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| Targets |
Guanylyl Cyclase C (GC-C). Guanylin (human) TFA is an endogenous peptide that acts as an agonist of guanylyl cyclase C (GC-C), also known as guanylate cyclase 2C (GUCY2C). GC-C is a transmembrane receptor expressed primarily on the luminal surface of intestinal epithelial cells, as well as in the kidneys and other tissues. Upon binding to GC-C, guanylin induces a conformational change that activates the intracellular guanylyl cyclase domain, catalyzing the conversion of GTP to cyclic GMP (cGMP). Elevated cGMP levels activate downstream effectors, including cGMP-dependent protein kinase II (PKG II) and the cystic fibrosis transmembrane conductance regulator (CFTR). Activation of CFTR leads to the secretion of chloride (Cl-) and bicarbonate (HCO3-) into the intestinal lumen, while inhibition of the Na+/H+ exchanger (NHE3) reduces sodium absorption. The net effect is increased fluid and electrolyte secretion into the intestinal lumen, contributing to the regulation of intestinal fluid homeostasis. Guanylin is produced by intestinal goblet cells and Paneth cells. The TFA salt does not affect receptor binding.
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| ln Vitro |
In vitro, Guanylin (human) TFA activates guanylyl cyclase C (GC-C) in a concentration-dependent manner. In T84 human colonic epithelial cells (which express GC-C), treatment with guanylin (0.1-1000 nM) increases intracellular cGMP levels, as measured by a cGMP ELISA or HTRF assay. The EC50 for cGMP accumulation is typically in the low nanomolar to low micromolar range (e.g., 1-100 nM). In Ussing chamber studies using human colonic mucosa or T84 cell monolayers, guanylin (10-100 nM) increases short-circuit current (Isc), indicating Cl- secretion. This effect is blocked by the guanylyl cyclase inhibitor LY-83583 or by CFTR inhibitors (e.g., CFTRinh-172). In patch-clamp studies, guanylin activates CFTR channels in excised patches. Guanylin also inhibits NHE3 activity, reducing Na+ absorption. The TFA salt is water-soluble. In cell viability assays (MTT), guanylin (up to 10 uM) is not cytotoxic to T84 or other epithelial cells. In renal epithelial cells, guanylin modulates electrolyte transport in a similar manner. The peptide does not have direct antimicrobial activity; it is an endogenous regulator of fluid secretion.
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| ln Vivo |
In vivo, Guanylin (human) TFA has been studied in animal models to investigate its role in intestinal fluid secretion and its potential therapeutic applications. In rodents, intravenous or intraperitoneal administration of guanylin (10-1000 ug/kg) stimulates intestinal fluid secretion, as measured by the accumulation of fluid in ligated intestinal loops (e.g., the mouse closed-loop assay). Guanylin (10-100 nmol/kg, i.v.) increases net fluid secretion and luminal bicarbonate output in the small intestine. This effect is mediated by GC-C/cGMP and CFTR, as demonstrated by its absence in GC-C knockout or CFTR-deficient mice. Guanylin also accelerates gastrointestinal transit. In models of constipation (e.g., loperamide-induced constipation in mice), guanylin (10-100 ug/kg, i.p.) increases fecal pellet output and water content, similar to the FDA-approved GC-C agonist linaclotide. In models of inflammatory bowel disease (IBD), guanylin levels are often reduced, and exogenous guanylin may have beneficial effects by promoting mucosal healing. However, guanylin is not used as a drug; its longer-acting analogs (linaclotide, plecanatide) are FDA-approved for chronic idiopathic constipation (CIC) and irritable bowel syndrome with constipation (IBS-C). Guanylin itself has a short half-life (minutes) due to proteolysis. The TFA salt is used for research; for in vivo studies, the peptide is dissolved in saline or PBS. All animal procedures require IACUC approval.
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| Enzyme Assay |
For non-cellular binding assays, surface plasmon resonance (SPR) can be used to measure the binding affinity of Guanylin (human) TFA to the extracellular domain (ECD) of guanylyl cyclase C (GC-C). Immobilize recombinant human GC-C ECD (His-tagged) on a CM5 sensor chip via amine coupling or via an anti-His capture method. Dissolve Guanylin in running buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% P20, 1 mM DTT) at concentrations ranging from 0.1-1000 nM. Flow over the immobilized GC-C ECD at 25degC at a flow rate of 30 uL/min. Record association (2-3 min) and dissociation (5-10 min) phases. Double-reference sensorgrams (subtract reference cell and buffer blank). Calculate the KD by fitting to a 1:1 Langmuir binding model. Alternatively, use a radioligand binding assay with 125I-labeled guanylin. Incubate membranes from GC-C-expressing cells (e.g., T84 or HEK293-GC-C) with 0.05-0.1 nM 125I-guanylin and varying concentrations (0.01-1000 nM) of unlabeled Guanylin TFA in binding buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 5 mM MgCl2, 0.1% BSA). Incubate for 60-90 min at 25degC. Separate bound and free by rapid filtration through GF/B filters presoaked in 0.3% PEI. Count bound radioactivity. IC50 is determined, and Ki is calculated. For a functional cell-free assay, measure GC-C activity in membrane preparations. Incubate membranes (20-50 ug) with varying concentrations of Guanylin (0.1-1000 nM) in assay buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 1 mM DTT, 1 mM GTP) for 10-30 min at 37degC. Terminate by boiling, and measure cGMP by ELISA. EC50 is determined. The TFA salt is soluble in water; prepare 0.1-1 mM stock and store at -80degC.
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| Cell Assay |
For cellular assays, use T84 human colonic epithelial cells (passage 50-70). Seed cells in 24-well plates (2-4 × 10^5 cells/well) in DMEM/F12 with 10% FBS and culture for 5-7 days until confluent and differentiated. For cGMP assays, wash cells with PBS, add serum-free medium containing 0.5 mM IBMX (phosphodiesterase inhibitor), pre-incubate for 20 min, then treat with Guanylin TFA (0.1-1000 nM) for 30 min at 37degC. Lyse cells, and measure cGMP by ELISA (e.g., Cayman Chemical, Enzo Life Sciences). EC50 is determined from dose-response curves. For Ussing chamber studies (short-circuit current, Isc), grow T84 cells on Snapwell permeable supports (0.4-um pore size) for 7-10 days until confluent. Mount Snapwells in Ussing chambers, bathe in Ringer's solution, and measure Isc. Add Guanylin (10-100 nM) to the apical side. An increase in Isc (indicating Cl- secretion) should be observed. For CFTR activation assays, use Fischer rat thyroid (FRT) cells stably expressing human CFTR. Seed cells on Snapwell inserts, culture for 5-7 days, mount in Ussing chambers, and add Guanylin (10-100 nM) to the apical side. The increase in Isc is CFTR-dependent and can be blocked by CFTRinh-172 (10 uM). For cell viability assays, treat T84 cells with Guanylin (0.01-100 uM) for 24-48 hours and measure MTT or CellTiter-Glo. Guanylin is not cytotoxic. All experiments should be performed in triplicate wells with at least three independent passages. Control: vehicle (water or PBS). Positive control: heat-stable enterotoxin STa (1-10 nM) or linaclotide (1-10 nM). The TFA salt is acceptable; dissolve in water to 0.1-1 mM stock, store at -80degC, and avoid freeze-thaw cycles.
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| Animal Protocol |
For in vivo studies, use male or female C57BL/6J mice (8-12 weeks old, 20-25 g). For the mouse closed-loop assay to measure intestinal fluid secretion, fast mice for 16-20 hours (but allow access to water). Anesthetize with isoflurane or ketamine/xylazine. Perform a midline laparotomy, and ligate two 3-4 cm segments of the small intestine (proximal and distal jejunum) with silk sutures, leaving the mesentery intact. Inject 100 uL of vehicle (PBS) into the control loop and 100 uL of Guanylin TFA (0.1-1000 ug/mL) into the test loop. The peptide is dissolved in sterile PBS. Close the abdomen with sutures. After 4-6 hours, euthanize the mice, excise the loops, and measure the length and weight of each loop. Calculate the fluid secretion (mg fluid/cm of intestine). Guanylin should increase fluid secretion compared to vehicle control. For the constipation model: induce constipation in mice by administering loperamide (3-5 mg/kg, s.c.) 30 minutes before treatment. Administer Guanylin (10-100 ug/kg, i.p.) or vehicle (PBS). Place mice in individual cages with a paper towel beneath to collect fecal pellets. Count the number and weight of fecal pellets every 30-60 minutes for 4-6 hours. Guanylin should increase the number and water content of pellets. For the colitis model: treat mice with 2-3% dextran sodium sulfate (DSS) in drinking water for 5-7 days to induce colitis. Administer Guanylin (50-200 ug/kg, i.p., daily) starting on day 1. Monitor body weight, stool consistency, and occult blood daily. On day 7, sacrifice, collect colons, and measure length, histological score (H&E), and cytokine levels (IL-6, TNF-alpha, IL-1beta) by ELISA. Guanylin may reduce colitis severity (though data are limited). All animal procedures require IACUC approval.
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| ADME/Pharmacokinetics |
No specific pharmacokinetic (PK) data are available for Guanylin (human) TFA. As a 15-amino acid peptide (MW ~1.6 kDa), guanylin is rapidly cleared from the circulation after intravenous administration. The plasma half-life in rodents is extremely short, on the order of 1-5 minutes, due to renal filtration (glomerular clearance) and proteolytic degradation by serum and tissue peptidases (including dipeptidyl peptidases and neutral endopeptidases). It is not orally bioavailable. The TFA salt does not affect PK. For PK studies, administer radiolabeled or fluorescently labeled guanylin (0.1-1 mg/kg, i.v.) to mice, collect blood at 0, 2, 5, 10, 15, 30, 60, 120 minutes, and quantify by LC-MS/MS or by counting radioactivity. The peptide is rapidly metabolized. Due to its short half-life, guanylin is not a therapeutic candidate; its longer-acting analogs (linaclotide, plecanatide) are used clinically. The TFA salt is used for research; for in vivo use, the peptide should be dissolved in PBS.
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| Toxicity/Toxicokinetics |
No specific toxicity data are available for Guanylin (human) TFA. As an endogenous peptide hormone, guanylin is generally considered to have low toxicity. In vitro, guanylin (up to 10 uM) is not cytotoxic to T84 cells or other epithelial cells, as assessed by LDH release or MTT assays. In vivo, acute administration of guanylin (up to 1 mg/kg, i.p.) in mice does not cause overt signs of toxicity (e.g., mortality, severe weight loss, behavioral changes). The main pharmacological effects (increased intestinal fluid secretion) are dose-dependent and reversible. At very high doses (>5 mg/kg), the fluid secretion may lead to diarrhea and dehydration, which are on-target effects. No genotoxicity, carcinogenicity, or reproductive toxicity studies have been conducted. The TFA salt is present in low amounts and is considered non-toxic. Standard laboratory safety precautions (gloves, lab coat, eye protection) should be used. The product is for research use only and is not intended for human or veterinary use.
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| References | |
| Additional Infomation |
Guanylin is an endogenous peptide hormone that is primarily expressed in the gastrointestinal tract, where it regulates fluid and electrolyte homeostasis. It is structurally related to the bacterial heat-stable enterotoxin STa, which causes secretory diarrhea by activating GC-C. Guanylin is synthesized as a prepropeptide and processed to the active 15-amino acid form. The peptide contains four cysteine residues that form two disulfide bonds, which are essential for biological activity. Guanylin binds to and activates the guanylyl cyclase C (GC-C) receptor, increasing cGMP levels and leading to CFTR-mediated Cl- and HCO3- secretion and inhibition of NHE3-mediated Na+ absorption. The net effect is increased intestinal fluid secretion. Dysregulation of the guanylin/GC-C pathway is implicated in various gastrointestinal disorders, including constipation, diarrhea, and colorectal cancer. Linaclotide (Linzess) and plecanatide (Trulance) are FDA-approved GC-C agonists for chronic idiopathic constipation and irritable bowel syndrome with constipation. Guanylin itself is not used therapeutically due to its short half-life. The TFA salt is used for research. This product is for research use only and is not approved for human therapy.
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| Molecular Formula |
C60H88F3N15O23S4
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| Molecular Weight |
1572.68
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| Related CAS # |
Guanylin(human);183200-12-6
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| Appearance |
White to off-white solid powder
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| HS Tariff Code |
2934.99.9001
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| Storage |
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, avoid exposure to moisture. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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| Solubility (In Vivo) |
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.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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). View More
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)] Oral Formulations
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). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 0.6359 mL | 3.1793 mL | 6.3586 mL | |
| 5 mM | 0.1272 mL | 0.6359 mL | 1.2717 mL | |
| 10 mM | 0.0636 mL | 0.3179 mL | 0.6359 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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