P2T Receptor Research Articles

Challenges in developing P2 purinoceptor-based therapeutics.

Advances in the molecular cloning, expression and functional characterization of the P2 purinoceptor superfamily have provided a wealth of data to support a diverse functional role for ATP and related nucleotides in the regulation of tissue function. As with other receptor superfamilies, it is likely that distinct subtypes of each receptor will subserve discrete functions depending on tissue distribution and disease pathophysiology. At the present time, ATP is being evaluated as an anticancer agent and as an anaesthesia adjunct whereas UTP is studied as a novel treatment for cystic fibrosis. ARL67085 is a potent and selective P2T receptor antagonist that has potential as a novel antithrombotic agent. The key to exploiting the P2 purinoceptor area to enhance understanding of disease aetiology and concurrent therapeutic potential will be to focus efforts on the identification of novel pharmacophores that have potent and selective interactions with the various receptor subtypes as potential new leads. To this end, the use of high-throughput screening in conjunction with combinatorial chemical, conventional chemical and natural product library compound sources will be critical.

Facilitated angioplasty with combined ADP P2T receptor blockade and fibrinolysis for the treatment of acute myocardial infarction: results from the STEP-AMI trial

Facilitated angioplasty with combined ADP P2T receptor blockade and fibrinolysis for the treatment of acute myocardial infarction: results from the STEP-AMI trial

P2 receptors and platelet activation.

Adenosine diphosphate (ADP) plays a crucial role in hemostasis and thrombosis by activating platelets. In platelets, the classical P2T receptor is now resolved into three P2 receptor subtypes: the P2Y1, the P2Y12, and the P2X1 receptors. Both pharmacological and molecular biological approaches have confirmed the role of the P2Y1 and P2Y12 receptors in the ADP-induced platelet fibrinogen receptor activation. The P2Y1 and the P2X1 receptors independently contribute to platelet shape change. Whereas the P2Y12 receptor mediates the potentiation of dense granule release reaction, both the P2Y1 and P2Y12 receptors play an important role in the ADP-induced phospholipase A2 activation. The signaling events downstream of these receptors leading to the physiological effects remain elusive, and they are yet to be delineated.

The C6-2B glioma cell P2Y(AC) receptor is pharmacologically and molecularly identical to the platelet P2Y(12) receptor.

P2Y receptor activation in many cell types leads to phospholipase C activation and accumulation of inositol phosphates, while in blood platelets, C6-2B glioma cells, and in B10 microvascular endothelial cells a P2Y receptor subtype, which couples to inhibition of adenylyl cyclase, historically termed P2Y(AC), (P2T(AC) or P(2T) in platelets) has been identified. Recently, this receptor has been cloned and designated P2Y(12) in keeping with current P2 receptor nomenclature. Three selective P(2T) receptor antagonists, with a range of affinities, inhibited ADP-induced aggregation of washed human or rat platelets, in a concentration-dependent manner, with a rank order of antagonist potency (pIC(50), human: rat) of AR-C78511 (8.5 : 9.1)>AR-C69581 (6.2 : 6.0)>AR-C70300 (5.4 : 5.1). However, these compounds had no effect on ADP-induced platelet shape change. All three antagonists had no significant effect on the ADP-induced inositol phosphate formation in 1321N1 astrocytoma cells stably expressing the P2Y(1) receptor, when used at concentrations that inhibit platelet aggregation. These antagonists also blocked ADP-induced inhibition of adenylyl cyclase in rat platelets and C6-2B cells with identical rank orders of potency and overlapping concentration - response curves. RT - PCR and nucleotide sequence analyses revealed that the C6-2B cells express the P2Y(12) mRNA. These data demonstrate that the P2Y(AC) receptor in C6-2B cells is pharmacologically identical to the P2T(AC) receptor in rat platelets.

Clinical experience with antithrombotic drugs acting on purine receptor pathways

AbstractRupture of atherosclerotic plaques and subsequent thrombus formation in coronary arteries are key events in the progression of coronary artery disease and the pathogenesis of acute coronary syndromes. Platelets play a crucial role in this thrombus formation and the success of aspirin in reducing cardiovascular morbidity and mortality has spurred on the search for antiplatelet agents that have an alternative mechanism of action and may, therefore, add to the therapeutic effects of aspirin. The thienopyridines, ticlopidine and clopidogrel, act via metabolites on the platelet ADP receptor subtype designated P2T, and these agents have proven clinical efficacy, either as alternatives to aspirin or, in prevention of coronary stent thrombosis, in combination with aspirin. Potent P2T receptor antagonists have been developed that act directly on the receptor and have a rapid onset of action. One such antagonist, AR‐C69931MX, is being developed for clinical use. AR‐C69931MX is a potent antagonist of ADP‐induced platelet activation, aggregation, and secretion and also antagonises platelet responses to all other agonists in view of the central role of the P2T receptor in amplifying platelet responses. Phase II studies of intravenous AR‐C69931MX in patients with acute coronary syndromes show that this agent has a rapid onset of action, rapidly achieving steady‐state inhibition of platelet aggregation, with a half‐life of only a few minutes. AR‐C69931MX appears to be safe and well tolerated as adjunctive therapy in these patients, and more effective inhibition of platelet function is achieved than with the thienopyridine clopidogrel. Orally active antagonists are also being developed that may be more effective than the thienopyridines. Drug Dev. Res. 52:202–212, 2001. © 2001 Wiley‐Liss, Inc.

Antiplatelet action of R-99224, an active metabolite of a novel thienopyridine-type G(i)-linked P2T antagonist, CS-747.

1. CS-747 is a novel thienopyridine-type platelet ADP inhibitor which lacks in vitro activity. This study examined pharmacological profiles of R-99224, a hepatic metabolite of CS-747. 2. R-99224 produced a concentration-dependent inhibition of in vitro platelet aggregation in washed human platelets (0.03 - 1 microg ml(-1)), which was relatively specific to ADP compared to collagen and thrombin. 3. R-99224 (0.1 - 3 microg ml(-1)) also elicited a similar inhibition of ADP-induced aggregation in rat platelets. The inhibition by R-99224 (10 microg ml(-1)) persisted even after platelets were washed three times. Intravenous injection of R-99224 (0.1 - 3 mg kg(-1)) to rats resulted in a dose-dependent inhibition of ex vivo ADP-induced platelet aggregation. 4. R-99224 (0.1 - 100 microM) decreased binding of [(3)H]-2-methylthio-ADP ([(3)H]-2-MeS-ADP), a stable ligand for platelet ADP receptors, to washed human platelets. The inhibition by R-99224 reached a plateau at a concentration of 3 microM (1.4 microg ml(-1)), but complete inhibition was not achieved even at the highest concentration used (100 microM). 5. R-99224 (10 microM) in combination with ARL-66096 (0.3 microM), an ATP analogue-type G(i)-linked P2T receptor antagonist, produced no additional inhibition of [(3)H]-2-MeS-ADP binding. In contrast, [(3)H]-2-MeS-ADP binding was completely abolished by R-99224 (10 microM) in combination with A3P5PS (300 microM), a selective P2Y(1) antagonist, suggesting that R-99224 selectively binds to the G(i)-linked P2T receptor. 6. R-99224 (0.01 - 3 microg ml(-1)) inhibited ADP-induced [(125)I]-fibrinogen binding to human platelets in a concentration-dependent manner. R-99224 (0.1 - 1 microg ml(-1)) also inhibited the ADP-induced decrease in cyclic AMP levels in PGE(1)-stimulated platelets, whereas the agent did not affect ADP (10 microM)-induced Ca(2+) mobilization. 7. These findings suggest that R-99224 is a selective and irreversible antagonist of G(i)-linked P2T receptors and that R-99224 is a responsible molecule for in vivo actions of CS-747.

Stromal cell–derived factor-1 and macrophage-derived chemokine: 2 chemokines that activate platelets

Platelets play roles in both thrombosis and inflammation, and chemokines that are released at sites of inflammation could potentially activate platelets. Among the chemokine receptors expressed on platelets, the CXCR4 is the receptor for chemokine stromal cell-derived factor-1 (SDF-1), and the CCR4 is the receptor for macrophage-derived chemokine (MDC). Of the chemokines tested, SDF-1 and MDC were the only 2 that activated platelets. Both are weak agonists, but they enhanced response to low-dose adenosine 5'-diphosphate (ADP), epinephrine, or serotonin. When SDF-1 and MDC were added together, full and brisk platelet aggregation occurred. Platelet activation by these 2 chemokines appears to involve distinct pathways: SDF-1 inhibited an increase in cyclic adenosine monophosphate (cAMP) following prostaglandin (PG) I(2), while MDC had no effect. In contrast, MDC, but not SDF-1, lead to Ca(++) mobilization by platelets. Further, second-wave aggregation induced by MDC in platelet-rich plasma was inhibited by aspirin, ADP scavenger creatine phosphate/creative phosphokinase (CP/CPK), and ARL-66096, an antagonist of the ADP P2T(AC) receptor involved in adenylyl cyclase inhibition. But the aggregation was not affected by A3P5PS, an inhibitor of the ADP P2Y receptor. SDF-1-induced aggregation was inhibited by aspirin, but it was only slightly affected by CP/CPK, ARL-66096, or A3P5PS. Finally, the presence of chemokines in platelets was determined. Reverse transcriptase-polymerase chain reaction studies with platelet RNA did not detect the presence of SDF-1 or MDC. In summary, SDF-1 and MDC are platelet agonists that activate distinct intracellular pathways. Their importance in the development of thrombosis at sites of inflammation needs to be further evaluated.

The in vivo pharmacological profile of CS-747, a novel antiplatelet agent with platelet ADP receptor antagonist properties.

1. CS-747 is a novel antiplatelet agent that generates an active metabolite, R-99224, in vivo. CS-747 itself was totally inactive in vitro. This study examined in vivo pharmacological profiles of CS-747 after single oral administration to rats. 2. Orally administered CS-747 (0.3 - 10 mg kg(-1)) partially but significantly decreased [(3)H]-2-methylthio-ADP binding to rat platelets. CS-747 (3 mg kg(-1), p.o.) treatment neutralized ADP-induced decreases of cyclic AMP concentrations induced by prostaglandin E(1), suggesting that metabolites of CS-747 interfere with G(i)-linked P2T receptor. 3. CS-747 (0.3 and 3 mg kg(-1), p.o.) markedly inhibited ex vivo washed platelet aggregation in response to ADP but not to thrombin. CS-747 also exhibited a marked inhibition of ADP-induced ex vivo platelet aggregation in PRP with a rapid onset (<0.5 h) and long duration (>3 days) of action (ED(50) at 4 h=1.2 mg kg(-1)). 4. R-99224 (IC(50)=45 microM) inhibited in vitro PRP aggregation in a concentration-related manner. 5. CS-747 prevented thrombus formation in a dose-related manner with an ED(50) value of 0.68 mg kg(-1). CS-747 was more potent than clopidogrel (6.2 mg kg(-1)) and ticlopidine (>300 mg kg(-1)). 6. CS-747, clopidogrel, and ticlopidine prolonged the bleeding time. The order of potency of these agents in this activity was the same as that in antiaggregatory and antithrombotic activities. 7. These findings indicate that CS-747 is an orally active and a potent antiplatelet and antithrombotic agent with a rapid onset and long duration of action, and warrants clinical evaluations of the agent.

ADP can induce aggregation of human platelets via both P2Y(1) and P(2T) receptors.

1. In the present study we have investigated the roles of P2Y(1) and P(2T) receptor subtypes in adenosine 5'-diphosphate (ADP)-induced aggregation of human platelets in heparinized platelet rich plasma. 2. The response to ADP can be characterized as the initial rate or the maximum or final extent of aggregation. The response profile is determined by the concentration of ADP used, being transient at lower and sustained at higher concentrations. 3. The P2Y(1) receptor antagonist, adenosine-3'-phosphate-5'-phosphate (A3P5P) competitively antagonized the initial rate of aggregation (pK(B) 5. 47) and transformed the response profile to a slowly developing but sustained response. Both maximum and final extents were also inhibited by A3P5P although not in a competitive manner (Schild slope <1). 4. The P(2T) receptor antagonist, AR-C67085, competitively antagonized the final extent of aggregation (pK(B) 8.54), transforming the response profile to one of rapid, transient aggregation. Its effect on maximum extent (the most widely used index of aggregation) was complex, and further supported the involvement of both receptor subtypes in the aggregation response. 5. ADP-induced aggregation is a complex phenomenon, the nature of which is determined by the relative occupancy of the two receptor subtypes. While P2Y(1) receptor activation causes a rapid and transient aggregation, the extent of sustained aggregation is determined by the level of P(2T) receptor occupancy. Hence, detailed analysis of the aggregation response is essential to correctly define the purinergic pharmacology of the platelet and interpretation of results is critically dependent on the response index chosen.

Molecular Mechanism of Thromboxane A2-induced Platelet Aggregation: ESSENTIAL ROLE FOR P2TAC and α2ARECEPTORS

Thromboxane A(2) is a positive feedback lipid mediator produced following platelet activation. The G(q)-coupled thromboxane A(2) receptor subtype, TPalpha, and G(i)-coupled TPbeta subtype have been shown in human platelets. ADP-induced platelet aggregation requires concomitant signaling from two P2 receptor subtypes, P2Y1 and P2T(AC), coupled to G(q) and G(i), respectively. We investigated whether the stable thromboxane A(2) mimetic, (15S)-hydroxy-9, 11-epoxymethanoprosta-5Z,13E-dienoic acid (U46619), also causes platelet aggregation by concomitant signaling through G(q) and G(i), through co-activation of TPalpha and TPbeta receptor subtypes. Here we report that secretion blockade with Ro 31-8220, a protein kinase C inhibitor, completely inhibited U46619-induced, but not ADP- or thrombin-induced, platelet aggregation. Ro 31-8220 had no effect on U46619-induced intracellular calcium mobilization or platelet shape change. Furthermore, U46619-induced intracellular calcium mobilization and shape change were unaffected by A3P5P, a P2Y1 receptor-selective antagonist, and/or cyproheptadine, a 5-hydroxytryptamine subtype 2A receptor antagonist. Either Ro 31-8220 or AR-C66096, a P2T(AC) receptor selective antagonist, abolished U46619-induced inhibition of adenylyl cyclase. In addition, AR-C66096 drastically inhibited U46619-mediated platelet aggregation, which was further inhibited by yohimbine, an alpha(2A)-adrenergic receptor antagonist. Furthermore, inhibition of U46619-induced platelet aggregation by Ro 31-8220 was relieved by activation of the G(i) pathway by selective activation of either the P2T(AC) receptor or the alpha(2A)-adrenergic receptor. We conclude that whereas thromboxane A(2) causes intracellular calcium mobilization and shape change independently, thromboxane A(2)-induced inhibition of adenylyl cyclase and platelet aggregation depends exclusively upon secretion of other agonists that stimulate G(i)-coupled receptors.

Antagonists of the platelet P2T receptor: a novel approach to antithrombotic therapy.

The platelet P2T receptor plays a major role in platelet aggregation, and its antagonists are predicted to have significant therapeutic potential as antithrombotic agents. We have explored analogues of adenosine triphosphate (ATP), which is a weak, nonselective but competitive P2T receptor antagonist. Modification of the polyphosphate side chain to prevent breakdown to the agonist adenosine diphosphate (ADP) and substitution of the adenine moiety to enhance affinity and selectivity for the P2T receptor led to the identification of 10e (AR-C67085MX), having an IC50 of 2.5 nM against ADP-induced aggregation of human platelets. Compound 10e was the first very potent antagonist of the P2T receptor, with a selectivity for that subtype of the P2 receptor family of >1000-fold. Further modification of the structure produced compound 10l (AR-C69931MX) having an IC50 of 0.4 nM. In vivo, at maximally effective antithrombotic doses, there is little prolongation of bleeding time (1.4-fold), which is in marked contrast to the 5-6-fold found with GPIIb/IIIa antagonists.

Chapter 3 Molecular biology of P2Y receptors expressed in the nervous system

Since the cloning of the first two members of the P2Y receptor family—the P2Y 1 and P2Y 2 receptors—a number of related sequences have been isolated from species ranging from Xenopus laevis to Homo sapiens . On the basis of sequence analysis alone 12 subtypes of P2Y receptor have been proposed across these species boundaries. This family of receptors can be divided into four subtypes: the adenine nucleotide specific receptors, P2Y 1 and P2Y 11 ; the uridine nucleotide preferring receptors, P2Y 3 and P2Y 6 ; those at which both UTP and ATP are highly active, P2Y 2 and P2Y 4; and finally those which are activated by all triphosphate nucleotides, xlp2y and tp2y. At least two further subtypes of P2Y receptor have yet to be identified at the molecular level. The first of these has long been known as the “P2T receptor,” most extensively characterized on platelets. The second is the “P2D” receptor activated by diadenosine polyphosphates.

P2 receptor subtypes in the cardiovascular system.

Extracellular nucleotides have been implicated in a number of physiological functions. Nucleotides act on cell-surface receptors known as P2 receptors, of which several subtypes have been cloned. Both ATP and ADP are stored in platelets and are released upon platelet activation. Furthermore, nucleotides are also released from damaged or broken cells. Thus during vascular injury nucleotides play an important role in haemostasis through activation of platelets, modulation of vascular tone, recruitment of neutrophils and monocytes to the site of injury, and facilitation of adhesion of leucocytes to the endothelium. Nucleotides also moderate these functions by generating nitric oxide and prostaglandin I2 through activation of endothelial cells, and by activating different receptor subtypes on vascular smooth muscle cells. In the heart, P2 receptors regulate contractility through modulation of L-type Ca2+ channels, although the molecular mechanisms involved are still under investigation. Classical pharmacological studies have identified several P2 receptor subtypes in the cardiovascular system. Molecular pharmacological studies have clarified the nature of some of these receptors, but have complicated the picture with others. In platelets, the classical P2T receptor has now been resolved into three P2 receptor subtypes: the P2Y1, P2X1 and P2TAC receptors (the last of these, which is coupled to the inhibition of adenylate cyclase, is yet to be cloned). In peripheral blood leucocytes, endothelial cells, vascular smooth muscle cells and cardiomyocytes, the effects of classical P2X, P2Y and P2U receptors have been found to be mediated by more than one P2 receptor subtype. However, the exact functions of these multiple receptor subtypes remain to be understood, as P2-receptor-selective agonists and antagonists are still under development.

Molecular physiology of platelet ADP receptors

Extracellular nucleotides have been implicated in a number of physiologic functions. Nucleotides act on cell surface receptors known as P2 receptors of which several subtypes have been cloned. Both ATP and ADP are stored in platelets and are released upon platelet activation. During vascular injury, nucleotides play an important role in hemostasis by activating platelets. Classic pharmacologic studies have identified ADP receptors on platelets, designated P2T receptor. The P2T receptor is now resolved into three P2 receptor subtypes, the P2Y 1 , the P2X 1 , and the P2T AC receptor, which remains to be cloned. Although the P2Y 1 receptor solely mediates ADP-induced platelet shape change, both the P2Y 1 and P2T AC receptors are essential for ADP-induced fibrinogen receptor activation on platelets. The function of the P2X 1 receptor remains to be elucidated. Thus the complexities in the functional characterization of platelet ADP receptors now appear to be resolved.

The P2Y1 Receptor Is Necessary for Adenosine 5′-Diphosphate–Induced Platelet Aggregation

The human P2Y1 receptor heterologously expressed in Jurkat cells behaves as a specific adenosine 5'-diphosphate (ADP) receptor at which purified adenosine triphosphate (ATP) is an ineffective agonist, but competitively antagonizes the action of ADP. This receptor is thus a good candidate to be the elusive platelet P2T receptor for ADP. In the present work, we examined the effects on ADP-induced platelet responses of two selective and competitive P2Y1 antagonists, adenosine-2'-phosphate-5'-phosphate (A2P5P) and adenosine-3'-phosphate-5'-phosphate (A3P5P). Results were compared with those for the native P2Y1 receptor expressed on the B10 clone of rat brain capillary endothelial cells (BCEC) and for the cloned human P2Y1 receptor expressed on Jurkat cells. A2P5P and A3P5P inhibited ADP-induced platelet shape change and aggregation (pA2 = 5) and competitively antagonized calcium movements in response to ADP in fura-2-loaded platelets, B10 cells, and P2Y1-Jurkat cells. In contrast, these compounds had no effect on ADP-induced inhibition of adenylyl cyclase in platelets or B10 cells, whereas known antagonists of platelet activation by ADP such as Sp-ATPalphaS were effective. These identical signaling responses and pharmacologic properties suggest that platelets and BCEC share a common P2Y1 receptor involved in ADP-induced aggregation and vasodilation, respectively. This P2Y1 receptor coupled to the mobilization of intracellular calcium stores was found to be necessary to trigger ADP-induced platelet aggregation. The present results, together with data from the literature, also point to the existence of another as yet unidentified ADP receptor, coupled to adenylyl cyclase and responsible for completion of the aggregation response. Thus, the term, P2T, should no longer be used to designate a specific molecular entity.

Molecular Basis for ADP-induced Platelet Activation: I. EVIDENCE FOR THREE DISTINCT ADP RECEPTORS ON HUMAN PLATELETS

Acting through cell surface receptors, ADP activates platelets resulting in shape change, aggregation, thromboxane A2 production, and release of granule contents. ADP also causes a number of intracellular events including inhibition of adenylyl cyclase, mobilization of calcium from intracellular stores, and rapid calcium influx in platelets. However, the receptors that transduce these events remain unidentified and their molecular mechanisms of action have not been elucidated. The receptor responsible for the actions of ADP on platelets has been designated the P2T receptor. In this study we have used ARL 66096, a potent antagonist of ADP-induced platelet aggregation, and a P2X ionotropic receptor agonist, alpha,beta-methylene adenosine 5'-triphosphate, to distinguish the ADP-induced intracellular events. ARL 66096 blocked ADP-induced inhibition of adenylyl cyclase, but did not affect ADP-mediated intracellular calcium increases or shape change. Both ADP and 2-methylthio-ADP caused a 3-fold increase in the level of inositol 1,4,5-trisphosphate over control levels which peaked in a similar fashion to the Ca2+ transient. The increase in inositol 1,3,4-trisphosphate was of similar magnitude to that of inositol 1,4,5-trisphosphate. alpha,beta-Methylene adenosine 5'-triphosphate did not cause an increase in either of the inositol trisphosphates. These results clearly demonstrate the presence of two distinct platelet ADP receptors in addition to the P2X receptor: one coupled to adenylyl cyclase and the other coupled to mobilization of calcium from intracellular stores through inositol trisphosphates.

The P2Y 1 receptor is an ADP receptor antagonized by ATP and expressed in platelets and megakaryoblastic cells

The human P2Y 1 purinoceptor has been expressed in Jurkat cells and the effects of HPLC purified nucleotides on calcium movements were measured. The most potent agonist was 2-methylthio-ADP followed by ADP. ATP, Sp-ATPαS and β,γ-methylene-ATP were competitive antagonists. Suramin and PPADS inhibited the effects of ADP. This pharmacological profile is the same as that of the so-called P2T purinoceptor responsible for platelet aggregation, which has not yet been identified. Using PCR we found the P2Y 1 receptor to be present in blood platelets and megakaryoblastic cell lines. These data suggest that the P2Y 1 receptor may be the elusive P2T receptor. © 1997 Federation of European Biochemical Societies.

Adenine dinucleotides: a novel class of signalling molecules.

1. Adenine dinucleotides (Ap3A, Ap4A, Ap5A, Ap6A) are stored in secretory granules of thrombocytes, chromaffin cells and neuronal cells. After release into the extracellular space, the dinucleotides exhibit divergent biological effects on a variety of target cells and organs. The dinucleotides are metabolized by soluble enzymes in the blood plasma as well as by membrane-bound ectoenzymes of endothelial cells, smooth muscle cells, and other cell types. 2. The enzymatic cleavage of the dinucleotides plays a dual role for their biological function: (a) termination of the signal; and (b) generation of purinergically active products such as ATP, ADP and finally adenosine. In contrast to ATP the dinucleotides are long-lived purine nucleotides in the blood. 3. The potential role of the dinucleotides as signalling molecules has been demonstrated in several systems. The adenosine polyphosphates have autocrine function for thrombocytes. Ap3A at low concentration reversibly activates isolated platelets. The mechanism of activation has been elucidated by showing a continuous cleavage of Ap3A, leading to the formation of ADP which is a known agonist of the P2T receptor on thrombocytes. Ap4A and other dinucleotides act as antagonists and inhibit platelet activation. 4. The vasotone of perfused isolated arteries as well as of resistance vessels in the beating heart is differentially influenced by adenine dinucleotides. While Ap3A and Ap4A exhibit relaxing effects at micromolar concentrations, Ap5A and Ap6A elicit vasoconstriction in these vessels. 5. In rat kidney mesangial cells adenine dinucleotides efficiently promote growth. Stimulation of DNA synthesis by various growth factors is enhanced synergistically. ApnA significantly increase the expression of the early growth response gene Egr-1. 6. The specificity and, in some tissues, the uniqueness of effects evoked by dinucleotides may be mediated by genuine dinucleotide receptors (P4) or by specialized P2 receptors (P2D).

P2 purinergic receptors potentiate parathyroid hormone receptor-mediated increases in intracellular calcium and inositol trisphosphate in UMR-106 rat osteoblasts.

The PTH receptor has been cloned and shown to activate both adenylate cyclase and phospholipase C. Evidence exists that both signaling pathways are important for mediating the net physiological effects of this hormone on bone remodeling. We have shown previously that UMR-106 osteoblastic sarcoma cells express two calcium-signaling P2 purinergic receptors, a P2U and a unique P2T receptor. Neither receptor modulates PTH receptor-mediated activation of adenylate cyclase. We now report that stimulation of either P2 receptor will, however, potentiate the magnitude of the calcium signal observed after subsequent addition of human (h) PTH-(1-34) to fluo-3-loaded UMR-106 cells. Results from experiments with staurosporine and phorbol 12-myristate 13-acetate argue against a role for protein kinase C as a mediator of this potentiating effect of P2 receptor ligands. The P2 receptor-mediated intracellular calcium elevation itself cannot account for the potentiating mechanism, because addition of ionomycin will not replicate the effect of P2 receptor ligands on hPTH-(1-34) signaling. Addition of EGTA after exposure to P2 ligands does not prevent the potentiation of hPTH-(1-34), indicating that P2 ligands potentiate the release of intracellular calcium after PTH receptor stimulation. Inositol trisphosphate production is potentiated in response to hPTH-(1-34) after first priming [3H]inositol-labeled cells with a P2 agonist. We conclude that UMR-106 cells express PTH receptors that are capable of activating adenylate cyclase, but may be unable to activate phospholipase C until cells receive a signal as a consequence of P2 receptor activation. The nature of the signal is unclear, but appears not to be mediated by either calcium or protein kinase C.

Purinoceptors on blood platelets: further pharmacological and clinical evidence to suggest the presence of two ADP receptors.

Platelet aggregation by ADP plays a major role in the development and extension of arterial thrombosis. The antithrombotic thienopyridine compounds ticlopidine and clopidogrel have proved useful tools to investigate the mechanisms of ADP-induced platelet activation. In essence, although clopidogrel has been shown to completely and selectively block ADP-induced platelet aggregation, G protein activation and inhibition of adenylyl cyclase, this drug does not affect shape change and Ca2+ influx. Binding studies, using the non-hydrolysable ligand [33P]2MeSADP, have shown that human platelets contain about 600 high-affinity binding sites for 2MeSADP (Kd approximately 5 nM). These sites present pharmacological characteristics of a P2T receptor. Clopidogrel treatment reduces the number of sites by 70% on rat platelets (from 1200 to 450) and leaves the residual binding sites resistant to clopidogrel. Moreover, patients with congenital impairment of ADP-induced platelet aggregation but normal shape change display very low levels of [33P]2MeSADP binding sites. The current data thus strongly suggest the presence of two ADP receptors, one responsible for shape change and rapid Ca2+ influx and the other a Gi protein-coupled receptor responsible for Ca2+ mobilization from internal stores, inhibition of adenylyl cyclase and platelet aggregation.