P2Y2 Receptor

MRS2768 (0.01-10000 μM; 24 hours) greatly boosts the proliferation of PANC-1 cells[1].

---

Incubation of PANC-1 cells with UTP or MRS2768, a selective P2Y2 receptor agonist, resulted in a dose- and time-dependent increase of proliferation. The messenger RNA transcript and protein of P2Y2 receptor were expressed in PANC-1 cells. P2 receptor antagonist suramin and small interfering RNA against P2Y2 receptor significantly decreased the proliferative effect of UTP and MRS2768. Activation of P2Y2 receptor by UTP transduced to phospholipase C, inositol 1,4,5-triphosphate (IP3), and protein kinase C. Uridine triphosphate-induced proliferation was mediated by protein kinase D, Src-family tyrosine kinase, Ca/calmodulin-dependent protein kinase II, phosphatidylinositol 3-kinase (PI3K), Akt, and phospholipase D. Uridine triphosphate increased phosphorylation of Akt through protein kinase C, Src-family tyrosine kinase, Ca/calmodulin-dependent protein kinase II, and PI3K. Conclusions: Uridine triphosphate increases proliferation of human pancreatic duct epithelial cells by activation of P2Y2 receptor and PI3K/Akt pathway. This could be helpful for discovering the long-term roles of P2Y2 receptor in pancreatic cells. [1]

---

Cultured rat cardiomyocytes pretreated with MRS2768 displayed protection from hypoxia [as revealed by lactate dehydrogenase (LDH) release and propidium iodide (PI) binding], which was reduced by P2Y2R antagonist, AR-C118925 (5-((5-(2,8-dimethyl-5H-dibenzo[a,d][7]annulen-5-yl)-2-oxo-4-thioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)-N-(1H-tetrazol-5-yl)furan-2-carboxamide) [2].

In mice, myocardial injury is reduced by pretreatment with MRS2768 (4.44 μg/kg iv). Heart muscle cells are shielded from ischemia injury in vivo by MRS2768[2].

---

In vivo, echocardiography and infarct size staining of triphenyltetrazolium chloride (TTC) in 3 groups of mice 24 h post-MI: sham, MI, and MI+MRS2768 indicated protection. Fractional shortening (FS) was higher in MRS2768-treated mice than in MI alone (40.0 ± 3.1 % vs. 33.4 ± 2.7 %, p < 0.001). Troponin T and tumor necrosis factor-α (TNF-α) measurements demonstrated that MRS2768 pretreatment reduced myocardial damage (p < 0.05) and c-Jun phosphorylation increased. Thus, P2Y2R activation protects cardiomyocytes from hypoxia in vitro and reduces post-ischemic myocardial damage in vivo [2].

---

Assessment of LV function post-MI using echocardiography [2]
There were no significant differences between groups in the echocardiographic measurements of cardiac structure or function at baseline. Induction of MI resulted in a more pronounced increase of left ventricular systolic diameter compared with the MRS2768-pretreated mice (p < 0.05, Fig. 3a, b). The infarcted mice also demonstrated a significantly reduced FS (33.4 ± 2.7 %) compared to MRS2768-treated mice (40.0 ± 3.13 %), or sham mice (51.8 ± 1.7 %, Fig. 3c, p < 0.001). Representative M mode pictures of the echo measurements are presented in Fig. 3e. Ischemic damage was measured using several markers as follows.

---

Biochemical marker of ischemic damage [2]
Troponin T: The level of Troponin T, a specific marker denoting damage in the heart muscle, was significantly elevated in mice post-MI (13.4 ± 1.4 ng/ml) compared with the sham group. MRS2768 pretreatment significantly lowered Troponin T levels in the serum (10.6 ± 0.8 ng/ml, p < 0.05, Fig. 4a).

P2Y2 Receptor siRNA Inhibition Assay [1]
Small interfering RNA against human P2Y2 receptor was used. The cells were grown in 24-well or 96-well plates at 70% to 80% confluence overnight and then transfected with predesigned siRNA (50 nmol/L) using Lipofectamine RNAiMAX transfection reagent as instructed by the manufacturer. A no-target siRNA (scrambled siRNA) was used as a negative control. Forty-eight hours later, cells were treated with UTP or MRS2768. The proliferation was measured after an additional 24-hour incubation period. P2Y2 messenger RNA (mRNA) expression was detected by quantitative RT-PCR and/or Western blotting to demonstrate successful silencing of the mRNA. The siRNA decreased the mRNA expression of the P2Y2 receptor more than 80% in the PANC-1 cells.

Cell Proliferation Assay[1]
Cell Types: Human pancreatic duct epithelial cells PANC-1
Tested Concentrations: 0.01, 0.1, 1, 10, 100, 1000, 10000 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: The effect on proliferation of PANC-1 cells was dependent on concentration (0.1 μM to 1 mM). The concentration that elicited a half-maximal response (EC50) in the stimulation of proliferation was 0.8±1.7 μM. Resulted in a dose- and time-dependent increase of proliferation in PANC-1 cells

Animal/Disease Models: Male wild-type mice (C57BL)[2]
Doses: 4.44 μg/ kg
Route of Administration: Injected iv; 1 h before myocardial infarct (MI)
Experimental Results: Pretreatment decreased myocardial damage. The damage was Dramatically smaller in mice compared to the untreated mice (25.6±4.5% vs. 39.2±6.3%).

---

Animals and experimental protocol [2]
Male wild-type mice (C57BL) were used. Three experimental groups were used:
1. sham without ligation of the left anterior descending coronary artery (LAD)
2. myocardial infarction (MI) = LAD ligation
3. animals injected with MRS2768 (4.44 μg/kg i.v.) 1 h before MI

[1]. Uridine Triphosphate Increases Proliferation of Human Cancerous Pancreatic Duct Epithelial Cells by Activating P2Y2 Receptor. Pancreas. 2013 May;42(4):680-6.

[2]. P2Y2 Receptor Agonist With Enhanced Stability Protects the Heart From Ischemic Damage in Vitro and in Vivo. Purinergic Signal. 2013 Dec;9(4):633-42.

In this study, the P2Y2 receptor mediated the proliferative action of UTP in PANC-1 cells. Uridine triphosphate, a potent agonist of the P2Y2/P2Y4 receptors, and MRS2768, a selective P2Y2 receptor agonist, regulated the proliferation of pancreatic duct epithelial cells in a concentration-dependent manner. The potency order was MRS2768 was greater than UTP, which indicates that UTP regulates proliferation through activation of the P2Y2 receptor. In addition, the P2 receptor antagonist suramin significantly inhibited UTP- and MRS2768-induced regulation of proliferation. The siRNA against the P2Y2 receptor also significantly inhibited UTP- and MRS2768-induced proliferation, supporting P2Y2 receptor involvement. Based on P2Y receptor mRNA, the expression level of the P2Y2 receptor was higher than those of P2Y1 and P2Y4 receptor subtypes in pancreatic cells. The regulation of cell proliferation by UTP is mediated by the P2Y2 receptor. Studies on P2Y2 receptor activation in ovarian cancer15,36 and colorectal cancer21,27 showed stimulation of proliferation in some cell lines and inhibition of proliferation in other cell lines. This may be due to differences in the cancer cell lines, the expression level of P2 receptors, and the associated signaling pathways. Furthermore, the proliferative action was dependent on serum because UTP without serum had no effect on pancreatic cell proliferation (data not shown).[1]

---

Since no differences in IκB levels were observed between the ischemic groups, we can assume that NFκB degradation is not part of the cardioprotective mechanism achieved by MRS2768. With respect to activation of c-Jun N-terminal kinases (JNKs), there are conflicting results. Several studies suggested that activation of JNK occurs specifically on reperfusion. We and others found that ischemia alone can activate this pathway. Moreover, the role of JNK in myocardial ischemia/reperfusion (I/R) remains controversial, since equally robust studies have reported dichotomous results suggesting both cardioprotective and detrimental effects. In contrast to our previous report, we have found a significant reduction in the expression of phospho-c-Jun in wild type (WT) mice pretreated with UTP compared to MI alone (p < 0.05). This difference might be the result of a difference in in vivo half-life, i.e., the rapidly degraded UTP compared to MRS2768. However, the in vivo pharmacokinetics and pharmacodynamics of MRS2768 remain to be determined. It seems that cardioprotective effects of MRS2768 are related in part to the phosphorylation of c-Jun.
In conclusion, we have demonstrated that activation of the P2Y2 receptor with MRS2768 protects the heart from ischemia, minimizes infarct size and improves cardiac function post-MI. Although the precise mechanism for the inhibition of myocardial ischemic injury remains unclear, therapeutic targeting of P2YRs for protection against ischemic myocardial damage is very promising and justifies further exploration. [2]

C15H16N2NA4O18P4

728.14

727.893936

2567869-47-8

90488893

Colorless to light yellow liquid

3

18

12

43

1120

4

C1=CC=C(C=C1)OP(=O)([O-])OP(=O)([O-])OP(=O)([O-])OP(=O)([O-])OC[C@@H]2[C@H]([C@H]([C@@H](O2)N3C=CC(=O)NC3=O)O)O.[Na+].[Na+].[Na+].[Na+]

ASYFBNZFGLWLNC-YYXHNCPRSA-J

InChI=1S/C15H20N2O18P4.4Na/c18-11-6-7-17(15(21)16-11)14-13(20)12(19)10(31-14)8-30-36(22,23)33-38(26,27)35-39(28,29)34-37(24,25)32-9-4-2-1-3-5-9;;;;/h1-7,10,12-14,19-20H,8H2,(H,22,23)(H,24,25)(H,26,27)(H,28,29)(H,16,18,21);;;;/q;4*+1/p-4/t10-,12-,13-,14-;;;;/m1..../s1

tetrasodium;[[(2R,3S,4R,5R)-5-(2,4-dioxopyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-oxidophosphoryl] [oxido-[oxido(phenoxy)phosphoryl]oxyphosphoryl] phosphate

MRS2768 tetrasodium salt; 2567869-47-8;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Shipping with dry ice.

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.

---

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).

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)]
*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)

---

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
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

---

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.

---

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