cyclic-gmp and Inflammation

Vascular cognitive impairment (VCI) describes neurodegenerative disorders characterized by a vascular component. Pathologically, it involves decreased cerebral blood flow (CBF), white matter lesions, endothelial dysfunction, and blood-brain barrier (BBB) impairments. Molecularly, oxidative stress and inflammation are two of the major underlying mechanisms. Nitric oxide (NO) physiologically stimulates soluble guanylate cyclase (sGC) to induce cGMP production. However, under pathological conditions, NO seems to be at the basis of oxidative stress and inflammation, leading to a decrease in sGC activity and expression. The native form of sGC needs a ferrous heme group bound in order to be sensitive to NO (Fe(II)sGC). Oxidation of sGC leads to the conversion of ferrous to ferric heme (Fe(III)sGC) and even heme-loss (apo-sGC). Both Fe(III)sGC and apo-sGC are insensitive to NO, and the enzyme is therefore inactive. sGC activity can be enhanced either by targeting the NO-sensitive native sGC (Fe(II)sGC), or the inactive, oxidized sGC (Fe(III)sGC) and the heme-free apo-sGC. For this purpose, sGC stimulators acting on Fe(II)sGC and sGC activators acting on Fe(III)sGC/apo-sGC have been developed. These sGC agonists have shown their efficacy in cardiovascular diseases by restoring the physiological and protective functions of the NO-sGC-cGMP pathway, including the reduction of oxidative stress and inflammation, and improvement of vascular functioning. Yet, only very little research has been performed within the cerebrovascular system and VCI pathology when focusing on sGC modulation and its potential protective mechanisms on vascular and neural function. Therefore, within this review, the potential of sGC as a target for treating VCI is highlighted.

Topics: Cognitive Dysfunction; Cyclic GMP; Heme; Humans; Inflammation; Soluble Guanylyl Cyclase; Vascular Diseases

Misregulation of gut function and homeostasis impinges on the overall well-being of the entire organism. Diarrheal disease is the second leading cause of death in children under 5 years of age, and globally, 1.7 billion cases of childhood diarrhea are reported every year. Accompanying diarrheal episodes are a number of secondary effects in gut physiology and structure, such as erosion of the mucosal barrier that lines the gut, facilitating further inflammation of the gut in response to the normal microbiome. Here, we focus on pathogenic bacteria-mediated diarrhea, emphasizing the role of cyclic adenosine 3',5'-monophosphate and cyclic guanosine 3',5'-monophosphate in driving signaling outputs that result in the secretion of water and ions from the epithelial cells of the gut. We also speculate on how this aberrant efflux and influx of ions could modulate inflammasome signaling, and therefore cell survival and maintenance of gut architecture and function.

Topics: Animals; Cholera; Cyclic AMP; Cyclic GMP; Diarrhea; Escherichia coli Infections; Gastrointestinal Microbiome; Humans; Inflammasomes; Inflammation; NLR Family, Pyrin Domain-Containing 3 Protein; Pyroptosis; Salmonella Infections

Iron is necessary for the survival of almost all aerobic organisms. In the mammalian host, iron is a required cofactor for the assembly of functional iron-sulfur (Fe-S) cluster proteins, heme-binding proteins and ribonucleotide reductases that regulate various functions, including heme synthesis, oxygen transport and DNA synthesis. However, the bioavailability of iron is low due to its insolubility under aerobic conditions. Moreover, the host coordinates a nutritional immune response to restrict the accessibility of iron against potential pathogens. To counter nutritional immunity, most commensal and pathogenic bacteria synthesize and secrete small iron chelators termed siderophores. Siderophores have potent affinity for iron, which allows them to seize the essential metal from the host iron-binding proteins. To safeguard against iron thievery, the host relies upon the innate immune protein, lipocalin 2 (Lcn2), which could sequester catecholate-type siderophores and thus impede bacterial growth. However, certain bacteria are capable of outmaneuvering the host by either producing "stealth" siderophores or by expressing competitive antagonists that bind Lcn2 in lieu of siderophores. In this review, we summarize the mechanisms underlying the complex iron tug-of-war between host and bacteria with an emphasis on how host innate immunity responds to siderophores.

Topics: Cyclic GMP; Ferrous Compounds; Host Microbial Interactions; Humans; Immunity, Innate; Inflammation; Iron; Lipocalin-2; Neutrophils; Peptides; Reactive Oxygen Species; Siderophores

Inflammatory bowel disease (IBD) symbolizes a group of intestinal disorders in which prolonged inflammation occur in the digestive tract (esophagus, large intestine, small intestine mouth, stomach). Both genetic and environmental factors (infections, stress, diet) are involved in the development of IBD. As we know that bacteria are found in the intestinal mucosa of human and clinical observations revealed bacterial biofilms associated with patients of IBD. Various factors and microbes are found to play an essential role in biofilm formation and mucosal colonization during IBD. Biofilm formation in the digestive tract is dependent on an extracellular matrix synthesized by the bacteria and it has an adverse effect on the immune response of the host. There is no satisfactory and safe treatment option for IBD. Therefore, the current research aims to disrupt biofilm in IBD and concentrates predominantly on improving the drug. Here, we review the literature on bacterial biofilm and IBD to gather new knowledge on the current understanding of biofilm formation in IBD, host immune deregulation and dysbiosis in IBD, molecular mechanism, bacteria involved in biofilm formation, current and future regimen. It is urgently required to plan new ways to control and eradicate bacteria in biofilms that will open up novel diagnostic and therapeutic avenues for IBD. This article includes the mechanism of signaling molecules with respect to the biofilm-related genes as well as the diagnostic methods and new technologies involved in the treatment of IBD.

Topics: Anti-Inflammatory Agents; Bacteria; Biofilms; Cyclic GMP; Dysbiosis; Gastrointestinal Microbiome; Gastrointestinal Tract; Humans; Immunosuppressive Agents; Inflammation; Inflammatory Bowel Diseases; Intestinal Mucosa; Intestines; Quorum Sensing; Stomach

First discovered in prokaryotes and more recently in eukaryotes, cyclic dinucleotides (CDNs) constitute a unique branch of second messenger signaling systems. Within prokaryotes CDNs regulate a wide array of different biological processes, whereas in the vertebrate system CDN signaling is largely dedicated to activation of the innate immune system. In this book chapter we summarize the occurrence and signaling pathways of these small-molecule second messengers, most importantly in the scope of the mammalian immune system. In this regard, our main focus is the role of the cGAS-STING axis in the context of microbial infection and sterile inflammation and its implications for therapeutic applications.

Topics: Adjuvants, Immunologic; Animals; Bacterial Infections; Cyclic AMP; Cyclic GMP; Evolution, Molecular; Humans; Immune System; Immunity, Innate; Inflammation; Membrane Proteins; Molecular Structure; Second Messenger Systems

Upon blood vessel injury, platelets are exposed to adhesive proteins in the vascular wall and soluble agonists, which initiate platelet activation, leading to formation of hemostatic thrombi. Pathological activation of platelets can induce occlusive thrombosis, resulting in ischemic events such as heart attack and stroke, which are leading causes of death globally. Platelet activation requires intracellular signal transduction initiated by platelet receptors for adhesion proteins and soluble agonists. Whereas many platelet activation signaling pathways have been established for many years, significant recent progress reveals much more complex and sophisticated signaling and amplification networks. With the discovery of new receptor signaling pathways and regulatory networks, some of the long-standing concepts of platelet signaling have been challenged. This review provides an overview of the new developments and concepts in platelet activation signaling.

Topics: Animals; Blood Platelets; Cyclic GMP; Humans; Inflammation; Integrins; Mice; Platelet Activation; Platelet Adhesiveness; Signal Transduction; Thrombosis

Colorectal cancer (CRC) is a major cause of cancer-related mortality and morbidity worldwide. While improved treatments have enhanced overall patient outcome, disease burden encompassing quality of life, cost of care, and patient survival has seen little benefit. Consequently, additional advances in CRC treatments remain important, with an emphasis on preventative measures. Guanylyl cyclase C (GUCY2C), a transmembrane receptor expressed on intestinal epithelial cells, plays an important role in orchestrating intestinal homeostatic mechanisms. These effects are mediated by the endogenous hormones guanylin (GUCA2A) and uroguanylin (GUCA2B), which bind and activate GUCY2C to regulate proliferation, metabolism and barrier function in intestine. Recent studies have demonstrated a link between GUCY2C silencing and intestinal dysfunction, including tumorigenesis. Indeed, GUCY2C silencing by the near universal loss of its paracrine hormone ligands increases colon cancer susceptibility in animals and humans. GUCY2C's role as a tumor suppressor has opened the door to a new paradigm for CRC prevention by hormone replacement therapy using synthetic hormone analogs, such as the FDA-approved oral GUCY2C ligand linaclotide (Linzess™). Here we review the known contributions of the GUCY2C signaling axis to CRC, and relate them to a novel clinical strategy targeting tumor chemoprevention.

Topics: Animals; Carcinogenesis; Cell Cycle; Colonic Neoplasms; Cyclic GMP; Enterotoxins; Gastrointestinal Hormones; Genomics; Homeostasis; Hormones; Humans; Inflammation; Ligands; Mutation; Natriuretic Peptides; Paracrine Communication; Receptors, Enterotoxin; Receptors, Guanylate Cyclase-Coupled; Receptors, Peptide; Signal Transduction; Treatment Outcome

Neuronal inflammation is a systematically organized physiological step often triggered to counteract an invading pathogen or to rid the body of damaged and/or dead cellular debris. At the crux of this inflammatory response is the deployment of nonneuronal cells: microglia, astrocytes, and blood-derived macrophages. Glial cells secrete a host of bioactive molecules, which include proinflammatory factors and nitric oxide (NO). From immunomodulation to neuromodulation, NO is a renowned modulator of vast physiological systems. It essentially mediates these physiological effects by interacting with cyclic GMP (cGMP) leading to the regulation of intracellular calcium ions. NO regulates the release of proinflammatory molecules, interacts with ROS leading to the formation of reactive nitrogen species (RNS), and targets vital organelles such as mitochondria, ultimately causing cellular death, a hallmark of many neurodegenerative diseases. AD is an enervating neurodegenerative disorder with an obscure etiology. Because of accumulating experimental data continually highlighting the role of NO in neuroinflammation and AD progression, we explore the most recent data to highlight in detail newly investigated molecular mechanisms in which NO becomes relevant in neuronal inflammation and oxidative stress-associated neurodegeneration in the CNS as well as lay down up-to-date knowledge regarding therapeutic approaches targeting NO.

Topics: Alzheimer Disease; Animals; Cyclic GMP; Humans; Inflammation; Nitric Oxide; Nitric Oxide Synthase; Oxidative Stress; Signal Transduction

Preeclampsia is an autoimmune disorder characterized by hypertension. It begins with abnormal cytotrophoblast apoptosis, which leads to inflammation and an increase in the levels of anti-angiogenic factors followed by the disruption of the angiogenic status. Increased levels of fetal DNA and RNA coming from the placenta, one of the most commonly affected organs in pregnancies complicated by preeclampsia, have been found in pregnant women with the condition. However, it remains unknown as to whether this is a cause or a consequence of preeclampsia. Few studies have been carried out on preeclampsia in which an animal model of preeclampsia was induced by an injection of different types of DNA that are mimic fetal DNA and provoke inflammation through Toll-like receptor 9 (TLR9) or cyclic guanosine monophosphate-adenosine monophosphate (cGAMP). The specific mechanisms involved in the development of preeclampsia are not yet fully understood. It is hypothesized that the presence of different fragments of fetal DNA in maternal plasma may cause for the development of preeclampsia. The function of DNase during preeclampsia also remains unresolved. Studies have suggested that its activity is decreased or the DNA is protected against its effects. Further research is required to uncover the pathogenesis of preeclampsia and focus more on the condition of patients with the condition.

Topics: Animals; Cyclic GMP; Disease Models, Animal; DNA; Female; Fetus; Humans; Inflammation; Pre-Eclampsia; Pregnancy; RNA; Toll-Like Receptor 9

Well over 2 decades have passed since the endothelium-derived relaxation factor was reported to be the gaseous molecule nitric oxide (NO). Although soluble guanylyl cyclase (which generates cyclic guanosine monophosphate, cGMP) was the first identified receptor for NO, it has become increasingly clear that NO exerts a ubiquitous influence in a cGMP-independent manner. In particular, many, if not most, effects of NO are mediated by S-nitrosylation, the covalent modification of a protein cysteine thiol by an NO group to generate an S-nitrosothiol (SNO). Moreover, within the current framework of NO biology, endothelium-derived relaxation factor activity (ie, G protein-coupled receptor-mediated, or shear-induced endothelium-derived NO bioactivity) is understood to involve a central role for SNOs, acting both as second messengers and signal effectors. Furthermore, essential roles for S-nitrosylation have been implicated in virtually all major functions of NO in the cardiovascular system. Here, we review the basic biochemistry of S-nitrosylation (and denitrosylation), discuss the role of S-nitrosylation in the vascular and cardiac functions of NO, and identify current and potential clinical applications.

Topics: Animals; Apoptosis; Cardiovascular Diseases; Cardiovascular System; Cyclic GMP; Endothelium-Dependent Relaxing Factors; Endothelium, Vascular; Humans; Inflammation; Myocardium; Neovascularization, Physiologic; Nitric Oxide; Protein Processing, Post-Translational; S-Nitrosothiols; Signal Transduction; Vascular Resistance

Topics: Aminopyridines; Anti-Inflammatory Agents; Benzamides; Cilia; Cyclic AMP; Cyclic GMP; Cyclopropanes; Humans; Inflammation; Isoenzymes; Mucins; Oxidative Stress; Phosphodiesterase 4 Inhibitors; Phosphoric Diester Hydrolases; Pulmonary Circulation; Pulmonary Disease, Chronic Obstructive; Pulmonary Emphysema; Second Messenger Systems

Diabetes mellitus (DM) is highly prevalent and is an important risk factor for congestive heart failure (HF). Increased left ventricular (LV) diastolic stiffness is recognized as the earliest manifestation of DM-induced LV dysfunction, but its pathophysiology remains incompletely understood. Mechanisms whereby DM increases LV diastolic stiffness differ between HF with normal LV ejection fraction (EF) (HFNEF) and HF with reduced LVEF (HFREF). In diabetic HFREF, fibrosis and deposition of advanced glycation end products (AGEs) are the most important contributors to high LV diastolic stiffness, whereas in diabetic HFNEF, elevated resting tension of hypertrophied cardiomyocytes is the most important contributor to high LV diastolic stiffness. As HF mortality remains high in DM despite proven efficacy of current treatments, better understanding of the pathophysiology of high LV diastolic stiffness could be beneficial for novel therapeutic strategies.

Topics: Cyclic GMP; Diabetic Angiopathies; Diastole; Extracellular Matrix; Glycation End Products, Advanced; Heart; Heart Failure, Diastolic; Humans; Incidence; Inflammation; Myocardium; Nitric Oxide; Oxidative Stress; Ventricular Dysfunction, Left

The nitric oxide (NO)-soluble guanylate cyclase (sGC)-cyclic 3',5'-guanosine monophosphate (cGMP) pathway plays an important role in cardiovascular regulation by promoting vasodilation and inhibiting vascular smooth muscle cell growth, platelet aggregation, and leukocyte adhesion. In pathophysiological states with endothelial dysfunction this signaling pathway is impaired. Activation of sGC has traditionally been achieved with nitrovasodilators; however, these drugs are associated with the development of tolerance and potentially deleterious cGMP-independent actions. In this review the actions of BAY 41-2272, the prototype of a new class of NO-independent sGC stimulators, in cardiovascular disease models is discussed. BAY 41-2272 binds to a regulatory site on the alpha-subunit of sGC and stimulates the enzyme synergistically with NO. BAY 41-2272 had antihypertensive actions and attenuated remodeling in models of systemic arterial hypertension. It also unloaded the heart in experimental congestive heart failure. BAY 41-2272 reduced pulmonary vascular resistance in acute and chronic experimental pulmonary arterial hypertension. Furthermore, BAY 41-2272 inhibited platelet aggregation in vitro and leukocyte adhesion in vivo. These findings make direct sGC stimulation with BAY 41-2272 a promising new therapeutic strategy for cardiovascular diseases and warrant further studies. Finally, the significance of the novel NO- and heme-independent sGC activator BAY 58-2667, which activates two forms of NO-insensitive sGC, is briefly discussed.

Topics: Animals; Antihypertensive Agents; Benzoates; Cardiovascular Diseases; Cell Adhesion; Cyclic GMP; Disease Models, Animal; Endothelium, Vascular; Enzyme Activation; Enzyme Activators; Guanylate Cyclase; Heart Failure; Heme; Humans; Hypertension; Hypertension, Pulmonary; Inflammation; Leukocytes; Nitric Oxide; Platelet Aggregation; Pyrazoles; Pyridines; Receptors, Cytoplasmic and Nuclear; Soluble Guanylyl Cyclase; Vasodilator Agents

Peripheral inflammation enhances the antinociceptive effects of opioid receptor agonists through the activation of peripheral opioid receptors whose expression also increases during inflammatory pain. Similarly, intestinal inflammation also increases the antitransit and antiexudative effects of opioids as well as the expression of neuronal and extra-neuronal opioid receptors located in the gut. Nitric oxide has been described either as pro- or antiinflammatory and could produce both pro- and antinociceptive effects. In addition, numerous studies have shown that the L-arginine-nitric oxide-cGMP system participates in the antinociceptive and in the intestinal effects produced by opioids during peripheral inflammation by enhancing their effects. Thus, substances capable of inhibiting cyclic guanosine-3',5'-monophosphate (cGMP) degradation or nitric oxide donors increase the analgesic effects of opioid receptor agonists during peripheral inflammation. At the same time, the administration of nitric oxide synthase (NOS) or guanylate cyclase inhibitors decreases those effects. In accordance with these results, different clinical trials have also demonstrated that the co-administration of nitric oxide donors with opioids is highly beneficial in the treatment of pain in patients. In the gut, nitric oxide has a further pro- and antiinflammatory action. It is also involved in the enhanced antitransit and antiexudative effects produced by opioids and in the up-regulation of the mu-opioid receptor gene transcription observed in the inflamed intestine. To sum up, a better knowledge of the involvement of the L-arginine-nitric oxide-cGMP pathway in the opioid mechanisms of action and a better understanding of the pathways that regulate the expression of opioid receptors during peripheral inflammation are essential to developing improved analgesic/antiinflammatory therapies.

Topics: Analgesics, Opioid; Anti-Inflammatory Agents; Arginine; Cyclic GMP; Enzyme Inhibitors; Gene Expression Regulation; Guanylate Cyclase; Humans; Inflammation; Intestinal Mucosa; Neurons; Nitric Oxide; Nitric Oxide Synthase; Nociceptin Receptor; Receptors, Opioid

In the CNS, NO is an important physiological messenger involved in the modulation of brain development, synaptic plasticity, neuroendocrine secretion, sensory processing, and cerebral blood flow [Annu. Rev. Physiol. 57 (1995) 683]. These NO actions are largely mediated by cyclic GMP (cGMP) formed by stimulation of soluble guanylyl cyclase (sGC). NO has also been recognized as a neuropathological agent in conditions such as epilepsy, stroke and neurodegenerative disorders. In these conditions, NO may contribute to excitotoxic cell death and neuroinflammatory cell damage [Brain Res. Bull. 41 (1996) 131; Glia 29 (2000) 1]. NO can be formed in every type of CNS parenchymal cell, however, cGMP appears to be formed mainly in neurons and astroglia [Annu. Rev. Physiol. 57 (1995) 683]. There is a large body of information about the regulation of NO formation in brain cells under both normal and pathological conditions but much less is known about the control of cGMP generation, in particular during neuroinflammation when there is a high NO output. Here we briefly review our present knowledge on the regulation of NO-dependent cGMP formation in brain cells under inflammatory conditions.

Topics: Animals; Cyclic GMP; Humans; Inflammation; Neurons; Nitric Oxide

During systemic inflammation, body temperature is either increased (fever) or decreased (anapyrexia). Either response depends on the dose of the inflammatory agent, e.g., lipopolysaccharide (LPS), and on the ambient temperature. Under thermoneutrality, LPS always produces fever; under subthermoneutral conditions, LPS evokes fever at lower doses and anapyrexia at higher doses. Because of the diagnostic and adaptive values of these responses, understanding their mechanisms is of interest. Recently, the intracellular mechanisms that occur in the preoptic region (PO), the thermointegrative site of the brain, to produce fever and anapyrexia have begun to be clarified. In response to febrigenic doses of LPS, an increased production of prostaglandin E2 and an inhibition of nitric oxide synthesis produce fever respectively by decreasing the intracellular content of cyclic AMP (cAMP) and cyclic GMP (cGMP) in the PO. Although the role of preoptic cAMP and cGMP has not been directly assessed in the anapyrexia induced by LPS, it has been studied in that induced by hypoxia. The likeness between the thermoregulatory responses to hypoxia and to a high dose of LPS suggests that they may have similar mechanisms. In contrast to fever, hypoxia-induced anapyrexia seems to be mediated by a simultaneous increase in the levels of cAMP and cGMP in the PO as the result of an enhanced production and/or release of serotonin and nitric oxide, respectively. This article reviews the recent advances in the understanding of the role of preoptic cAMP and cGMP signaling cascades in fever and anapyrexia.

Topics: Animals; Cyclic AMP; Cyclic GMP; Fever; Humans; Inflammation; Signal Transduction

Nitric oxide (NO) plays diverse roles in physiological and pathological processes. During immune and inflammatory responses, for example in asthma, NO is generated at relatively high and sustained levels by the inducible form of nitric oxide synthase (NOS-2). NOS-2 derived NO regulates the function, growth, death and survival of many immune and inflammatory cell types. In the case of mast cells, NO suppresses antigen-induced degranulation, mediator release, and cytokine expression. The action of NO on mast cells is time dependent, requiring several hours, and noncGMP mediated, most probably involving chemical modification of proteins. NO inhibits a number of mast cell-dependent inflammatory processes in vivo, including histamine mediated vasodilatation, vasopermeation and leucocyte-endothelial cell attachment. In human asthma and animal models of lung inflammation the role of NO is harder to define. However, although there are conflicting data, the balance of evidence favours a predominantly protective role for NO. Mimicking or targeting NO dependent pathways may prove to be a valuable therapeutic approach to mast cell mediated diseases.

Topics: Animals; Asthma; Cyclic GMP; Cytokines; Cytoplasmic Granules; Disease Models, Animal; Endopeptidases; Heparin; Histamine Release; Humans; Hypersensitivity; Inflammation; Mast Cells; Mice; Mice, Knockout; Models, Biological; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Oxidative Stress; Signal Transduction; Swine

A pathological glia activation, stimulated by inflammatory proteins, beta-amyloid, or brain ischemia, is discussed as a common pathogenic factor for progressive nerve cell damage in vascular and Alzheimer dementia. A critical point seems to be reached, if the cytokine-controlled microglial upregulation causes a secondary activation of astrocytes which loose the negative feedback control, are forced to give up their physiological buffering function, and may add to neuronal damage by the release of nitric oxide (NO) and by promoting toxic beta-amyloid formation. A strengthening of the cyclic adenosine-5',3'-monophosphate (cAMP) signaling exerted a differential inhibition of the stimulatory cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) released from cultured rat microglia, but maintained the negative feedback signal IL-6; cAMP inhibited also the release of free oxygen radicals (OR) but not of NO. Reinforcement of the NO-induced cyclic guanosine monophosphate (cGMP) increase by blockade of the phosphodiesterase (PDE) subtype-5 with propentofylline counterbalanced the toxic NO action that causes with OR neuronal damage by peroxynitrate formation. In rat cultured astrocytes, a prolonged cAMP elevation favored cell differentiation, the expression of a mature ion channel patter, and an improvement of the extracellular glutamate uptake. Cyclic AMP signaling could be strengthened by PDE blockade and by raising extracellular adenosine, which stimulates A2 receptor-mediated cAMP synthesis. Via an A1 receptor-mediated effect, elevated adenosine was found to overcome a deficient intracellular calcium mobilization resulting from an impaired muscarinic signaling at pathologically decreased acetylcholine concentrations. We suggest that pharmaca, which elevate extracellular adenosine and/or block the degradation of cyclic nucleotides, may be used to counteract glia-related neuronal damage in dementing processes.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Astrocytes; Cyclic AMP; Cyclic GMP; Dementia, Vascular; Humans; Inflammation; Microglia; Models, Neurological; Neuroglia; Rats; Signal Transduction

We are progressing towards an understanding of the mechanism of peripherally acting analgesics. Knowledge of the participation of the cytokines in inflammatory hyperalgesia and the involvement of the arginine-NO-cGMP pathway in the mechanism of agents which directly down-regulate ongoing hyperalgesia may provide for better therapeutic approaches and permit the development of novel analgesics.

Topics: Analgesics; Arginine; Cyclic GMP; Cyclooxygenase Inhibitors; Cytokines; Down-Regulation; Humans; Hyperalgesia; Inflammation; Nitric Oxide; Nociceptors; Sympatholytics

Topics: Animals; Ascorbic Acid; Candida albicans; Cell Division; Chemotaxis, Leukocyte; Copper; Cyclic GMP; Extracellular Matrix; Extracellular Space; Histamine; Horseradish Peroxidase; Humans; Hydrogen Peroxide; Hypersensitivity; Immunization; Inflammation; Iodides; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Phagocytosis; Prostaglandins; SRS-A

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acids; Cell Movement; Chemotaxis, Leukocyte; Cyclic GMP; Humans; Inflammation; Lipoxygenase; Macrophages; Neutrophils; Receptors, Complement; Subcellular Fractions

Topics: Animals; Antibody Formation; B-Lymphocytes; Cyclic AMP; Cyclic GMP; Cytotoxicity Tests, Immunologic; Humans; Hypersensitivity; Immunity, Cellular; Immunosuppression Therapy; Indomethacin; Inflammation; Lymphocyte Activation; Lymphocytes; Macrophages; Neoplasms; Neutrophils; Prostaglandins; Receptors, Prostaglandin; T-Lymphocytes

All leukocytes are capable of responding chemotactically (oriented locomotion) and chemokinetically (stimulated nondirected or random locomotion) to a variety of chemical agents. A brief review of the in vitro and in vivo methods of studying neutrophil movement and our present knowledge of chemotactic factors is presented as well as a discussion on the mechanisms of stimulated movement. Two clinically important instances of defects in neutrophil movement, ie, the Chédiak-Higashi syndrome and a case of actin dysfunction, are herein described.

Topics: Ascorbic Acid; Calcium; Cell Movement; Chediak-Higashi Syndrome; Chemotaxis, Leukocyte; Complement C3; Complement C5; Cyclic AMP; Cyclic GMP; Cytological Techniques; Cytoskeleton; Humans; Inflammation; Kallikreins; Lysosomes; Microtubules; Neutrophils; Oligopeptides; Plasminogen Activators; Receptors, Drug; Skin Window Technique; Sodium; Structure-Activity Relationship

Current knowledge of the mechanism of inflammatory mediator release from mast cells is reviewed with particular reference to the role of cyclic nucleotides and calcium and their interrelationship with one another as defined by studies in highly purified rat peritoneal mast cells. Data are presented indicating an important role for intracellular cAMP and calcium in the mediation or modulation of release, as well as evidence for a close relationship between these two regulatory systems. Releasing agents which clearly act at the level of the plasma membrane (concanavalin A and anti-IgE antibody) are shown to differ from releasing agents that may not (48/80 and the ionophore A23187) in regard to the early cellular cAMP response, dependency of the release reaction on phosphatidyl serine, and kinetics of release. Pharmacologic modulators of release are discussed; these include: PGE1 and theophylline, which raise cAMP and inhibit release; and diazoxide, adenine, and carbachol which lower cAMP and potentiate release. Adenosine was also found to enhance release with marked effects at concentrations in the low nanomolar range.

Topics: Animals; Antibodies; Calcimycin; Calcium; Catecholamines; Cell Membrane; Concanavalin A; Cyclic AMP; Cyclic GMP; Histamine Release; Humans; Immunoglobulin E; Inflammation; Kinetics; Mast Cells; p-Methoxy-N-methylphenethylamine; Prostaglandins E; Rats; Theophylline

The evidence supporting the role of prostaglandins and cyclic nucleotides "in vitro" has been reviewed. The pro-inflammatory role of prostaglandins of the E series (PGE) is typified by its ability to induce increased vascular permeability. Prostaglandins of the F series (PGF) may be anti-inflammatory via their inhibitory effect on increased vascular permeability. However, a paradox exists which suggests that PGE may also be anti-inflammatory via its stimulatory effect on cyclic AMP synthesis as shown "in vitro" (e.g. decreased leucocyte lysosomal enzyme secretion; decreased lymphocyte cytotoxicity and mitosis; decreased release of mediators from sensitized tissues during anaphylaxis). Conversely PGF is capable of stimulating cyclic GMP which augments the processes listed above, and may therefore be termed pro-inflammatory. An attempt has been made to correlate these findings with "in vivo" studies which support the anti-inflammatory role of cyclic GMP. However, the significance of PGE and PGF in the inflammatory response "in vivo" appears to be more complex. It is suggested that greater emphasis should be placed on the "in vivo" study of beta-adrenergic and cholinergic mediators, substances which induce the anti- and pro-inflammatory effects of cyclic AMP and cyclic GMP "in vitro", respectively.

Topics: Animals; Capillary Permeability; Cyclic AMP; Cyclic GMP; Humans; Inflammation; Leukocytes; Lymphocytes; Lysosomes; Models, Biological; Nucleotides, Cyclic; Prostaglandins; Prostaglandins E; Prostaglandins F; Receptors, Cyclic AMP

Topics: Adenylyl Cyclases; Animals; Bucladesine; Concanavalin A; Cyclic AMP; Cyclic GMP; DNA Replication; Enzyme Activation; Inflammation; Lectins; Mitogens; Mononuclear Phagocyte System

Topics: Animals; Calcium; Cyclic GMP; Glucuronidase; Humans; Inflammation; Leukocytes; Lysosomes; Neurotransmitter Agents; Neutrophils; Nucleotides, Cyclic; Peptide Hydrolases; Phagocytes; Prostaglandins

Topics: Animals; Arthus Reaction; Cations, Divalent; Chemotaxis; Chick Embryo; Complement C3; Cyclic AMP; Cyclic GMP; Cytoplasmic Granules; Energy Metabolism; Enzyme Precursors; Esterases; Histamine Release; Humans; Inflammation; Leukocytes; Mast Cells; Microtubules; Neutrophils; p-Methoxy-N-methylphenethylamine

Topics: Anti-Inflammatory Agents; Arthritis, Rheumatoid; Calcium; Cartilage; Cell Membrane; Connective Tissue; Cyclic AMP; Cyclic GMP; Epinephrine; Glucocorticoids; Humans; Hydrogen-Ion Concentration; In Vitro Techniques; Inflammation; Kinetics; Leukocytes; Lysosomes; Neutrophils; Osmotic Fragility; Phagocytosis; Prostaglandins

Tadalafil, the phosphodiesterase type 5 inhibitor (PDE5I), has been shown to reduce visceral adipose tissue in rabbit and to improve lean mass content in non-obese men. In order to clarify this effect in humans, in the present study we determined the impact of an acute oral tadalafil administration on lipolysis by evaluating plasma free fatty acids (FFAs) and glycerol. FFAs are potential modulator of inflammation response that we evaluated through tumor necrosis factor alpha (TNFα), interleukin 6 (IL6), interleukin 8 (IL8) and interleukin 10 (IL10) plasma levels. Moreover, we determined whether the effects of tadalafil would be reflected in variation of plasma levels of cGMP and NO, two important molecules involved in PDE5Is signaling.. Twelve healthy subjects were supplemented with 20 mg of tadalafil or a placebo, in a double-blind, randomized, cross-over design. Blood samples were collected immediately before, and at 2, 6, and 24 hours post ingestion, and assayed for biochemical analysis.. A condition effect was noted for FFAs and glycerol, with values higher for tadalafil when compared to the placebo group, at 2 and 6 hours post ingestion. No statistically significant effects were noted for glucose, cGMP, nitrate and nitrite. No inflammatory response was induced by tadalafil.. Tadalafil, in human subjects, increases lipolysis as evidenced by a significant increase in circulating FFAs and glycerol, without affecting the plasma cGMP and NO levels; noticeably, the increase in FFAs did not develop an inflammatory response. Further well-controlled studies are warranted to assess the impact of tadalafil administration on weight/fat loss.

Topics: Adult; Blood Glucose; Cyclic GMP; Double-Blind Method; Fatty Acids; Glycerol; Humans; Inflammation; Interleukin-6; Interleukin-8; Male; Nitric Oxide; Phosphodiesterase 5 Inhibitors; Tadalafil; Tumor Necrosis Factor-alpha

Activating PKG-1α induces a long-term hyperexcitability (LTH) in nociceptive neurons. Since the LTH correlates directly with chronic pain in many animal models, we tested the hypothesis that inhibiting PKG-1α would attenuate LTH-mediated pain. We first synthesized and characterized compound N46 (N-((3R,4R)-4-(4-(2-fluoro-3-methoxy-6-propoxybenzoyl)benzamido)pyrrolidin-3-yl)-1H-indazole-5-carboxamide). N46 inhibits PKG-1α with an IC50 of 7.5 nmol, was highly selective when tested against a panel of 274 kinases, and tissue distribution studies indicate that it does not enter the CNS. To evaluate its antinociceptive potential, we used 2 animal models in which the pain involves both activated PKG-1α and LTH. Injecting complete Freund's adjuvant (CFA) into the rat hind paw causes a thermal hyperalgesia that was significantly attenuated 24 hours after a single intravenous injection of N46. Next, we used a rat model of osteoarthritic knee joint pain and found that a single intra-articular injection of N46 alleviated the pain 14 days after the pain was established and the relief lasted for 7 days. Thermal hyperalgesia and osteoarthritic pain are also associated with the activation of the capsaicin-activated transient receptor protein vanilloid-1 (TRPV1) channel. We show that capsaicin activates PKG-1α in nerves and that a subcutaneous delivery of N46 attenuated the mechanical and thermal hypersensitivity elicited by exposure to capsaicin. Thus, PKG-1α appears to be downstream of the transient receptor protein vanilloid-1. Our studies provide proof of concept in animal models that a PKG-1α antagonist has a powerful antinociceptive effect on persistent, already existing inflammatory pain. They further suggest that N46 is a valid chemotype for the further development of such antagonists.

Topics: Adenosine Triphosphate; Animals; Biphenyl Compounds; Chronic Disease; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Disease Models, Animal; Double-Blind Method; Enzyme Inhibitors; Freund's Adjuvant; Gene Expression Regulation, Enzymologic; Hyperalgesia; Inflammation; Male; Models, Molecular; Osteoarthritis; Pain; Pain Threshold; Pyridines; Rats; Rats, Sprague-Dawley; Thionucleotides; Time Factors

Heart failure (HF) patients are at an increased risk of thrombotic events. Here, we investigated the effects of exercise training on platelet function and factors involved in its modulation in HF.. Thirty HF patients were randomized to 6 months of supervised exercise training or to a control group that remained sedentary. Exercise training consisted of 30 min of moderate-intensity treadmill exercise, followed by resistance and stretching exercises, performed three times a week. Blood was collected before and after the intervention for platelet and plasma obtainment.. Peak VO2 increased after exercise training (18.0 ± 2.2 vs. 23.8 ± 0.5 mlO2/kg/min; p < 0.05). Exercise training reduced platelet aggregation induced by both collagen and ADP (approximately -6%; p < 0.05), as well as platelet nitric oxide synthase activity (0.318 ± 0.030 vs. 0.250 ± 0.016 pmol/10(8) cells; p < 0.05). No difference in the above-mentioned variables were observed in the control group. No significant difference was observed in intraplatelet cyclic guanosine monophosphate levels among groups. There was a significant increase in the activity of the antioxidant enzymes superoxide dismutase and catalase in plasma and platelets, resulting in a decrease in both lipid and protein oxidative damage. Systemic levels of the inflammatory markers C-reactive protein, fibrinogen, and tumour necrosis factor α were also reduced in HF after training.. Our results suggest that regular exercise training is a valuable adjunct to optimal medical management of HF, reducing platelet aggregation via antioxidant and anti-inflammatory effects, and, therefore, reducing the risk of future thrombotic events.

Topics: Antioxidants; Biomarkers; Blood Platelets; Brazil; Cyclic GMP; Exercise Therapy; Female; Heart Failure; Humans; Inflammation; Inflammation Mediators; Lipids; Male; Middle Aged; Motor Activity; Muscle Stretching Exercises; Nitric Oxide Synthase; Oxidative Stress; Oxygen Consumption; Platelet Aggregation; Platelet Function Tests; Predictive Value of Tests; Prospective Studies; Recovery of Function; Resistance Training; Risk Factors; Thrombosis; Time Factors; Treatment Outcome

Metabolic syndrome (MetS) and benign prostate hyperplasia (BPH)/low urinary tract symptoms (LUTS) are often comorbid. Chronic inflammation is one of the putative links between these diseases. Phosphodiesterase type 5 inhibitors (PDE5i) are recognized as an effective treatment of BPH-related LUTS. One proposed mechanism of action of PDE5 is the inhibition of intraprostatic inflammation. In this study we investigate whether PDE5i could blunt inflammation in the human prostate.. Evaluation of the effect of tadalafil and vardenafil on secretion of interleukin 8 (IL-8, a surrogate marker of prostate inflammation) by human myofibroblast prostatic cells (hBPH) exposed to different inflammatory stimuli. We preliminary evaluate histological features of prostatic inflammatory infiltrates in BPH patients enrolled in a randomized, double bind, placebo controlled study aimed at investigating the efficacy of vardenafil (10 mg/day, for 12 weeks) on BPH/LUTS.. In vitro treatment with tadalafil or vardenafil on hBPH reduced IL-8 secretion induced by either TNFα or metabolic factors, including oxidized low-density lipoprotein, oxLDL, to the same extent as a PDE5-insensitive PKG agonist Sp-8-Br-PET-cGMP. These effects were reverted by the PKG inhibitor KT5823, suggesting a cGMP/PKG-dependency. Treatment with tadalafil or vardenafil significantly suppressed oxLDL receptor (LOX-1) expression. Histological evaluation of anti-CD45 staining (CD45 score) in prostatectomy specimens of BPH patients showed a positive association with MetS severity. Reduced HDL-cholesterol and elevated triglycerides were the only MetS factors significantly associated with CD45 score. In the MetS cohort there was a significant lower CD45 score in the vardenafil-arm versus the placebo-one.

Topics: Aged; Aged, 80 and over; Carbolines; Cyclic GMP; Double-Blind Method; Humans; Imidazoles; Inflammation; Interleukin-8; Lower Urinary Tract Symptoms; Male; Middle Aged; Myofibroblasts; Phosphodiesterase 5 Inhibitors; Pilot Projects; Piperazines; Prostate; Prostatic Hyperplasia; Sulfones; Tadalafil; Treatment Outcome; Triazines; Vardenafil Dihydrochloride

The binding of nitric oxide (NO) to heme in the β

Topics: Cyclic GMP; Dental Pulp; Guanylate Cyclase; Heme; Humans; Inflammation; Nitric Oxide; Soluble Guanylyl Cyclase

Interleukin-1 receptor-associated kinase-3 (IRAK3) is a critical checkpoint molecule of inflammatory responses in the innate immune system. The pseudokinase domain of IRAK3 contains a guanylate cyclase (GC) centre that generates small amounts of cyclic guanosine monophosphate (cGMP) associated with IRAK3 functions in inflammation. However, the mechanisms of IRAK3 actions are poorly understood. The effects of low cGMP levels on inflammation are unknown, therefore a dose-response effect of cGMP on inflammatory markers was assessed in THP-1 monocytes challenged with lipopolysaccharide (LPS). Sub-nanomolar concentrations of membrane permeable 8-Br-cGMP reduced LPS-induced NFκB activity, IL-6 and TNF-α cytokine levels. Pharmacologically upregulating cellular cGMP levels using a nitric oxide donor reduced cytokine secretion. Downregulating cellular cGMP using a soluble GC inhibitor increased cytokine levels. Knocking down IRAK3 in THP-1 cells revealed that unlike the wild type cells, 8-Br-cGMP did not suppress inflammatory responses. Complementation of IRAK3 knockdown cells with wild type IRAK3 suppressed cytokine production while complementation with an IRAK3 mutant at GC centre only partially restored this function. Together these findings indicate low levels of cGMP form a critical component in suppressing cytokine production and in mediating IRAK3 action, and this may be via a cGMP enriched nanodomain formed by IRAK3 itself.

Topics: Cyclic GMP; Cytokines; Guanylate Cyclase; Humans; Inflammation; Interleukin-1 Receptor-Associated Kinases; Lipopolysaccharides; Monocytes; Nitric Oxide

Titin phosphorylation contributes to left ventricular (LV) diastolic dysfunction. The independent effects of inflammation on the molecular pathways that regulate titin phosphorylation are unclear.. We investigated the effects of collagen-induced inflammation and subsequent tumor necrosis factor-α (TNF-α) inhibition on mRNA expression of genes involved in regulating titin phosphorylation in 70 Sprague-Dawley rats. LV diastolic function was assessed with echocardiography. Circulating inflammatory markers were quantified by enzyme-linked immunosorbent assay and relative LV gene expression was assessed by Taqman® polymerase chain reaction. Differences in normally distributed variables between the groups were determined by two-way analysis of variance (ANOVA), followed by Tukey post-hoc tests. For non-normally distributed variables, group differences were determined by Kruskal-Wallis tests.. Collagen inoculation increased LV relative mRNA expression of vascular cell adhesion molecule 1 (VCAM1), pentraxin 3 (PTX3), and inducible nitric oxide synthase (iNOS) compared to controls, indicating local microvascular inflammation. Collagen inoculation decreased soluble guanylate cyclase alpha-2 (sGCα2) and soluble guanylate cyclase beta-2 (sGCβ2) expression, suggesting downregulation of nitric oxide-soluble guanylate cyclase-cyclic guanosine monophosphate (NO-sGC-cGMP) signaling. Inhibiting TNF-α prevented collagen-induced changes in VCAM1, iNOS, sGCα2 and sGCβ2 expression. Collagen inoculation increased protein phosphatase 5 (PP5) expression. Like LV diastolic dysfunction, increased PP5 expression was not prevented by TNF-α inhibition.. Inflammation-induced LV diastolic dysfunction may be mediated by a TNF-α-independent increase in PP5 expression and dephosphorylation of the N2-Bus stretch element of titin, rather than by TNF-α-induced downregulation of NO-sGC-cGMP pathway-dependent titin phosphorylation. The steady rise in number of patients with inflammation-induced diastolic dysfunction, coupled with low success rates of current therapies warrants a better understanding of the systemic signals and molecular pathways responsible for decreased titin phosphorylation in development of LV diastolic dysfunction. The therapeutic potential of inhibiting PP5 upregulation in LV diastolic dysfunction requires investigation.

Topics: Animals; Collagen; Cyclic GMP; Inflammation; Rats; Rats, Sprague-Dawley; RNA, Messenger; Soluble Guanylyl Cyclase; Tumor Necrosis Factor-alpha; Ventricular Dysfunction, Left

Activating mutations in receptor guanylyl cyclase C (GC-C), the target of gastrointestinal peptide hormones guanylin and uroguanylin, and bacterial heat-stable enterotoxins cause early-onset diarrhea and chronic inflammatory bowel disease (IBD). GC-C regulates ion and fluid secretion in the gut via cGMP production and activation of cGMP-dependent protein kinase II. We characterize a novel mouse model harboring an activating mutation in Gucy2c equivalent to that seen in an affected Norwegian family. Mutant mice demonstrated elevated intestinal cGMP levels and enhanced fecal water and sodium content. Basal and linaclotide-mediated small intestinal transit was higher in mutant mice, and they were more susceptible to DSS-induced colitis. Fecal microbiome and gene expression analyses of colonic tissue revealed dysbiosis, up-regulation of IFN-stimulated genes, and misregulation of genes associated with human IBD and animal models of colitis. This novel mouse model thus provides molecular insights into the multiple roles of intestinal epithelial cell cGMP, which culminate in dysbiosis and the induction of inflammation in the gut.

Topics: Animals; Colitis; Colon; Cyclic GMP; Cyclic GMP-Dependent Protein Kinase Type II; Disease Models, Animal; Dysbiosis; Gene Expression; Inflammation; Inflammatory Bowel Diseases; Intestinal Mucosa; Intestines; Mice; Mutation; Receptors, Enterotoxin; Signal Transduction

Ferulic acid (FA) is a polyphenol pertaining to the class of hydroxycinnamic acids present in numerous foods of a plant origin. Its dietary consumption leads to the formation of several phase I and II metabolites in vivo, which represent the largest amount of ferulates in the circulation and in the intestine in comparison with FA itself. In this work, we evaluated their efficacy against the proinflammatory effects induced by lipopolysaccharide (LPS) in intestinal Caco-2 cell monolayers, as well as the mechanisms underlying their protective action. LPS-induced overexpression of proinflammatory enzymes such as inducible nitric oxide synthase (iNOS) and the consequent hyperproduction of nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) were limited by physiological relevant concentrations (1 µM) of FA, its derivatives isoferulic acid (IFA) and dihydroferulic acid (DHFA), and their glucuronidated and sulfated metabolites, which acted upstream by limiting the activation of MAPK p38 and ERK and of Akt kinase, thus decreasing the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-ĸB) translocation into the nucleus. Furthermore, the compounds were found to promote the expression of Nrf2, which may have contributed to the downregulation of NF-ĸB activity. The overall data show that phase I/II metabolites retain the efficacy of their dietary free form in contrasting inflammatory response.

Topics: Caco-2 Cells; Cell Survival; Coumaric Acids; Cyclic GMP; Enterocytes; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation; Humans; I-kappa B Proteins; Inflammation; Lipopolysaccharides; NF-E2-Related Factor 2; Nitric Oxide; Nitric Oxide Synthase Type II; p38 Mitogen-Activated Protein Kinases; Proto-Oncogene Proteins c-akt

Curli, a major component of the bacterial biofilms in the intestinal tract, activates pattern recognition receptors and triggers joint inflammation after infection with Salmonella enterica serovar Typhimurium. The factors that allow

Topics: Bacterial Proteins; Biofilms; Cues; Cyclic GMP; Flagella; Gene Expression Regulation, Bacterial; Humans; Inflammation; Nitrates; Salmonella enterica; Salmonella typhimurium; Serogroup

Nitric oxide (NO) binds to soluble guanylyl cyclase (sGC), activates it in a reduced oxidized heme iron state, and generates cyclic Guanosine Monophosphate (cGMP), which results in vasodilatation and inhibition of osteoclast activity. In inflammation, sGC is oxidized and becomes insensitive to NO. NO- and heme-independent activation of sGC requires protein expression of the α

Topics: Animals; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Cyclic GMP; Gene Expression Regulation; Heme; Humans; Inflammation; Iron; Nitric Oxide; Osteoclasts; Oxidation-Reduction; Periodontal Ligament; Periodontium; Soluble Guanylyl Cyclase

Topics: Alternaria; Alternariosis; Animals; Cyclic GMP; Cytokines; Immunity, Innate; Inflammation; Lung; Membrane Proteins; Mice; Mice, Knockout; Pneumonia; Signal Transduction

Topics: Animals; Cyclic GMP; Diet, High-Fat; Hyperlipidemias; Inflammation; Male; Nitric Oxide; Nitric Oxide Synthase Type III; Nucleotides, Cyclic; Obesity; Orlistat; Propolis; Rats; Rats, Sprague-Dawley; Signal Transduction; Vasodilation

Bacteria that colonize animals must overcome, or coexist, with the reactive oxygen species products of inflammation, a front-line defense of innate immunity. Among these is the neutrophilic oxidant bleach, hypochlorous acid (HOCl), a potent antimicrobial that plays a primary role in killing bacteria through nonspecific oxidation of proteins, lipids, and DNA. Here, we report that in response to increasing HOCl levels, Escherichia coli regulates biofilm production via activation of the diguanylate cyclase DgcZ. We identify the mechanism of DgcZ sensing of HOCl to be direct oxidation of its regulatory chemoreceptor zinc-binding (CZB) domain. Dissection of CZB signal transduction reveals that oxidation of the conserved zinc-binding cysteine controls CZB Zn

Topics: Bacteria; Bacterial Adhesion; Biofilms; Cyclic GMP; Escherichia coli; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Genome, Bacterial; Hypochlorous Acid; Inflammation; Signal Transduction

Reduced nitric oxide (NO) and a decrease in cGMP signaling mediated by soluble guanylate cyclase (sGC) has been linked to the development of several cardiorenal diseases. Stimulation of sGC is a potential means for enhancing cGMP production in conditions of reduced NO bioavailability. The purpose of our studies was to determine the effects of praliciguat, a clinical-stage sGC stimulator, in a model of cardiorenal failure. Dahl salt-sensitive rats fed a high-salt diet to induce hypertension and organ damage were treated with the sGC stimulator praliciguat to determine its effects on hemodynamics, biomarkers of inflammation, fibrosis, tissue function, and organ damage. Praliciguat treatment reduced blood pressure, improved cardiorenal damage, and attenuated the increase in circulating markers of inflammation and fibrosis. Notably, praliciguat affected markers of renal damage at a dose that had minimal effect on blood pressure. In addition, liver fibrosis and circulating markers of tissue damage were attenuated in praliciguat-treated rats. Stimulation of the NO-sGC-cGMP pathway by praliciguat attenuated or normalized indicators of chronic inflammation, fibrosis, and tissue dysfunction in the Dahl salt-sensitive rat model. Stimulation of sGC by praliciguat may present an effective mechanism for treating diseases linked to NO deficiency, particularly those associated with cardiac and renal failure. Praliciguat is currently being evaluated in patients with diabetic nephropathy and heart failure with preserved ejection fraction.

Topics: Animals; Biomarkers; Blood Pressure; Chemokine CCL2; Cyclic GMP; Fibrosis; Guanylyl Cyclase C Agonists; Inflammation; Kidney; Male; Natriuretic Peptide, Brain; Nitric Oxide; Osteopontin; Peptide Fragments; Pyrazoles; Pyrimidines; Rats; Rats, Inbred Dahl; Renal Insufficiency; Signal Transduction; Soluble Guanylyl Cyclase; Tissue Inhibitor of Metalloproteinase-1

Topics: alpha7 Nicotinic Acetylcholine Receptor; Animals; Choline; Cyclic GMP; Dinoprostone; Freund's Adjuvant; Hyperalgesia; Inflammation; KATP Channels; Male; Mice; Mice, Inbred BALB C; Nitric Oxide

The nucleosome is the basic structural repeating unit of chromatin. DNA damage and cell apoptosis release nucleosomes into the blood circulatory system, and increased levels of circulating nucleosomes have been observed to be related to inflammation and autoimmune diseases. However, how circulating nucleosomes trigger immune responses has not been fully elucidated. cGAS (cGMP-AMP synthase) is a recently discovered pattern recognition receptor that senses cytoplasmic double-stranded DNA (dsDNA). In this study, we employed in vitro reconstituted nucleosomes to examine whether extracellular nucleosomes can gain access to the cytoplasm of mammalian cells to induce immune responses by activating cGAS. We showed that nucleosomes can be taken up by various mammalian cells. Additionally, we found that in vitro reconstituted mononucleosomes and oligonucleosomes can be recognized by cGAS. Compared to dsDNA, nucleosomes exhibit higher binding affinities to cGAS but considerably lower potency in cGAS activation. Incubation of monocytic cells with reconstituted nucleosomes leads to limited production of type I interferons and proinflammatory cytokines via a cGAS-dependent mechanism. This proof-of-concept study reveals the cGAS-dependent immunogenicity of nucleosomes and highlights the potential roles of circulating nucleosomes in autoimmune diseases, inflammation, and antitumour immunity.

Topics: Adenosine Monophosphate; Animals; Apoptosis; Cell Line; Chromatin; Cyclic GMP; Cytokines; Cytosol; DNA; DNA Damage; Extracellular Vesicles; HeLa Cells; Hep G2 Cells; Humans; Immunity, Innate; Inflammation; Membrane Proteins; Mice; Nucleosomes; Nucleotidyltransferases; Signal Transduction; THP-1 Cells

Pulmonary hypertension (PH) is a life-threatening lung disease. PH with concomitant lung diseases, e.g., idiopathic pulmonary fibrosis, is associated with poor prognosis. Development of novel therapeutic vasodilators for treatment of these patients is a key imperative. We evaluated the efficacy of dual activation of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) using an active, small-molecule phosphodiesterase (PDE4)/PDE5 dual inhibitor (Compound A). Compound A increased both cAMP and cGMP levels in WI-38 lung fibroblasts and suppressed the expressions of type-1 collagen α1 chain and fibronectin. Additionally, compound A reduced right ventricular weight/left ventricular weight+septal weight ratio, brain natriuretic peptide expression levels in right ventricle, C─C motif chemokine ligand 2 expression levels in lung, and plasma surfactant protein D. Our data indicate that dual activation of cAMP/cGMP pathways may be a novel treatment strategy for PH.

Topics: Animals; Brain-Derived Neurotrophic Factor; Cells, Cultured; Collagen Type I; Collagen Type I, alpha 1 Chain; Cyclic AMP; Cyclic GMP; Disease Models, Animal; Epithelium; Fibronectins; Gene Expression Regulation; Humans; Hypertension, Pulmonary; Inflammation; Lung; Monocrotaline; Phosphodiesterase 5 Inhibitors; Rats, Wistar; Transforming Growth Factor beta

Interleukin-1 beta (IL-1β) is a pleiotropic mediator of inflammation and is produced in response to a wide range of stimuli. During infection, IL-1β production occurs in parallel with the onset of innate antimicrobial defenses, but the contribution of IL-1β signaling to cell-intrinsic immunity is not defined. Here, we report that exogenous IL-1β induces interferon regulatory factor 3 (IRF3) activation in human myeloid, fibroblast, and epithelial cells. IRF3 activation by IL-1β is dependent upon the DNA-sensing pathway adaptor, stimulator of interferon genes (STING), through the recognition of cytosolic mtDNA by cyclic guanosine monophosphate (GMP)-AMP synthase (cGAS). IL-1β treatment results in interferon (IFN) production and activation of IFN signaling to direct a potent innate immune response that restricts dengue virus infection. This study identifies a new function for IL-1β in the onset or enhancement of cell-intrinsic immunity, with important implications for cGAS-STING in integrating inflammatory and microbial cues for host defense.

Topics: Cyclic GMP; Dengue; Dengue Virus; DNA, Mitochondrial; Host-Pathogen Interactions; Humans; Immunity, Innate; Inflammation; Interferon Regulatory Factor-3; Interferons; Interleukin-1beta; Membrane Proteins; Myeloid Cells; Nucleotidyltransferases; Signal Transduction

Non-alcoholic steatohepatitis (NASH) is the hepatic manifestation of metabolic syndrome and is characterized by steatosis, inflammation and fibrosis. Soluble guanylate cyclase (sGC) stimulation reduces inflammation and fibrosis in experimental models of lung, kidney and heart disease. Here, we tested whether sGC stimulation is also effective in experimental NASH.. Mice with NASH showed reduced cGMP levels and sGC expression, increased steatosis, inflammation, fibrosis, TNF-α and MCP-1 levels and up-regulated collagen types I α1 and α2, MMP2, TGF-β1 and tissue metallopeptidase inhibitor 1 expression. IW-1973 restored hepatic cGMP levels and sGC expression resulting in a dose-dependent reduction of hepatic inflammation and fibrosis. IW-1973 levels were ≈40-fold higher in liver tissue than in plasma. IW-1973 also reduced hepatic steatosis and adipocyte hypertrophy secondary to enhanced autophagy in HFD-induced obese mice.. Our data indicate that sGC stimulation prevents hepatic steatosis, inflammation and fibrosis in experimental NASH. These findings warrant further evaluation of IW-1973 in the clinical setting.

Topics: Animals; Chromatography, Liquid; Cyclic GMP; Cytokines; Diet, High-Fat; Disease Models, Animal; Dose-Response Relationship, Drug; Inflammation; Liver Cirrhosis; Male; Mice; Mice, Inbred C57BL; Non-alcoholic Fatty Liver Disease; Polymerase Chain Reaction; Soluble Guanylyl Cyclase; Tandem Mass Spectrometry

Hyperammonemia is a main contributor to cognitive impairment and motor in-coordination in patients with hepatic encephalopathy. Hyperammonemia-induced neuroinflammation mediates the neurological alterations in hepatic encephalopathy. Intracerebral administration of extracellular cGMP restores some but not all types of cognitive impairment. Motor in-coordination, is mainly due to increased GABAergic tone in cerebellum. We hypothesized that extracellular cGMP would restore motor coordination in hyperammonemic rats by normalizing GABAergic tone in cerebellum and that this would be mediated by reduction of neuroinflammation. The aims of this work were to assess whether chronic intracerebral administration of cGMP to hyperammonemic rats: 1) restores motor coordination; 2) reduces neuroinflammation in cerebellum; 3) reduces extracellular GABA levels and GABAergic tone in cerebellum; and also 4) to provide some advance in the understanding on the molecular mechanisms involved. The results reported show that rats with chronic hyperammonemia show neuroinflammation in cerebellum, including microglia and astrocytes activation and increased levels of IL-1b and TNFa and increased membrane expression of the TNFa receptor. This is associated with increased glutaminase expression and extracellular glutamate, increased amount of the GABA transporter GAT-3 in activated astrocytes, increased extracellular GABA in cerebellum and motor in-coordination. Chronic intracerebral administration of extracellular cGMP to rats with chronic hyperammonemia reduces neuroinflammation, including microglia and astrocytes activation and membrane expression of the TNFa receptor. This is associated with reduced nuclear NF-κB, glutaminase expression and extracellular glutamate, reduced amount of the GABA transporter GAT-3 in activated astrocytes and reduced extracellular GABA in cerebellum and restoration of motor coordination. The data support that extracellular cGMP restores motor coordination in hyperammonemic rats by reducing microglia activation and neuroinflammation, leading to normalization of extracellular glutamate and GABA levels in cerebellum and of motor coordination.

Topics: Animals; Astrocytes; Bicuculline; Cerebellum; Cyclic GMP; GABA-A Receptor Antagonists; gamma-Aminobutyric Acid; Glutaminase; Hyperammonemia; Inflammation; Male; Microglia; Motor Skills; Rats; Rats, Wistar

Aircraft noise causes endothelial dysfunction, oxidative stress, and inflammation. Transportation noise increases the incidence of coronary artery disease, hypertension, and stroke. The underlying mechanisms are not well understood. Herein, we investigated effects of phagocyte-type NADPH oxidase (Nox2) knockout and different noise protocols (around-the-clock, sleep/awake phase noise) on vascular and cerebral complications in mice.. C57BL/6j and Nox2-/- (gp91phox-/-) mice were exposed to aircraft noise (maximum sound level of 85 dB(A), average sound pressure level of 72 dB(A)) around-the-clock or during sleep/awake phases for 1, 2, and 4 days. Adverse effects of around-the-clock noise on the vasculature and brain were mostly prevented by Nox2 deficiency. Around-the-clock aircraft noise of the mice caused the most pronounced vascular effects and dysregulation of Foxo3/circadian clock as revealed by next generation sequencing (NGS), suggesting impaired sleep quality in exposed mice. Accordingly, sleep but not awake phase noise caused increased blood pressure, endothelial dysfunction, increased markers of vascular/systemic oxidative stress, and inflammation. Noise also caused cerebral oxidative stress and inflammation, endothelial and neuronal nitric oxide synthase (e/nNOS) uncoupling, nNOS mRNA and protein down-regulation, and Nox2 activation. NGS revealed similarities in adverse gene regulation between around-the-clock and sleep phase noise. In patients with established coronary artery disease, night-time aircraft noise increased oxidative stress, and inflammation biomarkers in serum.. Aircraft noise increases vascular and cerebral oxidative stress via Nox2. Sleep deprivation and/or fragmentation caused by noise triggers vascular dysfunction. Thus, preventive measures that reduce night-time aircraft noise are warranted.

Topics: Aircraft; Animals; Brain; Circadian Clocks; Cyclic GMP; Endothelium, Vascular; Gene Expression Regulation; Hemodynamics; Humans; Inflammation; Mice, Inbred C57BL; Mice, Knockout; Models, Animal; NADPH Oxidase 2; Nitric Oxide Synthase Type I; Noise, Transportation; Oxidative Stress; Signal Transduction; Sleep Deprivation

The recruitment of bone marrow (BM)-derived progenitor cells to the lung is related to pulmonary remodelling and the pathogenesis of pulmonary hypertension (PH). Although sildenafil is a known target in PH treatment, the underlying molecular mechanism is still elusive. To test the hypothesis that the therapeutic effect of sildenafil is linked to the reduced recruitment of BM-derived progenitor cells, we induced pulmonary remodelling in rats by two-week exposure to chronic hypoxia (CH, 10% oxygen), a trigger of BM-derived progenitor cells. Rats were treated with either placebo (saline) or sildenafil (1.4 mg/kg/day ip) during CH. Control rats were kept in room air (21% oxygen) with no treatment. As expected, sildenafil attenuated the CH-induced increase in right ventricular systolic pressure and right ventricular hypertrophy. However, sildenafil suppressed the CH-induced increase in c-kit

Topics: Animals; Blood Gas Analysis; Bone Marrow Cells; Cell Hypoxia; Cyclic GMP; Inflammation; Lung; Male; Muscles; Proto-Oncogene Proteins c-kit; Rats, Sprague-Dawley; Receptors, CXCR4; Sildenafil Citrate; Stem Cells; Vascular Endothelial Growth Factor Receptor-2

Current worldwide figures suggest that obesity is pandemic. Understanding the underlying molecular mechanisms would help develop viable anti-obesity therapies. Here, we assess the influence of obesity-induced inflammation on white adipocyte cyclic guanosine monophosphate (cGMP) signaling, which is beneficial for adipocyte differentiation and thermogenesis. We find that murine gonadal and not inguinal fat is prone to obesity-induced inflammation. Correspondingly, the cGMP cascade is dysregulated in gonadal but not in inguinal fat of obese mice. Analysis of two independent human cohorts reveals a defective cGMP pathway only in visceral fat of obese subjects. Congruently, cGMP signaling is dysregulated in tumor necrosis factor α (TNF-α)-treated primary white adipocytes. TNF-α-mediated suppression of sGCβ

Topics: Adipogenesis; Adipose Tissue, White; Animals; Cell Differentiation; Cells, Cultured; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Down-Regulation; Female; Gonads; Guanylate Cyclase; Humans; Inflammation; Inguinal Canal; Male; MAP Kinase Signaling System; Mice, Inbred C57BL; Mice, Obese; Models, Biological; NF-kappa B; Obesity; Phenotype; Signal Transduction; Solubility; Tumor Necrosis Factor-alpha

Traumatic injury or the pathogenesis of some neurological disorders is accompanied by inflammatory cellular mechanisms, mainly resulting from the activation of central nervous system (CNS) resident microglia. Under inflammatory conditions, microglia up-regulate the inducible isoform of NOS (iNOS), leading to the production of high concentrations of the radical molecule nitric oxide (NO). At the onset of inflammation, high levels of microglial-derived NO may serve as a cellular defense mechanism helping to clear the damaged tissue and combat infection of the CNS by invading pathogens. However, the excessive overproduction of NO by activated microglia has been suggested to govern the inflammation-mediated neuronal loss causing eventually complete neurodegeneration. Here, we investigated how NO influences phagocytosis of neuronal debris by BV-2 microglia, and how neurite outgrowth of human NT2 model neurons is affected by microglial-derived NO. The presence of NO greatly increased microglial phagocytic capacity in a model of acute inflammation comprising lipopolysaccharide (LPS)-activated microglia and apoptotic neurons. Chemical manipulations suggested that NO up-regulates phagocytosis independently of the sGC/cGMP pathway. Using a transwell system, we showed that reactive microglia inhibit neurite outgrowth of human neurons via the generation of large amounts of NO over effective distances in the millimeter range. Application of a NOS blocker prevented the LPS-induced NO production, totally reversed the inhibitory effect of microglia on neurite outgrowth, but reduced the engulfment of neuronal debris. Our results indicate that a rather simple notion of treating excessive inflammation in the CNS by NO synthesis blocking agents has to consider functionally antagonistic microglial cell responses during pharmaceutic therapy.

Topics: Animals; Apoptosis; Cell Line; Coculture Techniques; Cyclic GMP; Guanylate Cyclase; Humans; Inflammation; Lipopolysaccharides; Mice; Microglia; Neurites; Neuroimmunomodulation; Neuronal Outgrowth; Nitric Oxide; Phagocytes; Phagocytosis; Signal Transduction

In the present study, the effect and mechanism of action of the flavonoid naringenin were evaluated in superoxide anion donor (KO2)-induced inflammatory pain in mice. Naringenin reduced KO2-induced overt-pain like behavior, mechanical hyperalgesia, and thermal hyperalgesia. The analgesic effect of naringenin depended on the activation of the NO-cGMP-PKG-ATP-sensitive potassium channel (KATP) signaling pathway. Naringenin also reduced KO2-induced neutrophil recruitment (myeloperoxidase activity), tissue oxidative stress, and cytokine production. Furthermore, naringenin downregulated KO2-induced mRNA expression of gp91phox, cyclooxygenase (COX)-2, and preproendothelin-1. Besides, naringenin upregulated KO2-reduced nuclear factor (erythroid-derived 2)-like 2 (Nrf2) mRNA expression coupled with enhanced heme oxygenase (HO-1) mRNA expression. In conclusion, the present study demonstrates that the use of naringenin represents a potential therapeutic approach reducing superoxide anion-driven inflammatory pain. The antinociceptive, anti-inflammatory and antioxidant effects are mediated via activation of the NO-cGMP-PKG-KATP channel signaling involving the induction of Nrf2/HO-1 pathway.

Topics: Animals; Behavior, Animal; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Cytokines; Flavanones; Inflammation; Male; Mice; NF-E2-Related Factor 2; Nitric Oxide; Oxidative Stress; Pain; Potassium Channels; Signal Transduction; Superoxides

Patients with hepatic encephalopathy (HE) show working memory and visuo-spatial orientation deficits. Hyperammonemia is a main contributor to cognitive impairment in HE. Hyperammonemic rats show impaired spatial learning and learning ability in the Y maze. Intracerebral administration of extracellular cGMP restores learning in the Y-maze. The underlying mechanisms remain unknown. It also remains unknown whether extracellular cGMP improves neuroinflammation or restores spatial learning in hyperammonemic rats and if it affects differently reference and working memory. The aims of this work were: Spatial working and reference memory were assessed using the radial and Morris water mazes and neuroinflammation by immunohistochemistry and Western blot. Membrane expression of NMDA and AMPA receptor subunits was analyzed using the BS3 crosslinker. Extracellular cGMP was administered intracerebrally using osmotic minipumps. Chronic hyperammonemia induces neuroinflammation in hippocampus, with astrocytes activation and increased IL-1β, which are associated with increased NMDA receptors membrane expression and impaired working memory. This process is not affected by extracellular cGMP. Hyperammonemia also activates microglia and increases TNF-α, alters membrane expression of AMPA receptor subunits (increased GluA1 and reduced GluA2) and impairs reference memory. All these changes are reversed by extracellular cGMP. These results show that extracellular cGMP modulates spatial reference memory but not working memory. This would be mediated by modulation of TNF-α levels and of membrane expression of GluA1 and GluA2 subunits of AMPA receptors.

Topics: Animals; Cognitive Dysfunction; Cyclic GMP; Disease Models, Animal; Hippocampus; Hyperammonemia; Inflammation; Interleukin-1beta; Male; Memory, Short-Term; Rats; Rats, Wistar; Receptors, AMPA; Receptors, N-Methyl-D-Aspartate; Spatial Memory; Tumor Necrosis Factor-alpha

Peripheral inflammation contributes to the neurological alterations in hepatic encephalopathy (HE). Neuroinflammation and altered GABAergic neurotransmission mediate cognitive and motor alterations in rats with HE. It remains unclear (a) if neuroinflammation and neurological impairment in HE are a consequence of peripheral inflammation and (b) how neuroinflammation impairs GABAergic neurotransmission. The aims were to assess in rats with HE whether reducing peripheral inflammation with anti-TNF-α (1) prevents cognitive impairment and motor in-coordination, (2) normalizes neuroinflammation and extracellular GABA in the cerebellum and also (3) advances the understanding of mechanisms linking neuroinflammation and increased extracellular GABA.. Rats with HE due to portacaval shunt (PCS) were treated with infliximab. Astrocytes and microglia activation and TNF-α and IL-1β were analyzed by immunohistochemistry. Membrane expression of the GABA transporters GAT-3 and GAT-1 was analyzed by cross-linking with BS3. Extracellular GABA was analyzed by microdialysis. Motor coordination was tested using the beam walking and learning ability using the Y maze task.. PCS rats show peripheral inflammation, activated astrocytes, and microglia and increased levels of TNF-α and IL-1β. Membrane expression of GAT-3 and extracellular GABA are increased, leading to impaired motor coordination and learning ability. Infliximab reduces peripheral inflammation, microglia, and astrocyte activation and neuroinflammation and normalizes GABAergic neurotransmission, motor coordination, and learning ability.. Neuroinflammation is associated with altered GABAergic neurotransmission and increased GAT-3 membrane expression and extracellular GABA (a); peripheral inflammation is a main contributor to the impairment of motor coordination and of the ability to learn the Y maze task in PCS rats (b); and reducing peripheral inflammation using safe procedures could be a new therapeutic approach to improve cognitive and motor function in patients with HE

Topics: Animals; Anti-Inflammatory Agents; Cerebellum; Cyclic GMP; Cytokines; Dinoprostone; Disease Models, Animal; Extracellular Fluid; GABA Plasma Membrane Transport Proteins; gamma-Aminobutyric Acid; Glial Fibrillary Acidic Protein; Hepatic Encephalopathy; Inflammation; Infliximab; Learning Disabilities; Male; Maze Learning; Psychomotor Disorders; Rats; Rats, Wistar

Activators of soluble guanylyl cyclase (sGC) act preferentially in conditions of enzyme oxidation or haem group removal. This study was designed to investigate the effects of the sGC activator BAY 60-2770 in murine airways inflammation and human eosinophil chemotaxis.. C57Bl/6 mice treated or not with BAY 60-2770 (1 mg/kg/day, 14 days) were intranasally challenged with ovalbumin (OVA). At 48 h, bronchoalveolar lavage fluid (BALF) was performed, and circulating blood, bone marrow and lungs were obtained. Human eosinophils purified from peripheral blood were used to evaluate the cell chemotaxis.. OVA-challenge promoted marked increases in eosinophil number in BAL, lung tissue, circulating blood and bone marrow, all of which were significantly reduced by BAY 60-2770. The IL-4 and IL-5 levels in BALF were significantly reduced by BAY 60-2770. Increased protein expression of iNOS, along with decreases of expression of sGC (α1 and β1 subunits) and cGMP levels were detected in lung tissue of OVA-challenged mice. BAY 60-2770 fully restored to baseline the iNOS and sGC subunit expressions, and cGMP levels. In human isolated eosinophils, BAY 60-2770 (1-5 μM) had no effects on the cGMP levels and eotaxin-induced chemotaxis; however, prior incubation with ODQ (10 μM) markedly elevated the BAY 60-2770-induced cyclic GMP production, further inhibiting the eosinophil chemotaxis.. BAY 60-2770 reduces airway eosinophilic inflammation and rescue the sGC levels. In human eosinophils under oxidized conditions, BAY 60-2770 elevates the cGMP levels causing cell chemotaxis inhibition. BAY 60-2770 may reveal a novel therapeutic target for asthma treatment.

Topics: Animals; Anti-Asthmatic Agents; Asthma; Benzoates; Biphenyl Compounds; Bronchoalveolar Lavage Fluid; Chemotaxis; Cyclic GMP; Disease Models, Animal; Eosinophils; Humans; Hydrocarbons, Fluorinated; Inflammation; Male; Mice; Mice, Inbred C57BL; Ovalbumin; Soluble Guanylyl Cyclase

We hypothesized that direct AT2R stimulation improves albuminuria in diabetes by preventing renal inflammation and improving oxidative stress. Normoglycemic controls (NCs) and streptozotocin-induced diabetes Sprague-Dawley rats (DM) were treated for 4 weeks with vehicle (V) or the AT2R agonist Compound 21 (C21). At the end of study, we evaluated blood pressure, urinary albumin to creatinine ratio (UACR), renal interstitial fluid (RIF) levels of tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), nitric oxide (NO), cGMP, and 8-isoprostane, and renal expression of TNF-α, IL-6, and AT2R. There were no significant differences in blood pressure between different treatments. DM rats demonstrated increased UACR, RIF TNF-α, IL-6 and 8-isoprostane, and messenger RNA (mRNA) for TNF-α and IL-6. DM rats also had reduced RIF NO and cGMP. C21 treatment of DM rats limited the increase in UACR, normalized RIF TNF-α, IL-6 and 8-isoprostane, and in mRNA for TNF-α and IL-6, and increased RIF NO and cGMP. In NC rats, C21 treatment did not change these parameters. AT2R mRNA and protein expressions increased in DM rats compared with NC but were not influenced by C21 treatment. We conclude that direct AT2R stimulation in diabetic rats improves diabetic albuminuria through the prevention of renal inflammation and improved production of NO and cGMP.

Topics: Animals; Blood Pressure; Cyclic GMP; Diabetes Mellitus, Experimental; Dinoprost; Inflammation; Interleukin-6; Kidney; Rats; Rats, Sprague-Dawley; Receptor, Angiotensin, Type 2; Treatment Outcome; Tumor Necrosis Factor-alpha

LCZ696, an angiotensin receptor-neprilysin inhibitor, has recently been demonstrated to exert more beneficial effects on hypertensive or heart failure patients than conventional renin-angiotensin system blockers. However, the mechanism underlying the benefit of LCZ696 remains to be understood. The present study was undertaken to examine the effect of LCZ696 compared with valsartan on hypertension and cardiovascular injury.. (i) Using telemetry, we compared the hypotensive effect of LCZ696 and valsartan in spontaneously hypertensive rats (SHR) that were fed a high-salt diet followed by a low-salt diet. (ii) We also examined the comparative effect of LCZ696 and valsartan on salt loaded SHRcp, a model of metabolic syndrome.. (i) LCZ696 exerted a greater blood pressure (BP) lowering effect than valsartan in SHR regardless of high-salt or low-salt intake. Additive BP reduction by LCZ696 was associated with a significant increase in urinary sodium excretion and sympathetic activity suppression. (ii) LCZ696 significantly ameliorated cardiac hypertrophy and inflammation, coronary arterial remodeling, and vascular endothelial dysfunction in high-salt loaded SHRcp compared with valsartan.. LCZ696 caused greater BP reduction than valsartan in SHR regardless of the degree of salt intake, which was associated with a significant enhancement in urinary sodium excretion and sympathetic activity suppression. Furthermore, an additive BP lowering effect of LCZ696 led to greater cardiovascular protection in hypertensive rats.

Topics: Aminobutyrates; Angiotensin Receptor Antagonists; Animals; Biphenyl Compounds; Blood Pressure; Cardiomegaly; Circadian Rhythm; Cyclic GMP; Drug Combinations; Drug Evaluation, Preclinical; Endothelium, Vascular; Fibrosis; Heart; Hypertension; Inflammation; Male; Myocardium; Neprilysin; Oxidative Stress; Random Allocation; Rats, Inbred SHR; Sodium, Dietary; Tetrazoles; Valsartan; Vascular Remodeling

Pattern recognition receptor nucleotide-binding oligomerization domain 2 (NOD2) is well investigated in immunity, but its expression and function in platelets has never been explored.. Using reverse transcription polymerase chain reaction and Western blot, we show that both human and mouse platelets express NOD2, and its agonist muramyl dipeptide induced NOD2 activation as evidenced by receptor dimerization. NOD2 activation potentiates platelet aggregation and secretion induced by low concentrations of thrombin or collagen, and clot retraction, as well. These potentiating effects of muramyl dipeptide were not seen in platelets from NOD2-deficient mice. Plasma from septic patients also potentiates platelet aggregation induced by thrombin or collagen NOD2 dependently. Using intravital microscopy, we found that muramyl dipeptide administration accelerated in vivo thrombosis in a FeCl3-injured mesenteric arteriole thrombosis mouse model. Platelet depletion and transfusion experiments confirmed that NOD2 from platelets contributes to the in vivo thrombosis in mice. NOD2 activation also accelerates platelet-dependent hemostasis. We further found that platelets express receptor-interacting protein 2, and provided evidence suggesting that mitogen activated-protein kinase and nitric oxide/soluble guanylyl cyclase/cGMP/protein kinase G pathways downstream of receptor-interacting protein mediate the role of NOD2 in platelets. Finally, muramyl dipeptide stimulates proinflammatory cytokine interleukin-1β maturation and accumulation in human and mouse platelets NOD2 dependently.. NOD2 is expressed in platelets and functions in platelet activation and arterial thrombosis, possibly during infection. To our knowledge, this is the first study on NOD-like receptors in platelets that link thrombotic events to inflammation.

Topics: Acetylmuramyl-Alanyl-Isoglutamine; Animals; Bacteremia; Blood Platelets; Clot Retraction; Cyclic GMP; Dimerization; Hemostasis; Humans; Inflammation; Interleukin-1beta; MAP Kinase Signaling System; Mice; Mice, Inbred NOD; Nitric Oxide; Nod2 Signaling Adaptor Protein; Platelet Activation; Receptor-Interacting Protein Serine-Threonine Kinase 2; Receptor-Interacting Protein Serine-Threonine Kinases; Signal Transduction; Thrombosis

Although recent studies have underscored the role of the heme-oxygenase (HO) inducer hemin, on insulin-signaling and glucose metabolism, the underlying mechanisms are not completely understood. In this study, two-dimensional-gel electrophoresis, massspectrometry and MSACOT-analyses were used to identify and characterize novel proteins modulated by hemin in spontaneoushypertensive rat (SHR), a model of essential hypertension with insulin resistance/impaired glucose metabolism. In addition, the effects of hemin on endothelin-1 (ET-1), protein-tyrosine-phosphatase-1B (PTP-1B), atrial-natriuretic-peptide (ANP) and its surrogate-marker urinary cGMP, and inflammatory cytokines including TNF-α, IL-6 and IL-1β were investigated. In hemin-treated SHR, several proteins related to oxidative-stress and metabolism were modulated. Particularly, hemin enhanced aldolase- B, fumarylacetoacetate hydrolase, purine-nucleoside phosphorylase, adenosine-kinase, argininosuccinate synthetase and carbonic anhydrase-3 all of which are enzymes involved in glucose/energy metabolism and pH homeostasis. Similarly, hemin potentiated antioxidant pathways including, NADP(+)-dependant isocitrate-dehydrogenase, catalase, glutathione-S-transferase-Yb1 and hsp70, a pleiotropic agent that regulates protein-folding, oxidative/pro-inflammatory events. Hemin also increased enzymes implicated in cell-growth such as the nitrilase-protein-family, but reduced betaine-homocysteine methyltransferase, an enzyme associated with insulin resistance and dysfunctional glucose metabolism. Furthermore, hemin increased ANP and its surrogate marker, urinary cGMP, but reduced ET-1, PTP-1B, TNF-α, IL-6, IL-1β, whereas the HO-inhibitor, chromium-mesoporphyrin abolished the effects. The potentiation of ANP, urinary-cGMP, aldolade-B, fumarylacetoacetate hydrolase, purine-nucleoside phosphorylase, adenosine-kinase, argininosuccinate synthetase, carbonic anhydrase-3, hsp70 and the corresponding reduction of betaine-homocysteine methyltransferase, PTP-1B, TNF-α, IL-6, IL-1β, and ET-1 may be responsible for the improved glucose metabolism in hemin-treated animals. Collectively, these findings underscore the pleiotropic effects of the HO-system in cellular homeostasis with important roles in metabolism and defence.

Topics: Animals; Cyclic GMP; Cytokines; Electrophoresis, Gel, Two-Dimensional; Endothelin-1; Essential Hypertension; Female; Glucose; Heme Oxygenase (Decyclizing); Hemin; Hypertension; Inflammation; Inflammation Mediators; Insulin Resistance; Male; Mass Spectrometry; Oxidative Stress; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Rats, Sprague-Dawley

Recent publications have shed new light on the role of the adaptive and innate immune system in the pathogenesis of hypertension. However, there are limited data whether receptors of the innate immune system may influence blood pressure. Toll-like receptor 4 (TLR4), a pattern recognition receptor, is a key component of the innate immune system, which is activated by exogenous and endogenous ligands. Hypertension is associated with end-organ damage and thus might lead to the release of damage-associated molecular patterns (DAMPs), which are endogenous activators of TLR4 receptors. The present study aimed to elucidate whether TLR4 signalling is able to modulate vascular contractility in an experimental model of hypertension thus contributing to blood pressure regulation.. NG-nitro-l-arginine methyl ester (l-NAME)-induced hypertension was blunted in TLR4(-/-) when compared with wild-type mice. Treatment with l-NAME was associated with a release of DAMPs, leading to reactive oxygen species production of smooth muscle cells in a TLR4-dependent manner. As oxidative stress leads to an impaired function of the NO-sGC-cyclic GMP (cGMP) pathway, we were able to demonstrate that TLR4(-/-) was protected from sGC inactivation. Consequently, arterial contractility was reduced in TLR4(-/-).. Cell damage-associated TLR4 signalling might act as a direct mediator of vascular contractility providing a molecular link between inflammation and hypertension.

Topics: Animals; Blood Vessels; Cyclic GMP; Hypertension; Inflammation; Mice; Mice, Inbred C57BL; NG-Nitroarginine Methyl Ester; Signal Transduction; Toll-Like Receptor 4

The objective of this study was to examine whether genetically determined differences in the guanylyl cyclase/natriuretic peptide receptor-A gene (Npr1) affect cardiac expression of proinflammatory cytokines, hypertrophic markers, nuclear factor-κB (NF-κB), and activating protein-1 (AP-1) in am Npr1 gene-dose-dependent manner. In the present studies, adult male Npr1 gene-disrupted (Npr1(-/-)), wild-type (Npr1(+/+)), and gene-duplicated (Npr1(++/++)) mice were used. The Npr1(-/-) mice showed 41 mm Hg higher systolic blood pressure and 60% greater heart weight to body weight (HW/BW) ratio; however, Npr1(++/++) mice exhibited 15 mm Hg lower systolic blood pressure and 12% reduced HW/BW ratio compared with Npr1(+/+) mice. Significant upregulation of gene expression of proinflammatory cytokines and hypertrophic markers along with enhanced NF-κB/AP-1 binding activities were observed in the Npr1(-/-) mouse hearts. Conversely, hypertrophic markers and proinflammatory cytokines gene expression as well as NF-κB/AP-1 binding activities were markedly decreased in Npr1(++/++) mouse hearts compared with wild-type mice. The ventricular guanylyl cyclase activity and cGMP levels were reduced by 96% and 87%, respectively, in Npr1(-/-) mice; however, these parameters were amplified by 2.8-fold and 3.8-fold, respectively, in Npr1(++/++) mice. Echocardiographic analysis revealed significantly increased fractional shortening in Npr1(++/++) mice (P < .05) but greatly decreased in Npr1(-/-) mice (P < .01) hearts compared with Npr1(+/+) mice. The present findings suggest that Npr1 represses the expression of cardiac proinflammatory mediators, hypertrophic markers, and NF-κB/AP-1-mediated mechanisms, which seem to be associated in an Npr1 gene-dose-dependent manner.

Topics: Animals; Body Weight; Cell Nucleus; Cyclic GMP; Cytokines; Cytosol; Fibrosis; Guanylate Cyclase; Heart; Heart Ventricles; Hypertrophy; Inflammation; Male; Mice; Mice, Transgenic; Myocardium; NF-kappa B; Organ Size; Receptors, Atrial Natriuretic Factor; Systole; Transcription Factor AP-1

The best conceivable treatment for hypoxia-ischemia (HI) is the restoration of blood flow to the hypoxic-ischemic region(s). Our objective was to examine whether boosting NO-cGMP signaling using sildenafil citrate, a phosphodiesterase-type 5 inhibitor, could modify cerebral blood flow and reduce lesions in the developing brain.. HI was induced in P7 Sprague-Dawley rats by unilateral carotid artery occlusion and hypoxia, and followed by either PBS or sildenafil. Blood-flow velocities were measured by ultrasound imaging with sequential Doppler recordings to evaluate collateral recruitment. Cell death, blood-brain barrier integrity, and glial activation were analyzed by immunohistochemistry. Motor behavior was evaluated using an open-field device adapted to neonatal animals.. Sildenafil citrate (10 mg/kg) induced collateral patency, reduced terminal dUTP nick-end labeling-positive cells, reactive astrogliosis, and macrophage/microglial activation at 72 hours and 7 days post-HI. Sildenafil also reduced the number of terminal dUTP nick-end labeling-positive endothelial cells within lesion site. Seven days after HI and sildenafil treatment, tissue loss was significantly reduced, and animals recovered motor coordination.. Our findings strongly indicate that sildenafil citrate treatment, associated with a significant increase in cerebral blood flow, reduces HI damage and improves motor locomotion in neonatal rats. Sildenafil may represent an interesting therapeutic strategy for neonatal neuroprotection.

Topics: Animals; Animals, Newborn; Blood Gas Analysis; Blood Pressure; Cell Death; Cerebrovascular Circulation; Cyclic GMP; Functional Laterality; Hypoxia-Ischemia, Brain; Immunohistochemistry; In Situ Nick-End Labeling; Inflammation; Macrophage Activation; Microcirculation; Motor Activity; Neuroglia; Neuroprotective Agents; Nitric Oxide; Phosphodiesterase Inhibitors; Piperazines; Psychomotor Performance; Purines; Rats; Sildenafil Citrate; Sulfones

Microglia are the resident immune cells of the brain, which become rapidly activated and migrate to the site of insult in brain infection and disease. Activated microglia generate large amounts of the highly reactive messenger molecule nitric oxide (NO). NO is able to raise cyclic GMP levels via binding to soluble guanylyl cyclase. We investigated potential mechanistic links between inflammation, NO signaling, and microglial migration. To monitor cell migration, we used a scratch wound assay and compared results obtained in the BV-2 microglial line to primary microglia. Incubation with lipopolysaccharide (LPS) as stimulator of acute inflammatory processes enhanced migration of both microglial cell types. LPS activated NO production in BV-2 cells and application of an NO donor increased BV-2 cell migration while an NO scavenger reduced motility. Pharmacological inhibition of soluble guanylyl cyclase and the resulting decrease in motility can be rescued by a membrane permeant analog of cGMP. Despite differences in the threshold towards stimulation with the chemical agents, both BV-2 cells and primary microglia react in a similar way. The important role of NO/cGMP as positive regulator of microglial migration, the downstream targets of the signaling cascade, and resulting cytoskeletal changes can be conveniently investigated in a microglial cell line.

Topics: Animals; Cell Line; Cell Movement; Cyclic GMP; Inflammation; Microglia; Nitric Oxide; Rats; Rats, Sprague-Dawley; Signal Transduction

Nitric oxide (NO) is the main activator of the soluble guanylate cyclase (sGC)-guanosine 3'5' cyclic monophosphate (cGMP) pathway. The level of cGMP is regulated by phosphodiesterases (PDEs), which break down cGMP. It has been reported that levels of NO in the central nervous system (CNS) can greatly increase during demyelination and/or neuroinflammation. Controversially, in demyelination models, mice without iNOS may develop more severe cases of disease. Furthermore, cGMP accumulation caused by PDE inhibitors has an anti-inflammatory/neuroprotective effect in MS-models. The role of the NO-cGMP pathway in the nervous tissue is, therefore, complex and not fully understood. The aim of the present study was to contribute to existing knowledge of the role of this pathway in the CNS. Wild type (WT - C57BL/6) and iNOS(-/-) animals were treated with sildenafil (25mg/kg) for 8 weeks. Control animals were not treated. VCAM and ICAM (adhesion proteins), GFAP and Iba-1 (astrocyte and microglia markers, respectively), PKG (cGMP-dependent protein kinase), sGC, eNOS (constitutive endothelial NO sinthase) and GSTpi (a marker of mature oligodendrocytes) were evaluated in the cerebellum using immunohistochemistry or western blotting. Myelin was assessed by luxol fast blue staining and electron transmission microscopy. Treatment with sildenafil reduced ICAM and VCAM levels (anti-inflammatory effect) and increased GFAP and Iba-1 expression (clearance phenotype) in WT animals. The expression of VCAM, ICAM, GFAP, PKG and sGC was lower in iNOS(-/-) mice than in WT control animals. The treatment of iNOS(-/-) animals with sildenafil resulted in an increase of all proteins (pro-inflammatory effect). There was overexpression of eNOS in untreated iNOS(-/-) mice. The myelin structure of iNOS(-/-) animals was damaged in comparison with WT control. Sildenafil increased GSTpi and resulted in an improved myelin structure in iNOS(-/-) mice. In conclusion, NO-cGMP signaling plays a role in the regulation of inflammation and myelination processes. The accumulation of cGMP produced opposite effects in WT and iNOS(-/-) mice. This can be explained by the overexpression of eNOS in iNOS(-/-) mice, unbalancing cGMP signaling, or cGMP has a dual role in inflammation. Drugs that modulate the NO-sGC-cGMP pathway may be clinically beneficial in the treatment of neuroinflammatory/demyelinating disorders, but further studies of the regulation of this pathway are required.

Topics: Animals; Cerebellum; Cyclic GMP; Inflammation; Mice; Mice, Inbred C57BL; Mice, Knockout; Myelin Sheath; Neuroglia; Nitric Oxide; Nitric Oxide Synthase Type II; Signal Transduction

Phosphodiesterase 2A (PDE2A) is an evolutionarily conserved enzyme that catalyzes the degradation of the cyclic nucleotides, cyclic adenosine monophosphate, and/or cyclic guanosine monophosphate. Recent studies reported the expression of PDE2A in the dorsal horn of the spinal cord, pointing to a potential contribution to the processing of pain. However, the functions of PDE2A in spinal pain processing in vivo remained elusive.. Immunohistochemistry, laser microdissection, and quantitative real-time reverse transcription polymerase chain reaction experiments were performed to characterize the localization and regulation of PDE2A protein and messenger RNA in the mouse spinal cord. Effects of the selective PDE2A inhibitor, BAY 60-7550 (Cayman Chemical, Ann Arbor, MI), in animal models of inflammatory pain (n = 6 to 10), neuropathic pain (n = 5 to 6), and after intrathecal injection of cyclic nucleotides (n = 6 to 8) were examined. Also, cyclic adenosine monophosphate and cyclic guanosine monophosphate levels in spinal cord tissues were measured by liquid chromatography tandem mass spectrometry.. The authors here demonstrate that PDE2A is distinctly expressed in neurons of the superficial dorsal horn of the spinal cord, and that its spinal expression is upregulated in response to hind paw inflammation. Administration of the selective PDE2A inhibitor, BAY 60-7550, increased the nociceptive behavior of mice in animal models of inflammatory pain. Moreover, BAY 60-7550 increased the pain hypersensitivity induced by intrathecal delivery of cyclic adenosine monophosphate, but not of cyclic guanosine monophosphate, and it increased the cyclic adenosine monophosphate levels in spinal cord tissues.. Our findings indicate that PDE2A contributes to the processing of inflammatory pain in the spinal cord.

Topics: Animals; Behavior, Animal; Cyclic AMP; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 2; Drug Hypersensitivity; Imidazoles; Immunohistochemistry; Inflammation; Injections, Spinal; Male; Mice; Mice, Inbred C57BL; Microdissection; Neuralgia; Pain; Pain Measurement; Phosphodiesterase Inhibitors; Posterior Horn Cells; Real-Time Polymerase Chain Reaction; Spinal Cord; Triazines; Up-Regulation; Zymosan

Nitric oxide plays an important role in various biological processes including antinociception. The control of its local concentration is crucial for obtaining the desired effect and can be achieved with exogenous nitric oxide-carriers such as ruthenium complexes. Therefore, we evaluated the analgesic effect and mechanism of action of the ruthenium nitric oxide donor [Ru(HEDTA)NO] focusing on the role of cytokines, oxidative stress and activation of the cyclic guanosine monophosphate/protein kinase G/ATP-sensitive potassium channel signaling pathway. It was observed that [Ru(HEDTA)NO] inhibited in a dose-dependent (1-10 mg/kg) manner the acetic acid-induced writhing response. At the dose of 1 mg/kg, [Ru(HEDTA)NO] inhibited the phenyl-p-benzoquinone-induced writhing response, and formalin- and complete Freund's adjuvant-induced licking and flinching responses. Systemic and local treatments with [Ru(HEDTA)NO] also inhibited the carrageenin-induced mechanical hyperalgesia and increase of myeloperoxidase activity in paw skin samples. Mechanistically, [Ru(HEDTA)NO] inhibited carrageenin-induced production of the hyperalgesic cytokines tumor necrosis factor-α and interleukin-1β, and decrease of reduced glutathione levels. Furthermore, the inhibitory effect of [Ru(HEDTA)NO] in the carrageenin-induced hyperalgesia and myeloperoxidase activity was prevented by the treatment with ODQ (soluble guanylyl cyclase inhibitor), KT5823 (protein kinase G inhibitor) and glybenclamide (ATP-sensitive potassium channel inhibitor), indicating that [Ru(HEDTA)NO] inhibits inflammatory hyperalgesia by activating the cyclic guanosine monophosphate/protein kinase G/ATP-sensitive potassium channel signaling pathway, respectively. These results demonstrate that [Ru(HEDTA)NO] exerts its analgesic effect in inflammation by inhibiting pro-nociceptive cytokine production, oxidative imbalance and activation of the nitric oxide/cyclic guanosine monophosphate/protein kinase G/ATP-sensitive potassium channel signaling pathway in mice.

Topics: Animals; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Cytokines; Disease Models, Animal; Dose-Response Relationship, Drug; Edetic Acid; Hyperalgesia; Inflammation; KATP Channels; Male; Mice; Nitric Oxide; Nitric Oxide Donors; Nociception; Oxidative Stress; Ruthenium Compounds; Signal Transduction

Obesity-associated nonalcoholic fatty liver disease (NAFLD), covering from simple steatosis to nonalcoholic steatohepatitis (NASH), is a common cause of chronic liver disease. Aberrant production of adipocytokines seems to play a main role in most obesity-associated disorders. Changes in adipocytokines in obesity could be mediated by alterations in cyclic GMP (cGMP) homeostasis. The aims of this work were: (1) to study the role of altered cGMP homeostasis in altered adipocytokines in morbid obesity, (2) to assess whether these alterations are different in simple steatosis or NASH, and (3) to assess whether these changes reverse in obese patients after bariatric surgery.. In 47 patients with morbid obesity and 45 control subjects, the levels in blood of adipocytokines, cGMP, nitric oxide (NO) metabolites, and atrial natriuretic peptide (ANP) were studied. Whether weight loss after a bariatric surgery reverses the changes in these parameters was evaluated.. NO metabolites and leptin increase (and adiponectin decreases) similarly in patients with steatosis or NASH, suggesting that these changes are due to morbid obesity and not to liver disease. Inflammation and cGMP homeostasis are affected both by morbid obesity and by liver disease. The increases in interleukin 6 (IL-6), interleukin 18 (IL-18), plasma cGMP, ANP, and the decrease in cGMP in lymphocytes are stronger in patients with NASH than with steatosis. All these changes reverse completely after bariatric surgery and weight loss, except IL-18.. Altered cGMP homeostasis seems to contribute more than inflammation to changes in leptin and adiponectin in morbid obesity.

Topics: Adipokines; Adult; Bariatric Surgery; Body Mass Index; Case-Control Studies; Chronic Disease; Cyclic GMP; Fatty Liver; Female; Homeostasis; Humans; Inflammation; Interleukin-18; Interleukin-6; Leptin; Male; Middle Aged; Non-alcoholic Fatty Liver Disease; Obesity, Morbid

The activation of nitric oxide (NO) production is an analgesic mechanism shared by drugs such as morphine and diclofenac. Therefore, the controlled release of low amounts of NO seems to be a promising analgesic approach. In the present study, the antinociceptive effect of the ruthenium NO donor [Ru(bpy)2(NO)SO3](PF6) (complex I) was investigated. It was observed that complex I inhibited in a dose (0.3-10mg/kg)-dependent manner the acetic acid-induced writhing response. At the dose of 1mg/kg, complex I inhibited the phenyl-p-benzoquinone-induced writhing response and formalin- and complete Freund's adjuvant-induced licking and flinch responses. Additionally, complex I also inhibited transient receptor potential cation channel subfamily V member 1 (TRPV1)-dependent overt pain-like behavior induced by capsaicin. Complex I also inhibited the carrageenin-induced mechanical hyperalgesia and increase of myeloperoxidase activity (MPO) in paw skin samples. The inhibitory effect of complex I in the carrageenin-induced hyperalgesia, MPO activity and formalin was prevented by the treatment with ODQ, KT5823 and glybenclamide, indicating that complex I inhibits inflammatory hyperalgesia by activating the cGMP/PKG/ATP-sensitive potassium channel signaling pathway. The present study demonstrates the efficacy of a novel ruthenium NO donor and its analgesic mechanisms.

Topics: Adenosine Triphosphate; Animals; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Electron Transport Complex I; Inflammation; Mice; Nitric Oxide Donors; Pain; Potassium Channels; Ruthenium Compounds; Signal Transduction; TRPV Cation Channels

We recently demonstrated that sildenafil reduces the expression of cytokines, COX-2, and GFAP in a demyelinating model induced in wild-type (WT) mice. Herein, the understandings of the neuroprotective effect of sildenafil and the mediation of iNOS/NO system on inflammatory demyelination induced by cuprizone were investigated. The cerebella of iNOS(-/-) mice were examined after four weeks of treatment with cuprizone alone or combined with sildenafil. Cuprizone increased GFAP, Iba-1, TNF- α , COX-2, IL-1 β , and IFN- γ expression, decreased expression of glutathione S-transferase pi (GSTpi), and damaged myelin in iNOS(-/-) mice. Sildenafil reduced Iba-1, IFN- γ , and IL-1 β levels but had no effect on the expression of GFAP, TNF- α , and COX-2 compared to the cuprizone group. Sildenafil elevated GSTpi levels and improved the myelin structure/ultrastructure. iNOS(-/-) mice suffered from severe inflammation following treatment with cuprizone, while WT mice had milder inflammation, as found in the previous study. It is possible that inflammatory regulation through iNOS-feedback is absent in iNOS(-/-) mice, making them more susceptible to inflammation. Sildenafil has at least a partial anti-inflammatory effect through iNOS inhibition, as its effect on iNOS(-/-) mice was limited. Further studies are required to explain the underlying mechanism of the sildenafil effects.

Topics: Animals; Cuprizone; Cyclic GMP; Demyelinating Diseases; Glutathione S-Transferase pi; Inflammation; Mice; Mice, Knockout; Microscopy, Electron, Transmission; Microscopy, Fluorescence; Myelin Sheath; Neurons; Nitric Oxide; Nitric Oxide Synthase Type II; Phosphodiesterase 5 Inhibitors; Piperazines; Purines; Sildenafil Citrate; Sulfones

Various studies have demonstrated the role of the nitric oxide (NO)/cGMP pathway in pain processing. Our group has also shown that this system participates in opioid-induced antinociception during peripheral inflammation. We have previously observed that inflammation mobilizes an endogenous opioidergic system to control hyperalgesia. Here, we investigated whether the NO/cGMP pathway underlies peripheral endogenous nociception control during inflammation. In this study, a pharmacological approach was used in conjunction with the rat paw pressure test to assess the effects of intraplantar NO synthase inhibitor NG-Nitro-l-arginine (NOArg), guanylyl cyclase inhibitor methylene blue (MB), phosphodiesterase-5 inhibitor zaprinast (ZP), or NO precursor l-arginine injection on carrageenan-induced hyperalgesia, which mimics an inflammatory process, or by prostaglandin E(2) (PGE(2)), which directly sensitizes nociceptors. Intraplantar carrageenan (62.5, 125, 250 or 500μg) or PGE(2) (0.1, 0.5 or 2μg) administration produced hyperalgesia, which manifested as a reduction in the rat nociceptive threshold to mechanical stimuli. NOArg (25, 50 or 100μg/paw) and MB (125, 250 or 500μg/paw) induced significant and dose-dependent reductions in the nociceptive threshold of carrageenan-induced (125μg/paw) hyperalgesia, but not PGE(2)-induced (0.5μg/paw) hyperalgesia. This was a local effect because it did not produce any modifications in the contralateral paw. Both Zaprinast (100, 200 or 400μg/paw) and l-arginine (100, 200 or 400μg/paw) significantly counteracted carrageenan-induced hyperalgesia (250μg/paw), yielding an increase in the nociceptive threshold compared with the control. Zaprinast (200μg/paw) or l-arginine (400μg/paw) did not produce an antinociceptive effect in the contralateral paw, indicating local action. In addition, at the same dose that was able to modify carrageenan-induced hyperalgesia, neither zaprinast nor l-arginine modified PGE(2) (2μg) injection-induced hyperalgesia of the rat paw. Taken together, these results indicate that the l-arginine/NO/cGMP pathway functions as an endogenous modulator of peripheral inflammatory hyperalgesia.

Topics: Animals; Arginine; Carrageenan; Cyclic GMP; Dinoprostone; Hyperalgesia; Inflammation; Male; Nitric Oxide; Pain; Pain Measurement; Pressure; Rats; Rats, Wistar

Cardiovascular disease (CVD) is associated with vascular oxidative imbalance and inflammation. Increased reactive oxygen species (ROS) generation is associated with a functional inactivation of nitric oxide (NO) due to the reaction with O₂⁻, leading to peroxynitrite (ONOO⁻) formation and subsequent reduction in the beneficial effect of vascular NO bioavailability. Carotenoids'-rich diets have been associated with decreased risk of CVD, but the underlying mechanism is still unknown.. In human umbilical vein endothelial cells (HUVECs), both β-carotene (BC) or lycopene (Lyc) significantly affected tumor necrosis factor-α (TNF-α)-induced inflammation, being associated with a significant decrease in the generation of ROS (spectrofluorometry) and nitrotyrosine (an index of ONOO⁻ formation, cytofluorimetry), an increased NO/cGMP (cyclic guanosine monophosphate) levels (EIA), and a down-regulation of NF-κB-dependent adhesion molecule expression (Western blot and EMSA) and monocyte-HUVEC interaction (adhesion assay). Our results indicate that BC or Lyc treatment reduce the inflammatory response in TNF-α-treated HUVECs. This is due to the redox balance protection and to the maintenance of NO bioavailability.. Our observations provide background for a novel mechanism for carotenoids' anti-inflammatory activity in the vasculature and may contribute to a better understanding of the protective effects of carotenoid-rich diets against CVD risk.

Topics: Anti-Inflammatory Agents, Non-Steroidal; beta Carotene; Biological Availability; Carotenoids; Cell Adhesion; Cell Adhesion Molecules; Cyclic GMP; Human Umbilical Vein Endothelial Cells; Humans; Inflammation; Lycopene; Monocytes; NF-kappa B; Nitric Oxide; Oxidative Stress; Reactive Oxygen Species; Tumor Necrosis Factor-alpha; Tyrosine

Nitric oxide (NO) generated by vascular NO synthases can exert anti-inflammatory effects, partly through its ability to decrease leukocyte recruitment. Inorganic nitrate and nitrite, from endogenous or dietary sources, have emerged as alternative substrates for NO formation in mammals. Bioactivation of nitrate is believed to require initial reduction to nitrite by oral commensal bacteria. Here we investigated the effects of inorganic nitrate and nitrite on leukocyte recruitment in microvascular inflammation and in NSAID-induced small-intestinal injury. We show that leukocyte emigration in response to the proinflammatory chemokine MIP-2 is reduced by 70% after 7 days of dietary nitrate supplementation as well as by acute intravenous nitrite administration. Nitrite also reduced leukocyte adhesion to a similar extent and this effect was inhibited by the soluble guanylyl cyclase inhibitor ODQ, whereas the effect on emigrated leukocytes was not altered by this treatment. Further studies in TNF-α-stimulated endothelial cells revealed that nitrite dose-dependently reduced the expression of ICAM-1. In rats and mice subjected to a challenge with diclofenac, dietary nitrate prevented the increase in myeloperoxidase and P-selectin levels in small-intestinal tissue. Antiseptic mouthwash, which eliminates oral nitrate reduction, markedly blunted the protective effect of dietary nitrate on P-selectin levels. Despite attenuation of the acute immune response, the overall ability to clear an infection with Staphylococcus aureus was not suppressed by dietary nitrate as revealed by noninvasive IVIS imaging. We conclude that dietary nitrate markedly reduces leukocyte recruitment to inflammation in a process involving attenuation of P-selectin and ICAM-1 upregulation. Bioactivation of dietary nitrate requires intermediate formation of nitrite by oral nitrate-reducing bacteria and then probably further reduction to NO and other bioactive nitrogen oxides in the tissues.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Cell Adhesion; Cell Movement; Cells, Cultured; Chemokine CXCL2; Cyclic GMP; Diclofenac; Dietary Supplements; Endothelial Cells; Gene Expression; Humans; Inflammation; Intercellular Adhesion Molecule-1; Intestine, Small; Leukocyte Count; Male; Mice; Mice, Inbred C57BL; Microvessels; Mouthwashes; Neutrophil Infiltration; Nitrates; Nitrites; P-Selectin; Peroxidase; Rats; Rats, Sprague-Dawley; Staphylococcal Infections; Staphylococcus aureus

Bipolar disorder (BD) is associated with elevated cardiovascular mortality rates. We investigated the modulation of l-arginine-nitric oxide (NO) signaling in platelets from patients with BD at different phases.. Platelets obtained from 28 patients with BD and 10 healthy volunteers were analyzed for l-arginine transport, NO synthase (NOS) activity, cyclic guanosine monophosphate content, and biomarkers of oxidative stress. Expressions of NOS isoforms, soluble guanylyl cyclase, and arginase were also measured in platelets. Amino acid and C-reactive protein levels in plasma were assessed.. Plasma concentrations of l-arginine (mean [M] ± standard error of the mean [SEM] = 97 ± 10 versus 121 ± 10 µM) and its transport into platelets (median [interquartile range] = 26.0 [28.6] versus 26.5 [43.9] pmol/10(9) cells per minute) did not differ between patients with BD and controls (p > .05). Patients with BD showed reduced NOS activity (M ± SEM = 0.037 ± 0.003 versus 0.135 ± 0.022 pmol/10(8) cells, p < .001), but not endothelial NOS, inducible NOS, and arginase expression, compared with controls (p > .05). Cyclic guanosine monophosphate content was reduced (M ± SEM = 0.022 ± 0.003 versus 0.086 ± 0.020 pmol/10(8) cells, p < .05) despite the absence of changes in soluble guanylyl cyclase expression (median [interquartile range] = 21.6 [15.5] versus 9.5 [9.4] arbitrary units, p > .05) in patients with BD. Superoxide dismutase activity, but not catalase activity, was increased in patients with BD in the manic phase (M ± SEM = 2094 ± 335 versus 172 ± 17 U/mg protein, p < .001). C-reactive protein was elevated only in manic episodes (M ± SEM = 0.8 ± 0.2 versus 0.1 ± 0.02 mg/L, p < .001).. Impaired NO generation from platelets, inflammation, and oxidative stress may play pivotal roles in the multifaceted process of cardiovascular events in BD.

Topics: Adult; Arginase; Arginine; Bipolar Disorder; Blood Platelets; C-Reactive Protein; Cardiovascular Diseases; Case-Control Studies; Comorbidity; Cyclic GMP; Female; Humans; Inflammation; Male; Nitric Oxide; Nitric Oxide Synthase; Oxidative Stress; Signal Transduction; Superoxide Dismutase

Inflammation involves the cooperation of various cells and biologically active molecules. An important intracellular messenger molecule participating in the regulation of the process is cyclic GMP (cGMP), which is synthesized by guanylyl cyclases (GCs). The GC family comprises cytosolic (soluble) and membrane-bound (particulate) enzymes. The aim of this study was to determine whether and how the synthesis of cGMP by various forms of GC affects the expression of inflammatory cytokines depending on the activity of the transcription factors NF-κB (nuclear factor-κB) and AP-1 (activator protein-1). We established that in rat peripheral blood mononuclear cells (PBMCs), synthesis of cGMP was elevated by sodium nitroprusside (SNP), the activator of soluble GC, and by atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP), the activators of particulate GC-A and GC-B, respectively. Stimulation of various GCs differently affected the expressions of the cytokines IL-1β, IL-6, and TNF-α in control cells and in cells activated by bacterial endotoxin (LPS). In control PBMCs their expression was elevated by stimulation of soluble, but not particulate, GC. SNP caused an increase in NF-κB activity, but had no influence on the activity of AP-1. The cells treated with LPS decreased the expressions of IL-1β, IL-6, and TNF-α in response to stimulation of particulate GC-A, but not other guanylyl cyclases. This inhibitory effect was a result of suppression of the activities of NF-κB and AP-1. Both effects that of SNP and of ANP, were cGMP dependent, as shown using its membrane-permeable analog 8-Br-cGMP. The implementation of specific inhibitors showed that the stimulatory effect of SNP was mediated by soluble GC and cGMP-dependent protein kinase (PKG-I). However, PKG-I was not involved in the inhibition of NF-κB and AP-1 activities by ANP in LPS-activated cells. Taken together, these results for the first time indicate that various GCs and various cGMP-dependent signaling pathways can modulate the activity of AP-1 and/or NF-κB and thus affect the expressions of IL-1β, IL-6, and TNF-α, which play important roles in the development of inflammation.

Topics: Animals; Atrial Natriuretic Factor; Blotting, Western; Cyclic GMP; Cyclic GMP-Dependent Protein Kinase Type I; Cyclic GMP-Dependent Protein Kinases; Cytokines; Electrophoretic Mobility Shift Assay; Gene Expression Regulation; Guanylate Cyclase; Inflammation; Leukocytes, Mononuclear; Lipopolysaccharides; Natriuretic Peptide, C-Type; Nitroprusside; Rats; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Solubility; Transcription Factor AP-1

Angiotensin II type 2 receptor (AT₂R) counteracts most effects of angiotensin II type 1 receptor (AT(1)R). We hypothesized that direct AT₂R stimulation reduces renal production of the inflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and transforming growth factor-β1 (TGF-β1) and enhances the production of nitric oxide (NO) and cyclic guanosine 3',5'-monophosphate (cGMP) in the clipped kidney of 2-kidney, 1-clip (2K1C) hypertension rat model. We used Sprague-Dawley rats to evaluate changes in renal interstitial fluid recovery levels of TNF-α, IL-6, NO, and cGMP; renal expression of AT₁R, AT₂R, TGF-β1, TNF-α, and IL-6 in sham and 2K1C rats treated for 4 days with vehicle, AT₂R agonist compound 21 (C21), or AT₂R antagonist PD123319 (PD), alone and combined (n=6, each group). Systolic blood pressure increased significantly in 2K1C and was not influenced by any treatment. Clipped kidneys showed significant increases in renal expression of AT₁R, AT₂R, TNF-α, IL-6, TGF-β1 and decreases in NO and cGMP levels. These factors were not influenced by PD treatment. In contrast, C21 caused significant decrease in renal TNF-α, IL-6, TGF-β1 and an increase in NO and cGMP levels. Combined C21 and PD treatment partially reversed the observed C21 effects. Compared to sham, there were no significant changes in TNF-α, IL-6, TGF-β1, NO, or cGMP in the nonclipped kidneys of 2K1C animals. We conclude that direct AT₂R stimulation reduces early renal inflammatory responses and improves production of NO and cGMP in renovascular hypertension independent of blood pressure reduction.

Topics: Angiotensin II Type 2 Receptor Blockers; Animals; Blood Pressure; Blotting, Western; Body Weight; Cyclic GMP; Gene Expression; Hypertension, Renovascular; Imidazoles; Inflammation; Interleukin-6; Kidney; Male; Nitric Oxide; Organ Size; Pyridines; Rats; Rats, Sprague-Dawley; Receptor, Angiotensin, Type 1; Receptor, Angiotensin, Type 2; Reverse Transcriptase Polymerase Chain Reaction; Sulfonamides; Thiophenes; Transforming Growth Factor beta1; Tumor Necrosis Factor-alpha

Nitric oxide (NO) production occurs through oxidation of the amino acid L-arginine by NO synthase (NOS). NO inhibits platelet activation by increasing the levels of cyclic guanosine monophosphate (cGMP), thus maintaining vascular homeostasis. Our group previously demonstrated (da Silva et al. 2005) an enhancement of the L-arginine-NO-cGMP pathway in platelets taken from chronic renal failure (CRF) patients on haemodialysis associated with reduced platelet aggregation. We investigate the platelet L-arginine-NO-cGMP pathway, platelet function, and inflammation from patients in CRF on conservative treatment. A total of 42 CRF patients and 42 controls (creatinine clearance = 27 ± 3 vs. 93 ± 1 mL per min per 1.73 m2, respectively) participated in this study. NOS activity and expression and cGMP concentration were measured in platelets. Platelet aggregation induced by collagen or ADP was evaluated and plasma levels of fibrinogen were determined by the Clauss method. A marked increase in basal NOS activity was seen in undialysed CRF patients compared with controls, accompanied by an elevation of fibrinogen plasma levels. There were no differences in expression of NOS and in cGMP levels. In this context, platelet aggregation was not affected. We provide the first evidence of increased intraplatelet NO biosynthesis in undialysed CRF patients, which can be an early marker of future haemostatic abnormalities during dialysis treatment.

Topics: Adenosine Diphosphate; Arginine; Blood Platelets; Case-Control Studies; Collagen; Cyclic GMP; Female; Fibrinogen; Humans; Inflammation; Kidney Failure, Chronic; Male; Middle Aged; Nitric Oxide; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Platelet Aggregation

Interleukin 6 (IL-6) and nitric oxide (NO) are important mediators of the inflammatory response. We report that in human peripheral blood mononuclear cells (PBMCs), NO exerts a biphasic effect on the expression of IL-6. Using sodium nitroprusside (SNP) and S-nitrosoglutathione (GSNO) as NO-donating compounds, we observed that both mRNA and protein levels of IL-6 increased at lower (≤10μM) and decreased at higher (>100μM) concentrations of NO donors. Changes in the expression of IL-6 correlated with changes in the activity of NF-κB, which increased at lower and decreased at higher concentrations of both NO donors as shown by the electrophoretic mobility shift assay (EMSA). The effects of NO on NF-κB activity were cGMP-dependent because they were reversed in the presence of ODQ, the inhibitor of soluble guanylyl cyclase (sGC), and KT5823, the inhibitor of cGMP-dependent protein kinase (PKG). Moreover, the membrane permeable analog of cGMP (8-Br-cGMP) mimicked the effect of the NO donors. These observations show that NO, depending on its concentration, may act in human PBMCs as a stimulator of IL-6 expression involving the sGC/cGMP/PKG pathway.

Topics: Carbazoles; Cell Nucleus; Cyclic GMP; Cytokines; Genetic Vectors; Humans; Inflammation; Interleukin-6; Leukocytes, Mononuclear; NF-kappa B; Nitric Oxide; Nitric Oxide Donors; Nitroprusside; S-Nitrosoglutathione

Phosphodiesterase 2A (PDE2A) is stimulated by cGMP to hydrolyze cAMP, a potent endothelial barrier-protective molecule. We previously found that lung PDE2A contributed to a mouse model of ventilator-induced lung injury (VILI). The purpose of the present study was to determine the contribution of PDE2A in a two-hit mouse model of 1-day intratracheal (IT) LPS followed by 4 h of 20 ml/kg tidal volume ventilation. Compared with IT water controls, LPS alone (3.75 μg/g body wt) increased lung PDE2A mRNA and protein expression by 6 h with a persistent increase in protein through day 4 before decreasing to control levels on days 6 and 10. Similar to the PDE2A time course, the peak in bronchoalveolar lavage (BAL) neutrophils, lactate dehydrogenase (LDH), and protein concentration also occurred on day 4 post-LPS. IT LPS (1 day) and VILI caused a threefold increase in lung PDE2A and inducible nitric oxide synthase (iNOS) and a 24-fold increase in BAL neutrophilia. Compared with a control adenovirus, PDE2A knockdown with an adenovirus expressing a short hairpin RNA administered IT 3 days before LPS/VILI effectively decreased lung PDE2A expression and significantly attenuated BAL neutrophilia, LDH, protein, and chemokine levels. PDE2A knockdown also reduced lung iNOS expression by 53%, increased lung cAMP by nearly twofold, and improved survival from 47 to 100%. We conclude that in a mouse model of LPS/VILI, a synergistic increase in lung PDE2A expression increased lung iNOS and alveolar inflammation and contributed significantly to the ensuing acute lung injury.

Topics: Acute Lung Injury; Adenoviridae; Animals; Bronchoalveolar Lavage Fluid; Cyclic AMP; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 2; Inflammation; Lipopolysaccharides; Lung; Male; Mice; Mice, Inbred C57BL; Neutrophils; Nitric Oxide Synthase Type II; Proteins; Pulmonary Alveoli; RNA, Messenger; RNA, Small Interfering; Tidal Volume; Time Factors; Trachea; Ventilator-Induced Lung Injury

Type 17 helper T (Th17) cells are implicated in the pathogenesis many of human autoimmune diseases. Development of Th17 can be enhanced by the activation of aryl hydrocarbon receptor (AHR) whose ligands include the environmental pollutant dioxin, potentially linking environmental factors to the increased prevalence of autoimmune disease. We report here that nitric oxide (NO) can suppress the proliferation and function of polarized murine and human Th17 cells. NO also inhibits AHR expression in Th17 cells and the downstream events of AHR activation, including IL-22, IL-23 receptor, and Cyp1a1. Conversely, NO did not affect the polarization of Th17 cells from mice deficient in AHR. Furthermore, mice lacking inducible nitric oxide synthase (Nos2(-/-)) developed more severe experimental autoimmune encephalomyelitis than WT mice, with elevated AHR expression, increased IL-17A, and IL-22 synthesis. NO may therefore represent an important endogenous regulator to prevent overexpansion of Th17 cells and control of autoimmune diseases caused by environmental pollutants.

Topics: Animals; Autoimmune Diseases; Cell Proliferation; Cyclic GMP; Cytochrome P-450 CYP1A1; Humans; Inflammation; Interleukin-10; Interleukin-2; Interleukin-22; Interleukins; Ligands; Mice; Mice, Inbred BALB C; Models, Biological; Nitric Oxide; Nitric Oxide Synthase Type II; Proto-Oncogene Protein c-ets-1; Receptors, Interleukin; T-Lymphocytes, Helper-Inducer; Th17 Cells

A large body of evidence indicates that nitric oxide (NO) and cGMP contribute to central sensitization of pain pathways during inflammatory pain. Here, we investigated the distribution of cyclic nucleotide-gated (CNG) channels in the spinal cord, and identified the CNG channel subunit CNGA3 as a putative cGMP target in nociceptive processing. In situ hybridization revealed that CNGA3 is localized to inhibitory neurons of the dorsal horn of the spinal cord, whereas its distribution in dorsal root ganglia is restricted to non-neuronal cells. CNGA3 expression is upregulated in the superficial dorsal horn of the mouse spinal cord and in dorsal root ganglia following hindpaw inflammation evoked by zymosan. Mice lacking CNGA3 (CNGA3(-/-) mice) exhibited an increased nociceptive behavior in models of inflammatory pain, whereas their behavior in models of acute or neuropathic pain was normal. Moreover, CNGA3(-/-) mice developed an exaggerated pain hypersensitivity induced by intrathecal administration of cGMP analogs or NO donors. Our results provide evidence that CNGA3 contributes in an inhibitory manner to the central sensitization of pain pathways during inflammatory pain as a target of NO/cGMP signaling.

Topics: Analysis of Variance; Animals; Cyclic GMP; Cyclic Nucleotide-Gated Cation Channels; Disease Models, Animal; Enzyme Inhibitors; Ganglia, Spinal; Gene Expression Regulation; Glial Fibrillary Acidic Protein; Inflammation; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Microdissection; Naphthalenes; Natriuretic Peptides; Nitric Oxide; Pain; Pain Measurement; Pain Perception; Peripheral Nervous System Diseases; Physical Stimulation; RNA, Messenger; Signal Transduction; Spinal Cord; Stathmin; Statistics, Nonparametric; Thionucleotides; Triazenes; Vesicular Inhibitory Amino Acid Transport Proteins

Obesity is characterized by chronic inflammation of adipose tissue, which contributes to insulin resistance and diabetes. Although nitric oxide (NO) signaling has antiinflammatory effects in the vasculature, whether reduced NO contributes to adipose tissue inflammation is unknown. We sought to determine whether (1) obesity induced by high-fat (HF) diet reduces endothelial nitric oxide signaling in adipose tissue, (2) reduced endothelial nitric oxide synthase (eNOS) signaling is sufficient to induce adipose tissue inflammation independent of diet, and (3) increased cGMP signaling can block adipose tissue inflammation induced by HF feeding.. Relative to mice fed a low-fat diet, an HF diet markedly reduced phospho-eNOS and phospho-vasodilator-stimulated phosphoprotein (phospho-VASP), markers of vascular NO signaling. Expression of proinflammatory cytokines was increased in adipose tissue of eNOS-/- mice. Conversely, enhancement of signaling downstream of NO by phosphodiesterase-5 inhibition using sildenafil attenuated HF-induced proinflammatory cytokine expression and the recruitment of macrophages into adipose tissue. Finally, we implicate a role for VASP, a downstream mediator of NO-cGMP signaling in mediating eNOS-induced antiinflammatory effects because VASP-/- mice recapitulated the proinflammatory phenotype displayed by eNOS-/- mice.. These results imply a physiological role for endothelial NO to limit obesity-associated inflammation in adipose tissue and hence identify the NO-cGMP-VASP pathway as a potential therapeutic target in the treatment of diabetes.

Topics: Adipose Tissue; Animals; Cell Adhesion Molecules; Cyclic GMP; Dietary Fats; Disease Models, Animal; Endothelium, Vascular; Inflammation; Insulin Resistance; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Microfilament Proteins; Nitric Oxide; Nitric Oxide Synthase Type III; Obesity; Phosphodiesterase 5 Inhibitors; Phosphoproteins; Phosphorylation; Piperazines; Purines; Signal Transduction; Sildenafil Citrate; Sulfones

S-Nitrosylation, the reversible post-translational modification of reactive cysteine residues in proteins, has emerged as an important mechanism by which NO acts as a signaling molecule. We recently demonstrated that actin is a major S-nitrosylated protein in the spinal cord and suggested that NO directly attenuates dopamine release from PC12 cells by causing the breakdown of F-actin. However, the occurrence of S-nitrosylation of actin remained unclarified in animal pain model. Kinetic analysis of S-nitrosylation of actin in the present study was made by using NO-generating donors. The biotin-switch assay and purification on streptavidin-agarose were employed for identification of S-nitrosylated actin.. Dopamine release from PC12 cells was markedly attenuated by NOR1 (t1/2 = 1.8 min) and much less by NOR3 (t1/2 = 30 min), but not by S-nitroso-glutathione, an endogenous NO donor. A membrane-permeable cGMP analogue could not substitute for NOR1 as a suppressor nor could inhibitors of soluble guanylate cyclase and cGMP-dependent protein kinase attenuate the suppression. S-Nitrosylated actin was detected by the biotin-switch assay at 5 min after the addition of NOR1. Consistent with the kinetic analysis, actin in the spinal cord was rapidly and maximally S-nitrosylated in an inflammatory pain model at 5 min after the injection of 2% formalin into the hind paws. In vivo patch-clamp recordings of the spinal dorsal horn, NOR3 showed an inhibitory action on inhibitory synaptic transmission in interneurons of the substantia gelatinosa.. The present study demonstrates that rapid S-nitrosylation of actin occurred in vitro in the presence of exogenous NO-generating donors and in vivo in inflammatory pain model mice. Our data suggest that, in addition to the well-known cGMP-dependent protein kinase pathway, S-nitrosylation is involved in pain transmission via disinhibition of inhibitory neurons.

Topics: Actins; Animals; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Disease Models, Animal; Inflammation; Mice; Neurons; Nitric Oxide Donors; Pain; PC12 Cells; Pituitary Adenylate Cyclase-Activating Polypeptide; Rats

Isatin, an endogenous indole compound, prevents atrial natriuretic peptide (ANP) from signaling through its cell-surface receptor, NPRA. Allergic airway inflammation has been linked to natriuretic peptide signaling and blocking this signaling axis in the lung prevents allergen-induced pathology. In this study we encapsulated isatin in chitosan nanoparticles and tested them in a mouse model of allergic asthma by intranasal delivery to the lung. Isatin nanocapsules reduced lung pathology by blocking ANP signaling, but surprisingly also by reducing the expression of NPRA. Ovalbumin-allergic mice were treated intranasally with isatin-containing chitosan nanocapsules either before or after allergen challenge, and lung function, cytokine levels, histopathology and cellular infiltration were determined. ANP activity was quantitated by measuring changes in intracellular cyclic GMP and changes in NPRA levels were determined. For comparison with isatin's effects, the expression of the receptor was inhibited with small interfering RNA against NPRA mRNA. Isatin nanocapsules administered locally to the lung reduced cGMP production and NPRA expression and protected allergic mice from airway hyperreactivity and lung inflammation when given either before or after allergen challenge. Leukocyte infiltration was reduced and lung cytokine profiles showed a repolarization from a Th2-like to a Th1-like phenotype. Isatin nanocapsules administered locally to the lung inhibit NPRA signaling but also are capable of lowering the expression of NPRA, thus effectively reducing inflammation in a mouse model of allergic asthma. Pharmacological intervention to reduce NPRA activity through the inflammatory natriuretic peptide axis in the lung may be a useful adjunct therapy for treating lung disease.

Topics: Administration, Intranasal; Animals; Asthma; Bronchoalveolar Lavage Fluid; Chitosan; Cyclic GMP; Cytokines; Female; Inflammation; Isatin; Leukocytes; Lung; Mice; Mice, Inbred BALB C; Nanocapsules; Ovalbumin; Receptors, Atrial Natriuretic Factor

Diet-induced obesity (DIO) in mice causes vascular inflammation and insulin resistance that are accompanied by decreased endothelial-derived NO production. We sought to determine whether reduced NO-cGMP signaling contributes to the deleterious effects of DIO on the vasculature and, if so, whether these effects can be blocked by increased vascular NO-cGMP signaling.. By using an established endothelial cell culture model of insulin resistance, exposure to palmitate, 100 micromol/L, for 3 hours induced both cellular inflammation (activation of IKK beta-nuclear factor-kappaB) and impaired insulin signaling via the insulin receptor substrate-phosphatidylinositol 3-kinase pathway. Sensitivity to palmitate-induced endothelial inflammation and insulin resistance was increased when NO signaling was reduced using an endothelial NO synthase inhibitor, whereas endothelial responses to palmitate were blocked by pretreatment with either an NO donor or a cGMP analogue. To investigate whether endogenous NO-cGMP signaling protects against vascular responses to nutrient excess in vivo, adult male mice lacking endothelial NO synthase were studied. As predicted, both vascular inflammation (phosphorylated I kappaB alpha and intercellular adhesion molecule levels) and insulin resistance (phosphorylated Akt [pAkt] and phosphorylated eNOS [peNOS] levels) were increased in endothelial NO synthase(-/-) (eNOS(-/-)) mice, reminiscent of the effect of DIO in wild-type controls. Next, we asked whether the vascular response to DIO in wild-type mice can be reversed by a pharmacological increase of cGMP signaling. C57BL6 mice were either fed a high-fat diet or remained on a low-fat diet for 8 weeks. During the final 2 weeks of the study, mice on each diet received either placebo or the phosphodiesterase-5 inhibitor sildenafil, 10 mg/kg per day orally. In high-fat diet-fed mice, vascular inflammation and insulin resistance were completely prevented by sildenafil administration at a dose that had no effect in mice fed the low-fat diet.. Reduced signaling via the NO-cGMP pathway is a mediator of vascular inflammation and insulin resistance during overnutrition induced by high-fat feeding. Therefore, phosphodiesterase-5, soluble guanylyl cyclase, and other molecules in the NO-cGMP pathway (eg, protein kinase G) constitute potential targets for the treatment of vascular dysfunction in the setting of obesity.

Topics: Animals; Aorta, Thoracic; Aortic Diseases; Cell Adhesion Molecules; Cells, Cultured; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 5; Dietary Fats; Disease Models, Animal; Down-Regulation; Endothelial Cells; Enzyme Inhibitors; Humans; I-kappa B Kinase; Inflammation; Inflammation Mediators; Insulin; Insulin Receptor Substrate Proteins; Insulin Resistance; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; NF-kappa B; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase Type III; Palmitic Acid; Phosphatidylinositol 3-Kinases; Phosphodiesterase 5 Inhibitors; Phosphodiesterase Inhibitors; Phosphorylation; Piperazines; Proto-Oncogene Proteins c-akt; Purines; Signal Transduction; Sildenafil Citrate; Sulfones

Dioscorea bulbifera var sativa is a medicinal plant commonly used in Cameroonian traditional medicine to treat pain and inflammation.. The present work evaluated the effects of the methanol extract of the bulbs of Dioscorea bulbifera in inflammatory and neuropathic models of pain and further investigated its possible mechanism of action.. The effects of Dioscorea bulbifera administered orally at the doses of 250 and 500mg/kg were tested in mechanical hypernociception induced by intraplantar (i.pl.) injection of complete Freund's adjuvant (CFA), lipopolysaccharides (LPS) or prostaglandin-E(2) (PGE(2)), as well as in partial ligation sciatic nerve (PLSN), nociception induced by capsaicin and thermal hyperalgesia induced by i.pl. injection of CFA. The therapeutic effects of Dioscorea bulbifera on PGE(2)-induced hyperalgesia were evaluated in the absence and in the presence of l-NAME, an inhibitor of nitric oxide synthase (NOS) and glibenclamide, an inhibitor of ATP-sensitive potassium channels.. The extract showed significant antinociceptive effects in persistent pain induced by CFA and on neuropathic pain induced by PLSN. The effects of Dioscorea bulbifera persisted for 5 days after two administrations in CFA-induced hypernociception. Dioscorea bulbifera significantly inhibited acute LPS-induced pain but failed to reduce thermal hypernociception and capsaicin-induced spontaneous nociception. The antinociceptive effects of this plant extract in PGE(2) model was antagonized by either l-NAME or glibenclamide.. Present demonstrate the antinociceptive activities of Dioscorea bulbifera both in inflammatory and neuropathic models of pain and these effects may result, at least partially, from its ability to activate the NO-cGMP-ATP-sensitive potassium channels pathway.

Topics: Animals; Cyclic GMP; Female; Freund's Adjuvant; Glyburide; Hyperalgesia; Inflammation; KATP Channels; Male; Methanol; Mice; Neuralgia; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Pain; Phytotherapy; Plant Extracts; Plant Roots

To determine if niacin can confer cardiovascular benefit by inhibiting vascular inflammation and improving endothelial function independent of changes in plasma lipid and lipoprotein levels.. New Zealand white rabbits received normal chow or chow supplemented with 0.6% or 1.2% (wt/wt) niacin. This regimen had no effect on plasma cholesterol, triglyceride, or high-density lipoprotein levels. Acute vascular inflammation and endothelial dysfunction were induced in the animals with a periarterial carotid collar. At the 24-hour postcollar implantation, the endothelial expression of vascular cell adhesion molecule-1, intercellular adhesion molecule-1, and monocyte chemotactic protein-1 was markedly decreased in the niacin-supplemented animals compared with controls. Niacin also inhibited intima-media neutrophil recruitment and myeloperoxidase accumulation, enhanced endothelial-dependent vasorelaxation and cyclic guanosine monophosphate production, increased vascular reduced glutathione content, and protected against hypochlorous acid-induced endothelial dysfunction and tumor necrosis factor alpha-induced vascular inflammation.. Previous human intervention studies have demonstrated that niacin inhibits coronary artery disease. This benefit is thought to be because of its ability to reduce low-density lipoprotein and plasma triglyceride levels and increase high-density lipoprotein levels. The present study showed that niacin inhibits vascular inflammation and protects against endothelial dysfunction independent of these changes in plasma lipid levels.

Topics: Animals; Anti-Inflammatory Agents; Aortic Diseases; Carotid Artery Diseases; Chemokine CCL2; Cyclic GMP; Disease Models, Animal; Dose-Response Relationship, Drug; Endothelium, Vascular; Free Radical Scavengers; Glutathione; Inflammation; Intercellular Adhesion Molecule-1; Lipids; Neutrophil Infiltration; Niacin; Oxidation-Reduction; Peroxidase; Rabbits; Reactive Oxygen Species; Tumor Necrosis Factor-alpha; Vascular Cell Adhesion Molecule-1; Vasodilation; Vasodilator Agents

Apelin, a potent vasodilator and angiogenic factor, may be a novel therapeutic agent in neonatal chronic lung disease, including bronchopulmonary dysplasia.. To determine the beneficial effect of apelin in neonatal rats with hyperoxia-induced lung injury, a model for premature infants with bronchopulmonary dysplasia.. The cardiopulmonary effects of apelin treatment (62 μg/kg/d) were studied in neonatal rats by exposure to 100% oxygen, using two treatment strategies: early concurrent treatment during continuous exposure to hyperoxia for 10 days and late treatment and recovery in which treatment was started on Day 6 after hyperoxic injury for 9 days and continued during the 9-day recovery period. We investigated in both models the role of the nitric oxide-cyclic guanosine monophosphate (cGMP) pathway in apelin treatment by specific inhibition of the nitric oxide synthase activity with N(ω)-nitro-L-arginine methyl ester (L-NAME, 25 mg/kg/d).. Parameters investigated include survival, lung and heart histopathology, pulmonary fibrin deposition and inflammation, alveolar vascular leakage, lung cGMP levels, right ventricular hypertrophy, and differential mRNA expression in lung and heart tissue. Prophylactic treatment with apelin improved alveolarization and angiogenesis, increased lung cGMP levels, and reduced pulmonary fibrin deposition, inflammation, septum thickness, arteriolar wall thickness, and right ventricular hypertrophy. These beneficial effects were completely absent in the presence of L-NAME. In the injury-recovery model apelin also improved alveolarization and angiogenesis, reduced arteriolar wall thickness, and attenuated right ventricular hypertrophy.. Apelin reduces pulmonary inflammation, fibrin deposition, and right ventricular hypertrophy, and partially restores alveolarization in rat pups with neonatal hyperoxic lung injury via a nitric oxide synthase-dependent mechanism.

Topics: Animals; Animals, Newborn; Apelin; Apelin Receptors; Carrier Proteins; Cyclic GMP; Heart Injuries; Hyperoxia; In Situ Hybridization; Inflammation; Intercellular Signaling Peptides and Proteins; Lung; Lung Injury; Myocardium; NG-Nitroarginine Methyl Ester; Rats; Rats, Wistar; Receptors, G-Protein-Coupled; Reverse Transcriptase Polymerase Chain Reaction

Through activation of the A1 adenosine receptors (A1Rs) at both the central and peripheral level, adenosine produces antinociception in a wide range of tests. However, the mechanisms involved in the peripheral effect are still not fully understood. Therefore, the mechanisms by which peripheral activation of A1Rs reduces inflammatory hypernociception (a decrease in the nociceptive threshold) were addressed in the present study. Immunofluorescence of rat dorsal root ganglion revealed significant expression of A1Rs in primary sensory neurons associated with nociceptive pathways. Functionally, peripheral activation of A1Rs reduced inflammatory hypernociception because intraplantar (i.pl.) administration of an A1R antagonist (DPCPX) enhanced carrageenan-induced hypernociception. On the other hand, local (paw) administration of CPA (a selective A1R agonist) reversed mechanical hypernociception induced by carrageenan or by the directly acting hypernociceptive mediator prostaglandin E(2) (PGE(2)). Down-regulation of A1Rs expression in primary nociceptive neurons by intrathecal treatment with antisense oligodeoxinucleotides significantly reduced peripheral antinociceptive action of CPA. Direct blockade of PGE(2) inflammatory hypernociception by the activation of A1Rs depends on the nitric oxide/cGMP/Protein Kinase G/KATP signaling pathway because the peripheral antinociceptive effect of CPA was prevented by pretreatment with inhibitors of neuronal nitric oxide synthase (N-propyl-l-arginine), guanylyl cyclase (ODQ), and Protein Kinase G (KT5823) as well as with a KATP blocker (glibenclamide). However, this effect of CPA was not reduced by naloxone, excluding the participation of endogenous opioids. These results suggest that the peripheral activation of A1R plays a role in the regulation of inflammatory hypernociception by a mechanism that involves the NO/cGMP/PKG/KATP intracellular signaling pathway.

Topics: Adenosine A1 Receptor Antagonists; Analysis of Variance; Animals; Carrageenan; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Dinoprostone; Dose-Response Relationship, Drug; Down-Regulation; Drug Administration Routes; Drug Interactions; Enzyme Inhibitors; Hyperalgesia; Inflammation; KATP Channels; Male; Nerve Tissue Proteins; Nitric Oxide; Nociceptors; Pain Threshold; Posterior Horn Cells; Potassium Channel Blockers; Rats; Rats, Wistar; Receptor, Adenosine A1; Signal Transduction; Spinal Cord; TRPV Cation Channels; Xanthines

Excessive production of nitric oxide (NO) by microglia is at least in part responsible for the pathogenesis of various neurodegenerative disorders including Parkinson disease, but at the same time NO may also play a distinct role as a signaling molecule such as an activator of soluble guanylyl cyclase. Here we investigated potential roles of the NO-soluble guanylyl cyclase-cyclic GMP signaling pathway in the regulation of dopaminergic neurodegeneration. Activation of microglia by interferon-gamma (IFN-gamma) followed by lipopolysaccharide (LPS) caused dopaminergic cell death in rat midbrain slice cultures, which was dependent on NO production. 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a soluble guanylyl cyclase inhibitor, as well as KT5823, an inhibitor of cyclic GMP-dependent protein kinase, exacerbated dopaminergic cell death induced by IFN-gamma/LPS. Conversely, 8-bromo-cyclic GMP attenuated IFN-gamma/LPS cytotoxicity on dopaminergic neurons. Notably, although heme oxygenase-1 (HO-1) was expressed prominently in cells other than dopaminergic neurons in control cultures, robust expression of HO-1 was induced in surviving dopaminergic neurons challenged with IFN-gamma/LPS. ODQ and KT5823 decreased, whereas 8-bromo-cyclic GMP increased, the number of dopaminergic neurons expressing HO-1 after IFN-gamma/LPS challenge, without parallel changes in HO-1 expression in other cell populations. An NO donor 3-(4-morpholinyl)sydnonimine hydrochloride also induced HO-1 expression in dopaminergic neurons, which was abolished by ODQ and augmented by 8-bromo-cyclic GMP. Moreover, IFN-gamma/LPS-induced dopaminergic cell death was augmented by zinc protoporphyrin IX, an HO-1 inhibitor. The NO donor cytotoxicity on dopaminergic neurons was also augmented by ODQ and zinc protoporphyrin IX. These results indicate that the NO-cyclic GMP signaling pathway promotes the induction of HO-1 specifically in dopaminergic neurons, which acts as an endogenous protective system to limit inflammatory degeneration of this cell population.

Topics: Animals; Animals, Newborn; Cyclic GMP; Dopamine; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Inhibitors; Gene Expression Regulation; Heme Oxygenase-1; In Vitro Techniques; Inflammation; Interferon-gamma; Mesencephalon; Nerve Degeneration; Nitric Oxide; Polysaccharides; Rats; Rats, Wistar; Signal Transduction; Tyrosine 3-Monooxygenase

Airway inflammation with mucus overproduction is a distinguishing pathophysiological feature of many chronic respiratory diseases. Phosphodiesterase (PDE) inhibitors have shown anti-inflammatory properties. In the present study, the effect of sildenafil, a potent inhibitor of PDE5 that selectively degrades cyclic guanosine 3',5'-monophosphate (cGMP), on acrolein-induced inflammation and mucus production in rat airways was examined. Rats were exposed to acrolein for 14 and 28 days. Sildenafil or distilled saline was administered intragastrically prior to acrolein exposure. Bronchoalveolar lavage fluid (BALF) was acquired for cell count and the detection of pro-inflammatory cytokine levels. Lung tissue was examined for cGMP content, nitric oxide (NO)-metabolite levels, histopathological lesion scores, goblet cell metaplasia and mucin production. The results suggested that sildenafil pretreatment reversed the significant decline of cGMP content in rat lungs induced by acrolein exposure, and suppressed the increase of lung NO metabolites, the BALF leukocyte influx and pro-inflammatory cytokine release. Moreover, sildenafil pretreatment reduced acrolein-induced Muc5ac mucin synthesis at both mRNA and protein levels, and attenuated airway inflammation, as well as epithelial hyperplasia and metaplasia. In conclusion, sildenafil could attenuate airway inflammation and mucus production in the rat model, possibly through the nitric oxide/cyclic guanosine 3',5'-monophosphate pathway, and, thus, might have a therapeutic potential for chronic airway diseases.

Topics: Acrolein; Analysis of Variance; Animals; Blotting, Western; Bronchoalveolar Lavage; Cyclic GMP; Cytokines; Enzyme-Linked Immunosorbent Assay; Immunohistochemistry; Inflammation; Leukocytes; Lung Diseases; Male; Mucins; Nitric Oxide; Piperazines; Purines; Rats; Rats, Sprague-Dawley; Respiratory Mucosa; Reverse Transcriptase Polymerase Chain Reaction; Sildenafil Citrate; Sulfones

This study assessed the effect of the agonist 15d-PGJ(2) administered into the rat temporomandibular joint (TMJ) on nociceptive behavioral and the anti-inflammatory potential of this prostaglandin on TMJ. It was observed that 15-deoxy-(Delta12,14)-prostaglandin J(2) (15d-PGJ(2)) significantly reduced formalin-induced nociceptive behavior in a dose dependent manner, however injection of 15d-PGJ(2) into the contralateral TMJ failed to reduce such effects. This antinociceptive effect is dependent on peroxisome proliferator-activated receptors-gamma (PPAR-gamma) since pre-treatment with GW9662 (PPAR-gamma receptor antagonist) blocked the antinociceptive effect of 15d-PGJ(2) in the TMJ. In addition, the antinociceptive effect of 15d-PGJ(2) was also blocked by naloxone suggesting the involvement of peripheral opioids in the process. Confirming this hypothesis pre-treatment with kappa, delta, but not mu receptor antagonists significantly reduced the antinociceptive effect of 15d-PGJ(2) in the TMJ. Similarly to opioid agonists, the 15d-PGJ(2) antinociceptive action depends on the nitric oxide (NO)/guanilate cyclase (cGMP)/ATP-sensitive potassium channel blocker(K(+)(ATP)) channel pathway since it was prevented by the pre-treatment with the inhibitors of nitric oxide synthase (NOS; aminoguanidine), cGMP (ODQ), or the K(+)(ATP) (glibenclamide). In addition, 15d-PGJ(2) (100 ng/TMJ) inhibits 5-HT-induced TMJ hypernociception. Besides, TMJ treated with 15d-PGJ(2) showed lower vascular permeability, assessed by Evan's Blue extravasation, and also lower neutrophil migration induced by carrageenan administration. Taken together, these results demonstrate that 15d-PGJ(2) has a potential peripheral antinociceptive and anti-inflammatory effect in the TMJ via PPAR-gamma activation. The results also suggest that 15d-PGJ(2) induced-peripheral antinociceptive response in the TMJ is mediated by kappa/delta opioid receptors by the activation of the intracellular l-arginine/NO/cGMP/K(+)(ATP) channel pathway. The pharmacological properties of the peripheral administration of 15d-PGJ(2) highlight the potential use of this PPAR-gamma agonist on TMJ inflammatory pain conditions.

Topics: Analgesics; Animals; Cyclic GMP; Dose-Response Relationship, Drug; Formaldehyde; Inflammation; KATP Channels; Male; Nitric Oxide Synthase; Pain; PPAR gamma; Prostaglandin D2; Rats; Rats, Wistar; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu; Serotonin; Signal Transduction; Temporomandibular Joint

A large body of evidence indicates that the release of nitric oxide (NO) is crucial for the central sensitization of pain pathways during both inflammatory and neuropathic pain. Here, we investigated the distribution of NO-sensitive guanylyl cyclase (NO-GC) in the spinal cord and in dorsal root ganglia, and we characterized the nociceptive behavior of mice deficient in NO-GC (GC-KO mice). We show that NO-GC is distinctly expressed in neurons of the mouse dorsal horn, whereas its distribution in dorsal root ganglia is restricted to non-neuronal cells. GC-KO mice exhibited a considerably reduced nociceptive behavior in models of inflammatory or neuropathic pain, but their responses to acute pain were not impaired. Moreover, GC-KO mice failed to develop pain sensitization induced by intrathecal administration of drugs releasing NO or carbon monoxide. Surprisingly, during spinal nociceptive processing, cGMP produced by NO-GC may activate signaling pathways different from cGMP-dependent protein kinase I (cGKI), whereas cGKI can be activated by natriuretic peptide receptor-B dependent cGMP production. Together, our results provide evidence that NO-GC is crucially involved in the central sensitization of pain pathways during inflammatory and neuropathic pain.

Topics: Animals; Behavior, Animal; Carbon Monoxide; Cyclic GMP; Cyclic GMP-Dependent Protein Kinase Type I; Cyclic GMP-Dependent Protein Kinases; Female; Ganglia, Spinal; Guanylate Cyclase; Inflammation; Male; Mice; Mice, Knockout; Neuralgia; Nociceptors; Pain; Receptors, Cytoplasmic and Nuclear; Signal Transduction; Soluble Guanylyl Cyclase; Spinal Cord; Tissue Distribution

The synthesis of mono- and bisphosphorothioate analogues of 3',5'-cyclic diguanylic acid (cdiGMP) via the modified H-phosphonate chemistry is reported. The immunostimulatory properties of these analogues were compared with those of cdiGMP.

Topics: Bronchi; Chemistry, Pharmaceutical; Chromatography, High Pressure Liquid; Cyclic GMP; Dose-Response Relationship, Drug; Drug Design; Humans; Immune System; Inflammation; Models, Chemical; Neutrophils; Organophosphonates; Phosphates; Vaccines

Reduced bioavailability of nitric oxide (NO) is a hallmark of diabetes mellitus-induced vascular complications. In the present study we investigated whether a pharmacological increase of endothelial NO synthase (eNOS) production can restore the impaired hindlimb flow in a rat model of severe diabetes.. A model of diabetes mellitus was induced in male Sprague-Dawley rats by a single injection of streptozotozin. Rats were treated chronically with the eNOS transcription enhancer AVE3085 (10 mg [kg body weight](-1) day(-1); p.o.) or vehicle for 48 days and compared with controls. Endothelial function and arterial BP were investigated in vivo using an autoperfused hindlimb model and TIP-catheter measurement, respectively. Protein production of eNOS, total and phosphorylated vasodilator-stimulated phosphoprotein (VASP) were assessed in their quadriceps muscle tissue, whereas cyclic GMP (cGMP) concentrations were assessed in blood plasma. RNA levels of intracellular and vascular cell adhesion molecules (ICAM-1 and VCAM-1) were measured by real-time PCR.. Untreated diabetic rats showed significantly reduced quadriceps muscle contents of eNOS (-64%) and phosphorylated VASP (-26%) protein associated with impaired vascular function (maximum vasodilatation: -30%, p < 0.05) and enhanced production of ICAM-1 (+121%) and VCAM-1 (+156%). Chronic treatment with AVE3085 did not alter arterial BP or severe hyperglycaemia, but did lead to significantly increased production of eNOS (+95%), cGMP (+128%) and VASP phosphorylation (+65%) as well as to improved vascular function (+36%) associated with reduced production of ICAM-1 (-36%) and VCAM-1 (-58%).. In a rat model of severe diabetes, pharmacological enhancement of impaired eNOS production and NO-cGMP signalling by AVE3085 restores altered hindlimb blood flow and prevents vascular inflammation.

Topics: Animals; Cell Adhesion Molecules; Cyclic GMP; Diabetes Complications; Diabetes Mellitus, Experimental; Gene Expression Regulation; Hindlimb; Humans; Inflammation; Intracellular Signaling Peptides and Proteins; Lipid Peroxidation; Male; Microfilament Proteins; Muscles; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Phosphoproteins; Rats; Rats, Sprague-Dawley; Streptozocin; Vascular Cell Adhesion Molecule-1; Vascular Diseases

The lymphatic vascular system regulates tissue fluid homeostasis and the afferent phase of the immune response, and it is also involved in tumor metastasis. There is increasing evidence that lymphatic vessels also mediate acute and chronic inflammation. However, the mechanisms and functional consequences of lymphangiogenesis under inflammatory conditions are largely unknown. Here, we show that lymphatic endothelial cells (LECs) specifically express the alpha1beta1 isoform of soluble guanylate cyclase (sGC), that vascular endothelial growth factor-A potently induces sGCalpha1beta1, and that nitric oxide (NO) -induced LEC proliferation, migration, and cGMP production in LECs are specifically dependent on sGCalpha1beta1. Moreover, the specific sGC inhibitor NS-2028 completely prevents ultraviolet B-irradiation-induced lymphatic vessel enlargement, edema formation, and skin inflammation in vivo. These findings identify a crucial role of the NO/sGCalpha1beta1/cGMP pathway in modulating lymphatic vessel function. The blockade of sGCalpha1beta1 signaling might serve as a novel therapeutic strategy for inhibiting lymphangiogenesis and inflammation, in addition to its effects on the blood vasculature.

Topics: Cell Movement; Cell Proliferation; Cells, Cultured; Cyclic GMP; Edema; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Guanylate Cyclase; Humans; Immunity, Cellular; Inflammation; Lymphatic Vessels; Nitric Oxide; Nitric Oxide Synthase Type III; Oxadiazoles; Oxazines; Receptors, Cytoplasmic and Nuclear; S-Nitroso-N-Acetylpenicillamine; Soluble Guanylyl Cyclase

Nitric oxide (NO) has been associated with a spectrum of harmful to protective roles in inflammatory bowel disease. The involvement of soluble guanylate cyclase (sGC)--the downstream effector of NO--in the negative effect of NO in inflammatory models has been proposed but this has not been evaluated in inflammatory bowel diseases. The present study investigates therefore the influence of colonic inflammation on sGC activity, as well as the effect of in vivo sGC inhibition on colonic inflammation and on in vitro changes in colonic motility in the dextran sulfate sodium (DSS)-model of colitis in rat. Administration of 7% DSS in the drinking water for 6 days resulted in colonic inflammation as judged from histology and myeloperoxidase activity, accompanied by weight loss and bloody stools. Plasma and colonic tissue cyclic guanosine 3',5'-monophosphate (cGMP) levels were decreased in DSS-treated rats. Colonic levels of neuronal NO synthase (nNOS) mRNA and immunoreactivity were not influenced, while those of inducible NO synthase (iNOS) and colonic nitrite/nitrate levels were increased by DSS exposure. Circular muscle strips from inflamed distal colon showed decreased inhibitory responses towards electrical field stimulation and exogenous NO, while methacholine-induced phasic activity was suppressed. Inhibition of sGC by in vivo treatment with ODQ further reduced cGMP levels but did not prevent the inflammation and motility alterations. These results suggest that DSS-induced colitis in rats is accompanied by a reduced sensitivity of sGC, leading to reduced basal cGMP levels and decreased colonic responsiveness towards nitrergic stimuli, but pharmacological reduction of cGMP generation does not prevent the development of DSS-induced colitis.

Topics: Animals; Colitis; Cyclic GMP; Dextran Sulfate; Disease Models, Animal; Enzyme Inhibitors; Guanylate Cyclase; Inflammation; Male; Nitrates; Nitric Oxide; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type II; Nitrites; Oxadiazoles; Quinoxalines; Rats; Rats, Wistar; Receptors, Cytoplasmic and Nuclear; RNA, Messenger; Soluble Guanylyl Cyclase

Morphine can inhibit inflammatory edema in experimental animals. The mechanisms and sites by which opioids exert this effect are still under debate. Since the spinal level is a site for modulation of the neurogenic component of inflammation, we investigated the effect of intrathecal (i.t.) administration of morphine, and the involvement of spinal nitric oxide (NO)/cyclic-guanosine monophosphate-GMP pathway in carrageenan (CG)-induced paw edema.. Male Wistar rats received i.t. injections of drugs (20 microL) 30 min before paw stimulation with CG (150 microg). Edema was measured as paw volume increase (mL), and neutrophil migration was evaluated indirectly by myeloperoxidase (MPO) assay.. Morphine (37, 75, and 150 nmol) inhibited inflammatory edema, but had no effect on MPO activity. Coinjection with naloxone (64 nmol) reversed the effect of morphine. The corticosteroid synthesis inhibitor, aminoglutethimide (50 mg/kg, v.o.), administered 90 min before morphine injection did not modify its antiedematogenic effect. Low doses of the NO synthase inhibitor, N(omega)-nitro-L-arginine (L-NNA; 10 and 30 pmol) increased, while higher doses (3 and 30 nmol) inhibited edema. The guanylate cyclase inhibitor 1H-oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 21 and 42 nmol) increased, while the phosphodiesterase type 5 inhibitor sildenafil (0.15 and 1.5 nmol) inhibited paw edema. Coadministration of a subeffective dose of L-NNA (3 pmol) or ODQ (10 nmol) with morphine prevented its antiedematogenic effect, but sildenafil (0.15 nmol) rendered a subeffective dose of morphine effective (18 nmol). ODQ also prevented the antiedematogenic effect of the NO donor S-nitroso-N-acetyl-penicilamine.. These results support the idea that morphine can act on opioid receptors at the spinal level to produce antiedematogenic, and that the NO/cGMP pathway seems to be an important mediator in this effect.

Topics: Analgesics, Opioid; Animals; Anti-Inflammatory Agents, Non-Steroidal; Carrageenan; Cyclic GMP; Disease Models, Animal; Dose-Response Relationship, Drug; Edema; Enzyme Inhibitors; Guanylate Cyclase; Inflammation; Injections, Spinal; Male; Morphine; Naloxone; Narcotic Antagonists; Neutrophil Infiltration; Neutrophils; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitroarginine; Oxadiazoles; Peroxidase; Phosphodiesterase Inhibitors; Piperazines; Purines; Quinoxalines; Rats; Rats, Wistar; S-Nitroso-N-Acetylpenicillamine; Signal Transduction; Sildenafil Citrate; Spinal Cord; Sulfones; Time Factors

Aldosterone is a mineral corticoid hormone that is produced in response to angiotensin-II, and like angiotensin-II, stimulates inflammation, oxidative stress, and fibrosis by activating nuclear factor-kappaB and activating protein-1. Recent evidence, however, indicates that aldosterone stimulates phospholipase C and activates nuclear factor-kappaB and activating protein-1. Although the heme oxygenase system is cytoprotective, its effects on aldosterone-phospholipase C signaling in deoxycorticosterone acetate (DOCA-salt) hypertension, a model of aldosteronism, and spontaneously hypertensive rat, a genetic model of human essential hypertension, have not been fully characterized.. In the present study, the heme oxygenase inducer, hemin, was given to spontaneously hypertensive and deoxycorticosterone acetate hypertensive rats, and the effects on blood pressure, aldosterone, nuclear factor-kappaB, activating protein-1, phospholipase C, and inositol 1,4,5-triphosphate were examined.. Hemin therapy restored physiological blood pressure to spontaneously hypertensive rats (209.9 +/- 0.9 to 127.3 +/- 0.85 mmHg, n = 10, P < 0.01) and to deoxycorticosterone acetate salt hypertensive rats (195.7 +/- 1.8 vs.132.5 +/- 2.1 mmHg; P < 0.01, n = 10), but had no effect on age-matched normotensive Wistar-Kyoto or Sprague-Dawley strains. The antihypertensive effect was accompanied by enhanced heme oxygenase activity, upregulated cyclic guanosine monophosphate-protein kinase G signaling, increased superoxide dismutase activity, and the potentiation of total antioxidant capacity, whereas aldosterone, activating protein-1, and nuclear factor-kappaB were reduced. Furthermore, hemin suppressed phospholipase C activity, attenuated inositol 1,4,5-triphosphate, and reduced resting intracellular calcium in the aorta.. Collectively, our results suggest that the concomitant depletion of aldosterone, phospholipase C-inositol 1,4,5-triphosphate activity, resting intracellular calcium and the corresponding decline of inflammatory, and oxidative insults may account for the antihypertensive effects of hemin in deoxycorticosterone acetate hypertension and spontaneously hypertensive rats.

Topics: Aldosterone; Animals; Aorta; Blood Pressure; Calcium; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Down-Regulation; Heme Oxygenase (Decyclizing); Hemin; Hypertension; Inflammation; Inositol 1,4,5-Trisphosphate; Male; Oxidative Stress; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Rats, Sprague-Dawley; Receptor Cross-Talk; Second Messenger Systems; Type C Phospholipases; Up-Regulation

Soluble guanylyl cyclase (sGC) is a cGMP-generating enzyme implicated in the control of smooth muscle tone that also regulates platelet aggregation. Moreover, sGC activation has been shown to reduce leukocyte adherence to the endothelium. Herein, we investigated the expression of sGC in a murine model of LPS-induced lung injury and evaluated the effects of sGC inhibition in the context of acute lung injury (ALI). Lung tissue sGC alpha1 and beta1 subunit protein levels were determined by Western blot and immunohistochemistry, and steady-state mRNA levels for the beta1 subunit were assessed by real-time PCR. LPS inhalation resulted in a decrease in beta1 mRNA levels, as well as a reduction in both sGC subunit protein levels. Decreased alpha1 and beta1 expression was observed in bronchial smooth muscle and epithelial cells. TNF-alpha was required for the LPS-triggered reduction in sGC protein levels, as no change in alpha1 and beta1 levels was observed in TNF-alpha knockout mice. To determine the effects of sGC blockade in LPS-induced lung injury, mice were exposed to 1H-[1,2,4]oxodiazolo[4,3-a]quinoxalin-l-one (ODQ) prior to the LPS challenge. Such pretreatment led to a further increase in total cell number (mainly due to an increase in neutrophils) and protein concentration in the bronchoalveoalar lavage fluid; the effects of ODQ were reversed by a cell-permeable cGMP analog. We conclude that sGC expression is reduced in LPS-induced lung injury, while inhibition of the enzyme with ODQ worsens lung inflammation, suggesting that sGC exerts a protective role in ALI.

Topics: Aerosols; Animals; Blotting, Western; Bronchi; Bronchoalveolar Lavage Fluid; Cyclic GMP; Enzyme Inhibitors; Epithelial Cells; Guanylate Cyclase; Immunohistochemistry; Inflammation; Inhalation; Lipopolysaccharides; Lung Injury; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Muscle, Smooth; Oxadiazoles; Protein Subunits; Quinoxalines; Respiratory Distress Syndrome; RNA, Messenger; Solubility; Tumor Necrosis Factor-alpha

Myocarditis is an acute inflammatory disease of the myocardium for which there is currently no specific therapy. We investigated the therapeutic potential of C-type natriuretic peptide (CNP) in acute experimental autoimmune myocarditis. One week after injection of porcine myosin into male Lewis rats, CNP (0.05 microg/kg/min) was continuously administered for 2 weeks. CNP infusion significantly increased maximum dP/dt, decreased left ventricular end-diastolic pressure, and improved fractional shortening compared with vehicle administration. In vehicle-treated hearts, severe necrosis and marked infiltration of CD68-positive inflammatory cells were observed. Myocardial and serum levels of monocyte chemoattractant protein-1 were elevated in myocarditis. However, these changes were attenuated by CNP infusion. In addition, treatment with CNP significantly increased myocardial capillary density. Guanylyl cyclase-B, a receptor for CNP, was expressed in myocarditic heart, and cyclic guanosine monophosphate was elevated by CNP infusion. In conclusion, CNP infusion attenuated cardiac function in acute myocarditis through anti-inflammatory and angiogenic effects.

Topics: Animals; Blood Pressure; Blood Vessels; Chemokine CCL2; Cyclic GMP; Enzyme-Linked Immunosorbent Assay; Guanylate Cyclase; Heart; Heart Rate; Inflammation; Male; Myocarditis; Myocardium; Natriuretic Agents; Natriuretic Peptide, C-Type; Radioimmunoassay; Rats; Rats, Inbred Lew; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Swine; Ultrasonography; von Willebrand Factor

At present, the clinical management inflammatory vasoplegia associated to sepsis or anaphylaxis is symptomatic. Volume is expanded by means of administration of fluids, and low blood pressure is managed by means of administration of positive inotropes and vasoconstrictors. This therapeutic approach is mainly associated to the cyclic AMP (cAMP) and, many times the circulatory shock is refractory to high amines concentrations. However, beside of cAMP-dependent vasoreactivity mechanisms there are other two known vasoplegia involved mechanisms: cyclic GMP (cGMP) and hyperpolarization that is less clinically considered. Also, it is possible to speculate about 'probable vasopressin deficiency'. Methylene blue (MB) is the most useful and clinically safe cGMP blocker. We propose a decision tree for diagnosis and institution of this therapeutical approach many times underestimate by intensive care and emergency teams.

Topics: Animals; Blood Circulation; Blood Pressure; Catecholamines; Critical Care; Cyclic GMP; Emergency Medicine; Humans; Inflammation; Lactates; Methylene Blue; Models, Biological; Nitric Oxide; Norepinephrine; Shock

Feeding high fructose (Frc) to rats induces a moderate increase in blood pressure, which is associated with insulin resistance. The present study was designed to evaluate the effect of the methanol extract of Sorbus commixta cortex (MSC) on vascular inflammation in a rat model of the metabolic syndrome induced by a high Frc-diet. Male Sprague-Dawley rats were divided into 4 groups and treated for 7 weeks as follows: 1) control, 2) high Frc-diet group, 3) Frc/MSC1 group; high Frc-diet group treated with MSC (100 mg/kg/day), and 4) Frc/MSC2 group; high Frc-diet group treated with MSC (200 mg/kg/day). High Frc-induced decreases of the expression level of aortic endothelial nitric oxide synthase (ecNOS) while the production of cyclic GMP (cGMP) was restored by treatment with MSC. On the contrary, increases of the expression level of endothelin-1 (ET-1) in the aorta, the transcription factor, the cytokine related with vascular inflammation, and the adhesion molecules were suppressed by MSC treatment. Moreover, MSC treatment was shown to lessen the thickening noted in the aortic intima and media of the high Frc-diet group. Our findings suggest that MSC may have an anti-vascular inflammatory effect on rats with a high Frc-induced metabolic syndrome.

Topics: Animals; Aorta, Thoracic; Cell Adhesion Molecules; Cyclic GMP; Disease Models, Animal; Endothelin-1; Endothelium, Vascular; Fructose; Guanosine Monophosphate; Inflammation; Male; Metabolic Syndrome; Methanol; NF-kappa B; Nitrites; Plant Extracts; Rats; Rats, Sprague-Dawley; RNA, Messenger; Sorbus; Sweetening Agents; Triglycerides; Tunica Intima; Tunica Media

One of the neurological alterations in patients with minimal or overt hepatic encephalopathy is cognitive impairment. This impairment is reproduced in rats with chronic liver failure due to portacaval shunt (PCS). These rats show decreased ability to learn a conditional discrimination task in a Y-maze, likely due to reduced function of the glutamate-nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) pathway in brain. It has been proposed that inflammation exacerbates the neuropsychological alterations induced by hyperammonemia, suggesting that inflammation-associated alterations may contribute to cognitive impairment in hepatic encephalopathy. This study assessed whether treatment with an anti-inflammatory drug, ibuprofen, is able to restore the function of the glutamate-NO-cGMP pathway in cerebral cortex in brain in vivo and/or learning ability in PCS rats. We show that PCS rats have increased levels of interleukin-6 and increased activities of cyclooxygenase and of inducible NO synthase in cerebral cortex, indicating the presence of inflammation. Chronic treatment with ibuprofen normalizes cyclooxygenase and inducible NO synthase activities but not interleukin-6 levels. Moreover, ibuprofen normalizes the function of the glutamate-NO-cGMP pathway in cerebral cortex in vivo and completely restores the ability of rats with chronic liver failure to learn the Y-maze task. This supports that inflammation contributes to the cognitive impairment in hepatic encephalopathy.. the results reported point to the possible therapeutic utility of decreasing inflammation in the treatment of the cognitive deficits in patients with minimal or overt hepatic encephalopathy.

Topics: Ammonia; Animals; Anti-Inflammatory Agents, Non-Steroidal; Cyclic GMP; Hepatic Encephalopathy; Ibuprofen; Inflammation; Male; Maze Learning; Nitric Oxide; Nitric Oxide Synthase Type II; Portacaval Shunt, Surgical; Prostaglandin-Endoperoxide Synthases; Rats; Rats, Wistar

Topics: Ammonia; Animals; Anti-Inflammatory Agents, Non-Steroidal; Cyclic GMP; Cytokines; Hepatic Encephalopathy; Inflammation; Nitric Oxide; Nitric Oxide Synthase; Prostaglandin-Endoperoxide Synthases; Rats

Several clinical studies have suggested possible increase in cardiovascular risk during and in the first weeks after an acute inflammatory disease. Using influenza vaccine as inflammatory stimulus, we investigated whether arterial endothelial dysfunction could persist beyond the inflammatory state, and whether amplified oxidative modification of low-density lipoprotein (LDL) accompanies this vascular disturbance.. The brachial artery responses to hyperemia (flow-mediated dilatation (FMD), and to sublingual glyceryl trinitrate (GTN), and the carotid intima-media thickness were assessed by external ultrasound in eight healthy male volunteers (age 17-30 y) before, and 2 and 14 days after intramuscular administration of influenza vaccine. Plasma levels of high-sensitivity C-reactive protein (CRP), fibrinogen, cyclic guanosine monophosphate (cGMP), and antibodies against oxidized LDL (oxLDL) were measured at each time point. Data are means+/-standard errors of the mean (SEM).. Influenza vaccination caused a slight elevation in CRP (from 0.5+/-0.1 at baseline, to 2+/-0.6 mg/L, P = 0.01) and fibrinogen (from 2.3+/-0.1 to 2.7+/-0.1 g/L, P = 0.01) at 2 days, which completely resolved at 14 days (CRP: 0.6+/-0.2 mg/L, P = 0.9, and fibrinogen: 2.3+/-0.1 g/L, P = 0.8 versus baseline). OxLDL antibody levels rose significantly at 2 days (from 1+/-0.1 at baseline to 2+/-0.4, P = 0.04), and remained elevated at 14 days (1.7+/-0.3, P = 0.1 versus baseline). FMD of the brachial artery decreased at 2 days (from 8.3+/-1.2% at baseline, to 5.4+/-1%, P = 0.05) with a further decrease at 14 days (4.9+/-0.8%, P = 0.03 versus baseline). The dilatory responses to GTN and the carotid IMT remained unchanged throughout the study period (P>0.5).. Abnormalities in arterial function and LDL oxidation may persist for at least 2 weeks after a slight inflammatory reaction induced by influenza vaccination. These could explain in part the earlier reported increase in cardiovascular risk during the first weeks after an acute inflammatory disorder.

Topics: Adolescent; Adult; Biomarkers; Brachial Artery; C-Reactive Protein; Carotid Artery, Common; Cholesterol, HDL; Cyclic GMP; Endothelium, Vascular; Fibrinogen; Follow-Up Studies; Humans; Hyperemia; Inflammation; Influenza Vaccines; Lipoproteins, LDL; Male; Nitroglycerin; Research Design; Ultrasonography; Vasodilation; Vasodilator Agents

Nerve injury elicits both universal and limited responses. Among the former is regenerative growth, which occurs in most peripheral neurons, and among the latter is the long-term hyperexcitability that appears selectively in nociceptive sensory neurons. Since positive injury signals communicate information from the site of an injury to the cell body, we hypothesize that a nerve injury activates both universal and limited positive injury signals. Studies in Aplysia indicate that protein kinase G is a limited signal that is responsible for the induction of long-term hyperexcitability. Given that long-term hyperexcitability contributes to chronic pain after axotomy in rodent neuropathic pain models, we investigated its underlying basis in the rat peripheral nervous system. Using biochemical assays, Western blots, and immunocytochemistry we found that the Type 1alpha protein kinase G is the predominant isoform in the rat periphery. It is present primarily in axons and cell bodies of nociceptive neurons, including populations that are isolectin B4-positive, isolectin B4-negative, and those that express transient receptor potential vanilloid receptor-1. Surprisingly, protein kinase G is not present in the facial nerve, which overwhelmingly contains axons of motor neurons. Crushing the sciatic nerve or a cutaneous sensory nerve activates protein kinase G in axons and results in its retrograde transport to the neuronal somata in the DRG. Preventing the activation of protein kinase G by injecting Rp-8-pCPT-cGMPS into the crush site abolished the transport. The protein kinase A inhibitor Rp-8-pCPT-cAMPS had no effect. Extracellular signal-related kinases 42/44 are also activated and transported after nerve crush, but in both motor and sensory axons. Chronic pain has been linked to long-term hyperexcitability following a nerve inflammation in several rodent models. We therefore injected complete Freund's adjuvant into the hindpaw to induce an inflammation and found that protein kinase G was activated in the sural nerve and transported to the DRG. In contrast, the extracellular signal-related kinases in the sensory axons were not activated by the complete Freund's adjuvant. These studies support the idea that the extracellular signal-related kinases are universal positive axonal signals and that protein kinase G is a limited positive axonal signal. They also establish the association between protein kinase G, the induction of long-term hyperexcitability, and chronic

Topics: Animals; Axons; Blotting, Western; Cell Count; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Enzyme Activation; Enzyme Inhibitors; Freund's Adjuvant; Ganglia, Spinal; Immunohistochemistry; Inflammation; Male; Neurofilament Proteins; Neurons, Afferent; Nociceptors; Protein Transport; Rats; Rats, Sprague-Dawley; S100 Proteins; Sciatic Neuropathy; Thionucleotides; Time Factors; TRPV Cation Channels

The expression of inducible nitric-oxide synthase (iNOS) and subsequent "high-output" nitric oxide (NO) production underlies the systemic hypotension, inadequate tissue perfusion, and organ failure associated with septic shock. Therefore, modulators of iNOS expression and activity, both endogenous and exogenous, are important in determining the magnitude and time course of this condition. We have shown previously that NO from the constitutive endothelial NOS (eNOS) is necessary to obtain maximal iNOS expression and activity following exposure of murine macrophages to lipopolysaccharide (LPS). Thus, eNOS represents an important regulator of iNOS expression in vitro. Herein, we validate this hypothesis in vivo using a murine model of sepsis. A temporal reduction in iNOS expression and activity was observed in LPS-treated eNOS knock-out (KO) mice as compared with wild-type animals; this was reflected in a more stable hemodynamic profile in eNOS KO mice during endotoxaemia. Furthermore, in human umbilical vein endothelial cells, LPS leads to the activation of eNOS through phosphoinositide 3-kinase- and Akt/protein kinase B-dependent enzyme phosphorylation. These data indicate that the pathogenesis of sepsis is characterized by an initial eNOS activation, with the resultant NO acting as a co-stimulus for the expression of iNOS, and therefore highlight a novel pro-inflammatory role for eNOS.

Topics: Animals; Aorta; Blood Pressure; Blotting, Western; Bone Marrow Cells; Cells, Cultured; Chromones; Cyclic GMP; Endothelium, Vascular; Enzyme Inhibitors; Inflammation; Lipopolysaccharides; Macrophages; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Morpholines; Nitrates; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Nitrites; Phosphatidylinositol 3-Kinases; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Rotenone; Sepsis; Shock, Septic; Time Factors; Tissue Distribution

Mice carrying a targeted disruption of the Npr1 gene (coding for guanylyl cyclase/natriuretic peptide receptor A (NPRA)) exhibit increased blood pressure, cardiac hypertrophy, and congestive heart failure, similar to untreated human hypertensive patients. The objective of this study was to determine whether permanent ablation of NPRA signaling in mice alters the expression of matrix metalloproteinase (MMP)-2 and MMP-9 and pro-inflammatory mediators such as tumor necrosis factor-alpha (TNF-alpha), leading to myocardial collagen remodeling. Here, we report that expression levels of the MMP-2 and MMP-9 genes were increased by 3-5-fold and that the expression of the TNF-alpha gene was enhanced by 8-fold in Npr1 homozygous null mutant (Npr1-/-) mouse hearts compared with wild-type (Npr1+/+) control mouse hearts. Myocardial fibrosis, total collagen, and the collagen type I/III ratio (p < 0.01) were dramatically increased in adult Npr1-/- mice compared with age-matched wild-type counterparts. Hypertrophic marker genes, including the beta-myosin heavy chain and transforming growth factor-beta1, were significantly up-regulated (3-5-fold) in both young and adult Npr1-/- mouse hearts. NF-kappa B binding activity in ventricular tissues was enhanced by 4-fold with increased translocation of the p65 subunit from the cytoplasmic to nuclear fraction in Npr1-/- mice. Our results show that reduced NPRA signaling activates MMP, transforming growth factor-beta1, and TNF-alpha expression in Npr1-/- mouse hearts. The findings of this study demonstrate that disruption of NPRA/cGMP signaling promotes hypertrophic growth and extracellular matrix remodeling, leading to the development of cardiac hypertrophy, myocardial fibrosis, and congestive heart failure.

Topics: Animals; Antihypertensive Agents; Blood Pressure; Blotting, Northern; Blotting, Western; Cell Nucleus; Collagen; Cyclic GMP; Cytosol; Densitometry; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors; Extracellular Matrix; Fibrosis; Genetic Markers; Genotype; Guanylate Cyclase; Heart Ventricles; Homozygote; Hydroxyproline; I-kappa B Kinase; Inflammation; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Mice; Mice, Transgenic; Models, Biological; Mutation; Myocardium; NF-kappa B; Protein Isoforms; Protein Serine-Threonine Kinases; Receptors, Atrial Natriuretic Factor; Recombination, Genetic; Ribonucleases; Signal Transduction; Tumor Necrosis Factor-alpha; Up-Regulation

Prolidase [E.C. 3.4.13.9], a member of the matrix metalloproteinase (MMP) family, is a manganese-dependent cytosolic exopeptidase that cleaves imidodipeptides containing C-terminal proline or hydroxyproline. It plays an important role in collagen metabolism, matrix remodeling and cell growth. Nitric oxide (NO), a versatile signaling molecule, regulates many processes including collagen synthesis and matrix remodeling and, thereby, may modulate angiogenesis, tumor invasiveness, and metastasis. Thus, we considered that prolidase may be an important target of NO regulation. In our study, SIN I and DETA/NO were used as NO donors. Both donors increased prolidase activity in a time-dependent and dose-dependent manner. Prolidase activity increased not only with NO donors but also with endogenous NO in cells transfected with iNOS. The effect of iNOS was abolished by treatment with S-methylisothiourea (SMT), a selective inhibitor of iNOS. However, with either exogenous or endogenous sources of NO, the increase in prolidase activity was not accompanied by increased prolidase expression. Therefore, we suspected phosphorylation of prolidase as a potential mechanism regulating enzyme activation. We observed increased serine/threonine phosphorylation on prolidase protein in cells treated with NO donors and in cells transfected with iNOS. To determinate the pathways that may mediate prolidase induction by NO, we first used 8-Br-cGMP, a cGMP agonist, and found that 8-Br-cGMP strongly and rapidly stimulated prolidase activity accompanied by increased phosphorylation. Rp-8-Br-pCPT-cGMP, an inhibitor of cGMP, reduced NO donor-stimulated prolidase activity to control levels. To test whether the MAPK pathway is involved in this NO-dependent activation, we used an ERK1/2 inhibitor and found that it had no effect on prolidase activity increased by NO donors. These results demonstrate that NO stimulates prolidase activity by increasing serine/threonine phosphorylation through PKG-cGMP pathway, but independent of MAPK and suggest an interaction between inflammatory signaling pathways and regulation of the terminal step of matrix degradation.

Topics: Animals; Blotting, Western; Cyclic GMP; Dipeptidases; DNA, Complementary; Dose-Response Relationship, Drug; Gene Expression Regulation, Enzymologic; Immunoprecipitation; Inflammation; Matrix Metalloproteinases; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neovascularization, Pathologic; NIH 3T3 Cells; Nitric Oxide; Nitric Oxide Synthase Type II; Peptides; Phosphorylation; Plasmids; Serine; Signal Transduction; Thiourea; Threonine; Time Factors; Transfection; Wound Healing

Several peptides, including vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), activate the release of arachidonic acid (AA) and nitric oxide (NO) in endothelial cells (ECs). Both messengers are involved in EC proliferation and vascular permeability and control calcium homeostasis in different ways. Interestingly, it has been recently suggested that NO acts as a downstream mediator of AA-induced calcium entry in smooth muscle cells and isolated mouse parotid cells. In this paper, we have investigated the complex relationships that link intracellular calcium, AA, and NO in cultured endothelial cells. Using different experimental approaches, mainly simultaneous Ca2+ and NO fluorimetric confocal imaging, we provide evidence for a complex pathway leading to noncapacitative calcium entry (NCCE) in bovine aortic endothelial cells (BAECs). In particular, AA is able to induce NCCE through two different pathways: one dependent on eNOS recruitment and NO release, the other NO-independent. Finally, we show that NO increase is involved in the control of BAEC proliferation.

Topics: Analysis of Variance; Animals; Aorta; Arachidonic Acid; Biological Transport; Calcium; Calcium Channels; Cattle; Cell Proliferation; Cells, Cultured; Cyclic GMP; Electrophysiology; Endothelial Cells; Endothelium, Vascular; Fluorescent Dyes; Homeostasis; Inflammation; Microscopy, Confocal; Models, Biological; Myocytes, Smooth Muscle; Neovascularization, Physiologic; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase Type III; Patch-Clamp Techniques; Perfusion; Permeability; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Time Factors

The natriuretic peptides, including human B-type natriuretic peptide (BNP), have been implicated in the regulation of cardiac remodeling. Because transforming growth factor-beta (TGF-beta) is associated with profibrotic processes in heart failure, we tested whether BNP could inhibit TGF-beta-induced effects on primary human cardiac fibroblasts. BNP inhibited TGF-beta-induced cell proliferation as well as the production of collagen 1 and fibronectin proteins as measured by Western blot analysis. cDNA microarray analysis was performed on RNA from cardiac fibroblasts incubated in the presence or absence of TGF-beta and BNP for 24 and 48 hours. TGF-beta, but not BNP, treatment resulted in a significant change in the RNA profile. BNP treatment resulted in a remarkable reduction in TGF-beta effects; 88% and 85% of all TGF-beta-regulated mRNAs were affected at 24 and 48 hours, respectively. BNP opposed TGF-beta-regulated genes related to fibrosis (collagen 1, fibronectin, CTGF, PAI-1, and TIMP3), myofibroblast conversion (alpha-smooth muscle actin 2 and nonmuscle myosin heavy chain), proliferation (PDGFA, IGF1, FGF18, and IGFBP10), and inflammation (COX2, IL6, TNFalpha-induced protein 6, and TNF superfamily, member 4). Lastly, BNP stimulated the extracellular signal-related kinase pathway via cyclic guanosine monophosphate-dependent protein kinase signaling, and two mitogen-activated protein kinase kinase inhibitors, U0126 and PD98059, reversed BNP inhibition of TGF-beta-induced collagen-1 expression. These findings demonstrate that BNP has a direct effect on cardiac fibroblasts to inhibit fibrotic responses via extracellular signal-related kinase signaling, suggesting that BNP functions as an antifibrotic factor in the heart to prevent cardiac remodeling in pathological conditions.

Topics: Adolescent; Blotting, Western; Butadienes; Cell Division; Cells, Cultured; Cyclic GMP; Enzyme Inhibitors; Extracellular Matrix Proteins; Fibroblasts; Fibrosis; Flavonoids; Gene Expression Profiling; Gene Expression Regulation; Humans; Inflammation; Male; MAP Kinase Signaling System; Middle Aged; Muscle Proteins; Natriuretic Peptide, Brain; Nitriles; Oligonucleotide Array Sequence Analysis; Reverse Transcriptase Polymerase Chain Reaction; Transforming Growth Factor beta; Ventricular Remodeling

Nitric oxide (NO) production by the vascular endothelium maintains an essential antiinflammatory, cytoprotective influence on the blood vessel wall. A key component of this activity is attributed to prevention of leukocyte-endothelial cell interactions, yet the underlying mechanisms remain unclear. The NO receptor, soluble guanylate cyclase (sGC), is expressed in endothelial cells but fulfils an unknown function. Therefore, we used intravital microscopy in mesenteric postcapillary venules from WT and endothelial nitric oxide synthase (eNOS) knockout (eNOS(-/-)) mice, and an sGC activator (BAY 41-2272), to investigate a potential role for sGC in the regulation of adhesion molecule expression and leukocyte recruitment. Leukocyte rolling and adhesion was 6-fold greater in eNOS(-/-) than WT animals. BAY 41-2272 and the NO-donor, diethylamine-NONOate, reduced leukocyte rolling and adhesion in eNOS(-/-) mice to levels observed in WT animals. These effects were blocked by the sGC inhibitor ODQ [1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one], which itself caused a 6-fold increase in leukocyte rolling and adhesion in WT mice. Increased leukocyte rolling and adhesion in IL-1beta-treated mice was also inhibited by BAY 41-2272. Fluorescence-activated cell sorting analysis in vitro and a specific P-selectin neutralizing antibody in vivo revealed that selective down-regulation of P-selectin expression accounted for the antiadhesive effects of sGC activation. These data demonstrate that sGC plays a key antiinflammatory role by inhibiting P-selectin expression and leukocyte recruitment.

Topics: Animals; Antibodies, Monoclonal; Cell Communication; Cyclic GMP; Down-Regulation; Endothelial Cells; Gene Expression Regulation; Guanylate Cyclase; Inflammation; Intercellular Adhesion Molecule-1; Interleukin-1; Leukocytes; Male; Mice; Mice, Knockout; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; P-Selectin; Pyrazoles; Pyridines

cGMP-dependent protein kinase I (PKG-I) has been suggested to contribute to the facilitation of nociceptive transmission in the spinal cord presumably by acting as a downstream target of nitric oxide. However, PKG-I activators caused conflicting effects on nociceptive behavior. In the present study we used PKG-I(-/-) mice to further assess the role of PKG-I in nociception. PKG-I deficiency was associated with reduced nociceptive behavior in the formalin assay and zymosan-induced paw inflammation. However, acute thermal nociception in the hot-plate test was unaltered. After spinal delivery of the PKG inhibitor, Rp-8-Br-cGMPS, nociceptive behavior of PKG-I(+/+) mice was indistinguishable from that of PKG-I(-/-) mice. On the other hand, the PKG activator, 8-Br-cGMP (250 nmol intrathecally) caused mechanical allodynia only in PKG-I(+/+) mice, indicating that the presence of PKG-I was essential for this effect. Immunofluorescence studies of the spinal cord revealed additional morphological differences. In the dorsal horn of 3- to 4-week-old PKG-I(-/-) mice laminae I-III were smaller and contained fewer neurons than controls. Furthermore, the density of substance P-positive neurons and fibers was significantly reduced. The paucity of substance P in laminae I-III may contribute to the reduction of nociception in PKG-I(-/-) mice and suggests a role of PKG-I in substance P synthesis.

Topics: Animals; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Disease Models, Animal; Formaldehyde; Hot Temperature; Hyperalgesia; Inflammation; Mice; Mice, Knockout; Neurons; Pain; Reaction Time; Substance P; Thionucleotides

Elevated plasma homocysteine (HCy) concentration is a risk factor for cardiovascular diseases associated with endothelial dysfunction, including the human digital ischaemic disease, Raynaud's phenomenon.. HCy causes dysfunction of equine vascular endothelium and elevated plasma concentrations predispose to laminitis.. To determine 1) the concentration of HCy in vitro, which inhibits equine vascular endothelial cell function and 2) any association between risk of laminitis and plasma HCy concentration.. Endothelial function was studied by measuring endothelium-dependent vasodilatory responses of the equine isolated perfused digit and basal nitric oxide (NO) production by cultured equine digital vein endothelial cells (EDVECs). Total plasma HCy (tHCy) concentrations were measured in samples collected in the winter and spring from normal ponies and ponies predisposed to laminitis.. HCy (10 and 100 micromol/l) inhibited endothelial function and, at concentrations above 100 micromol/l, inhibited NO production by EDVECs. Plasma tHCy concentration ranged from 13 to 14.7 micromol/l. There was no effect of season or disease status on the concentration measured.. In vitro, HCy was shown to interfere with endothelial cell function at physiologically relevant concentrations. No evidence was found for an association between risk of laminitis and high plasma concentrations of HCy.. Elevated plasma HCy concentrations could adversely affect endothelial cell function and mangement regimens that lead to increases in plasma HCy concentration should be avoided in ponies predisposed to laminitis.

Topics: Animals; Case-Control Studies; Cells, Cultured; Chromatography, High Pressure Liquid; Cyclic GMP; Endothelial Cells; Endothelium, Vascular; Foot Diseases; Homocysteine; Hoof and Claw; Horse Diseases; Horses; Inflammation; Nitric Oxide; Risk Factors; Seasons; Vasodilation

The leukocyte adhesion molecule L-selectin has an important role in the initial steps of leukocyte extravasation during inflammation and lymphocyte homing. Its cytoplasmic domain is involved in signal transduction after L-selectin cross-linking and in the regulation of receptor binding activity in response to intracellular signals. However, the signaling events occurring at the level of the receptor are largely unknown. This study therefore addressed the question of whether protein kinases associate with the cytoplasmic domain of the receptor and mediate its phosphorylation. Using a glutathione S-transferase fusion protein of the L-selectin cytoplasmic domain, we isolated a kinase activity from cellular extracts of the human leukemic Jurkat T-cell line that phosphorylated L-selectin on serine residues. This kinase showed characteristics of the protein kinase C (PKC) family. Moreover, the Ca(2+)-independent PKC isozymes theta and iota were found associated with the cytoplasmic domain of L-selectin. Pseudosubstrate inhibitors of these isozymes abolished phosphorylation of the cytoplasmic domain, demonstrating that these kinases are responsible for the phosphorylation. Analysis of proteins specifically bound to the phosphorylated cytoplasmic tail of L-selectin revealed that PKCalpha and -theta are strongly associated with the phosphorylated cytoplasmic domain of L-selectin. Binding of these isozymes to L-selectin was also found in intact cells after phorbol ester treatment inducing serine phosphorylation of the receptor. Furthermore, stimulation of Jurkat T-cells by CD3 cross-linking induced association of PKCalpha and -theta with L-selectin, indicating a role of these kinases in the regulation of L-selectin through the T-cell receptor complex. The phosphorylation-regulated association of PKC isozymes with the cytoplasmic domain of L-selectin indicates an important role of this kinase family in L-selectin signal transduction.

Topics: Amino Acid Sequence; Calcium; CD3 Complex; Cross-Linking Reagents; Cyclic GMP; Cytoplasm; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Gene Expression Regulation; Glutathione Transferase; Humans; Inflammation; Isoenzymes; Jurkat Cells; L-Selectin; Lymphocytes; Mass Spectrometry; Molecular Sequence Data; Phosphorylation; Precipitin Tests; Protein Binding; Protein Isoforms; Protein Kinase C; Protein Kinase C-alpha; Protein Kinase C-theta; Protein Structure, Tertiary; Recombinant Fusion Proteins; Sequence Homology, Amino Acid; Serine; Signal Transduction; Threonine

Vascular smooth muscle cells (VSMC) undergo many phenotypic changes when placed in culture. Several studies have shown that the levels of expression of soluble guanylyl cyclase (sGC) or cGMP-dependent protein kinase (PKG) are altered in cultured VSMC. In this study the mechanisms involved in the coordinated expression of sGC and PKG were examined. Pro-inflammatory cytokines that increase the expression of type II NO synthase (inducible NO synthase, or iNOS) decreased PKG expression in freshly isolated, non-passaged bovine aortic SMC. However, in several passaged VSMC lines (i.e. bovine aortic SMC, human aortic SMC, and A7r5 cells), PKG protein expression was not suppressed by cytokines or NO. sGC was highly expressed in non-passaged bovine aortic SMC but not in passaged cell lines. Restoration of expression of sGC to passaged bovine SMC using adenovirus encoding the alpha1 and beta1 subunits of sGC restored the capacity of the cells to increase cGMP in response to NO. Furthermore, treatment of these sGC-transduced cells with NO donors for 48 h resulted in decreased PKG protein expression. In contrast, passaged rat aortic SMC expressed high levels of NO-responsive sGC but demonstrated reduced expression of PKG. Adenovirus-mediated expression of the PKG catalytically active domain in rat aortic SMC caused a reduction in the expression of sGC in these cells. These results suggest that there is a mechanism for the coordinated expression of sGC and PKG in VSMC and that prolonged activation of sGC down-regulates PKG expression. Likewise, the loss of PKG expression appears to increase sGC expression. These effects may be an adaptive mechanism allowing growth and survival of VSMC in vitro.

Topics: Adenoviridae; Animals; Aorta; Blotting, Northern; Blotting, Western; Cattle; Cells, Cultured; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Cytokines; Gene Expression Regulation; Guanylate Cyclase; Inflammation; Lipopolysaccharides; Muscle, Smooth, Vascular; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Phenotype; Protein Structure, Tertiary; Radioimmunoassay; Rats; RNA; Signal Transduction; Time Factors

Natriuretic peptides (NPs), which consist of atrial, brain, and C-type natriuretic peptides (ANP, BNP, and CNP, respectively), are characterized as cardiac or vascular hormones that elicit their biological effects by activation of the cGMPcGMP-dependent protein kinase (cGK) pathway. We recently reported that adenoviral gene transfer of CNP into rabbit blood vessels not only suppressed neointimal formation but also accelerated reendothelialization, a required step for endothelium-dependent vasorelaxation and antithrombogenicity. Accordingly, we investigated the therapeutic potential of the NPscGMPcGK pathway for vascular regeneration. In transgenic (Tg) mice that overexpress BNP in response to hindlimb ischemia, neovascularization with appropriate mural cell coating was accelerated without edema or bleeding, and impaired angiogenesis by the suppression of nitric oxide production was effectively rescued. Furthermore, in BNP-Tg mice, inflammatory cell infiltration in ischemic tissue and vascular superoxide production were suppressed compared with control mice. Ischemia-induced angiogenesis was also significantly potentiated in cGK type I Tg mice, but attenuated in cGK type I knockout mice. NPs significantly stimulated capillary network formation of cultured endothelial cells by cGK stimulation and subsequent Erk12 activation. Furthermore, gene transfer of CNP into ischemic muscles effectively accelerated angiogenesis. These findings reveal an action of the NPscGMPcGK pathway to exert multiple vasculoprotective and regenerative actions in the absence of apparent adverse effects, and therefore suggest that NPs as the endogenous cardiovascular hormone can be used as a strategy of therapeutic angiogenesis in patients with tissue ischemia.

Topics: Animals; Atrial Natriuretic Factor; Blood Vessels; Cells, Cultured; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Gene Expression; Gene Transfer Techniques; Humans; Inflammation; Ischemia; Mice; Mice, Knockout; Mice, Transgenic; Natriuretic Peptide, Brain; Natriuretic Peptide, C-Type; Neovascularization, Physiologic; Regeneration

Expression of inducible nitric-oxide (NO) synthase (iNOS) and "high-output" production of NO by macrophages mediates many cytotoxic actions of these immune cells. However, macrophages have also been shown to express a constitutive NOS isoform, the function of which remains obscure. Herein, bone marrow-derived macrophages (BMDMØs) from wild-type and endothelial NOS (eNOS) knock-out (KO) mice have been used to assess the role of this constitutive NOS isoform in the regulation of macrophage activation. BMDMØs from eNOS KO animals exhibited reduced nuclear factor-kappaB activity, iNOS expression, and NO production after exposure to lipopolysaccharide (LPS) as compared with cells derived from wild-type mice. Soluble guanylate cyclase (sGC) was identified in BMDMØs at a mRNA and protein level, and activation of cells with LPS resulted in accumulation of cyclic GMP. Moreover, the novel non-NO-based sGC activator, BAY 41-2272, enhanced BMDMØ activation in response to LPS, and the sGC inhibitor 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one attenuated activation. These observations provide the first demonstration of a pathophysiological role for macrophage eNOS in regulating cellular activation and suggest that NO derived from this constitutive NOS isoform, in part via activation of sGC, is likely to play a pivotal role in the initiation of an inflammatory response.

Topics: Animals; Cyclic GMP; Enzyme Inhibitors; Guanylate Cyclase; Homeostasis; In Vitro Techniques; Inflammation; Inflammation Mediators; Lipopolysaccharides; Macrophage Activation; Male; Mice; Mice, Knockout; NF-kappa B; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Oxadiazoles; Pyrazoles; Pyridines; Quinoxalines; RNA, Messenger; Solubility

There is recent evidence that nitric oxide blocks IL-6 signaling in hepatocytes by inhibiting activation of Stat3. If this effect is mediated by cGMP, then high-dose biotin--which can directly activate guanylyl cyclase--may have the potential to suppress hepatic production of acute phase proteins.

Topics: Acute-Phase Reaction; Animals; Biotin; Cells, Cultured; Cyclic GMP; DNA-Binding Proteins; Down-Regulation; Humans; Inflammation; Interleukin-6; Liver; Models, Theoretical; Nitric Oxide; Rats; Signal Transduction; STAT3 Transcription Factor; Trans-Activators

The molecular mechanisms responsible for sepsis-induced myocardial dysfunction remain undefined. CD14 mediates the inflammatory response to lipopolysaccharide (LPS) in various organs including the heart. In this study we investigated the role of CD14 in LPS-induced myocardial dysfunction in vivo.. Wild-type and CD14-deficient (CD14-D) mice were challenged with Escherichia coli LPS. Myocardial tumor necrosis factor, interleukin-1beta (IL-1beta), and NOS2 induction was measured before and 6 hours after LPS challenge. Echocardiographic parameters of left ventricular function were measured before and 6 hours after LPS administration. LPS challenge induced a significant increase in myocardial tumor necrosis factor and IL-1beta mRNA and protein expression in wild-type mice. In contrast, mRNA and protein levels for TNF and IL-1beta were significantly blunted in CD14-D mice. An increase in NOS2 protein was noted within 6 hours of LPS provocation only in the hearts of wild-type mice. This was associated with an increase in ventricular cGMP levels. Activation of nuclear factor-kappaB was observed within 30 minutes of LPS in the hearts of wild-type mice but not in CD14-D mice. In wild-type mice, LPS significantly decreased left ventricular fractional shortening, velocity of circumferential shortening, and dP/dt(max). LPS-treated CD14-D mice maintained normal cardiac function.. These results suggest that CD14 is important in mediating the proinflammatory response induced by LPS in the heart and that CD14 is necessary for the development of left ventricular dysfunction during LPS-induced shock in vivo.

Topics: Animals; Cyclic GMP; Female; Heart Ventricles; Hemodynamics; Inflammation; Interleukin-1; Lipopolysaccharide Receptors; Lipopolysaccharides; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Myocardium; NF-kappa B; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; RNA, Messenger; Shock, Septic; Tumor Necrosis Factor-alpha; Ultrasonography; Ventricular Dysfunction, Left

Nitric oxide (*NO) and *NO-derived reactive species rapidly react with lipids during both autocatalytic and enzymatic oxidation reactions to yield nitrated derivatives that serve as cell signaling molecules. Herein we report the synthesis, purification, characterization, and bioactivity of nitrolinoleate (LNO2). Nitroselenylation of linoleic acid yielded LNO2 that was purified by solvent extraction, silicic acid chromatography, and reverse-phase HPLC. Structural characterization was performed by IR spectroscopy, 15N-NMR, LC-negative ion electrospray mass spectroscopy (MS), and chemiluminescent nitrogen analysis. Quantitative MS analysis of cell and vessel LNO2 metabolism, using L[15N]O2 as an internal standard, revealed that LNO2 is rapidly metabolized by rat aortic smooth muscle (RASM) monolayers and rat thoracic aorta, resulting in nitrite production and up to 3-fold increases in cGMP (ED50 = 30 microM for RASM, 50 microM for aorta). LNO2 induced endothelium-independent relaxation of preconstricted rat aortic rings, which was unaffected by L(G)-nitro-l-arginine methyl ester addition and inhibited by the guanylate cyclase inhibitor 1H-[1,2,4] oxadiazole[4,3-a]quinoxalin-1-one and the *NO scavenger HbO2. These results reveal that synthetic LNO2, identical to lipid derivatives produced biologically by the reaction of *NO and *NO-derived species with oxidizing unsaturated fatty acids (e.g., linoleate), can transduce vascular signaling actions of *NO.

Topics: Animals; Aorta, Thoracic; Chromatography, High Pressure Liquid; Cyclic GMP; Endothelium, Vascular; Enzyme Inhibitors; Fatty Acids, Unsaturated; Free Radical Scavengers; Guanylate Cyclase; Inflammation; Linoleic Acids; Muscle, Smooth, Vascular; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitrites; Nitro Compounds; Oxadiazoles; Oxidation-Reduction; Oxyhemoglobins; Quinoxalines; Rats; Spectrometry, Mass, Electrospray Ionization; Spectrophotometry, Infrared; Vasodilation

Tetrahydrobiopterin(BH4) serves as an essential cofactor for the biosynthesis of nitric oxide (NO). BH4 is de novo synthesized from GTP and GTP cyclohydrolase I(GCH I) is the rate-limiting enzyme in the biosynthesis of BH4. Under inflammatory conditions, it is reported that endothelial cells release large amount of BH4. In this study, we examined the regulation mechanism of the biosynthesis of BH4 in human umbilical vein endothelial cells(HUVEC). Prostacyclin and forskolin, reagents of stimulation of cAMP signaling cascade, reduced cytokine induced biosynthesis of BH4 through the inhibition of expression of GCH I mRNA. On the other hand, stimulations of NO-cGMP signaling pathway inhibited GCH I activities through the post translational modification of GCH I enzyme. Both two signaling cascade lead to vasodilation. It is suggested that the biosynthesis of BH4 can be regulated by negative feed back regulation systems between endothelium and smooth muscle cells to prevent over stimulated vasodilation.

Topics: Biopterins; Cells, Cultured; Cyclic AMP; Cyclic GMP; Endothelium, Vascular; Epoprostenol; Feedback, Physiological; GTP Cyclohydrolase; Humans; Inflammation; Muscle, Smooth, Vascular; Nitric Oxide; Protein Processing, Post-Translational; Signal Transduction; Umbilical Veins; Vasodilation

Myeloperoxidase (MPO) is an abundant mammalian phagocyte hemoprotein thought to primarily mediate host defense reactions. Although its microbicidal functions are well established in vitro, humans deficient in MPO are not at unusual risk of infection. MPO was observed herein to modulate the vascular signaling and vasodilatory functions of nitric oxide (NO) during acute inflammation. After leukocyte degranulation, MPO localized in and around vascular endothelial cells in a rodent model of acute endotoxemia and impaired endothelium-dependent relaxant responses, to which MPO-deficient mice were resistant. Altered vascular responsiveness was due to catalytic consumption of NO by substrate radicals generated by MPO. Thus MPO can directly modulate vascular inflammatory responses by regulating NO bioavailability.

Topics: Animals; Aorta; Catalysis; Cattle; Cells, Cultured; Chromans; Coculture Techniques; Cyclic GMP; Endothelium, Vascular; Endotoxemia; Humans; Hydrogen Peroxide; Inflammation; Leukocytes; Mice; Mice, Inbred C57BL; Muscle, Smooth, Vascular; Mutation; Nitric Oxide; Oxidation-Reduction; Peroxidase; Rats; Rats, Sprague-Dawley; Signal Transduction; Transfection; Tumor Cells, Cultured; Vasodilation

This study investigated the regulatory effects of the major inflammatory mediator, nitric oxide (NO), on human neutrophil apoptosis in vitro. Co-culture of human neutrophils with the NO donors GEA 3162 (1,2,3,4-oxatriazolium,5-amino-3-(3,4-dichlorophenyl)-chloride) (10-100 microM) and 3-morpholino-sydnonimine (SIN-1) (0.3-3 mM) caused a dramatic and concentration-dependent induction of apoptosis. However, N-formyl-methionyl-leucyl-phenylalanine (FMLP)-induced neutrophil activation (actin reorganization and chemotaxis) was inhibited by GEA 3162 treatment. The pro-apoptotic effects of the NO donors were (i) unaffected by the soluble guanylate cyclase inhibitor LY-83583 (6-anilino-5,8-quinolinedione; 100 microM), (ii) antagonized by superoxide dismutase (6 microg/mL), (iii) mimicked by exogenous peroxynitrite (at concentrations >100 microM), and (iv) inhibited by the caspase inhibitor Z-Val-Ala-DL-Asp-fluoromethylketone (100 microM). The pro-apoptotic effect of the NO donors was not mimicked by the cell-permeable cyclic nucleotide analogue, N6,2-O-dibutyrylguanosine-3',5'-cyclic monophosphate (dibutyryl-cGMP) at concentrations < or =0.2 mM. Indeed, at high concentrations (> or =2 mM), dibutyryl-cGMP caused an inhibition of apoptosis. These results suggest that NO-mediated apoptosis, although caspase-dependent, is mediated by a cGMP-independent mechanism and involves the concurrent generation of oxygen free radicals and, potentially, peroxynitrite. Our data reveal a unique role for NO in inflammatory responses with differential effects upon neutrophil activation and survival, with important implications for the successful resolution of inflammation.

Topics: Adult; Apoptosis; Caspases; Cyclic GMP; Free Radicals; Guanylate Cyclase; Humans; In Vitro Techniques; Inflammation; Neutrophil Activation; Neutrophils; Nitric Oxide; Nitric Oxide Donors; Reactive Oxygen Species; Triazoles

The present study examined whether nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) can directly inhibit aerobic energy metabolism and impair cell function in interleukin (IL)-1beta,-stimulated cardiac myocytes.. Recent reports have indicated that excessive production of NO induced by cytokines can disrupt cellular energy balance through the inhibition of mitochondrial respiration in a variety of cells. However, it is still largely uncertain whether the NO-induced energy depletion affects myocardial contractility.. Primary cultures of rat neonatal cardiac myocytes were prepared, and NO2-/NO3- (NOx) in the culture media was measured using Griess reagent.. Treatment with IL-1beta (10 ng/ml) increased myocyte production of NOx in a time-dependent manner. The myocytes showed a concomitant significant increase in glucose consumption, a marked increase in lactate production, and a significant decrease in cellular ATP (adenosine 5'-triphosphate). These metabolic changes were blocked by co-incubation with N(G)-monomethyl-L-arginine (L-NMMA), an inhibitor of NO synthesis. Sodium nitroprusside (SNP), a NO donor, induced similar metabolic changes in a dose-dependent manner, but 8-bromo-cyclic guanosine 3',5'-monophosphate (8-bromo-cGMP), a cGMP donor, had no effect on these parameters. The activities of the mitochondrial iron-sulfur enzymes, NADH-CoQreductase and succinate-CoQreductase, but not oligomycin-sensitive ATPase, were significantly inhibited in the IL-1beta, or SNP-treated myocytes. Both IL-1beta and SNP significantly elevated maximum diastolic potential, reduced peak calcium current (I(Ca)), and lowered contractility in the myocytes. KT5823, an inhibitor of cGMP-dependent protein kinase, did not block the electrophysiological and contractility effects.. These data suggest that IL-1beta-induced NO production in cardiac myocytes lowers energy production and myocardial contractility through a direct attack on the mitochondria, rather than through cGMP-mediated pathways.

Topics: Adenosine Triphosphate; Animals; Animals, Newborn; Cells, Cultured; Cyclic GMP; Disease Models, Animal; Dose-Response Relationship, Drug; Energy Metabolism; Glucose; Glycolysis; Inflammation; Interleukin-1; Lactic Acid; Mitochondria, Heart; Myocardial Contraction; Myocardium; Nitric Oxide; Nitric Oxide Synthase; Nitroprusside; omega-N-Methylarginine; Rats

Topics: Amyloid beta-Peptides; Animals; Aorta; Cyclic GMP; Dipyridamole; Endothelin-1; In Vitro Techniques; Inflammation; Leukotriene B4; Microglia; Muscle, Smooth, Vascular; Nitroprusside; Peptide Fragments; Rats; Vasoconstriction

1. The objective of the present paper was to evaluate the relevance of neuronal balance of cyclic AMP and cyclic GMP concentration for functional regulation of nociceptor sensitivity during inflammation. 2. Injection of PGE2 (10-100 ng paw-1) evoked a dose-dependent hyperalgesic effect which was mediated via a cyclic AMP-activated protein kinase (PKA) inasmuch as hyperalgesia was blocked by the PKA inhibitor H89. 3. The PDE4 inhibitor rolipram and RP73401, but not PDE3 and PDE5 inhibitors potentiated the hyperalgesic effects of PGE2. The hyperalgesic effect of dopamine was also enhanced by rolipram. Moreover, rolipram significantly potentiated hyperalgesia induced by carrageenan, bradykinin, TNF alpha, IL-1 beta, IL-6 and IL-8. This suggests that neuronal cyclic AMP mediates the prostanoid and sympathetic components of mechanical hyperalgesia. Moreover, in the neuron cyclic AMP is mainly metabolized by PDE4. 4. To examine the role of the NO/cyclic GMP pathway in modulating mechanical hyperalgesia, we tested the effects of the soluble guanylate cyclase inhibitor, ODQ. This substance counteracts the inhibitory effects of the NO donor, SNAP, on the hyperalgesia induced by PGE2. 5. The ODQ potentiated hyperalgesia induced by carrageenan, bradykinin, TNF alpha, IL-1 beta, IL-6 and IL-8. In contrast, ODQ had no significant effect on the hyperalgesia induced by PGE2 and dopamine. This indicates that the hyperalgesic cytokines may activate soluble guanylate cyclase, which down-regulate the ability of these substances to cause hyperalgesia. This event appears not to be mediated by prostaglandin or dopamine. 6. In conclusion, the results presented in this paper confirm an association between (i) hyperalgesia and elevated levels of cyclic AMP as well as (ii) antinociception and elevated levels of cyclic GMP. The intracellular levels of cyclic AMP that enhance hyperalgesia are controlled by the PDE4 isoform and appear to result in activation of protein kinase A whereas the intracellular levels of cyclic GMP results from activation of a soluble guanylate cyclase.

Topics: Animals; Bradykinin; Carrageenan; Cyclic AMP; Cyclic GMP; Dinoprostone; Dopamine; Hyperalgesia; Inflammation; Interleukin-1; Interleukin-6; Interleukin-8; Isoquinolines; Male; Oxadiazoles; Phosphodiesterase Inhibitors; Pyrrolidinones; Quinoxalines; Rats; Rats, Wistar; Rolipram; Second Messenger Systems; Sulfonamides; Tumor Necrosis Factor-alpha

We examined the mechanisms by which nitric oxide (.NO) decreased vectorial Na+ transport across confluent monolayers of rat alveolar type II (ATII) cells grown on permeable supports. Amiloride (10 microM) applied to the apical side of monolayers inhibited approximately 90% of the equivalent (Ieq) and the short-circuit (Isc) current, with an half-maximal inhibitory concentration (IC50) of 0.85 microM, indicating that Na+ entry into ATII cells occurred through amiloride-sensitive Na+ channels. .NO generated by spermine NONOate and papa NONOate added to both sides of the monolayers decreased Ieq and increased transepithelial resistance in a concentration-dependent fashion (IC50 = 0.4 microM .NO). These changes were prevented or reversed by addition of oxyhemoglobin (50 microM). Incubation of ATII monolayers with 8-bromoguanosine 3',5'-cyclic monophosphate (400 microM) had no effect on transepithelial Na+ transport. When the basolateral membranes of ATII cells were permeabilized with amphotericin B (10 microM) in the presence of a mucosal-to-serosal Na+ gradient (145:25 mM), .NO (generated by 100 microM papa NONOate) inhibited approximately 60% of the amiloride-sensitive Isc. In addition, after permeabilization of the apical membranes, .NO inhibited the Isc [a measure of Na(+)-K(+)-adenosinetriphosphatase (ATPase) activity] by approximately 60%. We concluded that .NO at noncytotoxic concentrations decreased Na+ absorption across cultured ATII monolayers by inhibiting both the amiloride-sensitive Na+ channels and Na(+)-K(+)-ATPase through guanosine 3',5'-cyclic monophosphate-independent mechanisms.

Topics: Absorption; Adenosine Triphosphate; Animals; Cells, Cultured; Cyclic GMP; Inflammation; Male; Nitric Oxide; Pulmonary Alveoli; Rats; Rats, Sprague-Dawley; Sodium; Sodium Channels; Sodium-Potassium-Exchanging ATPase

Topics: Animals; Calcium; Carbon Monoxide; Cyclic AMP; Cyclic GMP; Fluorometry; Histamine Release; Inflammation; Male; Mast Cells; p-Methoxy-N-methylphenethylamine; Rats; Rats, Wistar

To clarify relationships between the (endothelial vasodilatory and vasoconstrictive function) and leukocyte inflammatory mediators in subjects with asymptomatic atherosclerosis, we measured (intraplatelet cyclic 3',5'-guanosine monophosphate [cGMP] and cyclic 3',5'-adenosine monophosphate [cAMP]), plasma endothelin (ET-1), and plasma neopterin in 197 subjects with asymptomatic atherosclerosis (median age 63 years, range 49-69 years). We measured neutrophil protease 4 (NP4), tumor necrosis factor (TNFmu), soluble tumor necrosis factor receptor-1 (sTNFR-1), and neutrophil gelatinase associated lipocalin (NGAL) in 152 of the 197 subjects. Intraplatelet cGMP correlated inversely with plasma ET-1 (r=-0.22; p=0.01), which confirms earlier in vitro data of the inhibitory effect of ET-1 on NO production and/or the cGMP mediated inhibitory effect of NO on ET-1 production. Plasma neopterin as well as NP4 correlated directly with intraplatelet cGMP (r=0.24; p<0.01 and r=0.33; p<0.001, respectively). Intraplatelet cAMP correlated directly with plasma TNFmu (r=0.17; p<0.05) and sTNFR-1 (r=0.20; p<0.05). The relationship between leukocyte derived inflammatory mediators and intraplatelet cyclic nucleotides suggest an antiaggregating effect of leukocytes upon platelets, which may constitute a negative feedback mechanism that inhibits platelet activation during the atherosclerotic inflammatory process.

Topics: Aged; Arteriosclerosis; Blood Platelets; Carotid Artery, Common; Carotid Stenosis; Cyclic AMP; Cyclic GMP; Cytokines; Endothelin-1; Female; Humans; Inflammation; Leukocytes; Male; Middle Aged; Myeloblastin; Neopterin; Platelet Aggregation; Prospective Studies; Risk Factors; Serine Endopeptidases; Ultrasonography

Using the indirect immunofluorescence method, the distribution of cyclic GMP (cGMP) and nitric oxide synthase (NOS) was investigated in lumbar 5 dorsal root ganglia (DRGs) of untreated rats 1, 3 and 7 days following sciatic nerve section (axotomy). Untreated and axotomized (7 days) rats were also studied after perfusion with the NO donor sodium nitroprusside (SNP). Moreover, rats were injected with carrageenan lambda into the unilateral hindpaw and studied after 6 h, 1 day or 2 days. An increase in the number of cGMP-positive satellite cell profiles was found in axotomized DRGs at 3 days with lower numbers after 7 days. In contrast, no change in cGMP-like immunoreactivity (LI) in satellite cell profiles was detected 1 day after axotomy or 6h, 1 day or 2 days after inflammation, as compared to controls. Axotomy induced a marked increase in the percentage of NOS-immunoreactive (IR) neuron profiles in the ipsilateral DRGs as follows: 3.0% at 1 day, 15% at 3 days and 25% at 7 days, whereas no significant change was found in the expression of NOS-LI in the inflamed DRGs as compared to untreated DRGs. Between 15 and 20% of all NOS-positive neuron profiles were surrounded by, or in partial contact with, cGMP-IR satellite cells in controls 1 and 3 days after axotomy, whereas the corresponding figure was around 5% after 7 days. After SNP perfusion 60-70% of all DRG neuron profiles were partly or totally associated with cGMP-positive satellite cell profiles, with no significant difference between untreated and axotomized ganglia. The nerve injury-induced, parallel upregulation of NOS in DRG neurons and cGMP in satellite cells in the initial phase after axotomy suggests an involvement of NO as a signalling molecule between neurons and satellite cells in DRGs, especially after peripheral nerve injury, perhaps exerting a survival effect as recently proposed by Thippeswamy and Morris (1997).

Topics: Animals; Axotomy; Cyclic GMP; Ganglia, Spinal; Hindlimb; Inflammation; Male; Neurons, Afferent; Nitric Oxide Donors; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Nitroprusside; Rats; Rats, Sprague-Dawley; Sciatic Nerve; Time Factors

Topics: Alprostadil; Arachidonic Acid; Bucladesine; Calcium-Calmodulin-Dependent Protein Kinases; Carbachol; Cell Adhesion; Cell Aggregation; Cyclic GMP; Dibutyryl Cyclic GMP; Enzyme Activation; Humans; In Vitro Techniques; Inflammation; Kinetics; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Salicylates; Salicylic Acid; Virulence Factors, Bordetella

The possible modulatory role of nitric oxide (NO) in neurogenic edema formation in rat paw skin, induced by electrical stimulation of the saphenous nerve, was investigated by using two NO synthase inhibitors, NG-nitro-L-arginine methyl ester (L-NAME) and 7-nitroindazole (7-NI). Both L-NAME (100 mg/kg IV, P < .05) and 7-NI (10 mg/kg IV, P < .05) caused an L-arginine (100 mg/kg IV, P < .01)-reversible inhibition of neurogenic edema as measured by 125I-albumin accumulation, whereas D-NAME (inactive enantiomer of L-NAME) and 6-aminoindazole (structurally similar to 7-NI) were without inhibitory effect. L-NAME produced the predicted vasopressor effect (before, 115 +/- 18 mm Hg; 5 minutes after, 174 +/- 18 mm Hg; n = 6; P < .05), whereas 7-NI showed no significant increase in blood pressure (before, 96 +/- 9 mm Hg; 5 minutes after, 102 +/- 10 mm Hg; n = 6), and neither L-NAME nor 7-NI had any effect on basal or vasodilator calcitonin gene-related peptide (CGRP, 10 pmol per site)-stimulated local blood flow in rat skin, as measured by laser Doppler flowmetry. Furthermore, systemic and local 7-NI had no effect on edema formation induced by local administration of substance P (with or without CGRP) and histamine (with or without CGRP) in rat skin. Since 7-NI blocks edema produced by stimulation of the saphenous nerve, it is suggested that release of NO is involved in neurogenic edema formation, but the vasodilator action of NO is unimportant in this context.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Arginine; Blood Pressure; Cyclic GMP; Endothelium, Vascular; Indazoles; Inflammation; Male; Microcirculation; NG-Nitroarginine Methyl Ester; Nitric Oxide; Rats; Rats, Wistar; Regional Blood Flow; Skin; Substance P

Unilateral intraplantar injection of Freund's complete adjuvant (FCA) into 1 hind paw of rats was used as a model of peripheral inflammation and persistent pain in order to examine time course effects of a continuous barrage of nociceptive input on the second-messenger transducing systems in the spinal cord. cAMP, cGMP and inositol 1,4,5-trisphosphate (insP3) were extracted from the lumbosacral cord at days 1, 7, 14, 21 and 42 following FCA injection and quantified by either radioreceptor-assay (RRA) or radioimmunoassay (RIA). The lumbosacral contents of cAMP and cGMP when quantified in whole lumbosacral cord segment were not significantly changed by FCA treatment at all time points. InsP3 accumulation was significantly increased on days 14, 21 and 42 following FCA injection relative to sham-treated time-matched controls. However, cGMP and insP3 contents were significantly increased in the left longitudinal half of the lumbar enlargement ipsilateral to the injected paw on day 21 following FCA treatment, but not in the sham-treated time-matched controls. With [3H]insP3 as a ligand, Scatchard (Rosenthal) analyses of the concentration-dependent saturation curves showed that the densities (Bmax) of insP3 receptors (insP3R) were significantly increased throughout the time course of adjuvant-induced peripheral inflammation. The binding affinities (KD) for insP3R were significantly decreased on days 7, 14 and 21 following FCA injection corresponding to the times of most stable and peak inflammation. InsP3R from the cerebelli of the same rats as used in the lumbosacral insP3R characterization was used as a positive control in this study and did not show any change in both Bmax and KD as a result of FCA treatment, thus demonstrating that the changes in lumbosacral insP3R characteristics might be specific to the nociceptive sensory pathway such as the spinal cord. Thus it appears that sustained afferent nociceptive input induced by FCA injection increased the accumulation of cGMP, insP3 and insP3R density in the spinal cord through increased neuronal activities of functional receptors coupled to major classes of chemical mediators of nociception including neuropeptides and excitatory aminoacids. Changes in insP3 accumulation in the lumbosacral cord following FCA injection were significantly correlated with changes in insP3R density. Changes in the ratios of lumbosacral insP3 contents and insP3R density were also significantly correlated with changes in body weight

Topics: Animals; Body Weight; Cyclic AMP; Cyclic GMP; Freund's Adjuvant; Inflammation; Inositol 1,4,5-Trisphosphate; Kinetics; Male; Membranes; Nociceptors; Rats; Rats, Sprague-Dawley; Second Messenger Systems; Spinal Cord; Thermodynamics

Hindpaw injections of carrageenan (CARRA) decreased paw withdrawal latencies to radiant heat and increased hindpaw edema. At the peak of hyperalgesia, the content of immunoreactive guanosine 3',5'-cyclic monophosphate (i-cGMP) was increased, while immunoreactive adenosine 3',5'-cyclic monophosphate (i-cAMP) was decreased in lumbar, but not cervical dorsal horn. In general, the content of i-cGMP in the dorsal horn was correlated with thermal hyperalgesia throughout the course of this model. These results indicate that a peripheral inflammation alters the content of i-cGMP and i-cAMP in the spinal cord and, further, suggest that these nucleotides are involved in the modulation of hyperalgesia in the spinal cord.

Topics: Animals; Carrageenan; Cyclic AMP; Cyclic GMP; Foot; Hyperalgesia; Inflammation; Injections; Lumbosacral Region; Male; Neck; Rats; Rats, Sprague-Dawley; Spinal Cord

Nitric oxide (NO.) is a short-lived mediator which can be induced in a variety of cell types and produces many physiologic and metabolic changes in target cells. The inducible or high-output NO. synthase (NOS) pathway was first characterized in macrophages activated by lipopolysaccharide (LPS) and interferon gamma (IFN-gamma). Hepatocytes also express an inducible NOS following exposure to the combination of endotoxin (LPS) and tumor necrosis factor (TNF), interleukin 1 (IL-1), and IFN-gamma. In this study, to identify which of these cytokines, if any, was acting to induce the gene expression for hepatocyte NOS, we measured the levels of rat hepatocyte NOS mRNA by Northern blot analysis after stimulation by various combinations of endotoxin and cytokines in vitro. We found the mRNA for hepatocyte NOS to be a single band at approximately 4.5 kilobases which was maximally up-regulated (approximately 70-fold) by the combination of TNF, IL-1, IFN-gamma, and LPS. Abundance of NOS mRNA peaked 6-8 hr after stimulation and then declined by 25% at 24 hr. Unstimulated hepatocytes in vitro showed only a trace mRNA band after prolonged autoradiographic exposure. As single agents, TNF and IL-1 were the most effective inducers of hepatocyte NOS mRNA. Combinations of two or three stimuli revealed strong synergy between TNF, IL-1, and IFN-gamma. The increased mRNA levels correlated with elevated nitrogen oxide release and cGMP levels in the culture supernatants. Dexamethasone and cycloheximide inhibited induction of mRNA for hepatocyte NOS in a dose-dependent fashion. The addition of NG-monomethyl-L-arginine had no effect on mRNA levels but effectively blocked NO. formation. The inducible hepatocyte NOS mRNA was also detected in rat hepatocytes following chronic hepatic inflammation triggered by Corynebacterium parvum injection in vivo. These data demonstrate that the inducible NOS is functional in rat hepatocytes both in vitro and in vivo and that this pathway is under complex control. Endotoxin and inflammatory cytokines act synergistically to up-regulate gene expression for hepatocyte NOS, whereas glucocorticoids down-regulate the mRNA.

Topics: Amino Acid Oxidoreductases; Animals; Cyclic GMP; Cycloheximide; Cytokines; Dexamethasone; Endotoxins; Enzyme Induction; Glucocorticoids; Guanylate Cyclase; Inflammation; Liver; Male; Nitric Oxide Synthase; Rats; Rats, Sprague-Dawley; RNA, Messenger; Up-Regulation

In this study the effects of cell-permeable 8-bromo-cAMP and 8-bromo-cGMP on intraocular pressure (IOP) and puncture-induced inflammatory response were investigated. Both 8-bromo-cAMP and 8-Bromo-cGMP reduced IOP when given subconjunctivally, but not topically. Subconjunctival administration of 8-bromo-cAMP induced a moderate disruption of the blood-aqueous barrier (BAB); in addition, subconjunctival 8-bromo-cAMP, but not topical 8-bromo-cAMP or subconjunctival 8-bromo-cGMP, reduced the disruption of the BAB and elevation of the aqueous PGE2 level after puncture trauma. It is concluded that the effects of 8-bromo-cAMP depend on the mode of administration, since this determines the concentration of 8-bromo-cAMP reached in the aqueous humor. It is suggested that 8-bromo-cAMP can partially suppress a slight inflammatory response by interference with the release of arachidonic acid from the tissues surrounding the aqueous humor.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Animals; Aqueous Humor; Biological Transport, Active; Conjunctiva; Corneal Injuries; Cyclic AMP; Cyclic GMP; Dinoprostone; Eye Diseases; Eye Proteins; Inflammation; Intraocular Pressure; Rabbits

The inflammatory mediator adenosine caused sustained Cl- secretion across monolayers of T84 cells. The effect was promptly reversed by the adenosine receptor antagonist 8-phenyltheophylline and appeared to be mediated through an adenosine A2-receptor [rank order of potency: 5'-(N-ethyl)-carboxamido-adenosine (NECA) greater than adenosine greater than (-)-N6-(phenylisopropyl)adenosine (PIA) greater than or equal to (+)-PIA]. High doses of adenosine and its analogues increased cellular adenosine 3',5'-cyclic monophosphate (cAMP) but not guanosine 3',5'-cyclic monophosphate (cGMP) or free cytosolic Ca2+. However, lower concentrations of adenosine had maximal effects on Cl- secretion with little or no effect on cAMP. In other respects, Cl- secretion resembled that induced by cAMP-mediated secretagogues such as vasoactive intestinal peptide (VIP). Addition of both low and high doses of NECA activated basolateral K+ and apical Cl- channels, exhibited synergism with Ca2(+)-mediated secretagogues, did not produce additive effects with VIP or Escherichia coli heat-stable enterotoxin, and was associated with cAMP-dependent protein kinase-mediated protein phosphorylation. The results suggest that either adenosine mobilizes an intracellular pool of cAMP that is extremely efficiently coupled to the cAMP-dependent protein kinase and is thereafter rapidly destroyed or that second messenger(s) other than cAMP, cGMP, or Ca2+ are able to activate Cl- secretion in the T84 cell line. In the latter case, such messenger(s), as yet unidentified, might represent a final common pathway for cyclic nucleotide-activated Cl- secretion.

Topics: 2-Chloroadenosine; Adenosine; Adenosine-5'-(N-ethylcarboxamide); Calcium; Cell Line; Chlorides; Cyclic AMP; Cyclic GMP; Cytosol; Electrolytes; Epithelium; Histamine; Humans; Inflammation; Kinetics; Phenylisopropyladenosine; Phosphorylation; Protein Kinases; Theophylline

Endothelial cell incubated with IL-1 have been shown adhere more lymphocytes than nontreated endothelial cells. Here we demonstrate that IL-1 can also increase lymphocyte penetration through endothelial monolayers in vitro. IL-1 induced a transient increase in the number of lymphocytes penetrated through the endothelial monolayer into a filter in a time- and dose-dependent manner. This effect could be mimicked by increasing the cytosolic cAMP levels in the endothelial cells either by forskolin or dibutyryl-cAMP. Concomitantly we were able to show that IL-1 increased the cytosolic cAMP levels in endothelial cells. An inhibitor of adenylate cyclase, ddAdo, decreased both the IL-1-induced cAMP elevation and lymphocyte penetration. A protein kinase A inhibitor HA 1004 could inhibit the IL-1-induced lymphocyte penetration, where as protein kinase C (N-(2-guamidino-ethyl)-5-isoquinolinesyl foamide hydrocloride) and calcium-calmodulin (N-(6-aminohexyl)-5-chloro-1-naphthalensulfanamide) inhibitors had no effect. Adding dibutyryl-cGMP or calcium ionophore to the endothelial cells could not mimic IL-1-induced penetration and finally IL-1 did not induce PKC translocation in endothelial cells. These data support the view that IL-1 acts via cAMP as a second messenger in regard to lymphocyte penetration through endothelial cells. The above data demonstrate that IL-1-induced lymphocyte penetration through endothelial cells and that this IL-1-induced signal is transduced via cAMP in endothelial cells.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Calcium; Cell Movement; Cells, Cultured; Colforsin; Cyclic AMP; Cyclic GMP; Cycloheximide; Dactinomycin; Endothelium, Vascular; Gene Expression; In Vitro Techniques; Inflammation; Interleukin-1; Isoquinolines; Lymphocytes; Piperazines; Protein Kinase C; Protein Kinase Inhibitors; Rats; Rats, Inbred Strains; Signal Transduction; Sulfonamides

Topics: Adult; Biopsy; Colitis, Ulcerative; Cyclic AMP; Cyclic GMP; Female; Humans; Inflammation; Male; Middle Aged; Nucleotides, Cyclic; Rectum; Wound Healing

Because ethanol consumption is associated with increased susceptibility to infection, we examined the effects of ethanol and its metabolite acetaldehyde on human T-lymphocyte migration, an important functional component of cellular inflammatory responses. With a modified Boyden chamber system, ethanol at 0.25% and 0.50% (vol/vol) inhibited spontaneous motility of human T-lymphocytes, in a noncytotoxic manner, to 65% +/- 7% (mean +/- SEM) and 62% +/- 7% of control values of migration, respectively. When T-lymphocyte migration was stimulated by colchicine (10(-5) mol/L), incubation with ethanol (0.25% and 0.50%, vol/vol) decreased migration to 80% +/- 4% and 66% +/- 8% of control values, respectively. Similar degrees of inhibition of migration were obtained with acetaldehyde at concentrations five to 10 times less than ethanol. Ethanol was similarly capable of inhibiting T cell migration induced by dibutyryl cyclic guanosine monophosphate, but it had no effect on stimulated migration induced by a human chemokinetic lymphokine. Our study demonstrates that ethanol, at concentrations achievable in vivo, is capable of depressing T-lymphocyte migration. This effect might contribute to the immunosuppression associated with ethanol consumption.

Topics: Acetaldehyde; Cell Movement; Chemokines, C; Colchicine; Cyclic GMP; Depression, Chemical; Ethanol; Humans; In Vitro Techniques; Inflammation; Lymphocyte Activation; Lymphokines; Sialoglycoproteins; T-Lymphocytes

All anesthetic agents studied, general and local, that controlled pain sufficiently to permit surgery, produced sharp and prolonged reductions in the output of small and blast lymphocytes into efferent lymph from levels encountered in unanesthetized animals. The depth and duration of the depression in lymphocyte traffic were related to the doses of anesthetic agents and had no relation to the surgical trauma involved. Inflammation from previous recent drainage area surgery, extensive intraoperative manipulation of edematous inflammatory tissue, and postoperative manipulation of inflammatory tissue by distention manipulation with injected fluid were found to exert a strong modulating effect on the anesthesia-associated depression in lymphocyte traffic with the capability of rather quick action in elevating lymphocyte traffic. Rapid infusion of bradykinin and 8-bromo cyclic guanosine monophosphate, both traffic enhancing agents, into cannulated afferent lymphatic vessels of the study nodes during anesthesia-associated depression in traffic also were found to exert a modulating effect with prompt increases in lymphocyte output into the efferent lymph.

Topics: Anesthesia, General; Animals; Barbiturates; Bradykinin; Cyclic GMP; Depression, Chemical; Drainage; Halothane; Inflammation; Ketamine; Leukocyte Count; Lymph; Lymph Nodes; Lymphatic System; Lymphocytes; Physical Stimulation; Sheep; Xylazine

The tissue damage during the inflammation is determined by the enzymes and the mediators of anaphylaxis released from polymorphonuclear cells (PMN), platelets, basophils and mastocytes. The control of this release involves the cAMP and cGMP. The cyclic nucleotides independently or synergically regulate the polymerisation of the microtubules and the microfilaments. Drugs increasing the intracellular concentration of cAMP or cGMP inhibit or enhance respectively the enzymatic release from PMN, basophils and mastocytes. In the platelets, cAMP plays a very important role, whereas the cGMP function is controversial.

Topics: Adenylyl Cyclases; Animals; Basophils; Blood Platelets; Calcium; Cyclic AMP; Cyclic GMP; Guanylate Cyclase; Humans; Inflammation; Mast Cells; Neutrophils

Polymorphonuclear (PMN) leukocytes mediate that phase of inflammation at which vascular responses become translated into tissue injury. After phagocytosis, the PMN leukocyte generates derivatives of molecular oxygen (O2-.,OH., and H2O2) that stimulate a metabolic burst and assist in the killing of microorganisms. They also release oxidation products of membrane fatty acids (e.g., arachidonate), which are detected as thromboxanes and protaglandins. After interaction of phagocytic ligands (immune complexes and C3b-opsonized particles), the PMN leukocyte secretes lysosomal enzymes from open phagocytic vacuoles, and, especially when phagocytosis is blocked by cytochalasin B, secretes them directly into the cell's surrounding fluids. Secretion is enhanced by agents that elevate intracellular levels of cyclic GMP, and inhibited by agents that raise cyclic AMP. These reciprocal changes are associated with assembly and disassembly (respectively) of cytoplasmic microtubules. These cytoskeletal structures, together with contractile elements, regulate in part the secretory events of inflammation in which lysosomal constituents (e.g., elastase, collagenase, and cathepsin G) are diverted from their intracellular depots to an inappropriate assault on the tissues of the host.

Topics: Blood Bactericidal Activity; Cyclic AMP; Cyclic GMP; Fatty Acids; Humans; Inflammation; Leukocytes; Lysosomes; Phagocytosis; Prostaglandins; Thromboxanes

Polymorphonuclear leukocytes secrete substances that defend against foreign invaders by mechanisms that bear remarkable resemblances to those of exocrine and endocrine glands. Similarly, leukocyte secretions can also damage the very host they are intended to protect. Like other secretory cells, leukocytes also bear surface receptors to detect signals that initiate secretion. Clinical implications are detailed.

Topics: Antigen-Antibody Complex; Cell Membrane; Cyclic AMP; Cyclic GMP; Inflammation; Neutrophils; Phagocytosis; Superoxides

Stimulated rabbit peritoneal polymorphonuclear leukocyte (PMN) preparations simultaneously produce prostaglandin-like material and mediators that induce metabolic alterations in experimental animals characteristic of the host's responses to inflammation. The alterations observed in rats include responses by: proteins, carbohydrates, hormones, trace metals, and total blood neutrophils. This study demonstrates a possible relationship between prostaglandins and PMN-derived substances that mediate plasma zinc depression, hepatic amino acid uptake, and increased numbers of blood neutrophils. Production of these mediators by stimulated-PMN preparations was prevented by 23 muM indomethacin or 93 muM aspirin. Conversely, morphine (2 mM or less) had no detrimental effect on production of these mediators, although, it consistently stimulated production of a substance stimulating total blood neutrophia. In addition, 2 muM prostaglandin E and F stimulated production of substances mediating hepatic amino acid uptake plasma zinc depression, respectively. At this concentration, neither prostaglandin significantly altered production of substances mediating increased numbers of total blood neutrophils. A partial-nitrogen atmosphere, dibutyryl cyclic analogs of AMP and GMP, or homogenization of the PMN had no effect on mediator production. The inhibitory effect of indomethacin and aspirin also was observed with PMN-homogenastes. These experimental observations suggest that prostaglandin synthesis has a function in production of mediators by stimulated-PMN preparations.

Topics: Amino Acids; Animals; Aspirin; Biological Assay; Bucladesine; Cells, Cultured; Cyclic GMP; Cyclooxygenase Inhibitors; Indomethacin; Inflammation; Leukocyte Count; Mixed Function Oxygenases; Morphine; Neutrophils; Prostaglandins; Prostaglandins E; Prostaglandins F; Rabbits; Zinc

The changes in leucocyte concentrations of cyclic AMP and cyclic GMP were monitored during pyrophosphate-induced pleurisy in rats. A classical 'Yin Yang' relationship was demonstrated.

Topics: Animals; Cyclic AMP; Cyclic GMP; Diphosphates; Inflammation; Male; Pleurisy; Rats; Time Factors

Topics: Atropine; Blood Platelets; Bucladesine; Carbachol; Cell Survival; Cells, Cultured; Colchicine; Cyclic AMP; Cyclic GMP; Deuterium; Epinephrine; Glucuronidase; Histamine; Humans; Inflammation; Isoproterenol; Leukocytes; Lymphocytes; Monocytes; Peroxidases; Phenylephrine; Prostaglandins; Theophylline