A simple and sensitive bioassay method for detection of EDRF with RFL-6 rat lung fibroblasts

Ishii, Kunio; Sheng, Hong; Warner, Timothy D.; Förstermann, Ulrich; Murad, Ferid

doi:10.1152/ajpheart.1991.261.2.h598

Cited by 66 publications

(77 citation statements)

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“…Thus, allosteric inhibition of sGC is mediated by a regulatory purine nucleotide binding site formed by residues in the ␣ and ␤ subunits that preferentially binds ATP compared to GTP, in contrast to the rigid nucleotide specificity of the substrate catalytic site. The role of sGC as an intracellular sensor of ATP, coupling metabolic and energetic fluxes to NO-dependent signaling was examined by using RFL-6 cells, a particularly responsive model of NO-induced [GMP] i accumulation (27). Regulation of sGC by [ATP] i , rather than extracellular ATP, was highlighted by the inability of ATP to alter [cGMP] i accumulation in response to SNP when added to intact RFL-6 cells, reflecting the impermeability of the plasma membrane to polyanionic nucleotides (Fig.…”

Section: Resultsmentioning

confidence: 99%

Guanylyl cyclase is an ATP sensor coupling nitric oxide signaling to cell metabolism

Ruiz-Stewart

Tiyyagura

Lin

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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Defending cellular integrity against disturbances in intracellular concentrations of ATP ([ATP]M etabolically active cells with high-energy requirements are particularly susceptible to structural and functional impairment when deprived of oxygen and substrates obligatory for generating ATP (1, 2). Maintenance of homeostasis requires these cells to respond to rapidly fluctuating energy demands in the context of varying supplies of metabolic substrates. Tight coordination between energy supply and demand is predicated on efficient communication and integration between metabolism, the steady-state and local availability of high-energy phosphates, and signaling mechanisms regulating cell functions. Although specific components and their interactions that transduce metabolic and energetic signaling have been described (1), molecular mechanisms mediating their coordination with cellular signaling remain incompletely understood.Production and release of NO represents one paracrine͞ autocrine mechanism coordinating energy supply and demand in tissues. Metabolic stress, such as ischemia, stimulates NO synthases to produce NO from arginine (3). In turn, NO regulates the supply of energy by improving the delivery of substrates and oxygen to deprived tissues (4-6), regulating the intracellular delivery of metabolic substrates (7-10), selection of substrates that support metabolism (6, 10-14), oxidation of metabolic substrates (15), generation of ATP by improving metabolic efficiency (16), and biogenesis of mitochondria (17). Conversely, NO reduces the demand for ATP by decreasing energyconsuming cell functions, for example, by reducing the activity of the contractile apparatus in muscle cells (ref. 18 and references therein). Moreover, in some models of severe hypoxic stress, NO induces complete and reversible metabolic stasis required to survive the insult (19). The importance of NO in defending against ischemia is underscored by its regulation of the transcription factor hypoxia inducible factor 1, a master regulator of cellular homeostasis in the context of oxygen insufficiency (20).Coordination of energy supply and demand is mediated, in part, by NO activation of soluble guanylyl cyclase (sGC) and the associated accumulation of intracellular cGMP concentration ([cGMP] i ) (9,10,12,15,16,18), yet the mechanisms coordinating cGMP-dependent and metabolic signaling, beyond the production of NO, remain undefined. Recently, a novel mechanism was identified by which adenine nucleotides inhibit GCs (21,22) that is analogous to P site inhibition of adenylyl cyclases (23). Indeed, the two-substituted adenine nucleotides 2-methylthio-ATP and 2-chloro-ATP inhibit NO-dependent cGMP production by directly binding to sGC (22). Synthetic two-substituted adenine nucleotides presumably exploit allosteric mechanisms regulated by endogenous cell products. The present study revealed that ATP is the native ligand mediating adenine nucleotide-dependent inhibition of sGC by binding directly to an allosteric purinergic site on the enzyme. More...

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Section: Resultsmentioning

confidence: 99%

Guanylyl cyclase is an ATP sensor coupling nitric oxide signaling to cell metabolism

Ruiz-Stewart

Tiyyagura

Lin

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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“…For flow cytometry, Abs against human CD2 (6F10.3), CD14 (RM052), CD19 (J4.119), CD80 (MAB104), CD83 (HB15A) (all Beckmann Coulter, Krefeld, Germany), CD86 (BU63, Serotec, Oxford, U.K.), HLA-DR (YD1/ 63.4.10, Serotec), nNOS (nNOS-AB, A-11, Santa Cruz Biotechnology, Santa Cruz, CA), CD40 (5c3), CD208-APC (110-1112) (all Becton Dickinson), CCR7-APC (150503; R&D Systems, Wiesbaden, Germany), mouse (MOPC-31-C, MOPC-173, [27][28][29][30][31][32][33][34][35], and rat (R35-95, MOPC-21) subclassspecific isotypes (all Beckmann Coulter) were used. Conjugated secondary reagents: FITC-conjugated goat-anti-rat-IgG (Biozol, Eching, Germany), PE-conjugated donkey anti-mouse-IgG (Jackson ImmunoResearch Laboratories, Suffolk, U.K.), for myeloid DC: anti-CD1c-PE (M241, Ancell, Cologne, Germany).…”

Section: Absmentioning

confidence: 99%

“…The cGMP content of the RFL-6 cells was determined by RIA as described previously (27). Basal cGMP values measured in RFL-6 cells incubated under the same conditions but not exposed to DCs were subtracted from the experimental values.…”

Section: Rfl-6 Reporter Cell Assaymentioning

confidence: 99%

Neuronal Nitric Oxide Synthase Modulates Maturation of Human Dendritic Cells

Adler¹,

Simon

Graulich³

et al. 2010

The Journal of Immunology

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Dendritic cells (DCs) are the most potent APCs of the immune system. Understanding the intercellular and intracellular signaling processes that lead to DC maturation is critical for determining how these cells initiate T cell-mediated immune processes. NO synthesized by the inducible NO synthase (iNOS) is important for the function of murine DCs. In our study, we investigated the regulation of the arginine/NO-system in human monocyte-derived DCs. Maturation of DCs induced by inflammatory cytokines (IL-1β, TNF, IL-6, and PGE2) resulted in a pronounced expression of neuronal NOS (nNOS) but only minimal levels of iNOS and endothelial NOS were detected in human mature DCs. In addition, reporter cell assays revealed the production of NO by mature DCs. Specific inhibitors of NOS (N-nitro-l-arginine methyl ester) or of the NO target guanylyl cyclase (H-(1,2,4)-oxadiazolo [4,3-a] quinoxalin-1-one) prevented DC maturation (shown by decreased expression of MHC class II, costimulatory and CD83 molecules and reduced IL-12 production) and preserved an immature phenotype, indicating an autocrine effect of nNOS-derived NO on human DC maturation. Notably, inhibitor-treated DCs were incapable of inducing efficient T cell responses after primary culture and generated an anergic T cell phenotype. In conclusion, our results suggest that, in the human system, nNOS-, but not iNOS-derived NO, plays an important regulatory role for the maturation of DCs and, thus, the induction of pronounced T cell responses.

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“…A large body of experimental evidence bears out this assumption. For example, endothelial nitric oxide can be detected extracellularly by either electrochemical means [1] or by using a reporter cell system [2]. It has been suggested repeatedly that nitric oxide requires a carrier system in order to reach its pharmacological destination because of its inherently high reactivity with oxygen [3].…”

Section: Introductionmentioning

confidence: 99%

The role of glutathione in the transport and catabolism of nitric oxide

1996

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Nitric oxide acts as a neuronal and vascular messenger implying diffusion through intracellular environments containing 5-10 mM glutathione. Nitric oxide reacts with glutathione under aerobic conditions generating S-nitrosoglutathione (GSNO). GSNO reacts with glutathione (k = 8.3 × 10 -3 M -1" S -1) to generate nitrous oxide and glutathione disulfide (GSSG). Anaerobically, glutathione reacts with nitric oxide generating nitrous oxide and GSSG (k = 4.8 X 10 -4 s -1 at 5 mM GSH). In both aerobic and anaerobic situations the nitroxyl anion may be an intermediate in the synthesis of nitrous oxide and, under aerobic conditions, nitroxyl anion may generate peroxynitrite. We present a hypothesis for the intraeellular interaction between nitric oxide and glutathione.

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A simple and sensitive bioassay method for detection of EDRF with RFL-6 rat lung fibroblasts

Cited by 66 publications

References 0 publications

Guanylyl cyclase is an ATP sensor coupling nitric oxide signaling to cell metabolism

Guanylyl cyclase is an ATP sensor coupling nitric oxide signaling to cell metabolism

Neuronal Nitric Oxide Synthase Modulates Maturation of Human Dendritic Cells

The role of glutathione in the transport and catabolism of nitric oxide

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