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 It has since been confirmed (J. Simon et al., unpublished data) by use of a specific antibody to the P2Y1 receptor that this protein is present at the cell surface of these B10 cells.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. A P2Y (nucleotide) receptor activity in a clonal population (B10) of rat brain capillary endothelial cells is coupled to inhibition of adenylyl cyclase and has functional similarities to the P2Y(T) (previously designated 'P2T') receptor for ADP of blood platelets. However, the only P2Y receptor which was detectable in a previous study of B10 cells by mRNA analysis was the P2Y(1) receptor, which elsewhere shows no transduction via cyclic nucleotides. We have sought here to clarify these issues. 2. The inhibition of forskolin-stimulated adenylyl cyclase induced by purified nucleotides was measured on B10 cells. The EC(50) value for 2-methylthioADP (2-MeSADP) was 2.2 nM and, surprisingly, 2-MeSATP was an almost equally strong agonist (EC(50)=3.5 nM). ATP and 2-ClATP were weak partial agonists (EC(50)=26 microM and 10 microM respectively) and under appropriate conditions could antagonise the activity on 2-MeSADP. 3. A known selective antagonist of the platelet P2Y(T) receptor, 2-propylthioadenosine-5'-(beta,gamma)-difluoromethylene) triphosphonate (AR-C 66096), was a competitive antagonist of this B10 cell receptor, with pK(B)=7.6. That ligand is inactive at the P2Y(1) receptor in the same cells. Conversely, the competitive P2Y(1) receptor antagonists, the 3', 5'- and 2', 5'-adenosine bis-monophosphates, are, instead, weak agonists at the adenylyl cyclase-inhibitory receptor. 4. The inhibition of adenylyl cyclase by 2-MeSADP was completely abolished by pertussis toxin. 5. In summary, these brain endothelial cells possess a P2Y(T)-type receptor in addition to the P2Y(1) receptor. The two have similarities in agonist profiles but are clearly distinguishable by antagonists and by their second messenger activations. The possible relationships between the B10 and platelet P2Y(T) receptors are discussed.[1]

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Researchers had previously noted (Webb et al., 1996) that alternatively a second, unknown P2Y receptor could be a possible origin of the adenylyl cyclase inhibition in B10 cells. That is now seen to be the case, since the adenylyl cyclase inhibition is sensitive to a specific antagonist of the platelet P2YT receptor, AR-C66096 (Figure 4a), which does not affect the activity of the P2Y1 receptor (Figure 5) in the B10 cells. The two receptors are also totally distinguished by the bis-monophosphate P2Y1 antagonists (Figure 3).
In platelets, the ADP-mediated adenylyl cyclase inhibition is due to the P2YT receptor (see Introduction) and we can conclude that this inhibition in the B10 cells is produced by an endothelial cell receptor which is functionally very similar, if not identical, to that platelet receptor. Thus, (i) both of those receptors respond to ADP with adenylyl cyclase inhibition and without intracellular Ca2+ mobilization (as just discussed), (ii) both are competitively antagonized by AR-C66096 (Daniel et al., 1998 (platelets) and Figure 4a) and also (iii) by BzATP (Vigne et al., 1999 (platelets) and Figure 4b), (iv) neither are blocked by the bis-phosphate antagonists of the P2Y1 receptor (Fagura et al., 1998; Hechler et al., 1998b; Jin & Kunapuli, 1998 (platelets) and Figure 3), (v) both involve a G-protein, sensitive to pertussis toxin (Ohlmann et al., 1995 (platelets) and Figure 6), unlike the P2Y1 receptor in the B10 cells, and (vi) both are (again unlike the P2Y1 receptor) not antagonized by 100 μM PPADS (Geiger et al., 1998 (platelets); Webb et al., 1996 (B10)). Further, in agonist potencies for adenylyl cyclase inhibition, ADP is 400 to 500 fold weaker than 2-MeSADP in both the rat platelet (Savi et al., 1994a) and the rat B10 cell (Table 1).
There are, however, differences in the activity of some adenosine triphosphate derivatives between the platelet P2YT receptor and the similar receptor in B10 cells. 2-ClATP, 2-MeSATP and ATP itself are clearly agonists for the adenylyl cyclase inhibition in rat B10 cells (Figure 1), but are well established to be antagonists of the ADP-induced adenylyl cyclase inhibition (as well as aggregation) in human platelets (Cusack & Hourani, 1982; Park & Hourani, 1999). There is no evidence as yet to indicate that the species difference here is likely to cause this major change. The P2YT receptor of platelets of another rodent, the mouse, behaves identically to that of man in its ADP responses and their AR-C66096 sensitivity (Kim et al., 1999). ADP-mediated inhibition of adenylyl cyclase shows only a 4 fold greater potency on the rat platelet (Savi et al., 1994a) than on the human platelet (with pure nucleotide: 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.)