In vivo lipopolysaccharide pretreatment inhibits cGMP release from the isolated-perfused rat lung

1995 ◽  
Vol 269 (5) ◽  
pp. L618-L624 ◽  
Author(s):  
M. M. Kurrek ◽  
W. M. Zapol ◽  
A. Holzmann ◽  
G. Filippov ◽  
M. Winkler ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Signal Transduction ◽  
Superoxide Dismutase ◽  
Soluble Guanylate Cyclase ◽  
No Synthase ◽  
Rat Lung ◽  
Signal Transduction System ◽  
Endothelial No Synthase ◽  
Cgmp Signal

Administration of bacterial lipopolysaccharide (LPS) to rats stimulates synthesis of nitric oxide (NO), a free radical molecule that activates soluble guanylate cyclase, thereby increasing intracellular guanosine 3',5'-cyclic monophosphate (cGMP) concentration and inducing systemic vasodilatation. To investigate the effect of endotoxemia on the pulmonary NO/cGMP signal transduction system, we measured the release of cGMP by isolated-perfused lungs of rats that received an intraperitoneal injection of LPS (1 mg/kg) or saline 2 days earlier. Over 90 min, 1.4 +/- 0.78 and 0.079 +/- 0.016 nmol cGMP accumulated in pulmonary perfusates of saline- and LPS-treated rats, respectively (P < 0.05). Despite addition to the perfusate of Zaprinast, superoxide dismutase, or A23187, markedly less cGMP was released from the lungs of rats exposed to LPS than from the lungs of control rats. In contrast, after ventilation with 100 parts per million NO gas, cGMP accumulating in the perfusate of the lungs of both groups of rats was markedly increased, and the quantity of cGMP released from the lungs of LPS-treated rats was similar to that released by control rat lungs (2.8 +/- 0.57 vs. 3.3 +/- 0.88 nmol, P = NS). With the use of immunoblot techniques, equal concentrations of constitutive endothelial NO synthase were detected in the lungs of rats treated with saline or LPS. These results demonstrate that the NO/cGMP signal transduction system is abnormal in the lungs of rats exposed to LPS, at least in part, at the level of endothelial NO synthase activation.

Inhaled nitric oxide does not alter endotoxin-induced nitric oxide synthase activity during rat lung perfusion

1995 ◽  
Vol 79 (4) ◽  
pp. 1088-1092 ◽  
Author(s):  
M. M. Kurrek ◽  
L. Castillo ◽  
K. D. Bloch ◽  
S. R. Tannenbaum ◽  
W. M. Zapol
Keyword(s):  
Nitric Oxide ◽  
Feedback Inhibition ◽  
Ex Vivo ◽  
Lung Perfusion ◽  
No Synthase ◽  
Rat Lung ◽  
Ex Vivo Lung Perfusion ◽  
Inducible No Synthase ◽  
Isolated Lung

Nitric oxide (NO) has been demonstrated to decrease its own synthesis in tissue preparations. We tested the hypothesis that endogenous NO synthesis induced by lipopolysaccharides (LPS) would be decreased by exogenous NO during isolated lung perfusion. Rats were pretreated with either saline or LPS 48 h before lung harvest. Endogenous NO synthase activity was measured as conversion of L-[14C]-arginine to L-[14C]citrulline during 90 min of perfusion. NO (100 ppm) was added to the ventilating gas during perfusion of lungs from one group of control or LPS-treated rats. A second group of control or LPS-treated rats was exposed chronically to 100 ppm NO for the 48 h before lung harvest, in addition to receiving 100 ppm NO added to the ventilating gas during lung perfusion. We conclude that conversion of L-[14C]arginine to L-[14C]citrulline was minimal in control lungs and increased in response to LPS pretreatment. NO added to the ventilating gas for the 90 min of ex vivo perfusion did not alter the rate of L-[14C]citrulline production. In vivo exposure to 100 ppm NO for 48 h did not alter the induction of inducible NO synthase activity as measured during ex vivo lung perfusion. This indicates that inhaled NO does not exert negative-feedback inhibition on inducible NO synthase in the ex vivo rat lung.

Nitric oxide synthase and guanylate cyclase levels in canine basilar artery after subarachnoid hemorrhage

1995 ◽  
Vol 82 (2) ◽  
pp. 250-255 ◽  
Author(s):  
Hidetoshi Kasuya ◽  
Bryce K. A. Weir ◽  
Masaki Nakane ◽  
Jennifer S. Pollock ◽  
Lydia Johns ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Subarachnoid Hemorrhage ◽  
Guanylate Cyclase ◽  
Basilar Artery ◽  
Soluble Guanylate Cyclase ◽  
No Synthase ◽  
Soluble Form ◽  
Control Group ◽  
Endothelial No Synthase ◽  
Basilar Arteries

✓ Endothelium-dependent vasodilation may be impaired during cerebral vasospasm following subarachnoid hemorrhage. Under normal circumstances nitric oxide (NO) released by endothelial cells induces relaxation of smooth muscle by activating the soluble form of guanylate cyclase within muscle cells. In this study the levels of both endothelial NO synthase, the enzyme that produces NO, and soluble guanylate cyclase were determined in canine basilar arteries in a double-hemorrhage model using Western blot immunoassays. Thirty dogs were assigned to three groups: Group D0, control; Group D2, dogs sacrificed 2 days after cisternal injection of blood; and Group D7, dogs given double cisternal injections of blood and sacrificed 7 days after the first injection. Constriction of the basilar artery was confirmed by arterial angiography. Portions of the affected arteries or the corresponding region in control animals were solubilized for sodium dodecylsulfate—polyacrylamide gel electrophoresis and Western blotting. A specific monoclonal antibody against endothelial NO synthase was used. The extract from basilar arteries showed two bands on the blots: 135 kD, characteristic of endothelial NO synthase, and 120 kD, which may be a degradation product of the enzyme. The densitometer values of the bands were presented as percentages of D0 control values. Although the total signal in the D7 group was less than that of the D0 control group (D2, 97% ± 22%; D7, 78% ± 40%), it was not statistically significant. The proportion of the 135-kD form decreased between Groups D0 and D7, but the difference was not significant. A single major band corresponding to the α-subunit of soluble guanylate cyclase was seen at 70 kD in the basilar artery extracts. The signals of D2 and D7 samples were 69%± 40% and 25% ± 18%, respectively. There was a significant difference between D7 and D0 (p < 0.001). The reduced expression of soluble guanylate cyclase may be related to the impairment of endothelium-dependent vasodilation in vasospasm.

scholarly journals Nitrite enhances RBC hypoxic ATP synthesis and the release of ATP into the vasculature: a new mechanism for nitrite-induced vasodilation

2009 ◽  
Vol 297 (4) ◽  
pp. H1494-H1503 ◽  
Author(s):  
Zeling Cao ◽  
Jeffrey B. Bell ◽  
Joy G. Mohanty ◽  
Enika Nagababu ◽  
Joseph M. Rifkind
Keyword(s):  
Nitric Oxide ◽  
Blood Pressure ◽  
Blood Cells ◽  
Purinergic Receptors ◽  
Atp Release ◽  
Atp Synthesis ◽  
No Synthase ◽  
Endothelial No Synthase ◽  
Rbc Membrane

A role for nitric oxide (NO) produced during the reduction of nitrite by deoxygenated red blood cells (RBCs) in regulating vascular dilation has been proposed. It has not, however, been satisfactorily explained how this NO is released from the RBC without first reacting with the large pools of oxyhemoglobin and deoxyhemoglobin in the cell. In this study, we have delineated a mechanism for nitrite-induced RBC vasodilation that does not require that NO be released from the cell. Instead, we show that nitrite enhances the ATP release from RBCs, which is known to produce vasodilation by several different methods including the interaction with purinergic receptors on the endothelium that stimulate the synthesis of NO by endothelial NO synthase. This mechanism was established in vivo by measuring the decrease in blood pressure when injecting nitrite-reacted RBCs into rats. The observed decrease in blood pressure was not observed if endothelial NO synthase was inhibited by Nω-nitro-l-arginine methyl ester (l-NAME) or when any released ATP was degraded by apyrase. The nitrite-enhanced ATP release was shown to involve an increased binding of nitrite-modified hemoglobin to the RBC membrane that displaces glycolytic enzymes from the membrane, resulting in the formation of a pool of ATP that is released from the RBC. These results thus provide a new mechanism to explain nitrite-induced vasodilation.

Direct and reversible inhibition of endothelial nitric oxide synthase by nitric oxide

1995 ◽  
Vol 268 (6) ◽  
pp. H2216-H2223 ◽  
Author(s):  
L. V. Ravichandran ◽  
R. A. Johns ◽  
A. Rengasamy
Keyword(s):  
Nitric Oxide ◽  
Enzyme Activity ◽  
Feedback Mechanism ◽  
No Synthase ◽  
Maximal Velocity ◽  
Dependent Manner ◽  
Reversible Inhibition ◽  
Endothelial No Synthase ◽  
Endothelial Nitric Oxide

The objective of this study was to investigate the regulation of endothelial nitric oxide (NO) synthase by NO. Partially purified endothelial NO synthase was exposed to authentic NO (10-200 microM) and to the nitrovasodilators sodium nitroprusside (SNP; 10-1,000 microM) and S-nitroso-N-acetylpenicillamine (SNAP; 100-1,000 microM), and enzyme activity was assayed by measuring the conversion of L-[3H]arginine to L-[3H]citrulline in the presence of added cofactors. NO, SNP, and SNAP inhibited NO synthase activity in a dose-dependent manner, NO being the most potent inhibitor. The Michaelis constant for L-arginine was not altered (4.87 microM) by NO (50 microM), whereas the maximal velocity of the enzyme decreased from 784 to 633 pmol.mg-1.min-1. Oxyhemoglobin (10 microM) partially prevented the inhibition of NO synthase by NO (50 microM). The data also suggest that NO inhibits endothelial NO synthase activity by directly interacting with the NO synthase and not by an indirect mechanism such as limitation of cofactor or oxygen availability. Dialysis of NO synthase treated with NO (50 microM) partially restored the enzyme activity. This study demonstrates a direct and reversible inhibition of NO synthase by NO, suggesting a feedback mechanism in vivo.

scholarly journals Dissection of a Hypoxia-induced, Nitric Oxide–mediated Signaling Cascade

2009 ◽  
Vol 20 (18) ◽  
pp. 4083-4090 ◽  
Author(s):  
Pascale F. Dijkers ◽  
Patrick H. O'Farrell
Keyword(s):  
Nitric Oxide ◽  
Signal Transduction ◽  
Signaling Cascade ◽  
S2 Cells ◽  
Signal Transduction System ◽  
Signaling Systems ◽  
Calcium Dependent ◽  
Drosophila S2 Cells ◽  
Oxide Synthase ◽  
Multiple Signaling

Befitting oxygen's key role in life's processes, hypoxia engages multiple signaling systems that evoke pervasive adaptations. Using surrogate genetics in a powerful biological model, we dissect a poorly understood hypoxia-sensing and signal transduction system. Hypoxia triggers NO-dependent accumulation of cyclic GMP and translocation of cytoplasmic GFP-Relish (an NFκB/Rel transcription factor) to the nucleus in Drosophila S2 cells. An enzyme capable of eliminating NO interrupted signaling specifically when it was targeted to the mitochondria, arguing for a mitochondrial NO signal. Long pretreatment with an inhibitor of nitric oxide synthase (NOS), L-NAME, blocked signaling. However, addition shortly before hypoxia was without effect, suggesting that signaling is supported by the prior action of NOS and is independent of NOS action during hypoxia. We implicated the glutathione adduct, GSNO, as a signaling mediator by showing that overexpression of the cytoplasmic enzyme catalyzing its destruction, GSNOR, blocks signaling, whereas knockdown of this activity caused reporter translocation in the absence of hypoxia. In downstream steps, cGMP accumulated, and calcium-dependent signaling was subsequently activated via cGMP-dependent channels. These findings reveal the use of unconventional steps in an NO pathway involved in sensing hypoxia and initiating signaling.

scholarly journals Ultraviolet B Radiation Acts through the Nitric Oxide and cGMP Signal Transduction Pathway to Stimulate Melanogenesis in Human Melanocytes

1996 ◽  
Vol 271 (45) ◽  
pp. 28052-28056 ◽  
Author(s):  
Christine Roméro-Graillet ◽  
Edith Aberdam ◽  
Naïma Biagoli ◽  
William Massabni ◽  
Jean-Paul Ortonne ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Signal Transduction ◽  
Signal Transduction Pathway ◽  
Ultraviolet B ◽  
Transduction Pathway ◽  
Ultraviolet B Radiation ◽  
Cgmp Signal ◽  
Human Melanocytes

scholarly journals Nitric Oxide as an Endogenous Mutagen for Sendai Virus without Antiviral Activity

2004 ◽  
Vol 78 (16) ◽  
pp. 8709-8719 ◽  
Author(s):  
Jun Yoshitake ◽  
Takaaki Akaike ◽  
Teruo Akuta ◽  
Fumio Tamura ◽  
Tsutomu Ogura ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Nitric Oxide ◽  
Viral Replication ◽  
Mutation Frequency ◽  
Sendai Virus ◽  
No Synthase ◽  
Mutagenic Potential ◽  
Gfp Gene ◽  
Sw480 Cells

ABSTRACT Nitric oxide (NO) may affect the genomes of various pathogens, and this mutagenesis is of particular interest for viral pathogenesis and evolution. Here, we investigated the effect of NO on viral replication and mutation. Exogenous or endogenous NO had no apparent antiviral effect on influenza A virus and Sendai virus. The mutagenic potential of NO was analyzed with Sendai virus fused to a green fluorescent protein (GFP) gene (GFP-SeV). GFP-SeV was cultured in SW480 cells transfected with a vector expressing inducible NO synthase (iNOS). The mutation frequency of GFP-SeV was examined by measuring loss of GFP fluorescence of the viral plaques. GFP-SeV mutation frequency in iNOS-SW480 cells was much higher than that in parent SW480 cells and was reduced to the level of mutation frequency in the parent cells by treatment with an NO synthase (NOS) inhibitor. Immunocytochemistry showed generation of more 8-nitroguanosine in iNOS-SW480 cells than in SW480 cells without iNOS transfection. Authentic 8-nitroguanosine added exogenously to GFP-SeV-infected CV-1 cells increased the viral mutation frequency. Profiles of the GFP gene mutations induced by 8-nitroguanosine appeared to resemble those of mutations occurring in mouse lungs in vivo. A base substitution that was characteristic of both mutants (those induced by 8-nitroguanosine and those occurring in vivo) was a C-to-U transition. NO-dependent oxidative stress in iNOS-SW480 cells was also evident. Together, the results indicate unambiguously that NO has mutagenic potential for RNA viruses such as Sendai virus without affecting viral replication, possibly via 8-nitroguanosine formation and cellular oxidative stress.

Abstract 1415: Activation of Soluble Guanylate Cyclase Regulates Phosphodiesterase 5A Localization and Restores Anti-adrenergic Action of Sildenafil despite Nitric Oxide Synthase Inhibition

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Takahiro Nagayama ◽  
Manling Zhang ◽  
Eiki Takimoto ◽  
David A Kass
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Guanylate Cyclase ◽  
Cyclic Gmp ◽  
Soluble Guanylate Cyclase ◽  
Synergistic Effects ◽  
Adrenergic Stimulation ◽  
Adrenergic Activity ◽  
Oxide Synthase

Background: We have shown that inhibition of cyclic GMP-phosphodiesterase 5A (PDE5A) by sildenafil (SIL) blunts cardiomyocyte β-adrenergic stimulation, but this effect depends on the activity of endothelial nitric oxide synthase (eNOS) to generate a specific pool of cyclic GMP. PDE5A normally localizes at Z-bands in myocytes, but localization is more diffuse in cells with eNOS chronically inhibited. Here, we tested whether the influence of eNOS on PDE5A localization and anti-adrenergic action depends upon cyclic GMP. Methods and Results: Mouse in vivo hemodynamics were assessed by pressure-volume analysis. Isoproterenol (ISO: 20 ng/kg/min, iv ) stimulated contractility was inhibited by SIL (100 μg/kg/min, iv ), however this did not occur in mice given N w -nitro-L-arginine methyl ester (L-NAME: 1 mg/mL in drinking water for 1 week) to inhibit NOS. Myocytes transfected with an adenoviral vector encoding a fusion protein (PDE5A-DSred) in vivo were subsequently isolated and examined for PDE5A/α-actinin localization. Normal cells showed strong co-localization, whereas L-NAME-treated cells had diffuse PDE5A distribution. If L-NAME was stopped for 1-wk washout, SIL regained anti-adrenergic activity, and PDE5A z-band localization was restored. If L-NAME was continued but combined with Bay 41– 8543 (BAY: 30 mg/kg/day, po ), a soluble guanylate cyclase (sGC) activator, both PDE5A localization and SIL anti-adrenergic action were also restored. Chronic L-NAME suppressed phosphorylation of vasodilator-stimulated protein (VASP), a marker of protein kinase G (PKG) activity, in hearts acutely exposed to ISO+SIL. After L-NAME washout or L-NAME+BAY, VASP phosphorylation with ISO+SIL was restored. Conclusion: NOS-dependent modulation of both PDE5A sarcomere localization and anti-adrenergic activity depends upon sGC-derived cyclic GMP, and is linked to PKG activation. This suggests sGC activators may have synergistic effects with PDE5A inhibitors.

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