Cyclic GMP directly activates several downstream effector proteins, most notably protein kinase G (PKG), as well as cyclic nucleotide-gated (CNG) ion channels and certain phosphodiesterases. By binding to the regulatory domain of PKG, cGMP induces a conformational change that activates the kinase. This leads to phosphorylation of downstream targets involved in smooth muscle relaxation, inhibition of platelet aggregation, and regulation of gene expression. Cyclic GMP also serves as a substrate for phosphodiesterases (PDEs) that hydrolyze it to GMP.

Cellular cGMP levels are typically measured in response to natriuretic peptides (e.g., atrial natriuretic peptide, ANP) or nitric oxide (NO) stimulation. In vitro, addition of exogenous cell-permeable cGMP analogs (e.g., 8-Br-cGMP) or non-hydrolyzable analogs (e.g., 8-pCPT-cGMP) to cultured vascular smooth muscle cells, endothelial cells, or neurons induces PKG activation, leading to reduced intracellular calcium concentrations, increased myosin light chain phosphatase activity (and thus smooth muscle relaxation), and modulation of gene transcription (e.g., c-fos). Direct addition of Cyclic GMP (TBAOH) to cell lysates or purified enzyme systems activates PKG (EC50 approx. 0.1-1 microM).

cGMP is the critical second messenger mediating the effects of nitric oxide (NO) and natriuretic peptides in vivo. In animal models, administration of NO donors (e.g., sodium nitroprusside, SNAP) or ANP leads to rapid elevation of tissue cGMP levels, resulting in vasodilation, lowering of blood pressure, and inhibition of platelet aggregation. In the erectile dysfunction pathway, NO activates soluble guanylyl cyclase (sGC) to produce cGMP, which relaxes corpus cavernosum smooth muscle, increasing blood flow. Phosphodiesterase type 5 (PDE5) inhibitors (e.g., sildenafil) block the breakdown of cGMP, prolonging its effects. Cyclic GMP (TBAOH) itself is not administered as a pharmacologic agent but is used as a substrate for in vitro assays.

Cyclic GMP (TBAOH) is used as a substrate or standard in enzyme activity assays. The activation of purified PKG is measured in vitro using 32P-ATP and a specific peptide substrate (e.g., BPDEtide or VASP-derived peptide). The reaction mixture (50 microL) contains 20 mM Tris-HCl (pH 7.4), 10 mM MgCl2, 0.5 mM ATP (including gamma-32P-ATP, approx. 100 cpm/pmol), 10 microM substrate peptide, 1 mM DTT, 20 nM PKG, and various concentrations of cGMP (0.01-10 microM). Reactions are initiated by adding PKG, incubated for 10 minutes at 30degC, and terminated by spotting onto P81 phosphocellulose paper. After washing, incorporated radioactivity is measured by liquid scintillation. To measure PDE activity, cGMP is used as the substrate. PDE enzyme (e.g., PDE5) is incubated with 1 microM [3H]cGMP in assay buffer (40 mM Tris-HCl, pH 8.0, 5 mM MgCl2) for 30 minutes at 30degC. 5‘-Nucleotidase is added to convert 5'-GMP to [3H]guanosine. The reaction is stopped, and [3H]guanosine is separated from unreacted [3H]cGMP using anion-exchange resin (e.g., AG1-X2). Radioactivity in the supernatant (containing guanosine) is quantified.

Cyclic GMP is a naturally occurring intracellular nucleotide and is not applied to live cells in its unmodified form because the free acid form (or the TBAOH salt) is membrane-impermeable. However, cell lysates or membrane preparations are used to study guanylyl cyclase activity. For example, to measure soluble guanylyl cyclase (sGC) activity, tissue homogenates (e.g., rat lung) are centrifuged at 100,000×g to obtain the cytosolic fraction. The assay mixture (200 microL) contains 50 mM triethanolamine (pH 7.4), 3 mM DTT, 0.1% BSA, 3 mM MnCl2, 1 mM GTP, and the test compound (e.g., NO donor). The reaction is initiated by adding the cytosolic fraction (50 microg protein) and incubated for 15 min at 37degC. cGMP produced is quantified by a specific enzyme immunoassay (EIA) or radioimmunoassay (RIA) using a commercially available cGMP ELISA kit. The TBAOH salt is used to prepare cGMP standard curves (0.1-1000 pmol/mL) for the immunoassay, as it is stable and accurately quantifiable.

Animal studies involving cGMP generally measure endogenous levels after pharmacological intervention rather than dosing animals with cGMP itself, as exogenous cGMP does not cross cell membranes effectively. A typical experimental design: Male Sprague-Dawley rats (200-250 g) are anesthetized, and the femoral artery is cannulated for blood pressure monitoring. The rats receive an intravenous injection of sodium nitroprusside (SNP, 1-10 microg/kg/min), a NO donor that activates sGC. Blood samples (0.5 mL) are collected via the arterial cannula into EDTA tubes containing 0.5 mM IBMX (a PDE inhibitor to prevent cGMP degradation). Plasma is separated by centrifugation, and cGMP is extracted using ethanol or acetonitrile. cGMP levels are quantified by ELISA. The cGMP concentration in control animals is typically 5-20 pmol/mL, while SNP treatment increases levels to 50-200 pmol/mL. Cyclic GMP (TBAOH) itself is used as the spiking standard in sample preparation for LC-MS/MS or EIA validation, not as a therapeutic agent.

Cyclic GMP is a small, water-soluble nucleotide (MW = 345 g/mol for free acid). Under physiological conditions, cGMP is rapidly degraded by cyclic nucleotide phosphodiesterases (PDE1, PDE2, PDE3, PDE5, PDE9, PDE10, PDE11) with a half-life in blood and tissues of seconds to minutes. For use as an analytical standard, the TBAOH salt form is provided as a powder or solution that is stable when stored at -20degC and protected from light. For an analytical standard (no cellular uptake expected), the in vitro PK concept does not apply; the compound is used solely as a reference for assay calibration.

Cyclic GMP (TBAOH) is a stable chemical reagent. The TBAOH (tetra-n-butylammonium hydroxide) counterion is a quaternary ammonium base that is irritating to skin, eyes, and the respiratory tract. The compound is not intended for in vivo administration to animals; systemic toxicity data are not applicable. For safe handling, standard laboratory safety precautions are recommended: wear protective gloves (nitrile), lab coat, and safety goggles. Avoid inhalation of dust or aerosols. Wash hands thoroughly after handling. Dispose of chemical waste according to local regulations. The material should be stored in a tightly sealed container away from light. Cyclic GMP itself is not classified as hazardous, but the organic base component (TBAOH) may exhibit some toxicity; however, at the small scale used for analytical work (mg quantities), the risk is minimal.

[1]. Wu J, et al. Cyclic GMP-AMP is an endogenous second messenger in innate immune signaling by cytosolic DNA. Science. 2013 Feb 15;339(6121):826-30.
[2]. Jenal U, et al. Cyclic di-GMP: second messenger extraordinaire. Nat Rev Microbiol. 2017 May;15(5):271-284.

CGMP is a 3',5'-cyclic purine nucleotide with a guanidine nucleobase. It is a mouse metabolite, a Saccharomyces cerevisiae metabolite, a plant metabolite, an Escherichia coli metabolite, and a human metabolite. It is a 3',5'-cyclic purine nucleotide and guanylate ribonucleotide. It is the conjugate acid of 3',5'-cyclic GMP(1-). Cyclic guanosine monophosphate (cGMP) is a guanine nucleotide with a phosphate group esterified at both the 3' and 5' positions. It is a cell regulator and is considered a second messenger. Its levels rise in response to various hormones, including acetylcholine, insulin, and oxytocin, and it has been found to activate specific protein kinases. (From Merck Index, 11th edition)
Cyclic guanosine monophosphate (cGMP) is present in or produced by Escherichia coli (K12 strain, MG1655 strain).
Cyclic guanosine monophosphate has been reported in jujube, jasmine, and other organisms with relevant data. Cyclic guanosine monophosphate (cGMP) is a cell regulator that acts as a second messenger. Its levels rise in response to various signals (acetylcholine, insulin, oxytocin) and activate specific protein kinases. 3',5'-Cyclic guanosine monophosphate (cGMP) is found in or produced by *Saccharomyces cerevisiae*. Cyclic guanosine 3',5'-(hydrogen phosphate) is a guanine nucleotide with esterification at both the 3' and 5' positions and contains a phosphate group. It is a cell regulator and is also considered a second messenger. Its levels rise in response to various hormones (including acetylcholine, insulin, and oxytocin) and have been found to activate specific protein kinases. (From *Merck Index*, 11th edition)

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Cyclic GMP (cGMP) is an endogenous cyclic nucleotide second messenger synthesized from GTP by the action of two families of guanylyl cyclase enzymes: the soluble (sGC) isoform (activated by nitric oxide) and the membrane-bound particulate (pGC) isoforms (activated by natriuretic peptides ANP, BNP, and CNP). cGMP is one of the two primary cyclic nucleotide second messengers (the other being cAMP). The TBAOH salt form is specifically prepared to enhance solubility and handling for research applications, especially for use as a standard in cGMP immunoassays, LC-MS/MS, and enzyme assays (PKG, PDE). Cyclic GMP (TBAOH) is not intended for human use (diagnostic or therapeutic). Its CAS number is 7665-99-8. The compound is stable for at least 2 years when stored desiccated at -20degC.

C10H12N5O7P

345.21

345.047

7665-99-8

Cyclic GMP sodium;40732-48-7

135398570

White to off-white solid powder

2.64g/cm3

792.7ºC at 760 mmHg

433.2ºC

2.064

0

4

9

1

23

616

4

C1[C@@H]2[C@H]([C@H]([C@@H](O2)N3C=NC4=C3N=C(NC4=O)N)O)OP(=O)(O1)O

ZOOGRGPOEVQQDX-UUOKFMHZSA-N

InChI=1S/C10H12N5O7P/c11-10-13-7-4(8(17)14-10)12-2-15(7)9-5(16)6-3(21-9)1-20-23(18,19)22-6/h2-3,5-6,9,16H,1H2,(H,18,19)(H3,11,13,14,17)/t3-,5-,6-,9-/m1/s1

9-[(4aR,6R,7R,7aS)-2,7-dihydroxy-2-oxo-4a,6,7,7a-tetrahydro-4H-furo[3,2-d][1,3,2]dioxaphosphinin-6-yl]-2-amino-1H-purin-6-one

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.

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

DMSO :~100 mg/mL (~165.38 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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