Regulation of death and survival in astrocytes by ADP activating P2Y1 and P2Y12 receptors

ADP is the endogenous agonist for both P2Y(1) and P2Y(12) receptors, which are important therapeutic targets. It was previously demonstrated that ADP and a synthetic agonist, 2-methylthioadenosine 5'-diphosphate (2MeSADP), can induce apoptosis by activating the human P2Y(1) receptor heterologously expressed in astrocytoma cells. However, it was not known whether the P2Y(12) receptor behaved similarly. We demonstrated here that, unlike with the G(q)-coupled P2Y(1) receptor, activation of the G(i)-coupled P2Y(12) receptor does not induce apoptosis. Furthermore, activation of the P2Y(12) receptor by either ADP or 2MeSADP significantly attenuates the tumor necrosis factor alpha (TNFalpha)-induced apoptosis in 1321N1 human astrocytoma cells. This protective effect was blocked by the P2Y(12) receptor antagonist 2-methylthioAMP and by inhibitors of phospholipase C (U73122) and protein kinase C (chelerythrin), but not by the P2Y(1) receptor antagonist MRS2179. Toward a greater mechanistic understanding, we showed that hP2Y(12) receptor activation by 10nM 2MeSADP, activates Erk1/2, Akt, and JNK by phosphorylation. However, at a lower protective concentration of 100pM 2MeSADP, activation of the hP2Y(12) receptor involves only phosphorylated Erk1/2, but not Akt or JNK. This activation is hypothesized as the major mechanism for the protective effect induced by P2Y(12) receptor activation. Apyrase did not affect the ability of TNFalpha to induce apoptosis in hP2Y(12)-1321N1 cells, suggesting that the endogenous nucleotides are not involved. These results may have important implications for understanding the signaling cascades that follow activation of P2Y(1) and P2Y(12) receptors and their opposing effects on cell death pathways.     

Identification of a potent inverse agonist at a constitutively active mutant of human P2Y12 receptor

Human platelets express two P2Y receptors: G(q)-coupled P2Y(1), and G(i)-coupled P2Y(12). Both P2Y(1) and P2Y(12) are ADP receptors on human platelets and are essential for ADP-induced platelet aggregation that plays pivotal roles in thrombosis and hemostasis. Numerous constitutively active G protein-coupled receptors have been described in natural or recombinant systems, but in the P2Y receptors, to date, no constitutive activity has been reported. In our effort to identify G protein coupling domains of the human platelet ADP receptor, we constructed a chimeric hemagglutinin-tagged human P2Y(12) receptor with its C terminus replaced by the corresponding part of human P2Y(1) receptor and stably expressed it in Chinese hamster ovary-K1 cells. It is interesting that the chimeric P2Y(12) mutant exhibited a high level of constitutive activity, as evidenced by decreased cAMP levels in the absence of agonists. The constitutive activation of the chimeric P2Y(12) mutant was dramatically inhibited by pertussis toxin, a G(i) inhibitor. The constitutively active P2Y(12) mutant retained normal responses to 2-methylthio-ADP, with an EC(50) of 0.15 +/- 0.04 nM. The constitutively active P2Y(12) mutant caused Akt phosphorylation that was abolished by the addition of pertussis toxin. Pharmacological evaluation of several P2Y(12) antagonists revealed (E)-N-[1-[7-(hexylamino)-5-(propylthio)-3H-1,2,3-triazolo-[4,5-d]-pyrimidin-3-yl]-1,5,6-trideoxy-beta-d-ribo-hept-5-enofuranuronoyl]-l-aspartic acid (AR-C78511) as a potent P2Y(12) inverse agonist and 5'-adenylic acid, N-[2-(methylthio)ethyl]-2-[(3,3,3-trifluoropropyl)thio]-, monoanhydride with (dichloromethylene)bis[phosphonic acid] (AR-C69931MX) as a neutral antagonist. In conclusion, this is the first report of a cell line stably expressing a constitutively active mutant of human platelet P2Y(12) receptor and the identification of potent inverse agonist.     

Synthesis of pyridoxal phosphate derivatives with antagonist activity at the P2Y13 receptor

We have synthesized a series of derivatives of the known P2 receptor antagonist PPADS (pyridoxal-5'-phosphate-6-azo-phenyl-2,4-disulfonate) and examined their ability to inhibit functional activity of the recombinant human P2Y13 nucleotide receptor expressed in 1321N1 human astrocytoma cells co-expressing G(alpha)16 protein (AG32). Analogues of PPADS modified through substitution of the phenylazo ring, including halo and nitro substitution, and 5'-alkyl phosphonate analogues were synthesized and tested. A 6-benzyl-5'-methyl phosphonate analogue was prepared to examine the effect of stable replacement of the azo linkage. The highest antagonistic potency was observed for 6-(3-nitrophenylazo) derivatives of pyridoxal-5'-phosphate. The 2-chloro-5-nitro analogue (MRS 2211) and 4-chloro-3-nitro analogue (MRS 2603) inhibited ADP (100 nM)-induced inositol trisphosphate (IP3) formation with pIC50 values of 5.97 and 6.18, respectively, being 45- and 74-fold more potent than PPADS. The antagonism of MRS 2211 was competitive with a pA2 value of 6.3. MRS2211 and MRS2603 inhibited phospholipase C (PLC) responses to 30 nM 2-methylthio-ADP in human P2Y1 receptor-mediated 1321N1 astrocytoma cells with IC50 values of >10 and 0.245 microM, respectively. Both analogues were inactive (IC50>10 microM) as antagonists of human P2Y12 receptor-mediated PLC responses in 1321N1 astrocytoma cells. Thus, MRS2211 displayed >20-fold selectivity as antagonist of the P2Y13 receptor in comparison to P2Y1 and P2Y12 receptors, while MRS2603 antagonized both P2Y1 and P2Y13 receptors.     

Cloning, pharmacological characterisation and distribution of the rat G-protein-coupled P2Y(13) receptor

The human P2Y(13) receptor is a new receptor characterized by coupling to Gi, responsiveness to adenine di-phospho-nucleotides and blockade by the P2Y antagonist AR-C69931MX. The mouse P2Y(13) ortholog has also been reported. Here we report, for the first time, the cloning of rat P2Y(13) receptor, its pharmacological analysis and tissue distribution. Rat P2Y(13) is 79% and 87% identical to human and mouse P2Y(13) receptors, respectively. Expression of rP2Y(13) receptor in 1321N1 cells induced the appearance of responses to the typical P2Y(13) receptor agonists ADP and 2MeSADP, as detected by stimulation of [(35)S]GTPgammaS binding. Agonist activities were higher in cells transfected with rP2Y(13) receptor in the presence of the Galpha(16) subunit; in all cases agonist effects were abolished by pertussis toxin pre-treatment. At variance from both human and mouse receptors, ADP was more potent than 2MeSADP. Other nucleotides and sugar-nucleotides were ineffective. Both in the absence and presence of Galpha(16), activation of rP2Y(13) receptor by ADP and 2MeSADP was completely inhibited by nM concentrations of AR-C69931MX. In contrast, no inhibition of rP2Y(13) receptor was induced by the selective P2Y(1) receptor antagonist MRS2179. rP2Y(13) receptor showed highest expression levels in spleen, followed by liver and brain (with particularly high levels in cortex and striatum as reported in man), suggesting important roles in the nervous and immune systems. Expression levels comparable to those of the other cloned P2Y receptors were found in primary rat astrocytes, indicating a possible role in reactive astrogliosis. Hence, rat P2Y(13) receptor displays several similarities but also interesting differences with its human and mouse orthologs, that will have to be taken into account when characterizing the pathophysiological roles of this receptor in the rat animal models.     

On the mechanism of plasmin-induced platelet aggregation. Implications of the dual role of granule ADP

Plasmin-induced platelet aggregation has been considered to be a cause of reocclusion after thrombolytic treatment with plasminogen activators. However, little is known regarding the mechanism and regulation of plasmin-induced platelet aggregation. In this study, we demonstrated that plasmin causes the degranulation of platelets, and that ADP released from granules plays a crucial role in the induction of platelet aggregation. This conclusion is supported by results showing that both ADP antagonists and ADPase can inhibit the effect of plasmin on platelets. We also demonstrated that pretreatment of platelets with ADP makes the platelets more sensitive to plasmin, and plasmin-induced platelet aggregation is, therefore, observed at lower concentrations where no aggregation occurs in quiescent platelets. In other words, it is thought that ADP potentiates the plasmin-induced aggregation. The effect of ADP was inhibited by N(6)-[2-(methylthio)-ethyl]-2-(3,3, 3-trifluoropropyl)thio-5'-adenylic acid, monoanhydride with dichloromethylenebisphosphonic acid (AR-C69931), a selective antagonist for the P2T(AC) subtype of P2 receptor, but not by the P2Y1 receptor-selective antagonist adenosine 3'-phosphate 5'-phosphosulfate (A3P5PS). The P2X1 receptor agonist alpha, beta-methylene adenosine 5'-triphosphate (alpha,beta-MeATP) did not mimic the action of ADP. These data indicate that ADP potentiates plasmin-induced platelet aggregation via the P2T(AC) receptor. In addition, epinephrine, a typical G(i) agonist against platelets, could potentiate the plasmin-induced platelet aggregation, suggesting that the signal via the G(i) protein is involved in potentiating the plasmin-induced platelet aggregation, ADP is secreted from platelet granules, and concomitantly works in conjunction with plasmin in a P2T(AC) receptor-mediated manner.     

Molecular cloning of the platelet P2T(AC) ADP receptor: pharmacological comparison with another ADP receptor, the P2Y(1) receptor

Platelet activation plays an essential role in thrombosis. ADP-induced platelet aggregation is mediated by two distinct G protein-coupled ADP receptors, Gq-linked P2Y(1), and Gi-linked P2T(AC), which has not been cloned. The cDNA encoding a novel G protein-coupled receptor, termed HORK3, was isolated. The HORK3 gene and P2Y(1) gene were mapped to chromosome 3q21-q25. HORK3, when transfected in the rat glioma cell subline (C6-15), responded to 2-methylthio-ADP (2MeSADP) (EC(50) = 0.08 nM) and ADP (EC(50) = 42 nM) with inhibition of forskolin-stimulated cAMP accumulation. 2MeSADP (EC(50) = 1.3 nM) and ADP (EC(50) = 18 nM) also induced intracellular calcium mobilization in P2Y(1)-expressing cells. These results show that HORK3 is a Gi/o-coupled receptor and that its natural ligand is ADP. AR-C69931 MX and 2MeSAMP, P2T(AC) antagonists, selectively inhibited 2MeSADP-induced adenylyl cyclase inhibition in HORK3-expressing cells. On the other hand, A3P5PS, a P2Y(1) antagonist, blocked only 2MeSADP-induced calcium mobilization in P2Y(1)-expressing cells. HORK3 mRNA was detected in human platelets and the expression level of HORK3 was equivalent to that of P2Y(1). These observations indicate that HORK3 has the characteristics of the proposed P2T(AC) receptor. We have also determined that [(3)H]2MeSADP binds to cloned HORK3 and P2Y(1). Competition binding experiments revealed a similarity in the rank orders of potency of agonists and the selectivity of antagonists as obtained in the functional assay. These results support the view that P2Y(1) functions as a high-affinity ADP receptor and P2T(AC) as a low-affinity ADP receptor in platelets.     

Differential coupling of the human P2Y(11) receptor to phospholipase C and adenylyl cyclase

1. The human P2Y(11) (hP2Y(11)) receptor was stably expressed in two cell lines, 1321N1 human astrocytoma cells (1321N1-hP2Y(11)) and Chinese hamster ovary cells (CHO-hP2Y(11)), and its coupling to phospholipase C and adenylyl cyclase was assessed. 2. In 1321N1-hP2Y(11) cells, ATP promoted inositol phosphate (IP) accumulation with low microM potency (EC(50)=8.5+/-0.1 microM), whereas it was 15 fold less potent (130+/-10 microM) in evoking cyclic AMP production. 3. In CHO-hP2Y(11) cells, ATP promoted IP accumulation with slightly higher potency (EC(50)=3.6+/-1.3 microM) than in 1321N1-hP2Y(11) cells, but it was still 15 fold less potent in promoting cyclic AMP accumulation (EC(50)=62.4+/-15.6 microM) than for IP accumulation. Comparable differences in potencies for promoting the two second messenger responses were observed with other adenosine nucleotide analogues. 4. In 1321N1-hP2Y(11) and CHO-hP2Y(11) cells, down regulation of PKC by chronic treatment with phorbol ester decreased ATP-promoted cyclic AMP accumulation by 60--80% (P<0.001) with no change in its potency. Likewise, chelation of intracellular Ca(2+) decreased ATP-promoted cyclic AMP accumulation by approximately 45% in 1321N1-hP2Y(11) cells, whereas chelation had no effect on either the efficacy or potency of ATP in CHO-hP2Y(11) cells. 5. We conclude that coupling of hP2Y(11) receptors to adenylyl cyclase in these cell lines is much weaker than coupling to phospholipase C, and that activation of PKC and intracellular Ca(2+) mobilization as consequences of inositol lipid hydrolysis potentiates the capacity of ATP to increase cyclic AMP accumulation in both 1321N1-hP2Y(11) and CHO-hP2Y(11) cells.     

ADP-sensitive purinoceptors induce steroidogenesis via adenylyl cyclase activation in bovine adrenocortical fasciculata cells

1. The role of P2Y receptors in the production of cAMP and the activation of protein kinase A (PKA) was studied with respect to the regulation of the steroidogenesis in primary cultures of bovine adrenocortical fasciculata cells (BAFCs). 2. ADP and ATP stimulated cAMP production with EC(50) values of 23.7+/-6.8 microM and 40.1+/-5.5 microM, respectively. In contrast, the EC(50) of BzATP for cAMP production was 153.0+/-37.4 microM. Adenosine and AMP (0.1-1000 microM) were much less effective than ADP and ATP. 2MeSADP and UTP did not exert detectable effects. ADP (10 and 100 microM) significantly stimulated steroidogenesis; the process was blocked by an adenylyl cyclase inhibitor SQ22536 (100 microM) but not by the P2Y(1) receptor antagonist MRS2179 (100 microM). 3. Real-time imaging of the PKA activity with the dye ARII, which became less fluorescent upon phosphorylation, revealed that ADP (100 microM) immediately activated PKA. These effects could be mimicked by forskolin (100 microM) and were blocked by the PKA inhibitor H89 (50 microM). UTP (100 microM) did not activate PKA. 4. The cytoplasm harvested from morphologically and electrophysiologically identified single BAFCs contained mRNA for P2Y(2) but not for P2Y(1), P2Y(4), P2Y(11) or P2Y(12) receptors, as confirmed by single-cell RT-PCR amplification (50 cycles). 5. These results suggest an expression of an ADP-sensitive G(s)-coupled purinoceptor in BAFCs. We propose that this not yet described type of P2Y receptor might mediate the extracellular purine-activated steroidogenesis via cAMP/PKA-mediated pathways, independently from the pathways involving InsP(3) production and consequent intracellular Ca(2+) increase.     

An arginine/glutamine difference at the juxtaposition of transmembrane domain 6 and the third extracellular loop contributes to the markedly different nucleotide selectivities of human and canine P2Y11 receptors.

The recently cloned canine P2Y11 receptor (cP2Y11) and its human homolog (hP2Y11) were stably expressed in Chinese hamster ovary cells (CHO-K1) and 1321N1 human astrocytoma cells, and their agonist selectivities and coupling efficiencies to phospholipase C and adenylyl cyclase were assessed. Adenosine triphosphate nucleotides were much more potent and efficacious at the hP2Y11 receptor than their corresponding diphosphates in promoting both inositol phosphate and cyclic AMP accumulation. In contrast, adenosine diphosphate nucleotides were considerably more potent at the cP2Y11 receptor than their corresponding triphosphate analogs. The tri- versus diphosphate specificity of the two receptors was further confirmed in studies using Ca(2+) mobilization as a measure of receptor activation under conditions that minimized nucleotide degradation. Moreover, 2-methylthioadenosine-5'-triphosphate and 2-methylthioadenosine-5'-diphosphate were 58- and 75-fold more potent than ATP and ADP, respectively, at the cP2Y11 receptor compared with only 2- to 3-fold more potent at the hP2Y11 receptor. Mutational analysis revealed that the change of Arg-265, which is located at the juxtaposition of transmembrane domain 6 and the third extracellular loop in the hP2Y11 receptor, to glutamine in the cP2Y11 receptor is at least partly responsible for the diphosphate selectivity but not the increased sensitivity to 2-thioether-substituted adenine nucleotides at the canine receptor. These results imply a key role for a positively charged arginine residue in contributing to the recognition of extracellular nucleotides by the P2Y11 receptor and perhaps other P2Y receptors.     

Second messenger cascade specificity and pharmacological selectivity of the human P2Y1-purinoceptor.

1. The coding sequence of the P2Y1-purinoceptor was cloned from a human genomic library. 2. The open reading frame encodes a protein of 373 amino acids that is 83% identical to the previously cloned chick and turkey P2Y1-purinoceptor and is > or = 95% homologous to the recently cloned rat, mouse, and bovine P2Y1-purinoceptors. 3. The human P2Y1-purinoceptor was stably expressed in 1321N1 human astrocytoma cells using a retroviral vector. Although the P2Y1-purinoceptor agonist, 2MeSATP, had no effect on inositol phosphate accumulation in cells infected with the P2Y1-purinoceptor virus. No effect of 2MeSATP on cyclic AMP accumulation was observed in P2Y1-receptor-expressing 1321N1 cells. 4. The pharmacological selectively of 18 purinoceptor agonists was established for the expressed human P2Y1-purinoceptor. 2MeSATp was more potent than ATP but less potent than 2MeSADP. ADP also was more potent than ATP. A similar maximal effect was observed with most agonists tested. However, alpha, beta-MeATP had no effect and 3'-NH2-3'-deoxyATP and A2P4 were partial agonists. The order of potency of agonists for activation of the turkey P2Y1-purinoceptor, also stably expressed in 1321N1 cells, was identical to that observed for the human P2Y1-purinoceptor. 5. C6 glioma cells express a P2Y-purinoceptor that inhibits adenylyl cyclase but does not activate phospholipase C. Expression of the human P2Y1-purinoceptor in C6 cells conferred 2MeSATP-stimulated inositol lipid hydrolysis to these cells. The phospholipase C-activating human P2Y1-purinoceptor could be delineated from the endogenous P2Y-purinoceptor of C6 glioma cells by use of the P2-purinoceptor antagonist, PPADS, which blocks the P2Y1-purinoceptor but does not block the endogenous P2Y-purinoceptor of C6 cells. P2-purinoceptor agonists also exhibited differential selectivities for activation of these two P2Y-purinoceptors.     

Lack of specificity of [35S]‐ATPγS and [35S]‐ADPβS as radioligands for ionotropic and metabotropic P2 receptor binding

Adenosine‐5′‐O‐3‐thio[³⁵S]triphosphate ([³⁵S]‐ATPγS) has been reported to specifically bind several P2X receptor subtypes, including P2X₁, P2X₂, P2X₃, and P2X₄. Similarly, adenosine‐5′‐O‐2‐thio[³⁵S]diphosphate ([³⁵S]‐ADPβS) has been reported to label putative P2Y receptors. To address whether these radioligands selectively label P2 receptors, the functional activity of various P2 ligands was compared with their ability to compete for [³⁵S]‐ATPγS and [³⁵S]‐ADPβS binding to cell membrane preparations from rat brain, HEK293 cells, and to native and P2X₄ transfected 1321N1 astrocytoma cells. [³⁵S]‐ATPγS (0.2 nM) and [³⁵S]‐ADPβS (0.1 nM) displayed a high percentage of specific binding to membranes prepared from 1321N1 human astrocytoma cells, which were found to be devoid of detectable P2X and P2Y functional activity. [³⁵S]‐ATPγS and [³⁵S]‐ADPβS also exhibited equivalent high percentages of specific binding to HEK293 cell membranes, which endogenously express the P2Y₁ and P2Y₂ receptor subtypes, to 1321N1 cells stably transfected with the human P2X₄ receptor, and to rat brain membranes, which have previously been shown to contain both P2X and P2Y receptor subtypes. The potency order of P2 agonists to compete for radioligand binding to these cell membrane preparations was significantly different from the functional rank order potencies determined in HEK293 cells and 1321N1 cells expressing the P2X₄ receptor, as measured by cytosolic calcium flux. These data indicate that [³⁵S]‐ATPγS and [³⁵S]‐ADPβS appear to bind sites that do not correspond to known functional P2 receptor subtypes. The apparent lack of specificity of these radioligands for labeling P2 receptors is similar to that reported for other radiolabeled nucleotides and illustrates the need for caution in interpreting the apparent pharmacology of native P2 receptors on the basis of binding data alone. Drug Dev. Res. 48:84–93, 1999. © 1999 Wiley‐Liss, Inc.     

Agonist binding and Gq-stimulating activities of the purified human P2Y1 receptor

The human P2Y1 receptor (P2Y1-R) was purified after high-level expression from a recombinant baculovirus in Sf9 insect cells. Quantification by protein staining and with a radioligand binding assay using the high-affinity P2Y1-R antagonist [3H]MRS2279 ([3H]2-chloro-N6-methyl-(N)-methanocarba-2'-deoxyadenosine 3',5'-bis-phosphate) indicated a nearly homogenous preparation of receptor protein. Ki values determined in [3H]MRS2279 binding assays for antagonists with the purified P2Y1-R were in good agreement with the Ki and KB values determined for these molecules in membrane binding and activity assays, respectively. Availability of P2Y1-R in purified form allowed direct determination of nucleotide agonist affinities under conditions not compromised by nucleotide metabolism/interconversion, and an order of affinities of 2-methylthio-ADP (2MeSADP) > ADP = 2-methylthioATP = adenosine-5'-O-(3-thio)triphosphate = adenosine-5'-O(2-thiodiphosphate) > ATP was obtained. The signaling activity of the purified P2Y1-R was quantified after reconstitution in proteoliposomes with heterotrimeric G proteins. Steady-state GTP hydrolysis in vesicles reconstituted with P2Y1-R and Galpha(q)beta(1)gamma(2) was stimulated by the addition of either 2MeADP or RGS4 alone and was increased by up to 50-fold in their combined presence. EC50 values of agonists for activation of the purified P2Y1-R were similar to their respective Ki values determined in radioligand binding experiments with the purified receptor. Moreover, ATP exhibited 20-fold higher EC50 and Ki values than did ADP and was a partial agonist relative to ADP and 2MeSADP under conditions in which no metabolism of the nucleotide occurred. Both RGS4 and PLC-beta1 were potent and efficacious GTPase-activating proteins for Galphaq and Galpha11 in P2Y1-R-containing vesicles. These results illustrate that the binding and signaling properties of the human P2Y1-R can be studied with purified proteins under conditions that circumvent the complications that occur in vivo.     

Oxidized ATP (oATP) attenuates proinflammatory signaling via P2 receptor-independent mechanisms

Periodate-oxidized ATP (oATP), which covalently modifies nucleotide-binding proteins, can significantly attenuate proinflammatory signaling. Although the P2X7 nucleotide receptor (P2X7R) is irreversibly antagonized by oATP, it is unclear whether anti-inflammatory actions of oATP are predominantly mediated via its actions on P2X7R. Here, we describe inhibitory effects of oATP on proinflammatory responses in three human cell types that lack expression of P2X7R: human umbilical vein endothelial cells (HUVEC), HEK293 cells, and 1321N1 astrocytes. oATP decreased by 40-70% the secretion of interleukin (IL)-8 stimulated by tumor necrosis factor-alpha (TNF-alpha) in all three cell types, by IL-1beta in HUVEC and 1321N1 cells, and by endotoxin in HUVEC. Attenuation of TNF-alpha-stimulated IL-8 secretion by oATP was similar in wild-type HEK cells or HEK cells stably expressing recombinant P2X7R. oATP also attenuated cytokine-stimulated expression of nuclear factor-kappaB-luciferase reporter genes expressed in HEK or 1321N1 cells, but did not affect the rapid downregulation of IkappaB. oATP had no effect on uridine triphosphate-induced activation of native P2Y2 receptors in HEK cells, but reduced the potency and efficacy of ADP as an agonist of native P2Y1 receptors. However, inhibition of P2Y1 receptors with the specific antagonist MRS2216 did not mimic the effects of oATP on TNF-alpha-stimulated IL-8 secretion. Although 1321N1 astrocytes lack expression of any known P2 receptor subtypes, oATP markedly inhibited ecto-ATPase activity in these cells, resulting in a significant accumulation of extracellular ATP. In summary, oATP can attenuate proinflammatory signaling by mechanisms independent of the expression or activation of known P2 receptor subtypes.     

2,2'-Pyridylisatogen tosylate antagonizes P2Y1 receptor signaling without affecting nucleotide binding

The effect of 2,2'-pyridylisatogen tosylate (PIT) on the human P2Y(1) receptor and on other recombinant P2Y receptors has been studied. We first examined the modulation by PIT of the agonist-induced accumulation of inositol phosphates. PIT blocked 2-methylthio-ADP (2-MeSADP)-induced accumulation of inositol phosphates in 1321N1 astrocytoma cells stably expressing human P2Y(1) receptors in a non-competitive and concentration-dependent manner. The IC(50) for reduction of the maximal agonist effect was 0.14microM. In contrast, MRS2179, a competitive P2Y(1) receptor antagonist, parallel-shifted the agonist concentration-response curve to the right. PIT also concentration-dependently blocked the P2Y(1) receptor signaling induced by the endogenous agonists, ADP and ATP. A simple structural analogue of PIT was synthesized and found to be inactive as a P2Y(1) receptor antagonist, suggesting that the nitroxyl group of PIT is a necessary structural component for P2Y(1) receptor antagonism. We next examined the possible modulation of the binding of the newly available antagonist radioligand for the P2Y(1) receptor, [3H] MRS2279. It was found that PIT (0.01-10microM) did not inhibit [3H] MRS2279 binding to the human P2Y(1) receptor. PIT (10microM) had no effect on the competition for [3H] MRS2279 binding by agonists, ADP and ATP, suggesting that its antagonism of the P2Y(1) receptor may be allosteric. PIT had no significant effect on agonist activation of other P2Y receptors, including P2Y(2), P2Y(4), P2Y(6), P2Y(11) and P2Y(12) receptors. Thus, PIT selectively and non-competitively blocked P2Y(1) receptor signaling without affecting nucleotide binding.     

Tumor necrosis factor alpha-induced apoptosis in astrocytes is prevented by the activation of P2Y6, but not P2Y4 nucleotide receptors

The physiological role of the uracil nucleotide-preferring P2Y(6) and P2Y(4) receptors is still unclear, although they are widely distributed in various tissues. In an effort to identify their biological functions, we found that activation by UDP of the rat P2Y(6) receptor expressed in 1321N1 human astrocytes significantly reduced cell death induced by tumor necrosis factor alpha (TNF alpha). This effect of UDP was not observed in non-transfected 1321N1 cells. Activation of the human P2Y(4) receptor expressed in 1321N1 cells by UTP did not elicit this protective effect, although both receptors were coupled to phospholipase C. The activation of P2Y(6) receptors prevented the activation of both caspase-3 and caspase-8 resulting from TNF alpha exposure. Even a brief (10-min) incubation with UDP protected the cells against TNF alpha-induced apoptosis. Interestingly, UDP did not protect the P2Y(6)-1321N1 cells from death induced by other methods, i.e. oxidative stress induced by hydrogen peroxide and chemical ischemia. Therefore, it is suggested that P2Y(6) receptors interact rapidly with the TNF alpha-related intracellular signals to prevent apoptotic cell death. This is the first study to describe the cellular protective role of P2Y(6) nucleotide receptor activation.     

Cloned and transfected P2Y4 receptors: characterization of a suramin and PPADS-insensitive response to UTP.

The P2Y family of receptors are G protein-coupled receptors for ATP, ADP, UTP and UDP. Recently several members of this family have been cloned, including the P2Y4, which is activated by UTP but not by ATP. In the present report, using receptors stably transfected into 1321N1 cells, we show that suramin acts as an antagonist at cloned P2Y1 and (less potently) P2Y2 receptors, but not at the cloned P2Y4 receptor. Furthermore, PPADS (pyridoxal-phosphate-6-azophenyl-2'',4''-disulphonic acid), a potent antagonist at the P2Y1 receptor, is a relatively inneffective antagonist at the cloned P2Y4 receptor. This work moves us closer to the goal of classifying the native P2Y receptors on the basis of agonist and antagonist profiles.     

Regulation of P2Y1 receptor-mediated signaling by the ectonucleoside triphosphate diphosphohydrolase isozymes NTPDase1 and NTPDase2

Ectonucleoside triphosphate diphosphohydrolases (NTPDases) control the concentration of released extracellular nucleotides, but the precise physiological roles played by these isozymes in modulation of P2 receptor signaling remain unclear. Activation of the human P2Y(1) receptor was studied in the presence of NTPDase1 or NTPDase2 expressed either in the same cell as the receptor or in P2Y(1) receptor-expressing cells cocultured with NTPDaseexpressing cells. Coexpression of NTPDase1 with the P2Y(1) receptor resulted in increases in the EC(50) for 2'-methylthioadenosine 5'-diphosphate (2MeSADP; 12-fold), ADP (50-fold), and ATP (10-fold) for activation of phospholipase C. Similar effects were observed when the P2Y(1) receptor and NTPDase1 were expressed on different cells. These results are explained by the capacity of NTPDase1 to hydrolyze both nucleoside triphosphates and diphosphates. NTPDase2 preferentially hydrolyzes nucleoside triphosphates, and the presence of NTPDase2 under either coexpression or coculture conditions did not change the EC(50) of 2MeSADP, ADP, or adenosine 5'-O-(2-thiodiphosphate) for activation of the P2Y(1) receptor. However, the EC(50) for ATP was 15-fold lower in the presence of NTPDase2 than in cells expressing the P2Y(1) receptor alone. Whereas expression of NTPDase1 decreased basal activity of the P2Y(1) receptor, the presence of the NTPDase2 resulted in P2Y(1) receptor-dependent increases in basal activity. These results suggest that basal activity of the P2Y(1) receptor is maintained by paracrine or autocrine release of receptor agonists and that the biological and/or pharmacological response mediated by P2Y receptors in target tissues is highly dependent on the types of ectonucleotidases expressed in the vicinity of the receptor.     

Pharmacological characterization of the human P2Y11 receptor

1 The human P2Y11 receptor is coupled to both the phosphoinositide and the cyclic AMP pathways. A pharmacological characterization of the recombinant human P2Y11 receptor has been conducted following stable expression in two different cell lines: the 1321N1 astrocytoma cells for inositol trisphosphate measurements and the CHO-K1 cells for cyclic AMP assays. The rank order of potency of a series of nucleotides was almost identical for the two pathways: ATPgammaS approximately BzATP > dATP > ATP > ADPbetaS > 2MeSATP. 2 ADPbetaS, AMPalphaS and A3P5PS behaved as partial agonists of the human P2Y11 receptor. At high concentrations, these three nucleotides were able to partially inhibit the ATP response. 3 Suramin was a more potent antagonist than reactive blue 2, whereas pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid was completely inactive. The P2Y11 receptor proved to be sensitive to suramin in a competitive way with an apparent Ki value of 0.82+/-0. 07 microM. 4 The ATP derivative AR-C67085 (2-propylthio-beta, gamma-dichloromethylene-D-ATP), a potent inhibitor of ADP-induced platelet aggregation, was the most potent agonist of the P2Y11 receptor, among the various nucleotides tested. 5 The pharmacological profile of the recombinant human P2Y11 receptor is closely similar to that of the cyclic AMP-coupled P2 receptor recently described in HL-60 cells, suggesting that it is the same receptor.     

2-Chloro N(6)-methyl-(N)-methanocarba-2'-deoxyadenosine-3',5'-bisphosphate is a selective high affinity P2Y(1) receptor antagonist

1. We reported previously that bisphosphate derivatives of adenosine are antagonists of the P2Y(1) receptor and that modification of the ribose in these analogues is tolerated in the P2Y(1) receptor binding pharmacophore. 2. Here we delineate the pharmacological activity of one such non-nucleotide molecule, 2-chloro N(6)-methyl-(N)-methanocarba-2'-deoxyadenosine-3',5'-bisphosphate (MRS2279), in which the ribose is replaced by a cyclopentane ring constrained in the (N)-conformation by a cyclopropane moiety. 3. MRS2279 antagonized 2MeSADP-stimulated inositol phosphate formation in turkey erythrocyte membranes with competitive kinetics (pK(B)=7.75). High affinity competitive antagonism by MRS2279 was also observed at the human P2Y(1) receptor (pK(B)=8.10) stably expressed in 1321N1 human astrocytoma cells. Antagonism was specific for the P2Y(1) receptor since MRS2279 had no effect on activation of the human P2Y(2), P2Y(4), P2Y(6), or P2Y(11) receptors by their cognate agonists. 4. MRS2279 also did not block the capacity of ADP to act through the Gi/adenylyl cyclase linked P2Y receptor of platelets to inhibit cyclic AMP accumulation. 5. In contrast, the P2Y(1) receptor is known to be obligatory in the process of ADP-induced platelet aggregation, and MRS2279 competitively inhibited ADP-promoted platelet aggregation with an apparent affinity (pK(B)=8.05) similar to that observed at the human P2Y(1) receptor heterologously expressed in 1321N1 cells. 6. Taken together these results illustrate selective high affinity antagonism of the P2Y(1) receptor by a non-nucleotide molecule that should prove useful for pharmacological delineation of this receptor in various tissues.