Vasodilator; nitric oxide (NO) donor

Strong vasodilator sodium nitroprusside (Ro 21-2498) is. Vasoles and venules are strongly vasodilatory when exposed to sodium nitroprusside. Nitric oxide (NO) is released during the breakdown of sodium nitroprusside in the bloodstream. In vascular smooth muscle, NO stimulates guanylate cyclase, which raises intracellular cGMP synthesis. Vascular smooth muscle relaxes as a result, enabling blood vessels to widen [5]. Vascular smooth muscle cell proliferation can be inhibited by sodium nitroprusside [6]. As a nitric oxide donor, sodium nitroprusside (5 mg/kg) can dramatically lessen intestinal ischemia-reperfusion injury in rats [2].

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Human gastric cancer cells are made more susceptible to TRAIL (tumor necrosis factor-related apoptosis-inducing ligand)-induced apoptosis by nitroprusside disodium de Hydro [4].
In this study, researchers found that TRAIL induced apoptosis and cell cycle arrest in human gastric cancer cell lines, and that this effect was mediated by NO production, and activation of both the extrinsic and intrinsic signaling pathways of apoptosis. In addition, we found that the NO-donor SNP sensitizes gastric cancer cells to TRAIL-mediated apoptosis. Treatment of cells with both TRAIL and SNP resulted in increased activation of caspase-8 and caspase-9 and NO release. Inhibition of caspase-8 blocked cell TRAIL-induced apoptosis, while a selective caspase-9 inhibitor was unable to prevent apoptosis induced by either TRAIL or TRAIL plus SNP. Inhibition of NOS could block the activation of caspase-9, but had no obvious effect on cell apoptosis.
Conclusions: SNP-sensitized gastric cancer cells to TRAIL-induced cytotoxicity by stimulating the release of NO, in turn facilitating the mitochondria-mediated signal transduction pathway. The engagement of the mitochondria signaling pathways along with the TRAIL death receptor signaling pathway synergistically increase levels of apoptosis in these cells.[4]

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Previous studies reported that high levels of nitric oxide (NO) induce apoptotic cell death in osteoblasts. In this study, researchers examined molecular mechanisms of cytotoxic injury induced by sodium nitroprusside (SNP), a NO donor, in both glutathione (GSH)-depleted and control U2-OS osteoblasts. Cell viability was reduced by much lower effective concentrations of SNP in GSH-depleted cells compared to normal cells. The data suggest that the level of intracellular GSH is critical in SNP-induced cell death processes of osteoblasts. The level of oxidative stress due to SNP treatments doubled in GSH-depleted cells when measured with fluorochrome H2DCFDA. Pretreatment with the NO scavenger PTIO preserved the viability of cells treated with SNP. Viability of cells treated with SNP was recovered by pretreatment with Wortmannin, an autophagy inhibitor, but not by pretreatment with zVAD-fmk, a pan-specific caspase inhibitor. Large increases of LC3-II were shown by immunoblot analysis of the SNP-treated cells, and the increase was blocked by pretreatment with PTIO or Wortmannin; this implies that under GSH-depleted conditions SNP induces different molecular signaling that lead to autophagic cell death. The ultrastructural morphology of SNP-treated cells in transmission electron microscopy showed numerous autophagic vacuoles. These data suggest NO produces oxidative stress and cellular damage that culminate in autophagic cell death of GSH-depleted osteoblasts. [3]

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At a concentration of 100 µM, Sodium Nitroprusside alone showed no cytotoxicity (100% plating efficiency) in V79 Chinese hamster lung fibroblasts. At 1000 µM, it reduced cell survival to 66% plating efficiency. [1]
Sodium Nitroprusside (100 µM and 1000 µM) markedly enhanced hydrogen peroxide (H₂O₂)-mediated cytotoxicity in V79 cells. [1]
Sodium Nitroprusside (1000 µM) also enhanced the cytotoxicity mediated by a hypoxanthine/xanthine oxidase (HX/XO) system in V79 cells. [1]

Sodium nitroprusside could be elegantly used to reduce intestinal ischemia reperfusion. The recent focus on ischemia-reperfusion injury has been on the interaction between neutrophils and endothelial cells. Transendothelial migration of neutrophils, with release of reactive oxygen species and cytokines, causes further damage to the injured tissue. However, key components in the pathogenesis of reperfusion syndrome include the up-regulation of surface adhesion molecules on the vascular endothelium and their subsequent interaction with activated neutrophils. The most important adhesion protein identified on neutrophils is the integrin lymphocyte function-associated antigen-1 (LFA-1; CD11a/CD18). This is the ligand for intercellular adhesion molecule-1 (ICAM-1), which is expressed on the endothelium. The LFA-1/ICAM-1 interaction is crucial for neutrofils to ingress into inflammatory sites. Sodium nitroprusside down-regulates ICAM-1 and LFA-1 expression, and interferes with the ICAM-1–LFA-1 interaction by binding to LFA-1. This important mechanism should be borne in mind as the major mechanism for sodium nitroprusside-induced inhibition of neutrophil activity.[2]

The NOS enzyme activity assay was performed using the Nitric Oxide Synthase Assay Kit, according to the manufacturer's instructions. Briefly, the cells were inoculated in 96-well plates and experiments were performed when cells on the plate reached 80% to 90% confluence. Cells were treated with TRAIL and/or sodium nitroprusside (SNP) for 24 h, the supernatant was discarded, and 100 μl/well of NOS detection buffer solution was added. Following an incubation period of 40 min, the reaction products were measured using microplate reader. The relative NOS activity was determined by detecting the absorbance in each well at the excitation wavelength of 495 nm and emission wavelength of 515 nm. All experiments were performed in triplicate and compared to the blank control (empty wells with no cells) and the treatment control (cells treated with His peptide). The relative NOS activity = (RFU in treated cells − RFU in blank well) / (RFU in His treated cells − RFU in blank well). (RFU represents relative fluorescence unit).
The NO production was determined by assaying for nitrite and nitrate accumulation in the culture media. Briefly, following cell treatment with TRAIL and/or SNP, culture media was treated with nitrate reductase and its cofactors to convert all of nitrate to nitrite before applying 100 μl of the Griess reagent. Absorbance was measured at 546 nm.[4]

The MTT assay and flow cytometry were used to detect cellular proliferation and markers of apoptosis, respectively. Expression levels of caspases-8, and 9 were determined by Western blot. Changes in Nitric Oxide Synthase (NOS) activity, NO production, and caspase activation were also evaluated.[4]

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A 0.5 M stock solution of sodium nitroprusside (SNP) dissolved in water was prepared immediately prior to its addition to cell media. Treatments of the gastric cancer cell lines with SNP were performed at final concentrations ranging from 0.5 to 2.0 mM, in medium supplemented with serum. A 500 ng/ml stock solution of TRAIL was prepared and the final concentrations for treating the gastric cancer cell lines ranged from 50 to 300 ng/ml in medium supplemented with serum. Equal volumes of PBS and control His peptide were added to untreated cells as controls. Cells were treated with either TRAIL alone or in combination with varying doses of SNP (50 ng/ml TRAIL with or without 0.5 mM SNP; 100 ng/ml TRAIL with or without 1.0 mM SNP; 200 ng/ml TRAIL with or without 1.5 mM SNP and 300 ng/ml TRAIL with or without 2.0 mM SNP) for 24 h. In the case of cells treated with pharmacologic inhibitors, cells were pre-incubated with the inhibitors for 45 min prior to the addition of TRAIL (300 ng/ml).[4]

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Western blot assay: Cells were harvested in ice-cold PBS after incubation with TRAIL and/or sodium nitroprusside (SNP), and immediately lysed with lysis buffer containing protease inhibitors. The protein concentrations for each sample were determined using the BCA protein assay kit. Equal amounts of protein from each sample were loaded in sodium dodecyl sulfate-polyacrylamide gels for electrophoresis and then transferred to a nitrocellulose membrane by electro blotting. The membranes were blocked with 5% skim milk, and incubated with the primary antibodies to caspase-8, -9 and β-actin, followed by HRP-conjugated secondary antibodies. An enhanced chemiluminescence detection system was used for visualization of target proteins. β-actin was used as a loading control.[4]

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The clonogenic assay was used to assess cell survival. Exponentially growing V79 Chinese hamster lung fibroblasts were trypsinized and plated. After 16 hours of incubation, cells were exposed to varying concentrations of hydrogen peroxide for 1 hour, or to a hypoxanthine/xanthine oxidase system for 30 minutes. Sodium Nitroprusside was added to the culture medium at final concentrations of 100 µM or 1000 µM immediately prior to the addition of hydrogen peroxide or the HX/XO system. Following treatment, cells were washed, trypsinized, counted, and plated for macroscopic colony formation. Plates were incubated for 7 days, after which colonies were fixed, stained, and counted. Colonies containing more than 50 cells were scored. [1]

Metabolism / Metabolites
Cyanide can be rapidly absorbed and distributed throughout the body via oral ingestion, inhalation, and skin contact. Cyanide is primarily metabolized to thiocyanate by thiocyanate oxidase or 3-mercaptopyruvate-thiotransferase. Cyanide metabolites are excreted in the urine. (L96)

Toxicity Summary
Sodium nitroprusside is a cholinesterase, or acetylcholinesterase (AChE) inhibitor. Cholinesterase inhibitors (or "anticholinesterases") inhibit the activity of acetylcholinesterase. Because acetylcholinesterase plays a vital physiological role, chemicals that interfere with its activity are potent neurotoxins; even low doses can cause excessive salivation and lacrimation, followed by muscle spasms and ultimately death. Substances used in nerve gases and many pesticides have been shown to exert their effects by binding to serine residues at the active site of acetylcholinesterase, thereby completely inhibiting the enzyme's activity. Acetylcholinesterase breaks down the neurotransmitter acetylcholine, which is released at the neuromuscular junction, causing muscle or organ relaxation. The mechanism of action of acetylcholinesterase inhibitors is to allow acetylcholine to accumulate and exert its sustained effect, ensuring the continuous transmission of nerve impulses and preventing muscle contraction from ceasing. The most common acetylcholinesterase inhibitors are phosphorus-containing compounds designed to bind to the enzyme's active site. Its structural requirements include a phosphorus atom with two lipophilic groups, a leaving group (such as a halogen or thiocyanate group), and a terminal oxygen atom.

[1]. The effect of various nitric oxide-donor agents on hydrogen peroxide-mediated toxicity: a direct correlation between nitric oxide formation and protection. Arch Biochem Biophys, 1996. 331(2): p. 241-8.

[2]. Garg, U.C. and A. Hassid, Nitric oxide-generating vasodilators and 8-bromo-cyclic guanosine monophosphate inhibit mitogenesis and proliferation of cultured rat vascular smooth muscle cells. J Clin Invest, 1989. 83(5): p. 1774-7.

[3]. Namazi, H., Sodium nitroprusside as a nitric oxide donor in a rat intestinal ischemia reperfusion model: a novel molecular mechanism. Clinics (Sao Paulo), 2008. 63(3): p. 405.

Sodium nitroprusside is an organic sodium salt, which is the disodium salt of sodium nitroprusside. It is used as a nitric oxide donor and a vasodilator. It contains sodium nitroprusside. Sodium nitroprusside is a compound of sodium and cyanide. It is used as a vasodilator. (L105) See also: Sodium nitroprusside (note moved to). Sodium nitroprusside (SNP) is one of the nitric oxide (NO) donor complexes originally used to study the biological effects of nitric oxide (NO). However, under the biological conditions of this study (cell culture medium at 37°C), electrochemical assays showed that 1000 µM SNP did not produce a considerable concentration of free NO (<0.3 µM). [1] This study concludes that the enhancement of hydrogen peroxide-mediated cytotoxicity by SNP is not due to the release of free NO. Potential mechanisms of its enhanced toxicity include the release of cyanide (CN⁻) from the complex and/or iron complexes involved in catalyzing Fenton-type reactions. Deferroamine (DF) is an iron chelating agent that can only partially resist SNP/H₂O₂-mediated toxicity, suggesting that its mechanism of action may be more than one. [1]

NO-.5[CN-].FE+4.2[NA+]

261.9177

261.928

14402-89-2

Nitroprusside disodium dihydrate;13755-38-9

11963579

Brown to reddish brown solid powder

1.72

0.298

0

12

0

15

119

0

PECOKVKJVPMJBN-UHFFFAOYSA-N

InChI=1S/5CN.Fe.NO.2Na/c5*1-2;;1-2;;/q5*-1;+4;-1;2*+1

disodium;iron(4+);nitroxyl anion;pentacyanide

disodium;iron(3+);nitroxyl anion;pentacyanide; disodium;iron(4+);nitroxyl anion;pentacyanide; Nitroprusside (disodium dihydrate); Ro 21-2498; Sodium nitroprusside dihydrate;Sodium Nitroferricyanide(III) Dihydrate; Nitroprusside (Sodium); SCHEMBL3020702;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : ~100 mg/mL (~381.80 mM)
DMSO : ~33.33 mg/mL (~127.25 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (9.54 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (9.54 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: 100 mg/mL (381.80 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)