Platelet P2YT receptor

Effects of antagonists of the platelet P2YT receptor on the P2YT-type and P2Y1 receptors in B10 cells [1]
The ability of two compounds, known to act as antagonists at the P2YT receptor of human platelets, to block ADP-induced adenylyl cyclase inhibition in B10 cells was examined. One of the compounds, AR-C66096 or PSFM-ATP, is an ATP derivative that has been shown on human platelets to block selectively ADP-induced aggregation or inhibition of adenylyl cyclase (Humphries et al., 1994; Jin et al., 1998). On B10 cells, we found that this compound was also a potent competitive antagonist of the 2-MeSADP-induced adenylyl cyclase inhibition, with a pKB value of 7.6 (Figure 4a and Table 1). Alone, it had no effect (up to at least 1 μM) on the forskolin-stimulated level of cyclic AMP (data not shown). The antagonist potency, while high, is about 10 fold lower than that reported for its block of ADP-induced inhibition of adenylyl cyclase in human platelets (Table 1), but this could perhaps be accounted for by the species difference.

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ADP and related nucleoside diphosphates exert an additional action on B10 cells, mobilizing intracellular Ca2+ (see Introduction). We also tested the aforementioned antagonist AR-C66096 for ability to prevent those nucleotides from activating that response. The mobilization of intracellular Ca2+ was produced by ADP with an EC50 value of 0.91±0.06 μM (in agreement with that found earlier by Hechler et al., 1998c). This was unchanged in the presence of AR-C66096 at 10 μM concentration (Figure 5). The latter compound also had no action alone on intracellular Ca2+ level (Figure 5). These results are in accord with the attribution of that Ca2+ response in B10 cells to the P2Y1 receptor, detected therein by its mRNA (Webb et al., 1996). It has since been confirmed (J. Simon et al., unpublished data) by use of a specific antibody to the P2Y1 receptor that this protein is present at the cell surface of these B10 cells.

Adenylyl cyclase assay [1]
The confluent cell layers were washed once with serum-free culture medium (with 25 mM HEPES and 5 mM glucose, pH=7.4) at 37°C and then preincubated in medium containing 100 or 200 μM IBMX for 10–15 min at 37°C (to inhibit breakdown of cyclic AMP by phosphodiesterases). The preincubation medium was then aspirated and the incubations were initiated by addition of medium containing forskolin (10 μM, subclone a; 1 μM, subclone b), IBMX and effectors as stated. After 5 min at 37°C the incubations were terminated by aspiration of the medium and acidification to pH 5.5 or below at 4°C, 30 min. After centrifugation (in the plates, for the 96-well series), aliquots were processed for the cyclic AMP determinations, performed for subclone a as described by Carruthers et al. (1999) using competition for the binding of [3H]-cyclic AMP to a binding fragment of protein kinase A. For the parallel analyses on subclone b (6-well plates) at Sophia Antipolis, a cyclic AMP kit was used according to the manufacturer's protocol. The basal level of cyclic AMP, measured in the absence of drugs and forskolin, was 46±2 fmoles cyclic AMP per well (96-well plates; n=9) or 2.3±0.1 pmoles well−1 (n=12) in the bulk case (6-well plate assay). For antagonism studies, either the cells were preincubated with AR-C66096 for 15 min (for full equilibration of that agent) at 37°C in medium containing 200 μM IBMX, or BzATP or A3P5P or A2P5P or purified ATP was added together with the agonist. For pertussis toxin (PTX) treatment, the B10 cells cultured on 96-well plates to near confluency were used. Half of the wells on each plate were pretreated with 100 ng ml−1 pertussis toxin at 37°C for 14–16 h, followed by agonist/10 μM forskolin treatment and cyclic AMP assay as before.

[1]. Activity of adenosine diphosphates and triphosphates on a P2Y(T) -type receptor in brain capillary endothelial cells. Br J Pharmacol. 2001 Jan;132(1):173-82.

1. In the rat brain capillary endothelial cell clonal population (B10), P2Y (nucleotide) receptor activity is associated with inhibition of adenylate cyclase, and its function is similar to that of the platelet ADP receptor P2Y(T) (previously referred to as “P2T”). However, in previous studies analyzing mRNA in B10 cells, the only detectable P2Y receptor was the P2Y(1) receptor, and other studies have shown that the P2Y(1) receptor is not involved in cyclic nucleotide signal transduction. This paper aims to clarify these issues. 2. The inhibitory effect of purified nucleotides on forosclerin-stimulated adenylate cyclase was determined in B10 cells. The EC50 value of 2-methylthioADP (2-MeSADP) was 2.2 nM, and surprisingly, 2-MeSATP was also an almost equally potent agonist (EC50 = 3.5 nM). ATP and 2-ClATP are weak partial agonists (EC50 26 μM and 10 μM, respectively) that can antagonize the activity of 2-MeSADP under appropriate conditions. 3. The known platelet P2Y(T) receptor selective antagonist 2-propylthioadenosine-5'-(β,γ)-difluoromethylene)triphosphonate (AR-C 66096) is a competitive antagonist of the B10 cell receptor with pK(B) = 7.6. This ligand is inactive against the P2Y(1) receptor in the same cell. In contrast, competitive P2Y(1) receptor antagonists, namely 3',5'- and 2',5'-adenosine bis(monophosphate), are weak agonists of the adenylate cyclase inhibitory receptor. 4. Pertussis toxin completely eliminates the inhibitory effect of 2-MeSADP on adenylate cyclase. 5. In summary, these brain endothelial cells possess both P2Y(T) type receptors in addition to P2Y(1) receptors. These two receptors are similar in terms of agonist spectrum, but can be clearly distinguished by antagonists and second messenger activation mechanisms. This article discusses the possible connection between B10 cells and platelet P2Y(T) receptors. [1]

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Researchers previously pointed out (Webb et al., 1996) that another unknown P2Y receptor might be a possible source of adenylate cyclase inhibition in B10 cells. This now appears to be the case, as adenylate cyclase inhibition is sensitive to the platelet P2YT receptor specific antagonist AR-C66096 (Fig. 4a), while AR-C66096 does not affect the activity of P2Y1 receptors in B10 cells (Fig. 5). In addition, bis(monophosphate) P2Y1 antagonists can also completely distinguish between the two receptors (Fig. 3). In platelets, ADP-mediated inhibition of adenylate cyclase is caused by the P2YT receptor (see Introduction). From this, we can conclude that this inhibition in B10 cells is produced by an endothelial cell receptor that is functionally very similar to, or even identical to, the platelet receptor. Therefore, (i) both receptors respond to ADP by inhibiting adenylate cyclase and do not induce intracellular Ca2+ mobilization (as previously stated); (ii) both can be competitively antagonized by AR-C66096 (Daniel et al., 1998 (platelets) and Fig. 4a); (iii) both can also be competitively antagonized by BzATP (Vigne et al., 1999 (platelets) and Fig. 4b); (iv) neither can be blocked by bisphosphate antagonists of the P2Y1 receptor (Fagura et al., 1998; Hechler et al., 1998b; Jin and Kunapuri, 1998 (platelets) and Fig. 3); and (v) both involve a G protein sensitive to pertussis toxin (Ohlmann et al., 1995 (platelets) and Fig. 6), which is different from the P2Y1 receptor. B10 cells, and (vi) neither (again, unlike the P2Y1 receptor) was antagonized by 100 μM PPADS (Geiger et al., 1998 (platelets); Webb et al., 1996 (B10)). Furthermore, in terms of agonist potency against adenylate cyclase inhibition, ADP was 400 to 500 times weaker than 2-MeSADP in both rat platelets (Savi et al., 1994a) and rat B10 cells (Table 1). However, platelet P2YT receptors and similar receptors in B10 cells exhibit different activities against certain adenosine triphosphate derivatives. 2-ClATP, 2-MeSATP, and ATP are clearly agonists of adenylate cyclase inhibition in mouse B10 cells (Fig. 1), but have been shown to be antagonists of ADP-induced adenylate cyclase inhibition (and aggregation) in human platelets (Cusack & Hourani, 1982; Park & Hourani, 1999). There is currently no evidence that species differences could lead to such significant changes. The platelet P2YT receptor in another rodent, the mouse, is identical to the human receptor in terms of ADP response and sensitivity to AR-C66096 (Kim et al., 1999). ADP-mediated adenylate cyclase inhibition is only 4-fold more potent in rat platelets than in human platelets (Savi et al., 1994a) (using pure nucleotides: Geiger et al., 1998). [1]

C14H18F2N5NA4O12P3S

703.261194705963

702.944

C, 27.33; H, 3.60; F, 6.18; N, 11.38; O, 31.20; P, 15.10; S, 5.21

145782-74-7

90488830

Typically exists as solid at room temperature

3

19

10

41

941

4

S(CCC)C1N=C(C2=C(N=1)N(C=N2)[C@H]1[C@@H]([C@@H]([C@@H](COP(=O)([O-])OP(C(F)(F)P(=O)([O-])[O-])(=O)[O-])O1)O)O)N.[Na+].[Na+].[Na+].[Na+]

IETVOLFQZIGNAQ-HVYRMSERSA-J

InChI=1S/C14H22F2N5O12P3S.4Na/c1-2-3-37-13-19-10(17)7-11(20-13)21(5-18-7)12-9(23)8(22)6(32-12)4-31-36(29,30)33-35(27,28)14(15,16)34(24,25)26;;;;/h5-6,8-9,12,22-23H,2-4H2,1H3,(H,27,28)(H,29,30)(H2,17,19,20)(H2,24,25,26);;;;/q;4*+1/p-4/t6-,8-,9-,12-;;;;/m1..../s1

tetrasodium;[[(2R,3S,4R,5R)-5-(6-amino-2-propylsulfanylpurin-9-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-oxidophosphoryl]oxy-[difluoro(phosphonato)methyl]phosphinate

145782-74-7; 2-(Propylthio)adenosine-5'-O-(beta,gamma-difluoromethylene)triphosphatetetrasodiumsalt; tetrasodium;[[(2R,3S,4R,5R)-5-(6-amino-2-propylsulfanylpurin-9-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-oxidophosphoryl]oxy-[difluoro(phosphonato)methyl]phosphinate; 2-propylthio-D-beta,gamma-difluoromethylene ATP;

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.)