GO:0035732 nitric oxide storage: Mechanisms, Genes, Functions and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0035732 nitric oxide storage describes the accumulation and maintenance of nitric oxide (NO) in cells or tissues, primarily in the form of dinitrosyl-iron complexes stabilized by glutathione S-transferase proteins.
• NO storage is critical for regulating vasodilation, blood pressure, and cardiovascular homeostasis, as stored NO can be released to modulate vascular tone.
• Glutathione S-transferases (GSTs) and multidrug resistance-related protein 1 (MRP1) are key proteins involved in NO transport and storage.
• Dysregulated NO storage contributes to pathologies such as hemolysis-induced storage lesion in banked blood and endothelial dysfunction.
• NO storage also occurs in plants, influencing postharvest physiology such as sphingolipid metabolism in peach fruit.
• Experimental models for studying NO storage include knockout and overexpression of GSTs, MRP1, and other related genes, as well as synthetic systems like Cu-BTC for controllable NO storage.
Description
Nitric oxide (NO) is a gaseous signaling molecule with pleiotropic roles in physiology, including vasodilation, neurotransmission, and immune defense. However, its short half-life and high reactivity necessitate mechanisms for its stabilization and storage. The Gene Ontology term GO:0035732, nitric oxide storage, describes the accumulation and maintenance of NO in cells or tissues, primarily through the formation of dinitrosyl-iron complexes that are stabilized by binding to glutathione S-transferase proteins. This process is essential for regulating NO bioavailability and signaling, particularly in the cardiovascular system. Understanding NO storage has broad implications for human health, as dysregulation is linked to conditions such as hemolytic disorders, cardiovascular diseases, and even plant postharvest physiology. Moreover, NO storage is exploited in biomedical applications, including the development of NO-releasing materials for therapeutic use. This article provides a comprehensive overview of the molecular mechanisms, key genes, research methods, and disease relevance of nitric oxide storage, based on authoritative QuickGO data and verified PubMed literature.
nitric oxide storage At A Glance
| GO ID | GO:0035732 |
|---|---|
| GO term | nitric oxide storage |
| Ontology | biological_process |
| Synonym | NO storage |
| Definition | The accumulation and maintenance in cells or tissues of nitric oxide (NO). Nitric oxide is stored in the form of dinitrosyl-iron complexes, which are stabilized, and possibly sequestered, by binding to glutathione S-transferase proteins. |
| Major function | Storage and stabilization of nitric oxide for regulated release and signaling. |
| Key proteins | Glutathione S-transferases (GSTs), multidrug resistance-related protein 1 (MRP1), and other dinitrosyl-iron complex carriers. |
| Related processes | Nitric oxide transport, nitric oxide homeostasis, vasodilation, and stress responses. |
What Is GO:0035732?
According to the Gene Ontology, GO:0035732 nitric oxide storage is defined as the accumulation and maintenance in cells or tissues of nitric oxide (NO). Nitric oxide is stored in the form of dinitrosyl-iron complexes, which are stabilized, and possibly sequestered, by binding to glutathione S-transferase proteins. This process ensures a reservoir of NO that can be mobilized for signaling or defense, and it is distinct from NO synthesis or degradation.
Why Is nitric oxide storage Important in Cell Biology?
Nitric oxide storage is crucial for maintaining NO bioavailability and preventing its toxic accumulation. By sequestering NO in stable dinitrosyl-iron complexes, cells can regulate NO release in response to physiological demands, such as vasodilation and immune responses. This process is particularly important in the cardiovascular system, where stored NO modulates blood pressure and protects against ischemia-reperfusion injury. In blood banking, disruption of NO homeostasis during storage leads to hemolysis and reduced quality of stored blood, highlighting the clinical relevance of NO storage. Additionally, NO storage mechanisms are conserved in plants, affecting postharvest fruit quality. Thus, understanding NO storage is essential for developing therapeutic strategies and improving storage technologies.
• Regulates vascular tone and blood pressure by providing a releasable pool of NO.
• Protects against oxidative stress by sequestering reactive NO species.
• Plays a role in the storage lesion of banked blood, affecting transfusion efficacy.
• Influences plant physiology, including sphingolipid metabolism and fruit ripening.
• Involved in immune defense through NO storage in macrophages and other cells.
• Contributes to neurotransmission by maintaining NO reserves in neuronal tissues.
• Dysregulation is linked to cardiovascular diseases, hemolytic disorders, and inflammation.
• Provides a target for therapeutic NO delivery systems in biomedical engineering.
• Affects drug resistance through MRP1-mediated transport of NO complexes.
• Serves as a model for studying gasotransmitter storage and release in synthetic materials.
What Happens During nitric oxide storage?
Formation of dinitrosyl-iron complexes
In simple terms: Nitric oxide binds to iron to form stable complexes that can be stored.
The primary mechanism of nitric oxide storage involves the formation of dinitrosyl-iron complexes (DNICs). NO reacts with iron-sulfur clusters or free iron to form DNICs, which are relatively stable and can be sequestered within cells. These complexes are often bound to thiol-containing molecules such as glutathione, forming dinitrosyl-iron-glutathione complexes. The formation of DNICs is a key step in preventing NO from diffusing away or reacting with other molecules, thus maintaining a storage pool.
Stabilization by glutathione S-transferases
In simple terms: Glutathione S-transferase proteins hold onto the NO-iron complexes, keeping them stable.
Glutathione S-transferases (GSTs) play a crucial role in stabilizing DNICs. GSTs bind to DNICs and possibly sequester them, preventing their degradation and allowing for controlled release. This binding is thought to involve the active site of GSTs, although the exact molecular interactions are still under investigation. The stabilization by GSTs is essential for maintaining NO storage in cells and tissues.
Transport and sequestration by MRP1
In simple terms: MRP1 helps move the stored NO complexes into or out of cells.
Multidrug resistance-related protein 1 (MRP1) is involved in the transport of NO storage complexes. MRP1, an ATP-binding cassette transporter, can transport glutathione-NO conjugates and DNICs across membranes, contributing to the sequestration of NO in intracellular compartments or its export. This transport function is important for regulating NO availability and for detoxification processes.
Release and mobilization of stored NO
In simple terms: When needed, the stored NO can be released to act on targets.
Stored NO can be released from DNICs in response to specific stimuli, such as changes in redox state, pH, or the presence of releasing agents. For example, photobiomodulation using an AlGaAs diode laser can induce vasodilation by modulating NO storage levels. The release of NO from storage pools is critical for its signaling functions, including vasodilation and neurotransmission. The mechanisms of release are tightly regulated to ensure NO is available when needed.
Regulation of NO storage levels
In simple terms: The amount of stored NO is controlled by various factors.
NO storage levels are regulated by the availability of iron, glutathione, and GSTs, as well as by the activity of transporters like MRP1. Additionally, physiological conditions such as temperature and oxidative stress can affect NO storage, as seen in postharvest peach fruit where NO interacts with storage temperature to modulate sphingolipid metabolism. In the cardiovascular system, NO storage is modulated by hemolysis and blood storage conditions, leading to storage lesion. These regulatory mechanisms ensure that NO storage is dynamic and responsive to cellular needs.
Key Genes Involved in GO:0035732 nitric oxide storage
The following genes and proteins are involved in nitric oxide storage, based on their roles in forming, stabilizing, transporting, or releasing stored NO.
| Gene | Major Role | Research Relevance |
|---|---|---|
| GSTA1 | Glutathione S-transferase, stabilizes dinitrosyl-iron complexes | Knockout reduces NO storage; overexpression increases storage capacity |
| GSTM1 | Glutathione S-transferase, binds NO complexes | Polymorphisms affect NO bioavailability and disease risk |
| GSTP1 | Glutathione S-transferase, sequesters NO | Target for modulating NO storage in cancer |
| ABCC1 (MRP1) | Transports glutathione-NO conjugates and DNICs | Knockout alters NO efflux and storage |
| ALB | Albumin, carries NO and forms S-nitrosoalbumin | Modulates NO storage in blood |
| HBB | Hemoglobin, binds NO and affects its stability | Mutations cause hemolysis and disrupt NO homeostasis |
| HBA1 | Hemoglobin subunit, interacts with NO | Affects NO storage in red blood cells |
| NOS1 | Neuronal nitric oxide synthase, produces NO for storage | Knockout reduces NO available for storage |
| NOS2 | Inducible nitric oxide synthase, produces NO during inflammation | Overexpression increases NO storage |
| NOS3 | Endothelial nitric oxide synthase, produces NO for vascular storage | Knockout impairs vasodilation |
| GCLC | Glutamate-cysteine ligase, synthesizes glutathione | Knockout reduces glutathione and NO storage |
| GCLM | Glutamate-cysteine ligase modifier subunit | Regulates glutathione levels for NO storage |
| SLC7A11 | Cystine/glutamate antiporter, affects glutathione synthesis | Modulates NO storage capacity |
| NQO1 | Quinone oxidoreductase, may interact with NO | Potential role in redox regulation of NO storage |
| TXN | Thioredoxin, reduces oxidized proteins | Affects NO release from storage |
| CBS | Cystathionine beta-synthase, produces H2S which interacts with NO | Modulates NO storage and release |
| MPO | Myeloperoxidase, consumes NO | Knockout increases NO storage |
How Is nitric oxide storage Regulated?
Nitric oxide storage is regulated at multiple levels. The availability of iron and glutathione directly affects the formation of dinitrosyl-iron complexes. Glutathione S-transferases (GSTs) stabilize these complexes, and their expression is regulated by oxidative stress and xenobiotics. The transporter MRP1 (ABCC1) regulates the sequestration and export of NO complexes, and its activity is modulated by ATP levels and cellular stress. In the cardiovascular system, hemolysis releases hemoglobin that scavenges NO, disrupting storage and leading to endothelial dysfunction. Additionally, physiological factors such as temperature and pH can influence NO storage, as observed in plant tissues. In synthetic systems, the storage and release of NO can be controlled by the material's structure, such as in Cu-BTC metal-organic frameworks. These regulatory mechanisms ensure that NO storage is dynamic and responsive to cellular and environmental cues.
nitric oxide storage and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| HBB | Sickle cell disease, hemolysis, NO depletion | Knockout or point mutation in HBB in erythroid cells |
| NOS3 | Hypertension, atherosclerosis | Endothelial-specific knockout of NOS3 in mice |
| GSTP1 | Cancer, drug resistance | Knockout or overexpression in cancer cell lines |
| ABCC1 (MRP1) | Multidrug resistance, inflammation | Knockout in macrophages or cancer cells |
| MPO | Cardiovascular disease, oxidative stress | Knockout in neutrophils or endothelial cells |
Cardiovascular diseases
Disruption of nitric oxide storage contributes to cardiovascular pathologies. In hemolytic disorders, free hemoglobin scavenges NO, reducing its bioavailability and leading to vasoconstriction and hypertension. Storage lesion in banked blood is characterized by hemolysis-dependent disruption of NO homeostasis, which impairs the quality of transfused blood. Furthermore, NO storage levels modulate vasodilation and the hypotensive effect induced by photobiomodulation, suggesting that targeting NO storage could be therapeutic for hypertension. Endothelial nitric oxide synthase (NOS3) produces NO that is stored and released to maintain vascular tone, and its dysfunction is linked to atherosclerosis.
Inflammation and immune response
Nitric oxide storage is important in inflammation and immune defense. Inducible nitric oxide synthase (NOS2) produces large amounts of NO during inflammation, which can be stored as dinitrosyl-iron complexes stabilized by GSTs. This stored NO can be released to kill pathogens or modulate immune responses. However, excessive NO storage can also contribute to tissue damage and chronic inflammation. The transport of NO complexes by MRP1 may affect the availability of NO in immune cells, influencing their function.
Plant physiology and postharvest biology
In plants, nitric oxide storage plays a role in postharvest physiology. For example, in peach fruit, the interaction between nitric oxide and storage temperature affects sphingolipid metabolism, which is important for fruit quality and shelf life. NO storage in plants may also be involved in stress responses and development, although the mechanisms are less understood than in animals.
Biomedical applications and drug delivery
Nitric oxide storage is exploited in biomedical applications, such as the development of NO-releasing materials for therapeutic use. For instance, (N-acetyl-S-nitrosopenicillaminyl)-S-nitrosopenicillamine-incorporated silicone rubber coatings exhibit long-term storage stability and controlled NO release, which could be used for antimicrobial or cardiovascular applications. Additionally, Cu-BTC metal-organic frameworks have been studied for controllable NO storage and release, with crystallographic insights and bioactivity. These applications highlight the translational potential of understanding NO storage mechanisms.
From nitric oxide storage-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does GSTP1 knockout reduce NO storage capacity? | GSTP1 knockout cell line (e.g., HEK293 or HeLa) |
| How does MRP1 point mutation affect NO transport? | MRP1 point mutant knock-in in epithelial cells |
| Can overexpression of NOS3 increase NO storage? | NOS3 overexpression in endothelial cells |
| What is the effect of HBB mutation on NO homeostasis? | HBB knock-in mutation in erythroid progenitor cells |
| Does tagged GSTA1 localize to NO storage compartments? | GSTA1 tagged knock-in with fluorescent protein |
| Can CRISPR library screening identify new NO storage regulators? | Genome-wide CRISPR knockout library in a NO-responsive reporter cell line |
How to Study the nitric oxide storage Process
| Method | What It Measures | Typical Application |
|---|---|---|
| EPR spectroscopy | Dinitrosyl-iron complexes | Quantification of NO storage in cells/tissues |
| Chemiluminescence | NO release | Real-time monitoring of NO from stored pools |
| Griess assay | Nitrite/nitrate (NO metabolites) | Indirect assessment of NO production |
| Fluorescence microscopy | NO with DAF-FM | Visualization of NO in live cells |
| CRISPR knockout | Gene function | Identifying genes required for NO storage |
| CRISPR knock-in | Tagged proteins | Localizing GSTs or MRP1 in NO storage compartments |
| GST activity assay | Enzyme activity | Measuring GST function in NO storage |
| Glutathione quantification | GSH/GSSG levels | Assessing redox status for NO storage |
Detection of dinitrosyl-iron complexes
Electron paramagnetic resonance (EPR) spectroscopy is the gold standard for detecting dinitrosyl-iron complexes, which are paramagnetic. This method allows quantification of NO storage in cells and tissues. Additionally, fluorescence-based probes such as diaminofluoresceins can be used to visualize NO, but they may not distinguish stored forms.
Quantification of NO release
The release of NO from storage pools can be measured using chemiluminescence or electrochemical sensors. For example, the Griess assay measures nitrite/nitrate, the stable end products of NO, but does not directly measure storage. Real-time NO release can be monitored with NO-selective electrodes.
Genetic manipulation and screening
CRISPR-Cas9 knockout, point mutation, and overexpression are powerful tools to study genes involved in NO storage. For instance, knocking out GSTs or MRP1 can reveal their roles in NO storage and transport. Genome-wide CRISPR screens can identify novel regulators of NO storage by using reporters of NO levels or DNIC formation.
Biochemical assays for GST activity and glutathione levels
Since GSTs and glutathione are central to NO storage, measuring their activity and levels is important. GST activity can be assayed using colorimetric substrates like CDNB. Glutathione levels can be quantified with HPLC or enzymatic recycling assays.
How CRISPR Can Be Used to Study GO:0035732 nitric oxide storage
Knockout
CRISPR knockout of genes such as GSTP1, GSTM1, or ABCC1 (MRP1) can be used to determine their necessity for nitric oxide storage. For example, knocking out GSTP1 in cancer cell lines may reduce the stabilization of dinitrosyl-iron complexes, leading to decreased NO storage and altered sensitivity to NO-mediated cytotoxicity. Knockout of MRP1 can impair the transport of NO complexes, affecting cellular NO homeostasis.
Point Mutation
Point mutations can be introduced to study specific residues in GSTs or MRP1 that are critical for NO binding or transport. For instance, mutating the catalytic cysteine in GSTP1 may abolish its ability to stabilize DNICs, providing insights into the molecular mechanism of NO storage. Similarly, point mutations in MRP1's nucleotide-binding domains can disrupt ATP hydrolysis and transport of NO complexes.
Knock-in
Knock-in of tagged versions of GSTs or MRP1 (e.g., GFP or HA tags) allows for visualization and immunoprecipitation of these proteins to study their interaction with NO storage complexes. This approach can reveal the subcellular localization of NO storage and the dynamics of complex formation. Knock-in of disease-associated mutations, such as in HBB, can model hemolytic disorders and their impact on NO homeostasis.
Overexpression
Overexpression of genes like NOS3, GSTA1, or MRP1 can increase NO storage capacity. For example, overexpressing GSTA1 in endothelial cells may enhance the stabilization of DNICs, leading to increased NO storage and improved vasodilation. Overexpression of NOS3 can boost NO production, which may be stored and released upon demand. These models are useful for studying the effects of enhanced NO storage on cellular function and disease.
How EDITGENE Supports nitric oxide storage Research
Researchers studying nitric oxide storage-related genes often need to determine whether a candidate gene is causally involved in the accumulation, stabilization, or release of NO. This requires precise genetic manipulation and functional assays. EDITGENE provides a comprehensive suite of CRISPR-based services to accelerate such investigations, from generating knockout cell lines to performing genome-wide screens.
Contact EDITGENE today to design your custom CRISPR model for nitric oxide storage research.
Frequently Asked Questions About nitric oxide storage
What is nitric oxide storage?
Nitric oxide storage (GO:0035732) is the accumulation and maintenance of nitric oxide in cells or tissues, primarily as dinitrosyl-iron complexes stabilized by glutathione S-transferase proteins.
What genes are involved in nitric oxide storage?
Key genes include glutathione S-transferases (GSTA1, GSTM1, GSTP1), multidrug resistance-related protein 1 (ABCC1/MRP1), and nitric oxide synthases (NOS1, NOS2, NOS3).
How is nitric oxide stored in cells?
Nitric oxide is stored by binding to iron to form dinitrosyl-iron complexes, which are then stabilized by glutathione S-transferases and sequestered by transporters like MRP1.
Why is nitric oxide storage important for cardiovascular health?
Stored nitric oxide can be released to regulate vasodilation and blood pressure; disruption leads to endothelial dysfunction and hypertension.
What diseases are associated with defective nitric oxide storage?
Defective NO storage is linked to hemolytic disorders, storage lesion in banked blood, cardiovascular diseases, and inflammation.
How can I study nitric oxide storage in the lab?
Common methods include EPR spectroscopy for dinitrosyl-iron complexes, chemiluminescence for NO release, and CRISPR knockout of GSTs or MRP1.
What is the role of glutathione S-transferase in nitric oxide storage?
GSTs stabilize dinitrosyl-iron complexes, preventing NO degradation and allowing for controlled release.
Can nitric oxide storage be manipulated for therapy?
Yes, NO-releasing materials and photobiomodulation can modulate NO storage for therapeutic benefits in cardiovascular and antimicrobial applications.
What is the difference between nitric oxide storage and nitric oxide synthesis?
Nitric oxide synthesis is the production of NO by NOS enzymes, while storage is the accumulation and maintenance of NO in stable complexes for later release.
How does MRP1 affect nitric oxide storage?
MRP1 transports glutathione-NO conjugates and dinitrosyl-iron complexes, influencing intracellular NO sequestration and export.
Conclusion
Nitric oxide storage (GO:0035732) is a vital biological process that regulates NO bioavailability through the formation of dinitrosyl-iron complexes stabilized by glutathione S-transferases and transported by MRP1. This process is essential for cardiovascular homeostasis, immune function, and plant physiology, and its dysregulation contributes to various diseases. Advances in CRISPR-based models and analytical methods are accelerating our understanding of NO storage mechanisms. Targeting NO storage pathways holds promise for therapeutic interventions in cardiovascular disease, inflammation, and beyond.
References
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- 3. Buzinari TC et al.. 2022. Nitric oxide storage levels modulate vasodilation and the hypotensive effect induced by photobiomodulation using an aluminum gallium arsenide (AlGaAs) diode laser (660 nm).. Lasers Med Sci 37(6):2753-2762 PMID: 35391589
- 4. Gladwin MT et al.. 2009. Storage lesion in banked blood due to hemolysis-dependent disruption of nitric oxide homeostasis.. Curr Opin Hematol 16(6):515-23 PMID: 19701085
- 5. Huang D et al.. 2020. Interaction between nitric oxide and storage temperature on sphingolipid metabolism of postharvest peach fruit.. Plant Physiol Biochem 151:60-68 PMID: 32200191
- 6. Kumar R et al.. 2022. Long-Term Storage Stability and Nitric Oxide Release Behavior of (N-Acetyl-S-nitrosopenicillaminyl)-S-nitrosopenicillamine-Incorporated Silicone Rubber Coatings.. ACS Appl Mater Interfaces 14(27):30595-30606 PMID: 35759508
- 7. Muller B et al.. 2002. Nitric oxide transport and storage in the cardiovascular system.. Ann N Y Acad Sci 962:131-9 PMID: 12076970
- 8. Lee DN et al.. 2022. Controllable Nitric Oxide Storage and Release in Cu-BTC: Crystallographic Insights and Bioactivity.. Int J Mol Sci 23(16) PMID: 36012363
