Molecular cloning and expression of an avian G protein-coupled P2Y receptor

A family of G protein-coupled P2Y receptors that are activated by adenine and uridine nucleotides has been identified recently. Degenerate primers based on conserved sequences in these P2Y receptors were used to amplify turkey DNA, which was used to isolate the complete coding sequence of a cDNA that encodes a novel G protein-coupled receptor. Stable expression of this avian cDNA in 1321N1 human astrocytoma cells resulted in the conveyance of marked inositol phosphate responses to various nucleotides. Although this cloned avian receptor exhibited its highest homology to the previously cloned mammalian P2Y4 receptor, its pharmacological selectivity was not consistent with the avian receptor's being a species homologue of the P2Y4 receptor. That is, whereas the P2Y4 receptor is selectively activated by UTP and is not activated by ATP or Ap4A, the novel avian receptor was potently activated by ATP and Ap4A as well as by UTP. Taken together, these results describe the identification of an avian phospholipase C-coupled P2Y receptor that, like the mammalian P2Y2 receptor, is activated by both adenine and uridine nucleotides.     

UTP induces vascular responses in the isolated and perfused canine epicardial coronary artery via UTP-preferring P2Y receptors

1. Vasoconstrictor responses of the isolated and perfused canine epicardial coronary artery to uridine 5'-triphosphate (UTP) were analysed pharmacologically. 2. At basal perfusion pressure, UTP induced vasoconstriction in a dose-related manner and the vasoconstriction was sometimes followed by a slight vasodilatation at large doses (more than 10 nmol). The rank order of potency for vasoconstriction was UTP = UDP > ATP > TTP > or = ITP > UMP. At raised perfusion pressure by 20 mM KCl, the vasoconstriction was not changed and a small vasodilatation was induced at large doses. The rank order of potency for vasodilatation was induced at large doses. The rank order of potency for vasodilatation was ATP > ITP > or = UDP > UTP > or = TTP. The maximal vasodilator response to UTP was much less than that to ATP. UMP did not induce vasodilatation. 3. The P2X receptor agonist and desensitizing agent alpha, beta-methylene ATP (1 microM) and the P2 receptor antagonist suramin (100 microM) inhibited the vasoconstrictor responses to ATP but not those to UTP and UDP. The P2 receptor antagonist reactive blue 2 (30 microM) did not inhibit the vascular responses to UTP. 4. UTP (200 microM) desensitized the vasoconstrictor responses to UTP, but not either the vasodilator responses to UTP or the vasoconstrictor responses to ATP and UDP. UDP (200 microM) did not desensitize the vascular responses to UTP. 5. Preincubating the UDP stock solution and arterial preparation with hexokinase (10 and 1 uml-1, respectively) did not change the vasoconstrictor responses to UDP. 6. The Ca channel blocker diltiazem (1 microM) inhibited the vasoconstrictor responses to UTP but not those to ATP and UDP. Incubation in a Ca(2+)-free solution containing 1 mM EGTA inhibited the vascular responses to ATP, UTP and UDP. 7. Removal of the endothelium by an intraluminal injection of saponin (1 mg) inhibited the vasodilator responses to UTP. Indomethacin, a cyclo-oxygenase inhibitor (1 microM), inhibited the vasodilator responses to UTP, but NG-nitro-L-arginine, a nitric oxide synthase inhibitor (300 microM), did not have an inhibitory effect. 8. The results suggest that (1) UTP induces vasoconstriction via UTP-preferring P2Y receptors on the smooth muscle and vasodilatation via receptors different from those mediating the vasoconstriction induced by UTP and mediating the vasodilatation by ATP on the endothelium, through mainly the release of prostacyclin in the canine epicardial coronary artery; (2) UDP induces vasoconstriction via UDP-preferring P2Y receptors; and (3) L-type Ca ion channels are involved in the vasoconstriction induced by UTP, but not in that induced by UDP.     

Types of purinoceptors and phospholipase A2 involved in the activation of the platelet-activating factor-dependent transacetylase activity and arachidonate release by ATP in endothelial cells

Acyl analogs of PAF are the major products synthesized during agonist stimulation of endothelial cells. We have previously shown that PAF: 1-acyl-2-lyso-sn-glycero-3-phosphocholine transacetylase in calf pulmonary artery endothelial cells is activated by ATP through protein phosphorylation, and the increase in transacetylase activity by ATP contributes to the biosynthesis of acyl analogs of PAF (J. Biol. Chem. 272, 17431-17437, 1997). To understand the mechanisms(s) by which ATP stimulates acyl analogs of PAF production, we have identified the subtypes of the purinergic receptor that are linked to the activation of two enzymes involved in the generation of acyl analogs of PAF, namely, transacetylase and phospholipase A2. Experiments with transient transfection of the cells with antisense and sense thio-oligonucleotide to cytosolic phospholipase A2 (cPLA2) were also performed to evaluate whether downstream activation of cPLA2 is involved in ATP-receptor mediated induction of arachidonate release and synthesis of radylacetyl-GPC. We found that the P2u/P2Y2 receptor, which recognizes a pyrimidine nucleotide, UTP, as well as purine nucleotides, shows a potency profile of UTP > ATP = ATP gamma S > 2-methylthio-ATP in mediating the activation of PAF: lysophospholipid transacetylase. On the other hand, ADP beta S and 2-methylthio-ATP have similar potencies as ATP but have lower potencies than UTP and ATP gamma S in stimulating the release of arachidonate. These results suggest that both P2u/P2Y2 and P2y/P2Y1 receptor subtypes promote arachidonate release. In addition, transient transfection of endothelial cells with cPLA2 antisense but not the sense thio-oligonucleotide inhibited the stimulation of arachidonate release and [3H]acetate incorporation into radyl[3H]acetyl-GPC. Thus, our data suggest that a receptor-mediated process is involved in the activation of transacetylase for the induced synthesis of acyl analogs of PAF in endothelial cells. Furthermore, it is likely that cPLA2 supplies the lysophospholipids as substrates for the transacetylation reaction.     

Selective inhibition of M-type potassium channels in rat sympathetic neurons by uridine nucleotide preferring receptors

1. UTP and UDP depolarize rat superior cervical ganglion neurons and trigger noradrenaline release from these cells. The present study investigated the mechanisms underlying this excitatory action of uridine nucleotides by measuring whole-cell voltage-dependent K+ and Ca2+ currents. 2. Steady-state outward (holding) currents measured in the amphotericin B perforated-patch configuration at a potential of -30 mV were reduced by 10 microM UTP in a reversible manner, but steady-state inward (holding) currents at -70 mV were not affected. This action of UTP was shared by the muscarinic agonist oxotremorine-M. In current-voltage curves between -20 and -100 mV, UTP diminished primarily the outwardly rectifying current components arising at potentials positive to -60 mV. 3. Slow relaxations of muscarinic K+ currents (IM) evoked by hyperpolarizations from -30 to -55 mV were also reduced by 10 microM UTP (37% inhibition) and oxotremorine-M (81% inhibition). In contrast, transient K+-currents, delayed rectifier currents, fast and slow Ca2+-dependent K+ currents, as well as voltage-dependent Ca2+ currents were not altered by UTP. 4. In conventional (open-tip) whole-cell recordings, replacement of GTP in the pipette by GDPbetaS abolished the UTP-induced inhibition of IM, whereas replacement by GTPgammaS rendered it irreversible. 5. The UTP-induced reduction of IM was half maximal at 1.5 microM with a maximum of 37% inhibition; UDP was equipotent and equieffective, while ADP was less potent (half maximal inhibition at 29 microM). ATP had no effect at < or = 30 microM. 6. The inhibition of IM induced by 10 microM UTP was antagonized by pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) at > or = 30 microM and by reactive blue 2 at > or = 10 microM, but not by suramin at concentrations up to 30 microM. 7. These results show that rat superior cervical ganglion neurons possess uridine nucleotide preferring P2Y receptors which inhibit KM channels. This effect presumably forms the basis of the excitatory action of uridine nucleotides in rat sympathetic neurons.     

P2Y2 nucleotide receptors expressed heterologously in sympathetic neurons inhibit both N-type Ca2+ and M-type K+ currents

The P2Y2 receptor is a uridine/adenosine triphosphate (UTP/ATP)-sensitive G-protein-linked nucleotide receptor that previously has been reported to stimulate the phosphoinositide signaling pathway. Messenger RNA for this receptor has been detected in brain tissue. We have investigated the coupling of the molecularly defined rat P2Y2 receptor to neuronal N-type Ca2+ channels and to M-type K+ channels by heterologous expression in rat superior cervical sympathetic (SCG) neurons. After the injection of P2Y2 cRNA, UTP inhibited the currents carried by both types of ion channel. As previously reported [Filippov AK, Webb TE, Barnard EA, Brown DA (1997) Inhibition by heterologously expressed P2Y2 nuerones. Br J Pharmacol 121:849-851], UTP inhibited the Ca2+ current (ICa(N)) by up to 64%, with an IC50 of approximately 0.5 microM. We now find that UTP also inhibited the K+M current (IK(M)) by up to 61%, with an IC50 of approximately 1.5 microM. UTP had no effect on either current in neurons not injected with P2Y2 cRNA. Structure-activity relations for the inhibition of ICa(N) and IK(M) in P2Y2 cRNA-injected neurons were similar, with UTP >/= ATP > ITP > GTP,UDP. However, coupling to these two channels involved different G-proteins: pretreatment with Pertussis toxin (PTX) did not affect UTP-induced inhibition of IK(M) but reduced inhibition of ICa(N) by approximately 60% and abolished the voltage-dependent component of this inhibition. In unclamped neurons, UTP greatly facilitated depolarization-induced action potential discharges. Thus, the single P2Y2 receptor can couple to at least two G-proteins to inhibit both Ca2+N and K+M channels with near-equal facility. This implies that the P2Y2 receptor may induce a broad range of effector responses in the nervous system.     

Metabotropic receptors for ATP and UTP: exploring the correspondence between native and recombinant nucleotide receptors

In the past five years, an extended series (P2Y1-n) of metabotropic nucleotide (P2) receptors has been cloned from vertebrate tissues; these receptors are activated by either ATP or UTP, or both nucleotides. While certain cloned P2Y receptors appear to correspond functionally to particular native P2 receptor phenotypes, such pharmacological phenotypes could be explained by either a combination of several members of the P2Y1-n series being coexpressed in the same tissue or the existence of novel, uncloned P2Y subtypes. Here, Brian King, Andrea Townsend-Nicholson and Geoffrey Burnstock review recent findings on the matter of pharmacological relationships between native P2 and cloned P2Y receptors.     

The regulation of aortic endothelial cells by purines and pyrimidines involves co-existing P2y-purinoceptors and nucleotide receptors linked to phospholipase C

1. We have examined the phospholipase C responses in bovine aortic endothelial cells to purines (ATP, ADP and analogues) and the pyrimidine, uridine triphosphate (UTP). 2. The cells responded to purines in a manner consistent with the presence of P2y purinoceptors; both 2-methylthioadenosine 5'-triphosphate (2MeSATP) and adenosine 5'-0-(2-thiodiphosphate) (ADP beta S) were potent agonists (EC50 0.41 microM and 0.85 microM respectively) while beta, gamma-methylene ATP at 300 microM was not. 3. The cells also responded to UTP. The maximal response to UTP was less than that for either 2MeSATP and ADP beta S while adenosine 5'-0-(3-thiotriphosphate) (ATP gamma S) gave the largest maximal response. 4. The concentration-effect curve to UTP was additive in the presence of either 2MeSATP or ADP beta S. However, the concentration-effect curves to ATP gamma S reached the same maximum in the presence or absence of UTP. 5. Suramin, at concentrations between 10 microM and 100 microM was a competitive antagonist for the response to ADP beta S and 2MeSATP but not the response to UTP. 6. The results show that there are two separate, co-existing, receptor populations: P2y-purinoceptors (responding to purines) and nucleotide receptors (responding to both purines and pyrimidines). We conclude that purines such as ATP/ADP may regulate aortic endothelial cells by interacting with two phospholipase C-linked receptors.     

Purine and pyrimidine nucleotides inhibit a noninactivating K+ current and depolarize adrenal cortical cells through a G protein-coupled receptor

Bovine adrenal zona fasciculata (AZF) cells express a noninactivating K+ current (IAC) that sets the resting membrane potential and may mediate depolarization-dependent cortisol secretion. External ATP stimulates cortisol secretion through activation of a nucleotide receptor. In whole-cell patch clamp recordings from bovine AZF cells, we found that ATP selectively inhibited IAC K+ current by a maximum of 75.7 +/- 3% (n = 13) with a 50% inhibitory concentration of 1.3 microM. A rapidly inactivating A-type K+ current was not inhibited by ATP. Other nucleotides, including ADP and the pyrimidines UTP and UDP, also inhibited IAC, whereas 2-methylthio-ATP (2-MeSATP) and CTP were completely ineffective. The rank order of potency for six nucleotides was UTP = ADP > ATP > UDP > 2-MeSATP = CTP. At maximally effective concentrations, UTP, ADP, and UDP inhibited IAC current by 81.4 +/- 5.2% (n = 7), 70.7 +/- 7.2% (n = 4), and 65.2 +/- 7.9% (n = 5), respectively. Inhibition of IAC by external ATP was reduced from 71. 3 +/- 3.2% to 22.8 +/- 4.5% (n = 18) by substituting guanosine 5'-O-2-(thio) diphosphate for GTP in the patch pipette. Inhibition of IAC by external ATP (10 microM) was markedly suppressed (to 17.3 +/- 5.5%, n = 9) by the nonspecific protein kinase antagonist staurosporine (1 microM) and eliminated by substituting the nonhydrolyzable ATP analog 5-adenylyl-imidodiphosphate or UTP for ATP in the pipette. ATP-mediated inhibition of IAC was not altered by the kinase C antagonist calphostin C, the calmodulin inhibitory peptide, or by buffering the intracellular (pipette) Ca++ with 20 mM 1,2-bis-(2-aminophenoxy)ethane-N, N,N',N'-tetraacetic acid. In current clamp recordings, ATP and UTP (but not CTP) depolarized AZF cells at concentrations that inhibited IAC K+ current. These results demonstrate that bovine AZF cells express a nucleotide receptor with a P2Y3 agonist profile that is coupled to the inhibition of IAC K+ channels through a GTP-binding protein. The inhibition of IAC K+ current and associated membrane depolarization are the first cellular responses demonstrated to be mediated through this receptor. Nucleotide inhibition of IAC proceeds through a pathway that is independent of phospholipase C, but that requires ATP hydrolysis. The identification of a new signaling pathway in AZF cells, whereby activation of a nucleotide receptor is coupled to membrane depolarization through inhibition of a specific K+ channel, suggests a mechanism for ATP-stimulated corticosteroid secretion that depends on depolarization-dependent Ca++ entry. This may be a means of synchronizing the stress-induced secretion of corticosteroids and catecholamines from the adrenal gland.     

Differential regulation of inositol 1,4,5-trisphosphate by co-existing P2Y-purinoceptors and nucleotide receptors on bovine aortic endothelial cells

1. We have examined the inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) responses in bovine aortic endothelial (BAE) cells to purines (ATP, ADP and analogues) and the pyrimidine, uridine triphosphate (UTP). 2. Exchange of medium on BAE cells in the absence of agonist was found to be a stimulus for Ins(1,4,5)P3 generation. BAE cells stimulated with 100 microM ATP, 30 microM 2MeSATP (an agonist at P2Y-purinoceptors but not nucleotide receptors) or 100 microM UTP (an agonist at nucleotide receptors but not P2Y-purinoceptors) gave Ins(1,4,5)P3 responses above that caused by exchange of medium. The time course was rapid, with peak response within the first 5 s and levels returning close to basal after 30 s of stimulation. 3. Significant differences in Ins(1,4,5)P3 responses to 100 microM UTP and 30 microM 2MeSATP stimulation were observed. The response to UTP was reproducibly more sustained than that to 2MeSATP. 4. Stimulation of BAE cells with 100 microM UTP plus 30 microM 2MeSATP produced a response statistically indistinguishable from that predicted by addition of the responses to the two agonists in isolation. 5. The Ins(1,4,5)P3 response to UTP was attenuated to 25% of control by pretreatment of BAE cells with pertussis toxin. Responses to 2MeSATP and ADP were essentially unaffected. ATP stimulation was reduced to 65% of control. 6. Activation of protein kinase C with tetradecanoyl phorbol acetate (TPA) profoundly inhibited Ins(1,4,5)P3 responses to 2MeSATP and ADP but had no effect on UTP stimulation. The protein kinase C inhibitor, Ro 31-8220, enhanced responses to 2MeSATP, ADP and ATP but no effect was observed on UTP stimulation. 7. These observations show that nucleotide and P2Y-receptors mobilise the second messenger Ins(1,4,5)P3 by separate routes resulting in different patterns of generation and suggest that while ATP activates both receptors, ADP principally influences these cells by interacting with the P2Y-purinoceptors.     

Nucleotide-evoked calcium signals and anion secretion in equine cultured epithelia that express apical P2Y2 receptors and pyrimidine nucleotide receptors

1. Experiments with a spontaneously transformed equine epithelial cell line showed that certain nucleotides increased intracellular free calcium ([Ca2+]i) in cells plated on glass coverslips. The rank order of potency was ATP UTP > 5-Br-UTP, whilst UDP and ADP were ineffective. The response thus appears to be mediated by P2Y2 receptors. 2. Nucleotides also increased short circuit current (Isc) in cells grown into epithelial monolayers and the rank order of potency was UDP> UTP > 5-Br-UTP > ATP > ADP. The increase in [Ca2+]i and the rise in ISC thus have different pharmacological properties. Cross-desensitization experiments indicated that, as well as P2Y2 receptors, the monolayer cultures express at least one additional receptor population that allowed nucleotides to increase ISC. 3. The UDP-evoked increase in ISC was essentially abolished in BAPTA-loaded epithelia suggesting that this response is dependent upon increased [Ca2+]i. Moreover, experiments in which ISC and [Ca2+]i were measured simultaneously showed that the UDP- and ADP-evoked increases in ISC were accompanied by increases in [Ca2+]i. 4. When grown under conditions which favour the development of a polarized phenotype, these epithelial cells thus appear to express [Ca2+]i-mobilizing receptors sensitive to UDP and ADP that are not present in non-polarized cells on coverslips.     

Ca2+ mobilization in bovine corneal endothelial cells by P2 purinergic receptors

PURPOSE: To characterize Ca2+ mobilization by P2 receptors in the bovine corneal endothelial cells (BCEC). METHODS: Changes in intracellular Ca2+ ([Ca2+]i) were measured by fluorescence imaging of cultured and fresh BCEC cells loaded with the Ca2+-sensitive dye Fura-PE3. Relative rates of Ca2+ influx were measured employing Mn2+ as a surrogate for Ca2+. RESULTS: Exposure of cultured cells to uridine 5'-triphosphate (UTP), 2-methyl-thio ATP (msATP) and ATP caused biphasic changes in [Ca2+]i consisting of a peak followed by a plateau phase. Based on the peak responses to 100 microM agonist, the magnitude of UTP responses were similar to that of ATP but greater than that of msATP or ADP. UTP and msATP stimulated Mn2+ influx following [Ca2+]i peak similar to that observed in response to cyclopiazonic acid (CPA), an inhibitor of ER Ca2+-ATPase. Under Ca2+-free conditions, peak responses were similar to those in the presence of external Ca2+, but reduced when the cells were pre-exposed to CPA. Reactive Blue-2 (RB2), inhibited msATP responses by 60.4 +/- 18.8% but UTP responses by only 10.6 +/- 9.5%. Repeated exposures to UTP or msATP reduced [Ca2+]i mobilization indicating homologous desensitization. Response to UTP was not affected by a prior exposure to msATP. However, response to msATP was reduced by a prior exposure to UTP indicating mixed heterologous desensitization. Fresh cells responded to UTP (50 microM) with temporal characteristics of [Ca2+]i mobilization similar to that of cultured cells. CONCLUSION: BCEC express P2 receptors belonging to the P2Y subfamily. The emptying of the IP3-sensitive stores, leading to the initial peak in [Ca2+]i response, subsequently caused capacitative Ca2+ influx leading to the onset of the plateau phase. A significant homologous desensitization to UTP and msATP, selective heterologous desensitization between UTP and msATP, and selective inhibition by RB2 indicate the coexistence of multiple P2Y receptors.     

Differential heterologous and homologous desensitization of two receptors for ATP (P2y purinoceptors and nucleotide receptors) coexisting on endothelial cells

Bovine aortic endothelial cells in culture contain two coexisting phosphoinositidase C-linked receptors for ATP, the P2y-purinoceptors [for which 2-methylthio-ATP (2MeSATP) is a selective agonist] and the nucleotide (or P2u) receptors (for which UTP is a selective agonist). Here we have investigated the occurrence of homologous and heterologous desensitization of these two receptors and the involvement of protein kinase C-dependent mechanisms. Measuring total [3H]inositol (poly)phosphate accumulation in the presence of lithium, we showed that with long (15-min) stimulations with UTP or 2MeSATP desensitization occurred to a maximum of 40% within several minutes of preexposure to either agonist, i.e., with this procedure there is no difference between the heterologous and the homologous experimental design. In the remainder of the experiments reported we measured inositol-1,4,5-trisphosphate mass levels, using a protocol of 5-min preincubation, 2-min wash, and 5-sec stimulation. We found that preincubation with either agonist led to desensitization of the response to the same agonist of about 40%. However, whereas preincubation with 2MeSATP did not affect the subsequent response to UTP, preincubation with UTP did attenuate the 2MeSATP response. These results demonstrate that homologous desensitization occurs with both P2Y and nucleotide receptors but that heterologous desensitization follows only from activation of the nucleotide receptors. Preincubation with the protein kinase C inhibitor Ro 31-8220 enhanced the subsequent inositol-1,4,5-trisphosphate response to 2MeSATP but did not affect the desensitization of this response by preincubation with the same agonist. However, whereas the response to UTP was not enhanced by preincubation with the protein kinase C inhibitor, the desensitization caused by preincubation with UTP was partially inhibited by Ro 31-8220. These results show that multiple desensitizing events occur during the first few minutes of receptor activation and that these events are different for each of the receptors for ATP.     

Purine and pyrimidine nucleotide receptors in the apical membranes of equine cultured epithelia

1. The short circuit current (ISC) technique was used to quantify electrolyte transport by equine cultured sweat gland epithelia. Adenosine 5'-triphosphate (ATP) and certain related compounds, caused transient increases in ISC when added to the apical solution. The order of potency was uridine triphosphate (UTP) > ATP > ADP > > AMP = adenosine. 2. The responses to apical nucleotides were due to chloride and bicarbonate secretion and were reduced in pertussis toxin-treated cells. P2-receptors sensitive to uridine 5'-triphosphate (UTP), that interact with inhibitory G proteins, therefore appear to be present in the apical membrane. 3. Responses to ATP and UTP were reduced in cells loaded with BAPTA, a calcium chelator. BAPTA attenuated the response to ATP more than the response to UTP suggesting that these nucleotides may not act via a common pathway. 4. Cross-desensitization experiments indicated that two populations of UTP-sensitive receptor were present. One was sensitive to UTP and ATP, whereas the second was sensitive only to UTP. Uridine diphosphate appeared to activate the ATP-insensitive receptor population selectively. 5. These data suggest that apical pyrimidinoceptors may be expressed by these cells. The physiological role of these receptors is unknown but they may allow the autocrine regulation of epithelial function.     

Pharmacological profile of a novel cyclic AMP-linked P2 receptor on undifferentiated HL-60 leukemia cells

1. Extracellular ATP (EC50=146+/-57 microM) and various ATP analogues activated cyclic AMP production in undifferentiated HL-60 cells. 2. The order of agonist potency was: ATPgammaS (adenosine 5'-O-[3-thiotriphosphate]) > or = BzATP (2'&3'O-(4-benzoylbenzoyl)-adenosine-5'-triphosphate) > or = dATP > ATP. The following agonists (in order of effectiveness at 1 mM) were all less effective than ATP at concentrations up to 1 mM: beta,gamma methylene ATP > or = 2-methylthioATP > ADP > or = Ap4A (P1, P4-di(adenosine-5') tetraphosphate) > or = Adenosine > UTP. The poor response to UTP indicates that P2Y2 receptors are not responsible for ATP-dependent activation of adenylyl cyclase. 3. Several thiophosphorylated analogs of ATP were more potent activators of cyclic AMP production than ATP. Of these, ATPgammaS (EC50=30.4+/-6.9 microM) was a full agonist. However, adenosine 5'-O-[1-thiotriphosphate] (ATPalphaS; EC50=45+/-15 microM) and adenosine 5'-O-[2-thiodiphosphate] (ADPbetaS; EC50=33.3+/-5.0 microM) were partial agonists. 4. ADPbetaS (IC50=146+/-32 microM) and adenosine 5'-O-thiomonophosphate (AMPS; IC50=343+/-142 microM) inhibited cyclic AMP production by a submaximal concentration of ATP (100 microM). Consistent with its partial agonist activity, ADPbetaS was estimated to maximally suppress ATP-induced cyclic AMP production by about 65%. AMPS has not been previously reported to inhibit P2 receptors. 5. The broad spectrum P2 receptor antagonist, suramin (500 microM), abolished ATP-stimulated cyclic AMP production by HL-60 cells but the adenosine receptor antagonists xanthine amine congener (XAC; 20 microM) and 8-sulpho-phenyltheophylline (8-SPT; 100 microM) were without effect. 6. Extracellular ATP also activated protein kinase A (PK-A) consistent with previous findings that PK-A activation is involved in ATP-induced differentiation of HL-60 cells (Jiang et al., 1997). 7. Taken together, the data indicate the presence of a novel cyclic AMP-linked P2 receptor on undifferentiated HL-60 cells.     

Inhibition of ecto-ATPase by PPADS, suramin and reactive blue in endothelial cells, C6 glioma cells and RAW 264.7 macrophages

1. Previous studies have shown that bovine pulmonary artery endothelium (CPAE) has P2Y and P2U purinoceptors, rat C6 glioma cells have P2U purinoceptors and mouse RAW 264.7 cells have pyrimidinoceptors, all of which are coupled to phosphoinositide-specific phospholipase C (PI-PLC). The dual actions of PPADS, suramin and reactive blue as antagonists of receptor subtypes and ecto-ATPase inhibitors were studied in these three cell types. 2. In CPAE, suramin, at 3-100 microM, competitively inhibited the PI responses induced by 2MeSATP and UTP, with pA2 values of 5.5 +/- 0.3 and 4.4 +/- 0.4, respectively. Reactive blue, at 1-3 microM, produced shifts to the right of the 2MeSATP and UTP curves, but no further right shift at 10 microM. PPADS, at 10 microM, caused a 3 fold right shift of the 2MeSATP curve, but no further shift at concentrations up to 100 microM. In contrast, a dose-dependent shift to the left of the UTP curve and a weak inhibition of the ATP response were seen with PPADS. 3. In RAW 264.7 cells, suramin and reactive blue, but not PPADS, competitively inhibited the UTP response, with pA2 values of 4.8 +/- 0.5 and 5.8 +/- 0.7, respectively. 4. In C6 glioma cells, although suramin and reactive blue inhibited the ATP response, a potentiation effect on ATP and UTP responses was seen with PPADS. 5. The ecto-ATPase inhibitory activity of these three receptor antagonists were determined. All three inhibited ecto-ATPase present in CPAE, C6 and RAW 264.7 cells, with IC50 values of 4, 4.8 and 4.7 for PPADS, 4, 4.4 and > > 4 for suramin, and 4.5, 4.7 and 4.7 for reactive blue. 6. This study indicates that PPADS, suramin and reactive blue ar ecto-ATPase inhibitors. This property, combined with their antagonistic selectivity for receptor subtypes, can result in inhibition of, potentiation of, or lack of effect on agonist-mediated PI responses. Reactive blue is a more potent antagonist than suramin on P2Y, P2U and pyrimidinoceptors, and PPADS is a weak antagonist for P2Y receptors.     

Coexpression of ligand-gated P2X and G protein-coupled P2Y receptors in smooth muscle. Preferential activation of P2Y receptors coupled to phospholipase C (PLC)-beta1 via Galphaq/11 and to PLC-beta3 via Gbetagammai3

P2 receptor subtypes and their signaling mechanisms were characterized in dispersed smooth muscle cells. UTP and ATP stimulated inositol 1,4,5-triphosphate formation, Ca2+ release, and contraction that were abolished by U-73122 and guanosine 5'-O-(3-thio)diphosphate, and partly inhibited (50-60%) by pertussis toxin (PTX). ATP analogs (adenosine 5'-(alpha, beta-methylene)triphosphate, adenosine 5'-(beta, gamma-methylene)triphosphate, and 2-methylthio-ATP) stimulated Ca2+ influx and contraction that were abolished by nifedipine and in Ca2+-free medium. Micromolar concentrations of ATP stimulated both Ca2+ influx and Ca2+ release. ATP and UTP activated Gq/11 and Gi3 in gastric and aortic smooth muscle and heart membranes, Gq/11 and Gi1 and/or Gi2 in liver membranes, and Go and Gi1-3 in brain membranes. Phosphoinositide hydrolysis stimulated by ATP and UTP was mediated concurrently by Galphaq/11-dependent activation of phospholipase (PL) C-beta1 and Gbetagammai3-dependent activation of PLC-beta3. Phosphoinositide hydrolysis was partially inhibited by PTX or by antibodies to Galphaq/11, Gbeta, PLC-beta1, or PLC-beta3, and completely inhibited by the following combinations (PLC-beta1 and PLC-beta3 antibodies; Galphaq/11 and Gbeta antibodies; PLC-beta1 and Gbeta antibodies; PTX with either PLC-beta1 or Galphaq/11 antibody). The pattern of responses implied that P2Y2 receptors in visceral, and probably vascular, smooth muscle are coupled to PLC-beta1 via Galphaq/11 and to PLC-beta3 via Gbetagammai3. These receptors co-exist with ligand-gated P2X1 receptors activated by ATP analogs and high levels of ATP.     

Purine- and pyrimidine-stimulated phosphoinositide breakdown and intracellular calcium mobilisation in astrocytes

Phosphoinositide breakdown in cultured cortical astrocytes was assessed by measuring the accumulation of [3H]inositol phosphates (IP's) following incubations with various purines and pyrimidines. Dose-response relationships gave the following order of potency: 2-methylthioadenosine triphosphate (2-MeSATP) > uridine 5'-triphosphate (UTP) > ATP = ADP > inosine 5' triphosphate (ITP). However, 2-MeSATP and UTP were only half as effective as either ATP or ADP in stimulating [3H]IP production. Astrocytes were also challenged with combined additions of maximally effective concentrations of agonists. Responses to ADP plus UTP and 2-MeSATP plus UTP were essentially additive whilst ATP plus UTP evoked a response which was only partially additive. ATP-stimulated [3H]IP accumulation was markedly reduced in the presence of 2-MeSATP suggesting that the latter may be a partial agonist at these receptors. We also examined the ability of ATP and UTP to increase intracellular Ca2+ concentrations in these cells. Greater than 90% of all cells tested responded to ATP with a release from internal Ca2+ stores but less than half of these responded similarly when challenged with UTP. Our results indicate that astrocytes possess both P2Y-purinoceptors and a population of receptors which are also coupled to phosphoinositide metabolism and intracellular Ca2+ mobilisation but recognise ATP and the pyrimidine nucleotide UTP.