Evidence for stereospecificity of the P1-purinoceptor.

1 The effects of adenosine, 5'-N-ethylcarboxamidoadenosine (NECA), 2-chloroadenosine, 2-azidoadenosine, and their L-enantiomers were examined on driven left atria, trachea and transmurally stimulated ileum of the guinea-pig. 2 In each tissue the order of potency of the D-enantiomers for producing inhibitory effects was NECA greater than 2-chloroadenosine greater than 2-azidoadenosine greater than adenosine. 3 The log concentration-response curve of each agonist was shifted to the right in the presence of the P1-purinoceptor antagonist, theophylline. 4 Dipyridamole, which blocks adenosine uptake, potentiated the effects of adenosine but not those of the D-enantiomers of adenosine analogues. 5 The greater potency of the adenosine analogues therefore, is at least partly due to their resistance to tissue uptake and subsequent enzymatic destruction. 6 The L-enantiomers of adenosine and its analogues did not produce inhibitory responses in the driven left atria or transmurally stimulated ileum. At high concentrations relaxations of the tracheal muscle were obtained, with the potency series L-NECA greater than 2-chloro-L-adenosine greater than 2-azido-L-adenosine greater than L-adenosine. 7 It is concluded that the postsynaptic P1-purinoceptors in the guinea-pig atria and trachea and the presynaptic P1-purinoceptors on cholinergic nerve terminals in guinea-pig ileum are stereospecific for the D-enantiomers of adenosine and its analogues.     

Studies on the stereoselectivity of the P2-purinoceptor.

ATP, 2-chloro-ATP, 2-methylthio-ATP, and their unnatural L-enantiomers, were synthesized and their effects tested on the guinea-pig taenia coli and urinary bladder, and the stimulated frog ventricle. The potent P2-purinoceptor agonists, 2-chloro-ATP and 2-methylthio-ATP were, respectively, 30 and 200 times more effective than ATP in relaxing the guinea-pig taenia, but approximately as effective as ATP in contracting the guinea-pig bladder and augmenting the force of contraction of the frog ventricle. A high degree of stereoselectivity was observed for relaxations of the guinea-pig taenia coli produced by the P2-purinoceptoragonists, and 2-methylthio-ATP was over 700 times more effective than its L-enantiomer. In contrast, stereoselectivity for contraction of the guinea-pig bladder was observed only at low concentrations with each pair of enantiomers, and a similar low stereoselectivity was displayed by the frog ventricle. These results show that P2-purinoceptors mediating inhibitory responses in the guinea-pig taenia coli can show a high degree of stereoselectivity, while P2-purinoceptors mediating excitatory responses in the guinea-pig bladder and in the frog ventricle show little stereoselectivity. The partial stereoselectivity of the P2-purinoceptor in smooth muscle contrasts with the absolute stereospecificity of P1-purinoceptors for adenosine on smooth muscle and autonomic nerve terminals and the absolute stereospecificity of the receptor for ADP on the human platelet.     

P2-purinoceptor antagonists: I. Blockade of P2-purinoceptor subtypes and ecto-nucleotidases by small aromatic isothiocyanato-sulphonates

Effects of eight small aromatic isothiocyanatosulphonates, of the aliphatic 2-isothiocyanatoethene-1-sulphonate (IES), and of the parent amines were studied on contractions of the rat vas deferens elicited by ,-methy-lene ATP (,-MeATP; mediated by P_2X-purinoceptors), relaxations of the carbachol-precontracted guinea-pig taenia coli elicited by adenosine 5-O-(2-thiodiphosphate) (ADPS; mediated by P_2Y-purinoceptors), and the degradation of ATP by rat vas deferens tissue.The aromatic isothiocyanato-sulphonates all reduced contractions of the rat vas deferens elicited by ,-methy-lene ATP. The antagonism was non-competitive, with depression of the maximum of the concentration-response curve of ,-MeATP and incomplete reversibility. The IC₅₀ values were between 11 and 54 M. In the guineapig taenia coli, the aromatic compounds shifted the concentration-response curve of ADPS to the right in a surmountable manner (one exception), and where three concentrations were tested, the Arunlakshana-Schild regression was linear and its slope did not differ from 1. The apparent K _d values were between 10 and 214 M. The removal of ATP from the medium by vas deferens tissue was decreased by the aromatic isothiocyanates with IC_25% values between 25 and 464 M. IES and the parent amines were inactive or almost inactive (parent amines not tested on ATP breakdown).The results indicate that the isothiocyanato residue as well as the aromatic core are essential for P₂-purinoceptor blockade. At the P_2X-purinoceptor, potency increases with the size of the molecules but is independent of the position of the isothiocyanato and sulphonate substituents. No simple structure-activity relationship for the P_2Y-purinoceptor and the ATP-degrading ecto-nucleotidases can be derived beyond the apparent lack of a major influence of the position of the substituents. 2-Isothiocyanatonaphthalene1-sulphonate (-INS) seems to be interesting because of relatively high P_2X-selectivity versus both the P_2Y-purinoceptor and ecto-nucleotidases.     

P2-purinoceptor antagonists: III. Blockade of P2-purinoceptor subtypes and ecto-nucleotidases by compounds related to suramin

Effects of suramin and five analogs or fragments of suramin were studied on contractions of the rat vas deferens elicited by ,-methylene ATP (,-McATP; mediated by P_2X-purinoceptors), relaxations of the carbachol-precontracted guinea-pig taenia coli elicited by adenosine 5-O-(2-thiodiphosphate) (ADPS; mediated by P_2Y-purinoceptors), and the degradation of ATP by rat vas deferens tissue. One compound, NF023, differed from suramin by removal of two p-methylbenzamido groups, whereas another, BSt101, differed from NF023 by additional removal of the three sulphonate residues from one of the terminal naphthalene rings.The compounds all shifted the concentration-response curve of ,-MeATP in the rat vas deferens to the right and simultaneously increased the maximum of the curve. Where three concentrations were tested, the Arunlakshana-Schild regression was linear, and the slope did not differ from 1. The apparent K _d values were between 1 and 3672 M. In the guinea-pig taenia coli, the compounds shifted the concentration-response curve of ADPS to the right in a parallel manner, but in the one case where three concentrations were tested, the slope of the Arunlakshana-Schild regression was lower than 1. Apparent K _d values were between 10 and 786 M. The removal of ATP from the medium by vas deferens tissue was decreased only by suramin, NF023 and BSt101, with IC_25% values between 170 and 590 M.The results indicate that P_2X-purinoceptor affinity, P_2Y-purinoceptor affinity and the ecto-nucleotidase effect all increase with the size of the molecule. BSt101 resembled NF023 in potency at all three sites, indicating that the possession of a second naphthalene-trisulphonate group is not a prerequisite for relatively high affinity. NF023 is interesting because it is P_2X- versus P_2Y-selective and, in addition, the compound with the highest P_2X- versus ecto-nucleotidase-selectivity presently available.     

Evidence for two P2-purinoceptor subtypes in human small pulmonary arteries.

1. P2-purinoceptors have not been characterized in human pulmonary vessels and we therefore examined the effects of adenosine 5'-triphosphate (ATP) and its analogues on human isolated small pulmonary arteries (SPA) in vitro. 2. Contractile responses were induced by all of the analogues, with the rank order of potency alpha,beta-methylene-ATP (alpha,beta-meATP) = beta,gamma-methylene-ATP (beta,gamma-meATP) greater than ATP greater than 2-methylthio-ATP, indicating the presence of vasoconstrictor P2x receptors. 3. In precontracted SPA, vasodilator responses were produced by all of the analogues. The rank order of potency for the analogues causing vasodilator responses was: 2-methylthio-ATP much greater than ATP much greater than beta,gamma-meATP = alpha,beta-meATP, indicating a vasodilator P2y receptor. 4. Removal of endothelial cells had no significant effect on either the contractile or relaxant responses to any of the analogues. 5. After pretreatment of the endothelium-denuded vessels with alpha,beta-meATP (to desensitize P2x receptors), the contractile response to beta,gamma-meATP (a potent P2x receptor agonist) was abolished. 6. We conclude that both P2x- and P2y-purinoceptors are present in human SPA and that both receptors reside on the vascular smooth muscle.     

P2-purinoceptor antagonists: II. Blockade of P2-purinoceptor subtypes and ecto-nucleotidases by compounds related to Evans blue and trypan blue

Effects of Evans blue and four derivatives as well as of trypan blue and four derivatives, mostly smaller fragments but two compounds with an additional ethylene bridge in the center of the molecule, were studied on contractions of the rat vas deferens elicited by ,-methylene ATP (,-McATP; mediated by P_2X-purinoceptors), relaxations of the carbachol-precontracted guinea-pig taenia coli elicited by adenosine 5-O-(2-thiodiphosphate) (ADPS; mediated by P_2Y-purinoceptors), and the degradation of ATP by rat vas deferens tissue.All compounds shifted the concentration-response curve of ,-MeATP in the rat vas deferens to the right, and most compounds increased the maximum of the curve. Each member of the Evans blue series was similar in potency to the corresponding member of the trypan blue series. Where three concentrations were tested, the Arunlakshana-Schild regression was linear, and the slope did not differ from 1. The apparent K _d values were between 0.8 and 385 M. In the guinea-pig taenia coli, only the members of the trypan blue group were relatively potent, shifting the concentration-response curve of ADPS to the right in a surmountable manner. In 2 of 3 cases where three concentrations were tested, the slope of the Arunlakshana-Schild regression was lower than 1. Apparent K _d values in the trypan blue group were between 5.2 and 324 M. The removal of ATP from the medium by vas deferens tissue was decreased mainly by the members of the Evans blue group, with IC_25% values between 13 and 158 (in 1 case >1000) M.The results indicate that the position of the sulphonate residues at the terminal naphthalene rings of these compounds hardly influences P_2X purinoceptor affinity but greatly influences P_2Y affinity and ecto-nucleotidase blockade. Among active compounds, apparent purinoceptor affinity and ecto-nucleotidase blockade increase with the size of the molecules up to Evans blue and trypan blue themselves; introduction of a central ethylene bridge does not result in a further gain in potency. NH01, the desmethyl derivative of Evans blue, seems to be interesting because it is the compound with the highest P_2X- versus P_2Y-selectivity presently available.     

Evidence for the presence of both pre- and postjunctional P2-purinoceptor subtypes in human isolated urinary bladder.

1. In order to characterize P2-purinoceptor(s) in human urinary bladder the contractile effects of ATP and its slowly-hydrolyzable analogues alpha, beta-methylene ATP (alpha, beta-MeATP) and beta, gamma-methylene ATP (beta, gamma-MeATP) were investigated on human detrusor strips taken from patients undergoing cystectomy for bladder carcinoma. 2. Serial concentration-response curves (SCRC) for ATP, alpha, beta-MeATP and beta, gamma-MeATP were constructed with an interval of 25 min between two successive doses to avoid tachyphylaxis. ATP (10 microM-10 mM) induced a phasic contraction, which was very rapid in onset. The dose-response curve to ATP appeared not to be monophasic: at the lower concentrations (10-300 microM) the curve was shallow, whilst at high concentrations (1-10 mM) the curve was steeper. The magnitude of the response obtained at the highest concentration tested (10 mM) was only 21.1 +/- 2.8% (mean +/- s.e. mean; n = 4) of the KCl (100 mM)-induced contraction. 3. alpha, beta-MeATP (0.3 microM-1 mM) and beta, gamma-MeATP (10 microM-1 mM) elicited a phasic contraction with a time course similar to that exhibited by ATP. The magnitude of the response obtained at the highest concentration tested (1 mM) was 70.3 +/- 6.3% for alpha, beta-MeATP (n = 10) and 27.9 +/- 4.5% for beta, gamma-MeATP (n = 8) of KCl (100 mM)-induced contraction. The rank order of potency was alpha, beta-MeATP > beta, gamma-MeATP > ATP. A plateau of response could not be achieved by any of these agonists.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synergistic action between inhibition of P2Y12/P2Y1 and P2Y12/thrombin in ADP- and thrombin-induced human platelet activation

The objective of this study was to investigate if there is a synergistic effect of a combination of P2Y(12) and P2Y(1) inhibition and P2Y(12) and thrombin inhibition, on ADP- and thrombin-induced platelet activation, respectively. The rationale being that these combinations will cause a concurrent inhibition of both G alpha(q) and Galpha (i) signalling. Blood from healthy volunteers was preincubated with AR-C69931MX, a reversible P2Y(12) antagonist; MRS2179, a reversible P2Y(1) antagonist; or melagatran, a direct reversible thrombin inhibitor; alone or in various combinations prior to activation with ADP or thrombin. Platelet function in whole blood was assessed by flow cytometry using the antibody PAC-1 to estimate the expression of active alpha (IIb)beta(3) (the fibrinogen receptor GPIIb/IIIa). A synergistic effect was evaluated by comparing the concentrations in the different combinations with those of corresponding equipotent concentrations of each single inhibitor alone. The equipotent single concentrations were experimentally obtained from concentration response curves performed in parallel. A synergistic effect regarding inhibition of ADP-induced platelet activation (10 microM) was obtained with different combinations of AR-C69931MX and MRS2179. Inhibition of thrombin-induced platelet activation (2 nM) with combinations of AR-C69931MX and the thrombin inhibitor melagatran did also result in a strong synergistic effect. To our knowledge, this is the first time that data supporting a synergistic effect has been published for the inhibitor combinations described. Whether this synergistic effect in vitro also results in an improved antithrombotic effect in vivo with or without an increased risk of bleeding remains to be studied in well-conducted clinical studies.     

Evidence that the P1-purinoceptor in the guinea-pig taenia coli is an A2-subtype.

The effects of 5'-N-ethylcarboxamidoadenosine (NECA), L-NECA, 2-chloroadenosine, N6-phenylisopropyladenosine (L-PIA and D-PIA), cyclohexyladenosine (CHA), and adenosine were examined on the guinea-pig taenia coli. All the analogues except L-NECA caused relaxations; the order of potency for the series was: NECA greater than 2-chloroadenosine greater than L-PIA greater than CHA greater than D-PIA greater than adenosine. L-PIA was twice as potent as D-PIA in inducing relaxations of the guinea-pig taenia coli. Adenosine and its analogues that induce relaxation all caused a slow membrane hyperpolarization; differences in the rates of hyperpolarization and latencies were apparent, although not statistically significant. The duration of the response to adenosine was significantly less than that for any adenosine analogue. Ion studies, using the sucrose gap, revealed that responses to the analogues were attenuated in elevated extracellular potassium or reduced extracellular chloride. 8-Phenyltheophylline, a potent P1-purinoceptor antagonist, caused a rightward shift of all the adenosine and analogue concentration-response curves. Dipyridamole, an adenosine uptake inhibitor, potentiated the relaxations to adenosine but had no significant effect on the relaxations induced by the analogues. It is concluded that NECA, 2-chloroadenosine, L-PIA, CHA, D-PIA and adenosine mediate their relaxant effects via an extracellular P1-purinoceptor which displays characteristics of the A2-subtype as determined by the rank order of agonist potency. Electrophysiological analysis of the responses to each of the analogues did not reveal any marked differences in the modes of action even between NECA and L-PIA (preferential A2- and A1-receptor agonists, respectively).     

The ecto-nucleotidase NTPDase1 differentially regulates P2Y1 and P2Y2 receptor-dependent vasorelaxation

Background and purpose:

Extracellular nucleotides produce vasodilatation through endothelial P2 receptor activation. As these autacoids are actively metabolized by the ecto-nucleotidase nucleoside triphosphate diphosphohydrolase-1 (NTPDase1), we studied the effects of this cell surface enzyme on nucleotide-dependent vasodilatation.

Experimental approach:

Vascular NTPDase expression and activity were evaluated by immunohistochemistry and histochemistry. The vascular effects of nucleotides were tested in vivo by monitoring mean arterial pressure, and in vitro comparing reactivity of aortic rings using wild-type and Entpd1^−/− (lacking NTPDase1) mice.

Key results:

The absence of NTPDase1 in Entpd1^−/− mice led to a dramatic drop in endothelial nucleotidase activity. This deficit was associated with an exacerbated decrease in blood pressure after nucleotide injection. Following ATP injection, mean arterial pressure was decreased in Entpd1^+/+ and Entpd1^−/− mice by 5.0 and 17%, respectively, and by 0.1 and 19% after UTP injection (10 nmole·kg⁻¹ both). In vitro, the concentration-response curves of relaxation to ADP and ATP were shifted to the left, revealing a facilitation of endothelial P2Y1 and P2Y2 receptor activation in Entpd1^−/− mice. EC₅₀ values in Entpd1^+/+ versus Entpd1^−/− aortic rings were 14 µM versus 0.35 µM for ADP, and 29 µM versus 1 µM for ATP. In Entpd1^−/− aortas, P2Y1 receptors were more extensively desensitized than P2Y2 receptors. Relaxations to the non-hydrolysable analogues ADPβS (P2Y1) and ATPγS (P2Y2) were equivalent in both genotypes confirming the normal functionality of these P2Y receptors in mutant mice.

Conclusions and implications:

NTPDase1 controls endothelial P2Y receptor-dependent relaxation, regulating both agonist level and P2 receptor reactivity.     

Differential effects of P2-purinoceptor antagonists on phospholipase C- and adenylyl cyclase-coupled P2Y-purinoceptors.

1. Stimulation of P2Y-purinoceptors on turkey erythrocytes and many other cell types results in activation of phospholipase C. In contrast, we have observed recently that P2Y-purinoceptors on C6 rat glioma cells are not coupled to phospholipase C, but rather, inhibit adenylyl cyclase. 2. In this study we investigated the pharmacological selectivity of the P2-purinoceptor antagonists, suramin, reactive blue 2, and pyridoxal phosphate 6-azophenyl 2',4'-disulphonic acid (PPADS) for phospholipase C- and adenylyl cyclase-coupled P2Y-purinoceptors. 3. In C6 glioma cells, suramin and reactive blue 2 competitively antagonized the inhibitory effect of 2MeSATP on adenylyl cyclase (pKB = 5.4 +/- 0.2 and 7.6 +/- 0.1, respectively), whereas PPADS at concentrations up to 100 microM had no effect. 4. In contrast, in the turkey erythrocyte preparation, PPADS at concentrations up to 30 microM was a competitive antagonist of P2Y-purinoceptor-stimulated phospholipase C activity (pKB = 5.9 +/- 0.1). Suramin and reactive blue 2 produced both a shift to the right of the concentration-effect of 2MeSATP for the activation of phospholipase C and a significant decrease in the maximal inositol phosphate response. 5. Turkey erythrocytes also express a phospholipase C-coupled beta-adrenoceptor. Concentrations of PPADS that competitively inhibited the P2Y-purinoceptor-mediated response had only minimal effects on the activation of phospholipase C by beta-adrenoceptors. In contrast, suramin and reactive blue 2 produced a non-competitive inhibition, characterized by decreases in the maximal response to isoprenaline with no change in the potency of this beta-adrenoceptor agonist.(ABSTRACT TRUNCATED AT 250 WORDS)     

Methanocarba modification of uracil and adenine nucleotides: high potency of Northern ring conformation at P2Y1, P2Y2, P2Y4, and P2Y11 but not P2Y6 receptors.

The potency of nucleotide antagonists at P2Y1 receptors was enhanced by replacing the ribose moiety with a constrained carbocyclic ring (Nandanan, et al. J. Med. Chem. 2000, 43, 829-842). We have now synthesized ring-constrained methanocarba analogues (in which a fused cyclopropane moiety constrains the pseudosugar ring) of adenine and uracil nucleotides, the endogenous activators of P2Y receptors. Methanocarba-adenosine 5'-triphosphate (ATP) was fixed in either a Northern (N) or a Southern (S) conformation, as defined in the pseudorotational cycle. (N)-Methanocarba-uridine was prepared from the 1-amino-pseudosugar ring by treatment with beta-ethoxyacryloyl cyanate and cyclization to form the uracil ring. Phosphorylation was carried out at the 5'-hydroxyl group through a multistep process: Reaction with phosphoramidite followed by oxidation provided the 5'-monophosphates, which then were treated with 1,1'-carbonyldiimidazole for condensation with additional phosphate groups. The ability of the analogues to stimulate phospholipase C through activation of turkey P2Y1 or human P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11 receptors stably expressed in astrocytoma cells was measured. At recombinant human P2Y1 and P2Y2 receptors, (N)-methanocarba-ATP was 138- and 41-fold, respectively, more potent than racemic (S)-methanocarba-ATP as an agonist. (N)-methanocarba-ATP activated P2Y11 receptors with a potency similar to ATP. (N)-Methanocarba-uridine 5'-triphosphate (UTP) was equipotent to UTP as an agonist at human P2Y2 receptors and also activated P2Y4 receptors with an EC(50) of 85 nM. (N)-Methanocarba-uridine 5'-diphosphate (UDP) was inactive at the hP2Y6 receptor. The vascular effects of (N)-methanocarba-UTP and (N)-methanocarba-UDP were studied in a model of the rat mesenteric artery. The triphosphate was more potent than UTP in inducing a dilatory P2Y4 response (pEC(50) = 6.1 +/- 0.2), while the diphosphate was inactive as either an agonist or antagonist in a P2Y6 receptor-mediated contractile response. Our results suggest that new nucleotide agonists may be designed on the basis of the (N) conformation that favors selectivity for P2Y1, P2Y2, P2Y4, and P2Y11 receptors.     

Studies on the stereoselectivity of the P2-purinoceptor on the guinea-pig vas deferens.

ATP, 2-chloro-ATP, 2-methylthio-ATP and their unnatural L-enantiomers, Rp and Sp diastereoisomers of the ATP phosphorothioate analogues, ATP alpha S and ATP beta S, were tested on the guinea-pig vas deferens. The 2-substituted analogues of ATP were no more effective than ATP in causing contraction of the vas deferens. However, stereoselectivity was observed with each pair of enantiomers of ATP, 2-chloro-ATP and 2-methylthio-ATP. No stereoselectivity was observed for the phosphorothioate analogues. Rp- and Sp-ATP beta S were more effective than ATP at eliciting contractions of the vas deferens. These results show that unlike the P2-purinoceptor mediating excitatory responses in the guinea-pig bladder, the P2-purinoceptor mediating contraction in the guinea-pig vas deferens displays stereoselectivity.     

2-Alkylthio-substituted platelet P2Y12 receptor antagonists reveal pharmacological identity between the rat brain Gi-linked ADP receptors and P2Y12

The Gi-linked platelet ADP receptor, now designated as P2Y12, accounts for ADP-induced inhibition of adenylyl cyclase in platelets and certain clonal rat cell lines. The pharmacology of this receptor is well characterized. Based on the functional approach of [35S]GTPgammaS autoradiography, we recently disclosed the widespread presence of Gi-linked ADP receptors in the rat nervous system. Based on initial pharmacological analysis, these receptors were strikingly similar with P2Y12. Here, we extend this analysis by comparing the potencies of six 2-alkylthio-substituted ATP analogues, including the adenosine-aspartate conjugate 2-hexylthio-AdoOC(O)Asp2 and five AR-C compounds (AR-C67085, AR-C69931, AR-C78511, AR-C69581, AR-C70300) with wide range of affinities towards P2Y12, in reversing 2-methylthio-ADP stimulated G protein activity in rat brain sections and human platelet membranes. Closely matching pIC50 values (r2=0.99) revealed pharmacological similarity between the two receptors with one exception: AR-C67085 more avidly recognized the platelet P2Y12. Further analysis of the rat brain pIC50 data against those available for three of the AR-C compounds in reversing P2Y12-mediated adenylyl cyclase inhibition in rat platelets (r2=0.96) and rat C6 glioma cells (r2=1.00) demonstrated that the three P2Y receptors are pharmacologically indistinguishable. We conclude that the rat brain Gi-linked ADP receptors, as revealed using [35S]GTPgammaS autoradiography, correspond to P2Y12.     

Effect of P2-purinoceptor antagonists on glutamatergic transmission in the rat hippocampus.

1. A study has been made of the effects of P2-purinoceptor antagonists on the evoked excitatory postsynaptic currents (e.p.s.cs) generated in CA1 pyramidal cells on stimulation of Schaffer collaterals and in CA3 pyramidal cells on stimulation of mossy fibres. The effects of these antagonists on currents generated in the cells on application of glutamate has also been determined. 2. Suramin blocked the evoked e.p.s.cs with an 50% inhibition (ID50) of 62 +/- 8 microM (mean +/- s.e.mean, n = 17), spontaneous miniature e.p.s.cs and the currents induced by application of 100 microM glutamate with an ID50 = 121 +/- 36 microM (n = 15) in all the cells studied. 3. Reactive Blue 2 (RB-2) in a concentration of 200 microM decreased the e.p.s.cs by 80 +/- 10% (n = 6) and the glutamate-activated currents by 83 +/- 3% (n = 6). 4. Pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) in the concentration-range of 40-500 microM decreased the amplitude of the e.p.s.cs in 12 out of 13 cells studied. PPADS at 200 microM reduced the amplitude of the e.p.s.cs by 60 +/- 10% (n = 3). PPADS did not affect the glutamate-induced currents in 4 cells and produced potentiation of the current amplitude by 60 +/- 10% in 4 other cells. 5. These results suggest that both presynaptic and postsynaptic P2-purinoceptors in the hippocampus can modulate the release and action of endogenous glutamate.     

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.