P2Y6 (EC50 = 15 nM)

Endosomal trafficking is intricately linked to G protein-coupled receptors (GPCR) fate and signaling. Extracellular uridine diphosphate (UDP) acts as a signaling molecule by selectively activating the GPCR P2Y6. Despite the recent interest for this receptor in pathologies, such as gastrointestinal and neurological diseases, there is sparse information on the endosomal trafficking of P2Y6 receptors in response to its endogenous agonist UDP and synthetic selective agonist 5-iodo-UDP (MRS2693). Confocal microscopy and cell surface ELISA revealed delayed internalization kinetics in response to MRS2693 vs. UDP stimulation in AD293 and HCT116 cells expressing human P2Y6. Interestingly, UDP induced clathrin-dependent P2Y6 internalization, whereas receptor stimulation by MRS2693 endocytosis appeared to be associated with a caveolin-dependent mechanism. Internalized P2Y6 was associated with Rab4, 5, and 7 positive vesicles independent of the agonist. We have measured a higher frequency of receptor expression co-occurrence with Rab11-vesicles, the trans-Golgi network, and lysosomes in response to MRS2693. Interestingly, a higher agonist concentration reversed the delayed P2Y6 internalization and recycling kinetics in the presence of MRS2693 stimulation without changing its caveolin-dependent internalization. This work showed a ligand-dependent effect affecting the P2Y6 receptor internalization and endosomal trafficking. These findings could guide the development of bias ligands that could influence P2Y6 signaling [1].

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The endogenous P2Y6 receptor agonist UDP and synthetic agonist MRS2693 protected C2C12 skeletal muscle cells against apoptosis in a concentration-dependent manner (0.1−10 nM) as determined by propidium iodide staining, histochemical analysis using hematoxylin and Hoechst 33258, and DNA fragmentation. The insurmountable P2Y6 receptor antagonist MRS2578 blocked the protection. TNFα-induced apoptosis in C2C12 cells correlated with activation of the transcription factor NF-κB. The NF-κB activation was attenuated by 10 nM MRS2693, which activated the antiapoptic ERK1/2 pathway[2].

In an in vivo mouse hindlimb model, MRS2693 protected against skeletal muscle ischemia/reperfusion injury. The P2Y6 receptor is a novel cytoprotective receptor that deserves further exploration in ameliorating skeletal muscle injury [2].

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Cytoprotection in an in vivo model of mouse skeletal muscle ischemia/reperfusion We used a mouse hindlimb ischemia/reperfusion model to test the in vivo protective ability of the selective P2Y6 agonist MRS2693. As demonstrated in a previous study of adenosine receptor-induced protection in the same model [16], ischemia induced by an external constrictor (90 min) followed by reperfusion (24 h) resulted in significant skeletal muscle injury in PBS vehicle–treated mice. The extent of injury was quantified by an increase in the staining of the skeletal myocytes with EBD, which binds to albumin and enters only damaged cells, and by a higher level of serum creatine kinase. Administration of MRS2693 (1 mg/kg, i.p. administration) prior to ischemia and reperfusion caused a significant reduction in the extent of injury (Figure 5). The compound reduced skeletal muscle injury with a significant decrease in serum CK level (3450 U/L ± 1660 U/L, n = 10, SE, vs. vehicle-treated 12,600 U/L ± 3300 U/L, n = 14, P = 0.037). Similarly, the percent EBD-stained area was also significantly reduced by MRS2693 (10.4% ± 2.0%, SE, n = 10 vs. vehicle-treated 28.3% ± 5.6%, n = 7, P=0.0038). The myocytes stained with EBD in treated and untreated mice in sections are shown in Figure 6 [2].

Induction and detection of apoptosis [2]
TNFα was used to induce apoptosis in C2C12 cells (5, 7 and 12 days old). After washing of the cells, the medium was replaced with fresh medium containing 5 μg/ml cycloheximide, which was present during the entire subsequent incubation to promote apoptosis as previously discussed. In cases of coadminstration of the P2Y6 receptor antagonist MRS2578, this was the next reagent to be added. A freshly prepared DMSO solution of MRS2578 (1 mM) was added to the incubation medium to reach a final concentration of 10 μM for an optional initial incubation of 20 min. The next reagent added to the cells was either a P2Y6 receptor agonist (MRS2693 or UDP), when present, or TNFα (10 ng/ml). When P2Y6 receptor agonists were used, TNFα was added 10 min after the agonist. The cells remained in the presence of TNFα and other agents for 4 h. The medium was then changed and the culture was left in the presence of cycloheximide for 16 h. Cell death was observed 20 h after the first exposure to TNFα.
DNA fragmentation of apoptotic cells was detected using the standard Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) method. Cell death or apoptosis was indicated by PI positive cells and by fluorescence of termini of DNA fragments labeled with 5-bromo-2′-deoxyuridine 5′-triphosphate (BrdUTP). The presence of live cells was detected by fluorescent labeling of DNA with the cell-permeant dye Hoechst 33258 for staining nuclear chromatin. For microscopic applications, the cells were deposited onto slides.

Protocol for in vivo administration of P2Y6 receptor agonist [2]
The P2Y6 receptor agonist MRS2693 (300 μM) or vehicle alone (0.1% DMSO in PBS) was administered in a sterile 0.1-ml volume by i.p. injection 2 h before the induction of ischemia. This protocol was used in the previous study of adenosine receptor agonists and antagonists in the same model. EBD (1% wt/vol solution to yield 1 mg of EBD/10 g body weight) was also given via a separate i.p. injection 2.5 h before the induction of ischemia.

[1]. Ligand-dependent intracellular trafficking of the G protein-coupled P2Y6 receptor. Biochim Biophys Acta Mol Cell Res. 2023 Jun;1870(5):119476.

[2]. Attenuation of apoptosis in vitro and ischemia/reperfusion injury in vivo in mouse skeletal muscle by P2Y6 receptor activation. Pharmacol Res. 2008 Sep-Oct;58(3-4):232-9.

For the investigation of the role of the P2Y6 receptor in skeletal muscle cell injury, we have chosen to use a novel, potent synthetic agonist, MRS2693. MRS2693 can be considered more selective for this subtype because: 1) MRS2693 itself has no activity at other P2Y subtypes and 2) the corresponding 5′-triphosphate derivative is a much weaker agonist than UTP at other P2Y receptor subtypes. We have demonstrated that this derivative is cytoprotective toward skeletal muscle cells in the mouse both in vitro and in vivo.
Activation of the endogenous P2Y6 receptor in C2C12 cells significantly attenuated TNFα-induced apoptosis. The protection occurred at very low agonist concentrations and correlated with the activation of ERK1/2. The potent antiapoptotic protection by the novel P2Y6 receptor agonist MRS2693 in the C2C12 cell line was concentration-dependent between 0.1 and 10 nM. Similar results were obtained in astrocytoma cells, in which the protection by the appropriate P2Y agonist was clearly P2Y6 receptor-dependent and was absent in untransfected control cells or cells expressing the P2Y4 receptor.
Members of the protein kinase C (PKC) family of serine/threonine protein kinases are involved in many cellular responses across a wide range of cell types. Each PKC isoenzyme may be involved in specific regulatory processes. Various PKC isoenzymes exhibit differences in tissue distribution, intracellular localization, and cofactor requirements, suggesting that they are freely regulated in response to discrete ligands, and that they may act on distinct protein substrates. Our results showed that activation of the P2Y6 receptor by MRS2693 increased the expression level of PKC θ, which might control stimulation of ERK1/2 activation. ERK1/2 is a contributing pathway in the protection by MRS2693 against TNFα-induced cell death in C2C12 cells. Thus, the protection in skeletal muscle cells mechanistically resembles protection in the astrocytoma cells.
There are no competitive antagonists that can be used as pharmacological probes of the P2Y6 receptor, so we used the diisothiocyanate derivative MRS2578 as P2Y6 receptor antagonist. This antagonist blocked the protection provided by MRS2693 against apoptosis in the C2C12 cell line. The reduced protection at higher concentrations of the agonists might be a result of interaction with other extracellular nucleotide binding sites or enzymes that act on nucleotides.
TNFα-induced apoptosis in C2C12 cells correlated with NF-κB activation. This was consistent with numerous previous reports in which TNFα has been noted to activate NF-κB in various systems. NF-κB elevation was also noted to induce damage accompanied by protein degradation in cultured rat skeletal muscle cells. Curiously, P2Y6 receptor activation was previously reported to induce translocation of NF-κB to the nucleus in certain cell types, including osteoblasts. In our study, P2Y6 receptor activation alone had little effect on NF-κB, yet substantially reduced the dramatic elevation of NF-κB induced by TNFα.
In an in vivo mouse model of hindlimb skeletal muscle ischemia/reperfusion injury, MRS2693 was also able to exert a potent cytoprotective effect. The same model was previously applied to study the protective effect of adenosine receptor agonists. We have not yet elucidated the mechanism of the P2Y6 receptor-induced protection in vivo.
In conclusion, the P2Y6 receptor is a novel cytoprotective receptor that warrants exploration for ameliorating skeletal muscle injury. This effort will be aided with the development of even more potent, selective, and stable agonists of the P2Y6 receptor.[2]

C9H10IN2NA3O12P2

596.003209590912

595.844

C, 18.14; H, 1.69; I, 21.29; N, 4.70; Na, 11.57; O, 32.21; P, 10.39

1448858-83-0

1448858-83-0;MRS 2693 trisodium salt;93906-48-0;MRS2693

90488768

Typically exists as solid at room temperature

3

12

5

29

704

4

IC1C(NC(N(C=1)[C@H]1[C@@H]([C@@H]([C@@H](COP(=O)([O-])OP(=O)([O-])[O-])O1)O)O)=O)=O.[Na+].[Na+].[Na+]

QWGVSYFNEAILDQ-FCIXCQMASA-K

InChI=1S/C9H13IN2O12P2.3Na/c10-3-1-12(9(16)11-7(3)15)8-6(14)5(13)4(23-8)2-22-26(20,21)24-25(17,18)19;;;/h1,4-6,8,13-14H,2H2,(H,20,21)(H,11,15,16)(H2,17,18,19);;;/q;3*+1/p-3/t4-,5-,6-,8-;;;/m1.../s1

trisodium;[[(2R,3S,4R,5R)-3,4-dihydroxy-5-(5-iodo-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-oxidophosphoryl] phosphate

MRS 2693 trisodium salt; 1448858-83-0; 5-Iodouridine-5'-O-diphosphate trisodium salt; 5-Iodo-UDP trisodium salt; trisodium;[[(2R,3S,4R,5R)-3,4-dihydroxy-5-(5-iodo-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-oxidophosphoryl] phosphate;

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)

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

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.)