8-bromocyclic-gmp and Inflammation

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

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

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

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

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