p2x1 Receptor; P2X3 Receptor; P2X4 Receptor; P2X7 Receptor

BzATP (10-1000 μM; 24 h) stimulates U87 and U251 glioma cell migration and proliferation [3]. In human glioma cells, BzATP (100μM; 6-48h) promotes the production of P2X7R protein [3].

BzATP (5 mg/kg) dramatically increased P2X7R expression in the colon following cecal ligation and puncture (CLP) induction as compared to the control and sham operation groups [4].

Activation of rat P2X3 and P2X2/3 receptors in vitro [https://pubmed.ncbi.nlm.nih.gov/11156585/]
The recombinant rat P2X3 and rat P2X2/3 receptor cDNAs were identical to the previously published sequences used in the in vitro characterization of the pharmacology of the rat homomeric and heteromeric P2X3 receptors (Bianchi et al., 1999). 1321N1 human astrocytoma cells stably expressing rat P2X3, or rat P2X2/3 receptors were constructed using standard lipid-mediated transfection methods. All cell lines were maintained in D-MEM containing 10% FBS and antibiotics as follows: 300 μg ml−1 G418 for rat P2X3 containing cells; and 75 μg ml−1 hygromycin and 150 μg ml−1 G418 for rat P2X2/3 containing cells. Cells were grown at 37°C in a humidified atmosphere containing 5% CO2. P2X receptor function was determined on the basis of agonist-mediated increases in cytosolic Ca2+ concentration as previously described (Bianchi et al., 1999). BzATP (10 μM) and α,β-meATP (10 μM) were used to activate rat P2X3 and P2X2/3 receptors, respectively. Briefly, a fluorescent Ca2+ chelating dye (Fluo-4) was used as an indicator of the relative levels of intracellular Ca2+ in a 96-well format using a Fluorescence Imaging Plate Reader (FLIPR). Cells were grown to confluence in 96-well black-walled tissue culture plates and loaded with the acetoxymethylester (AM) form of Fluo-4 (1 μM) in D-PBS for 1 – 2 h at 23°C. Fluorescence data was collected at 1 – 5 s intervals throughout each experimental run. Concentration response data were analysed using a four-parameter logistic Hill equation in GraphPad Prism.https://pubmed.ncbi.nlm.nih.gov/11156585/

Cell Proliferation Assay[3]
Cell Types: U87 and U251 glioma cells
Tested Concentrations: 5, 10, 50, 100, 500 and 1000 μM
Incubation Duration: 2, 6, 12, 24, 48 and 72 hrs (hours)
Experimental Results: The proliferation of U87 and U251 glioma cell lines was Dramatically increased in the presence of 10-1000 uM and 100-1000 μM, respectively. The peak of cell proliferation of both U87 and U251 cell lines was at 100 μM. The optimal incubation time is 24 hrs (hours) in both U87 and U251 cells lines.

---

Western Blot Analysis[3]
Cell Types: U87 and U251 glioma cells
Tested Concentrations: 100 μM
Incubation Duration: 6-48 hrs (hours)
Experimental Results: Induced the upregulation of P2X7R.

Animal/Disease Models: Male 2-month-old C57BL/6 mice (each weighing between 20 and 25 g)[4]
Doses: 5 mg/kg
Route of Administration: Injected through the intraperitoneal (ip)route
Experimental Results: At 48 hrs (hours), mice in the treated group and The control group demonstrated mortalities of 91% and 86%, respectively.

[1]. Pharmacological characterization of recombinant human and rat P2X receptor subtypes. Eur J Pharmacol. 1999 Jul 2;376(1-2):127-38.

[2]. Amino acid residues in the P2X7 receptor that mediate differential sensitivity to ATP and BzATP. Mol Pharmacol. 2007 Jan;71(1):92-100.

[3]. Involvement of P2X 7 Receptor in Proliferation and Migration of Human Glioma Cells. Biomed Res Int. 2018 Jan 9;2018:8591397.

[4]. Systemic blockade of P2X7 receptor protects against sepsis-induced intestinal barrier disruption. Sci Rep. 2017 Jun 29;7(1):4364.

ATP functions as a fast neurotransmitter through the specific activation of a family of ligand-gated ion channels termed P2X receptors. In this report, six distinct recombinant P2X receptor subtypes were pharmacologically characterized in a heterologous expression system devoid of endogenous P2 receptor activity. cDNAs encoding four human P2X receptor subtypes (hP2X1, hP2X3, hP2X4, and hP2X7), and two rat P2X receptor subtypes (rP2X2 and rP2X3), were stably expressed in 1321N1 human astrocytoma cells. Furthermore, the rP2X2 and rP2X3 receptor subtypes were co-expressed in these same cells to form heteromultimeric receptors. Pharmacological profiles were determined for each receptor subtype, based on the activity of putative P2 ligands to stimulate Ca2+ influx. The observed potency and kinetics of each response was receptor subtype-specific and correlated with their respective electrophysiological properties. Each receptor subtype exhibited a distinct pharmacological profile, based on its respective sensitivity to nucleotide analogs, diadenosine polyphosphates and putative P2 receptor antagonists. Alphabeta-methylene ATP (alphabeta-meATP), a putative P2X receptor-selective agonist, was found to exhibit potent agonist activity only at the hP2X1, hP2X3 and rP2X3 receptor subtypes. Benzoylbenzoic ATP (BzATP, 2' and 3' mixed isomers), which has been reported to act as a P2X7 receptor-selective agonist, was least active at the rat and human P2X7 receptors, but was a potent (nM) agonist at hP2X1, rP2X3 and hP2X3 receptors. These data comprise a systematic examination of the functional pharmacology of P2X receptor activation. [1]

---

Agonist properties of the P2X7 receptor (P2X7R) differ strikingly from other P2X receptors in two main ways: high concentrations of ATP (> 100 microM) are required to activate the receptor, and the ATP analog 2',3'-O-(4-benzoyl-benzoyl)ATP (BzATP) is both more potent than ATP and evokes a higher maximum current. However, there are striking species differences in these properties. We sought to exploit the large differences in ATP and BzATP responses between rat and mouse P2X7R to delineate regions or specific residues that may be responsible for the unique actions of these agonists at the P2X7R. We measured membrane currents in response to ATP and BzATP at wild-type rat and mouse P2X7R, at chimeric P2X7Rs, and at mouse P2X7Rs bearing point mutations. Wild-type rat P2X7R was 10 times more sensitive to ATP and 100 times more sensitive to BzATP than wild-type mouse P2X7R. We found that agonist EC50 values were determined solely by the ectodomain of the P2X7R. Two segments (residues 115-136 and 282-288), when transposed together, converted mouse sensitivities to those of rat. Point mutations through these regions revealed a single residue, asparagine284, in the rat P2X7R that fully accounted for the 10-fold difference in ATP sensitivity, whereas the 100-fold difference in BzATP sensitivity required the transfer of both Lys127 and Asn284 from rat to mouse. Thus, single amino acid differences between species can account for large changes in agonist effectiveness and differentiate between the two widely used agonists at P2X7 receptors.[2]

---

Previous studies have demonstrated that activation of P2X7 receptors (P2X7R) results in the proliferation and migration of some types of tumor. Here, we asked whether and how the activated P2X7R contribute to proliferation and migration of human glioma cells. Results showed that the number of P2X7R positive cells was increasing with grade of tumor. In U87 and U251 human glioma cell lines, both expressed P2X7R and the expression was enhanced by 3'-O-(4-benzoylbenzoyl) ATP (BzATP), the agonist of P2X7R, and siRNA. Our results also showed that 10 μM BzATP was sufficient to induce the proliferation of glioma cell significantly, while the cell proliferation reached the peak with 100 μM BzATP. Also, the migration of U87 and U251 cells was significantly increased upon BzATP treatment. However, the number of apoptotic cells of U87 and U251 was not significantly changed by BzATP. In addition, the expression of ERK, p-ERK, and proliferating cell nuclear antigen (PCNA) protein was increased in BzATP-treated U87 and U251 glioma cells. PD98059, an inhibitor of the MEK/ERK pathway, blocked the increased proliferation and migration of glioma cells activated by BzATP. These results suggest that ERK pathway is involved in the proliferation and migration of glioma cells induced by P2X7R activation.[3]

C24H24N5O15P3.C18H45N3

1018.97

White to off-white solid powder

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, 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)

H2O :~50 mg/mL (~49.07 mM)

Solubility in Formulation 1: 100 mg/mL (98.14 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

---

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