atherosclerotic vascular disease.. Mice lacking TSP1 or CD47 show minimal loss of their resistance to ischemic injury with age and increased preservation of tissue perfusion immediately after injury. Treatment of WT and apolipoprotein E-null mice using therapeutic agents that decrease CD47 or enhance NO levels reverses the deleterious effects of age- and diet-induced vasculopathy and results in significantly increased tissue survival in models of ischemia.. With increasing age and diet-induced atherosclerotic vascular disease, TSP1 and its receptor CD47 become more limiting for blood flow and tissue survival after ischemic injury. Drugs that limit TSP1/CD47 regulation of blood flow could improve outcomes from surgical interventions in the elderly and ameliorate vascular complications attendant to aging.

Topics: Aging; Animals; Apolipoproteins E; Atherosclerosis; Blood Flow Velocity; CD47 Antigen; Cell Survival; Collateral Circulation; Cyclic GMP; Disease Models, Animal; Femoral Artery; Hindlimb; Ischemia; Ligation; Mice; Mice, Inbred C57BL; Mice, Knockout; Muscle, Skeletal; Necrosis; Nitric Oxide; Peripheral Vascular Diseases; Regional Blood Flow; Signal Transduction; Thrombospondin 1; Vasodilation

Preconditioning of livers with the atrial natriuretic peptide (ANP) markedly reduces hepatic ischemia-reperfusion injury. Aim of this study was to characterize the influence of ANP preconditioning on necrotic and apoptotic cell death and on proliferation.. Rat livers were perfused with Krebs-Henseleit buffer with or without ANP or its second messenger analogue 8-Bromo cyclic guanosine monophosphate (8-Br cGMP) for 20 min, stored in cold University of Wisconsin solution (24 h), and reperfused for up to 120 min. Apoptosis and necrosis were determined using biochemical and morphological criteria, proliferation was assessed by Ki67 histochemistry.. Apoptosis peaked after 24 h of cold ischemia. Preconditioning with both ANP and 8-Br-cGMP significantly reduced caspase-3-like activity and the number of triphosphate nick-end labelling-positive cells. Reduction of apoptosis was significant for hepatocytes, but not for endothelial cells. After ischemia, degenerative cell changes were clearly reduced in ANP pretreated livers. After reperfusion, ANP preconditioning led to a significant reduction of necrotic hepatocytes and endothelial cells in periportal zones. Cell proliferation was not affected by preconditioning.. ANP reduces necrotic and apoptotic cell death without affecting the proliferation status. The protection takes place mainly in the periportal area and seems to be most prominent against necrosis of hepatocytes and endothelial cells during reperfusion.

Topics: Animals; Apoptosis; Atrial Natriuretic Factor; Caspase 3; Caspase Inhibitors; Cell Division; Conditioning, Psychological; Cryopreservation; Cyclic GMP; In Situ Nick-End Labeling; Liver; Necrosis; Organ Preservation; Rats; Rats, Sprague-Dawley

In sepsis-induced acute renal failure, actin cytoskeletal alterations result in shedding of proximal tubule epithelial cells (PTEC) and tubular obstruction. This study examined the hypothesis that inflammatory cytokines, released early in sepsis, cause PTEC cytoskeletal damage and alter integrin-dependent cell-matrix adhesion. The question of whether the intermediate nitric oxide (NO) modulates these cytokine effects was also examined. After exposure of human PTEC to tumor necrosis factor-alpha, interleukin-1 alpha, and interferon-gamma, the actin cytoskeleton was disrupted and cells became elongated, with extension of long filopodial processes. Cytokines induced shedding of viable, apoptotic, and necrotic PTEC, which was dependent on NO synthesized by inducible NO synthase (iNOS) produced as a result of cytokine actions on PTEC. Basolateral exposure of polarized PTEC monolayers to cytokines induced maximal NO-dependent cell shedding, mediated in part through NO effects on cGMP. Cell shedding was accompanied by dispersal of basolateral beta(1) integrins and E-cadherin, with corresponding upregulation of integrin expression in clusters of cells elevated above the epithelial monolayer. These cells demonstrated coexpression of iNOS and apically redistributed beta(1) integrins. Attachment studies demonstrated that the major ligand involved in cell anchorage was laminin, probably through interactions with the integrin alpha(3)beta(1). This interaction was downregulated by cytokines but was not dependent on NO. These studies provide a mechanism by which inflammatory cytokines induce PTEC damage in sepsis, in the absence of hypotension and ischemia. Future therapeutic strategies aimed at specific iNOS inhibition might inhibit PTEC shedding after cytokine-induced injury and delay the onset of acute renal failure in sepsis.

Topics: Apoptosis; Cell Adhesion; Cell Membrane; Cells, Cultured; Cyclic GMP; Cytokines; Cytoskeleton; Dose-Response Relationship, Drug; Humans; Inflammation Mediators; Integrin beta1; Kidney Tubules, Proximal; Laminin; Necrosis; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II

Hypercontracture is an important mechanism of myocyte death during reperfusion. cGMP modulates the sensitivity of contractile myofilaments to Ca2+, and increasing cGMP concentration during the last minutes of anoxia prevents reoxygenation-induced hypercontracture in isolated cardiomyocytes. The purpose of this study was to determine whether stimulation of particulate guanylyl cyclase with the natriuretic peptide urodilatin, given at the time of reperfusion, reduces myocardial necrosis in the rat heart submitted to transient ischemia.. Isolated rat hearts (n = 38) were submitted to either 40 or 60 min of no-flow ischemia and 2 h of reperfusion, and were allocated to receive or not receive 0.05 microM urodilatin during the first 15 min of reperfusion or non-reperfusion treatment.. A marked reduction in myocardial cGMP concentration was observed in control hearts during reperfusion after 40 or 60 min of ischemia. Urodilatin significantly attenuated cGMP depletion during initial reperfusion, markedly improved contractile recovery after 40 min of ischemia (P < 0.0309), and reduced reperfusion-induced increase in left ventricular end-diastolic pressure (P = 0.0139), LDH release (P = 0.0263), and contraction band necrosis (P = 0.0179) after 60 min of ischemia. The beneficial effect of urodilatin was reproduced by the membrane permeable cGMP analog 8-Bromo-cGMP.. These results indicate that reduced cGMP concentration may impair myocyte survival during reperfusion. Stimulation of particulate guanylyl cyclase may appear as a new strategy to prevent immediate lethal reperfusion injury.

Topics: Analysis of Variance; Animals; Atrial Natriuretic Factor; Cyclic GMP; Enzyme Activators; Guanylate Cyclase; L-Lactate Dehydrogenase; Male; Myocardial Reperfusion Injury; Myocardium; Necrosis; Peptide Fragments; Perfusion; Rats; Rats, Sprague-Dawley

To better understand the mechanism(s) underlying nitric oxide (. NO)-mediated toxicity, in the presence and absence of concomitant oxidant exposure, postmitotic terminally differentiated NT2N cells, which are incapable of producing. NO, were exposed to PAPA-NONOate (PAPA/NO) and 3-morpholinosydnonimine (SIN-1). Exposure to SIN-1, which generated peroxynitrite in the range of 25-750 nM/min, produced a concentration- and time-dependent delayed cell death. In contrast, a critical threshold concentration (>440 nM/min) was required for. NO to produce significant cell injury. Examination of cells by electron microscopy shows a largely necrotic injury after peroxynitrite exposure but mainly apoptotic-like morphology after. NO exposure. Cellular levels of reduced thiols correlated with cell death, and pretreatment with N-acetylcysteine (NAC) fully protected from cell death in either PAPA/NO or SIN-1 exposure. NAC given within the first 3 h posttreatment further delayed cell death and increased the intracellular thiol level in SIN-1 but not. NO-exposed cells. Cell injury from. NO was independent of cGMP, caspases, and superoxide or peroxynitrite formation. Overall, exposure of non-. NO-producing cells to. NO or peroxynitrite results in delayed cell death, which, although occurring by different mechanisms, appears to be mediated by the loss of intracellular redox balance.

Topics: Acetylcysteine; Animals; Cell Death; Cell Differentiation; Cell Line; Cell Survival; Cyclic GMP; Hydrazines; Molsidomine; Necrosis; Neurons; Nitrates; Nitric Oxide; Nitric Oxide Donors; Oxidants; Sulfhydryl Compounds

Exposure of RINm5F cells to interleukin-1beta and to several chemical NO donors such as sodium nitroprusside (SNP), SIN-1 and SNAP induce apoptotic events such as the release of cytochrome c from mitochondria, caspase 3 activation, Bcl-2 downregulation and DNA fragmentation. SNP exposure led to transient activation of soluble guanylate cyclase (sGC) and prolonged protein kinase G (PKG) activation but apoptotic events were not attenuated by inhibition of the sGC/PKG pathway. Prolonged activation of the cGMP pathway by exposing cells to the dibutyryl analogue of cGMP for 12 h induced both apoptosis and necrosis, a response that was abolished by the PKG inhibitor KT5823. These results suggest that NO-induced apoptosis in the pancreatic beta-cell line is independent of acute activation of the cGMP pathway.

Topics: Animals; Apoptosis; Caspase 3; Caspases; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Cytochrome c Group; DNA Damage; Enzyme Activation; Guanylate Cyclase; Insulin; Islets of Langerhans; Mitochondria; Necrosis; Nitric Oxide; Nitroprusside; Protein Kinases; Proto-Oncogene Proteins c-bcl-2; Rats; Tumor Cells, Cultured

1. The biological actions of nitric oxide (NO), a highly diffusible and short-lived radical, range from signal transduction to cytotoxicity. The present study investigated whether NO is released in the course of liver necrosis and regeneration induced by a single necrogenic dose of thioacetamide (6.6 mmol kg-1 body wt) to rats. Samples of liver were obtained at 0, 3, 12, 24, 48, 72 and 96 h after thioacetamide administration. 2. Inducible nitric oxide synthase (iNOS) activity was determined in purified liver homogenates and a sharp 6 fold increase (P < 0.001) in iNOS activity was recorded at 48 h of intoxication, followed by a slight but progressive increase at 72 and 96 h. Changes in the expression of iNOS, as detected by its mRNA levels, were parallel to the NOS enzyme activity. Hepatocyte NO synthesis showed a progressive increase at 24, 48 and 72 h, to 8 (P < 0.001), 13 (P < 0.001) and 13 (P < 0.001) times the initial values, respectively. 3. In isolated Kupffer cells, where initial NO release was ten fold higher than in hepatocytes, a progressive increase was detected from 48 h which reached two fold of initial at 72 h of intoxication (192%; P < 0.001). Hepatic cyclic GMP concentration did not change significantly. However, mitochondrial aconitase activity decreased markedly at 12 and 24 h of intoxication showing a sharp increase towards normal values at 48 h which was maintained at 72 and 96 h. 4. As protein kinase C (PKC) is one of the likely candidates to mediate iNOS expression, translocation (activation) of PKC was assayed in hepatocytes, and a significant two fold increase (P < 0.001) between 48 and 96 h after thioacetamide intoxication was observed. When peritoneal macrophages from control rats were incubated with serum from thioacetamide-treated rats, a sharp increase in NO release was detected with serum obtained at 48 h, reaching at 96 h a value four fold (P < 0.001) that of the control. 5. These results suggest that iNOS activity and NO release play a role in the pathophysiological mechanisms that trigger post-necrotic hepatocellular regeneration following thioacetamide administration.

Topics: Aconitate Hydratase; Animals; Carcinogens; Cyclic GMP; Kupffer Cells; Liver; Male; Necrosis; Nitric Oxide; Nitric Oxide Synthase; Protein Kinase C; Rats; RNA, Messenger; Thioacetamide; Tumor Necrosis Factor-alpha

Our purpose was to determine the effects of nitric oxide synthase inhibition on maternal and fetal health in the last third of pregnancy.. Pregnant rats were treated from gestational day 13 to day 19 or 20 with the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester, which was administered in the drinking water ad libitum. Control animals received the inactive enantiomer NG-nitro-D-arginine methyl ester or no treatment. Maternal blood pressure, blood chemistry studies, and placenta and pup size were determined. A separate group of rats received nitroprusside sodium in conjunction with NG-nitro-L-arginine methyl ester.. NG-nitro-L-arginine methyl ester caused a dose-dependent reduction in placenta and pup size. Amniotic fluid levels of cyclic guanosine monophosphate were significantly reduced at 0.1 mg/ml but not at higher doses. Hemorrhagic necrosis of fetal hind limbs occurred only with treatment with NG-nitro-L-arginine methyl ester and was prevented by coadministration of nitroprusside sodium. Maternal blood pressure and blood and urine chemistry studies were unaffected by NG-nitro-L-arginine methyl ester.. Chronic reductions of nitric oxide production in the last third of pregnancy result in significant intrauterine growth retardation and hemorrhagic disruptions of hind limbs. Maternal complications were minimal and did not mimic preeclampsia.

Topics: Amniotic Fluid; Animals; Arginine; Cyclic GMP; Dose-Response Relationship, Drug; Female; Fetal Diseases; Fetal Growth Retardation; Fetus; Hemorrhage; Hindlimb; Necrosis; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitroprusside; Placenta; Pregnancy; Pregnancy, Animal; Rats; Rats, Sprague-Dawley

The plasma and cardiac levels of immunoreactive (IR) atrial natriuretic factor (ANF) were measured during the entire lifespan of cardiomyopathic hamsters, which eventually develop spontaneous congestive heart failure, and were correlated with immunohistochemical, ultrastructural, and immunocytochemical changes in the secretory apparatus of atrial and ventricular cardiocytes. Plasma IR-ANF rose in the early stages of the disease, reached a maximum in moderate heart failure, and declined thereafter but remained above control values. The peptide decreased constantly in the atria during the evolution of the disease but increased markedly in the ventricles. Its highest levels were found in the inner half of the left ventricle. In atrial cardiocytes, the size and complexity of the Golgi complex increased with the progression of the disease, whereas the number, size, and IR-ANF content (as assessed by the immunogold technique) of secretory granules decreased constantly. In ventricular cardiocytes, the size of the Golgi complex increased, and typical secretory granules were present in approximately 20% of these cells, regardless of their localization in the myocardium. The results suggest that stimulation of ANF secretion in atrial cardiocytes leads to a dissociation between synthesis and release, the latter being maximal according to ultrastructural and immunocytochemical criteria. In ventricular cardiocytes, the same stimulation culminates in increased synthesis and the possibility of release via two pathways: one constitutive, the other regulated. Thus, the elevated plasma levels of IR-ANF in congestive heart failure may be derived from secretion by both atrial and ventricular cardiocytes.

Topics: Animals; Atrial Natriuretic Factor; Cricetinae; Cyclic GMP; Heart Atria; Heart Failure; Heart Ventricles; Immunohistochemistry; Microscopy, Electron; Myocardium; Necrosis

In rat liver following white phorphorus poisoning a biphasic increase in cyclic AMP concentration was observed. After a lag period of 1 hour the cyclic AMP content rose to a first peak at 4 hours and to a second peak at 12 hours of intoxication. The cyclic AMP level fell to normal after 24 hours, by which time the cyclic nucleotide concentration was approaching control values. On the contrary, cyclic GMP content was found to the normal level during the different stages of intoxication. Only at 36 hours the cyclic GMP amount appeared significantly increased above the control values. Serum activity of alanine- and aspartate-amino transferases was found changed from 8 hours to 24 hours after poisoning. The serum level of the two enzymes was overlapping the control values after 36 hours. These results are discussed in relation to hepatocyte necrosis following white phosphorus intoxication.

Topics: Alanine Transaminase; Animals; Aspartate Aminotransferases; Cyclic AMP; Cyclic GMP; Liver; Necrosis; Phosphorus; Rats; Time Factors