Negative control for U-73122

Although U73343 has no inhibitory impact on K+-pNPPase or H+,K+-ATPase activity, it can effectively reduce acid secretion. Histamine (Hist), carbachol (CCh), and dbcAMP-stimulated aminopyrine accumulation in gastric glands are all inhibited by U73343 in a dose-dependent manner [1].

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In UMR-106 rat osteoblastic osteosarcoma cells, pretreatment with U-73343 at concentrations up to 8 μM for 3 minutes did not inhibit intracellular calcium transients induced by 100 nM endothelin-1 (ET-1) when measured in 1.8 mM calcium-containing medium. [2]
Similarly, pretreatment with U-73343 at concentrations up to 8 μM for 3 minutes did not inhibit intracellular calcium transients induced by 100 nM parathyroid hormone (PTH) in 1.0 mM calcium-containing medium. [2]
Preincubation of UMR-106 cells with 8 μM U-73343 for 3 minutes did not significantly inhibit the ET-1 (100 nM, 1 min)-stimulated production of inositol phosphates (IP1, IP2, IP3). The data showed that U-73343 did not affect the agonist-induced phosphatidylinositol turnover. [2]

Endothelin-1 (ET-1) and parathyroid hormone (PTH) increase calcium transients in rodent osteoblastic cells. To investigate the role of phospholipase C (PLC) in these hormone-stimulated calcium signals, the effects of U-73122 (1-[6-[[17 beta-3-methoxyestra-1,3,5(10)- trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione), a reported PLC inhibitor, and its inactive analog, U-73343 (1-[6[[17 beta-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]- 1H-pyrrolidine-2,5-dione), were determined. Intracellular calcium transients were measured in UMR-106 cells with the fluorescent indicator fluo-3. In normal calcium containing medium, prior exposure (3 min) to U-73122 inhibited ET-1 and PTH stimulated calcium transients in a dose-dependent (0.2-10 microM) manner with an IC50 of 1.5-1.8 microM. A concentration of 6-8 microM was required for complete inhibition of responses to 100 nM ET-1 or PTH. U-73343 elicited no effects over this concentration range. In cells in which external calcium was reduced to less than 1 microM by the addition of EGTA, ET-1 signals were completely inhibited by 4-6 microM U-73122 and the IC50 was 0.8 microM. In the low external calcium medium, the PTH response was abolished by 2 microM U-73122 (IC50 = 0.5 microM). U-73122, 8 microM, significantly (P < 0.01) inhibited the effect of ET-1 on inositol trisphosphate production at 3 min whereas U-73343 did not. Pertussis toxin (100 ng/ml) likewise significantly inhibited the effect of ET-1 on phosphoinositol turnover as well as on intracellular calcium concentration. In conclusion, the results support the hypothesis that PLC plays a role in the calcium transients elicited by ET-1 and PTH, and that ET-1 transmits its signal in part via a pertussis toxin sensitive G-protein coupled receptor. Furthermore they suggest that U-73122 is useful for investigating PLC-mediated process in osteoblastic cells.[2]

In order to elucidate the role of phospholipase C (PLC) in gastric acid secretion, we used U73122, a commonly employed specific inhibitor of receptor-mediated PLC, and its negative control, U73343. Although 10 microM U73122 inhibited the increase in [Ca++]i induced by U46619 in rabbit platelets, Ca++ transients in the rabbit parietal cells elicited by histamine and carbachol were both resistant to the inhibitor. U73122 augmented the acid secretion of isolated gastric glands stimulated by histamine, carbachol and dbcAMP, possibly through its indirect Ca++-releasing effect on the intracellular calcium store. U73122 potently inhibited K+-p-nitrophenylphosphatase without affecting overall H+,K+-ATPase activity. On the other hand, the negative control, U73343, strongly inhibited the acid secretion stimulated by all agonists tested. The inhibitory effect was also evident on digitonin-permeabilized glands and on the proton gradient of gastric vesicles. U73343 itself is not a proton pump inhibitor, so it was considered a protonophore. In conclusion, the widely used PLC-inhibitor, U73122, and its negative control, U73343, are both useless as tools for analyzing the role of PLC in rabbit parietal cells. The former is ineffective on gastric PLC and works as an intracellular calcium releaser, and the latter works as a protonophore.[1]

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Intracellular Calcium Measurement: UMR-106 cells were harvested by trypsin/EDTA treatment, loaded with 2 μM fluo-3 AM for 30 minutes at room temperature in a loading buffer, washed, and resuspended. The cell suspension was continuously stirred in a cuvette in a fluorometer. To test the effect of U-73343, cells were preincubated with varying concentrations (e.g., 8 μM) of U-73343 for 3 minutes before stimulation with 100 nM ET-1 or PTH. Fluorescence was measured (excitation 505 nm, emission 530 nm), and intracellular calcium concentration was calculated after calibration. U-73343 at 8 μM did not inhibit the calcium transients induced by either agonist. [2]
Inositol Phosphate Production Assay: UMR-106 cells were seeded and incubated with [³H]inositol for 48 hours to label phospholipids. Cells were preincubated with 5 mM LiCl for 10 minutes, then treated with 8 μM U-73343 for 3 minutes before stimulation with 100 nM ET-1 for 1 minute. The reaction was stopped with trichloroacetic acid (TCA). Inositol phosphates (IP1, IP2, IP3) were separated by anion-exchange FPLC using an ammonium formate gradient, and radioactivity in fractions was measured by liquid scintillation counting. U-73343 did not significantly inhibit ET-1-stimulated inositol phosphate production. [2]

The study noted that, similar to the active analog U-73122 and the solvent DMSO, when high concentrations of U-73343 were added to the cell suspension, transient peaks in fluorescence intensity were sometimes observed (which may reflect nonspecific interference), but this did not affect the subsequent agonist response. [2]

[1]. The putative phospholipase C inhibitor U73122 and its negative control, U73343, elicit unexpected effects on the rabbit parietal cell. J Pharmacol Exp Ther. 1997 Sep;282(3):1379-88.

[2]. U-73122, a phospholipase C antagonist, inhibits effects of endothelin-1 and parathyroid hormone on signal transduction in UMR-106 osteoblastic cells. Biochim Biophys Acta. 1994 Dec 30;1224(3):575-82.

To elucidate the role of phospholipase C (PLC) in gastric acid secretion, we used the commonly used receptor-mediated PLC-specific inhibitor U73122 and its negative control U73343. While 10 μM U73122 inhibited the U46619-induced increase in intracellular [Ca++]i in rabbit platelets, histamine- and carbachol-induced intracellular Ca++ transients in rabbit parietal cells were unaffected. U73122 enhanced gastric acid secretion from isolated gastric glands stimulated by histamine, carbachol, and dbcAMP, likely through its indirect Ca++ release effect on intracellular calcium stores. U73122 potently inhibited K+-p-nitrophenyl phosphatase without affecting overall H+,K+-ATPase activity. Conversely, the negative control U73343 strongly inhibited gastric acid secretion stimulated by all tested agonists. Inhibition was also observed along the proton gradient in glands and gastric pouch permeated with digitalis saponins. U73343 itself is not a proton pump inhibitor, and is therefore considered a proton carrier. In summary, neither the widely used PLC inhibitor U73122 nor its negative control U73343 can be used as a tool to analyze the role of PLC in rabbit parietal cells. The former is ineffective against gastric PLC and acts as an intracellular calcium releaser; the latter acts as a proton carrier. [1] Endothelin-1 (ET-1) and parathyroid hormone (PTH) can increase calcium transients in rodent osteoblasts. To investigate the role of phospholipase C (PLC) in these hormone-stimulated calcium signaling processes, we determined the effects of the reported PLC inhibitor U-73122 (1-[6-[[17β-3-methoxyestradiol-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrolo-2,5-dione) and its inactive analog U-73343 (1-[6-[[17β-3-methoxyestradiol-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrolo-2,5-dione). Intracellular calcium transients were measured in UMR-106 cells using the fluorescent indicator fluo-3. In normal calcium-containing media, pre-exposure to U-73122 (3 min) dose-dependently (0.2–10 μM) inhibited ET-1 and PTH-stimulated calcium transients, with an IC50 of 1.5–1.8 μM. Complete inhibition of the 100 nM ET-1 or PTH response required a concentration of 6–8 μM. U-73343 did not produce any effect within this concentration range. In cells where extracellular calcium concentrations were reduced to below 1 μM by adding EGTA, 4–6 μM of U-73122 completely inhibited ET-1 signaling, with an IC50 of 0.8 μM. In low-calcium media, 2 μM of U-73122 eliminated the PTH response (IC50 = 0.5 μM). U-73122 (8 μM) significantly inhibited the effect of ET-1 on inositol triphosphate production at 3 minutes (P < 0.01), while U-73343 had no such effect. Pertussis toxin (100 ng/ml) also significantly inhibited the effect of ET-1 on phosphatidylinositol turnover and intracellular calcium ion concentration. In summary, these results support the hypothesis that PLC plays a role in ET-1 and PTH-induced calcium transients, and that part of the ET-1 signaling is mediated through a pertussis toxin-sensitive G protein-coupled receptor. Furthermore, they indicate that U-73122 could be used to study PLC-mediated processes in osteoblasts. [2]

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U-73343 (1-[6-[[17β-3-methoxyestradiol-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrolidine-2,5-dione) is a structural analog of the aminosteroid PLC inhibitor U-73122. The key structural difference is thatU-73343 contains the pyrrolidine-2,5-dione moiety, while the active compound U-73122 contains the pyrrole-2,5-dione moiety. [2]
In this study,U-73343 served as an important negative control compound. At concentrations where U-73122 exhibited potent inhibitory activity (IC50 ~0.5-1.8 μM), U-73343 lacked activity, which helps confirm that the observed inhibitory effect of U-73122 on the ET-1 and PTH signaling pathways is a result of its specific action on the PLC pathway, rather than nonspecific or cytotoxic effects. [2]
This study concludes that U-73343 does not inhibit ET-1-stimulated calcium transients, PTH-stimulated calcium transients, or ET-1-stimulated inositol phosphate production, confirming that it is an inactive analogue in this cellular environment. [2]

C29H42N2O3

466.65538

466.319

142878-12-4

114825

White to off-white solid powder

1.2±0.1 g/cm3

641.5±55.0 °C at 760 mmHg

341.8±31.5 °C

0.0±1.9 mmHg at 25°C

1.578

5.49

1

4

9

34

725

5

C[C@]12CC[C@H]3[C@H]([C@@H]1CC[C@@H]2NCCCCCCN4C(=O)CCC4=O)CCC5=C3C=CC(=C5)OC

CJHWFIUASFBCKN-ZRJUGLEFSA-N

InChI=1S/C29H42N2O3/c1-29-16-15-23-22-10-8-21(34-2)19-20(22)7-9-24(23)25(29)11-12-26(29)30-17-5-3-4-6-18-31-27(32)13-14-28(31)33/h8,10,19,23-26,30H,3-7,9,11-18H2,1-2H3/t23-,24-,25+,26+,29+/m1/s1

1-(6-(((8R,9S,13S,14S,17S)-3-methoxy-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthren-17-yl)amino)hexyl)pyrrolidine-2,5-dione

U73343; u-73343; u73343; U 73343; S2C4J8704C; 1-[6-[[(8R,9S,13S,14S,17S)-3-methoxy-13-methyl-6,7,8,9,11,12,14,15,16,17-decahydrocyclopenta[a]phenanthren-17-yl]amino]hexyl]pyrrolidine-2,5-dione; 1-(6-(((8R,9S,13S,14S,17S)-3-methoxy-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthren-17-yl)amino)hexyl)pyrrolidine-2,5-dione; 1-(6-(((17beta)-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-2,5-Pyrrolidinedione; U-73343; U 73343

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

DMSO : ~3.33 mg/mL (~7.14 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.)