Br. J. Pharmacol. (1994), 113, 614-620 '." Macmillan Press Ltd, 1994

Differential effects of P2-purinoceptor antagonists on phospholipase C- and adenylyl cyclase-coupled P2y-purinoceptors Jose L. Boyer, Irene E. Zohn, *Kenneth A. Jacobson & IT. Kendall Harden

Department of Pharmacology, School of Medicine, University of North Carolina, Chapel Hill, NC 27599 and *Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, U.S.A.

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, , 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 concent- rations up to 100 JAM had no effect. 4 In contrast, in the turkey erythrocyte preparation, PPADS at concentrations up to 30 jaM 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 phosphate response. 5 Turkey erythrocytes also express a phospholipase C-coupled ,-adrenoceptor. Concentrations of PPADS that competitively inhibited the P2Y-purinoceptor-mediated response had only minimal effects on the activation of phospholipase C by P-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 P-adrenoceptor agonist. 6 The differential effect of PPADS on P2Y-purinoceptors of C6 glioma cells and turkey erythrocytes adds further support to the idea that different P2Y-purinoceptor subtypes mediate coupling to adenylyl cyclic and phospholipase C. Keywords: Reactive blue 2; suramin; PPADS; phospholipase C; inositol phosphates; P2y-purinoceptors; cyclic AMP accumula- tion; adenylyl cyclase inhibition; C6 rat glioma cells

Introduction 5'-triphosphate (ATP) is released from multiple useful as functional antagonists of ATP signalling responses. cell types including neurones, platelets, and endothelial cells Reactive blue 2 has been reported to antagonise ATP actions (Gordon, 1986; O'Connor et al., 1991). Extracellular ATP at the P2Y-purinoceptor subtype (Choo, 1981; Manzini et al., and its derivatives interact with specific receptors on the 1986; Reilly et al., 1987; Rice & Singleton, 1989), and the surface of target cells and regulate a number of biological trypanocidal drug suramin is an antagonist of multiple ATP- functions such as platelet aggregation, neurotransmission, stimulated responses (Dunn & Blakely, 1988; Hoyle cardiac performance, muscle contraction, and glucose et al., 1990; Leff et al., 1990). Pyridoxal phosphate 6- metabolism (for review see Dubyak & El-Motassim, 1993). azophenyl-2',4'-disulphonic acid (PPADS) recently was Purinoceptors originally were classified as PI- and P2- shown to inhibit ATP-receptor-stimulated mechanical res- purinoceptors (Burnstock, 1978). PI-purinoceptors, which are ponses of the rabbit vas deferens (Lambrecht et al., 1992) activated by adenosine, were further sub-classified into Al and rabbit urinary bladder detrusor muscle (Ziganshin et al., and A2 subtypes (Van Calker et al., 1979; Londos et al., 1993). 1980), and a third subtype (A3) has been The signalling mechanisms associated with P2- identified recently (Meyerhoff et al., 1991; Zhou et al., 1992). purinoceptors are only partially understood. Perhaps the P2-purinoceptors are activated by ATP and/or ADP. This most widely studied P2-purinoceptor signalling mechanism is receptor class originally was sub-divided into P2X- and P2y- the G-protein-regulated inositol lipid cascade and purinoceptors (Burnstock & Kennedy, 1985). However, addi- mobilization that results from activation of P2y- (Charest et tional receptor subtypes exist within this class including the al., 1985; Forsberg et al., 1987; Pirotton et al., 1987; Boyer et P2U-, P2z-, and P2T-purinoceptors (Gordon, 1986; O'Connor al., 1989; Berrie et al., 1989) and P2U-purinoceptors (Dubyak et al., 1991; Dubyak & El-Motassim, 1993). et al., 1988; Okajima et al., 1989; Fine et al., 1989; In contrast to the PI-purinoceptor class, classification of Stutchfield & Cockcroft, 1990; Brown et al., 1991). However, P2-purinoceptors has relied exclusively on the phar- activation of P2-receptors in a number of target tissues macological specificity of responses to agonist analogues of results in a decrease in adenosine 3':5'-cyclic monophosphate ATP and ADP. However, several compounds that are known (cyclic AMP) accumulation (Okajima et al., 1987; Pianet et to interact with multiple ATP binding proteins have proven al., 1989; Sato et al., 1992; Valeins et al., 1992; Lin & Chuang, 1993), and we recently reported that C6 rat glioma cells express a P2Y-purinoceptor subtype that inhibits adenylyl Author for correspondence. cyclase (Boyer et al., 1993). This observation contrasts with P,-PURINOCEPTOR ANTAGONISTS 615 the well-characterized effect of 2-methylthio-ATP The final resuspension was in 20 mM HEPES pH 7.0 to a (2MeSATP) on phospholipase C activity mediated by concentration of 6mg protein ml-'. This preparation was 'typical' P2Y-purinoceptors. Differences in the phar- used immediately for the phospholipase C assay. Twenty five macological specificity for analogues on J.l of [3H]-inositol-labelled ghost preparation (t 150 pg pro- the two receptor responses have been observed (Boyer et al., tein, 200,000 c.p.m.) were combined in a final volume of 1993; O'Tuel, Boyer & Harden, unpublished). The involve- 200il of a medium containing 0.91 mM MgSO4, 115 mM ment of a different signalling pathway and the possible KCI, 5 mM potassium phosphate, 0.424 mM CaC12, 2 mM existence of different pharmacological specificities are consis- EGTA, 10 mM HEPES, pH 7.0 (free Ca2" concentration was tent with the idea that a unique receptor subtype is involved t1 JIM). Since receptor-and G-protein activation of phos- in the regulation of adenylyl cyclase by P2y-purinoceptor pholipase C in turkey erythrocyte ghosts is strictly dependent agonists in C6 glioma cells. on the presence of (Boyer et al., 1989), In this study we compared the effect of the putative P2- the non-hydrolyzable analogue of GTP, GTPyS at 1 JIM was purinoceptor antagonists, reactive blue 2, suramin, and included in the assay. PPADS on 2MeSATP-promoted inhibition of adenylyl cyc- Ghosts were incubated for 5 min at 37°C with the lase in C6 glioma cells and on activation of phospholipase C indicated concentrations of 2MeSATP, in the presence or in in turkey erythrocytes. We present evidence of differences in the absence of antagonist. Incubations were terminated by apparent affinity of reactive blue 2 between the two test the addition of 1 ml of ice cold 5% trichloroacetic acid. systems and for a selective effect of PPADS for the phos- Samples were centrifuged, the supernatants were transferred pholipase C-coupled Py-purinoceptor. These results suggest to a fresh tube, and the trichloroacetic acid was extracted that different P2y-purinoceptors regulate adenylyl cyclase and three times with 3 ml portions of ethyl ether. Neutralized phospholipase C activities. samples were diluted with 8 ml of water and transferred into Dowex AG-X8 columns (formate form). Columns were washed with 8 ml of 200 mM ammonium formate, 100 mM formic acid, and the eluant was discarded. Total inositol Methods phosphates (UP2 through IP4) were eluted with 5 ml of 1.2 M ammonium formate, 100 mM formic acid, and collected in Cell culture scintillation vials. [3H]-inositol phosphates were quantitated by scintillation spectroscopy as previously described in detail C6 rat glioma cells were grown in Dulbecco's Modified (Harden et al., 1988, Boyer et al., 1989). Eagle's Medium supplemented with 5% foetal calf serum in a humidified atmosphere of 95% air and 5% CO2. Cells were Materials passaged by trypsinization. Experiments were carried out in confluent cultures 2-4 days after plating in 12-well plates as 2MeSATP was obtained from Research Biochemicals Inc. previously described (Frederich et al., 1983). (Natick, MA, U.S.A.); reactive Blue 2 (Cibacron Blue) was from Fluka (Ronkonkowa, NY, U.S.A.); PPADS was Cyclic AMP accumulation from Cookson Chemical Ltd (Southampton, England); isoprenaline was from Sigma Chemical Co. (St. Louis, MO, Cells were labelled for 2 h with 1-2 piCi ml-' [3H]-adenine U.S.A.); 2-[3H]-myo-inositol (20 Ci mmolh') and [8-3H]- (Meeker & Harden, 1982; Frederich et al., 1983). Cells were adenine (27 Ci mmol ') were obtained from American washed twice with HEPES (20 mM, pH 7.5) buffered Eagle's Radiolabeled Chemicals, Inc. (St. Louis, MO, U.S.A.); medium at 37°C, and then preincubated for 10 min with inositol-free DMEM was from Gibco BRL, (Grand Island, HEPES Eagle's medium containing 200 JM isobutyl methyl- NY, U.S.A.); suramin was a generous gift from the Depart- (IBMX). Antagonists were added to the cells 10 min ment of Drug Services from the Center for Disease Control before challenge with agonist. Agonist incubations were (Atlanta, GA, U.S.A.). The sources of other materials have initiated by the simultaneous addition of 10 JM isoprenaline been previously reported (Harden et al., 1988; Boyer et al., and the indicated concentrations of the P2Y-purinoceptor 1989; 1993). agonist, 2MeSATP. The reactions were stopped after 10 min by aspiration of the drug-containing medium and addition of Data analysis I ml of ice-cold 5% trichloroacetic acid. [3H]-cyclic AMP accumulation was determined by Dowex and alumina Agonist potencies were calculated using a four parameter chromatography as previously described (Harden et al., logistic equation using the GraphPad software package. In 1982). experiments where antagonists produced parallel dis- placements in the concentration-effect curves, dose-ratios Turkey erythrocyte labelling were calculated from the K0o5 estimates for each pair of curves and analysed according to Arunlakshana & Schild Fresh blood was obtained from female turkeys by venous (1959). puncture and collected into a heparinized syringe. Eryth- rocytes were washed twice by centrifugation and resuspension with sterile DMEM, followed by a final wash in inositol-free DMEM. One ml of washed packed erythrocytes was Results resuspended in a final volume of 4.2ml of inositol-free DMEM in the presence of 0.5-1 mCi [3H]-inositol. Cells Incubation of C6 glioma cells with the P2Y-purinoceptor were incubated in a stirred glass vial for 16-20 h at 37°C in a agonist 2MeSATP resulted in a concentration-dependent humidified atmosphere of 95% air, 5% CO2 as previously inhibition of isoprenaline-stimulated cyclic AMP accumula- described in detail (Harden et al., 1988; Boyer et al., 1989). tion (Boyer et al., 1993). Preincubation of cells with increas- ing concentrations of the putative P2-purinoceptor Phospholipase C assay antagonist, suramin, resulted in parallel shifts to the right of the concentration-effect curve of 2MeSATP (Figure la). Erythrocyte ghosts were prepared with [3H]-inositol-labelled Arunlakshana-Schild analysis of these data (Arunlakshana & cells by hypotonic lysis in 15 volumes of a buffer containing Schild, 1959) indicated that in intact C6 cells, suramin at 5 mM sodium phosphate, pH 7.4, 5 mM MgCl2, and 1 mM concentrations up to 100 JiM behaves as a competitive EGTA (lysis buffer). Erythrocyte ghosts were washed three antagonist (slope = 0.94 ± 0.04) of the P2y-purinoceptor times by centrifugation and resuspension with lysis buffer. coupled to the inhibition of adenylyl cyclase. The pKB for 616 J.L. BOYER et al. suramin derived from Arunlakshana-Schild plots (Figure Ib) In contrast to the P2Y-purinoceptor on C6 cells, activation was 5.4 ± 0.2 (n = 3 experiments). of P2Y-purinoceptors on turkey erythrocytes results in a rapid The effect of reactive blue 2 on 2MeSATP-induced inhibi- increase in the hydrolysis of inositol lipids by phospholipase tion of cyclic AMP accumulation is shown in Figure 2. As was observed with suramin, reactive blue 2 at concentrations up to 10 M produced parallel shifts to the right of the a concentration-effect curve of 2MeSATP (Figure 2a). The pKB value for reactive blue 2 obtained from four Arunlakshana- 0 Schild plots (slope = 1.07 ± 0.08) was 7.6 ± 0.1 (Figure 2b). These results indicate that at surprisingly low concentrations, U reactive blue 2 behaves as a competitive antagonist of the 0 P2y-purinoceptor on C6 rat glioma cells. Incubation of C6 cJ cells with concentrations of reactive blue 2 higher than 50 jM 0 significantly decreased the maximal responses of cyclic AMP I accumulation observed with isoprenaline. 1-0C._ Lambrecht et al. (1992) have reported recently that the - pyridoxal phosphate derivative, PPADS is a competitive antagonist of P2-purinoceptors in the vas deferens. In marked contrast to the effects of suramin and reactive blue 2, PPADS had no effect on the inhibition of cyclic AMP accumulation by 2MeSATP in C6 glioma cells when assayed at concentra- -log [2MeSATP1 M tions up to 100 gM (Figure 3).

a

0 0 (a a-cI1 0 0) 0_U) 2 0 CUU 0) -Y 01

0 6 5 4 -log [Reactive blue 21 M Figure 2 Effect of reactive blue 2 on P2y-purinoceptor-mediated 0- -log [2MeSATPI M inhibition of adenylyl cyclase activity in C6 glioma cells. Cyclic AMP T-.2 accumulation in [3H]-adenine-labelled C6 glioma cells was quan- ._ titated as described in Methods. (a) The concentration-dependence of 2MeSATP in inhibiting isoprenaline-stimulated cyclic AMP accumulation was determined in the absence (@), or in the presence of 0.03 (*), 0.1 (A), 1 (E), 3 (0) and IOgM (O) reactive blue 2. See legend of Figure 1 for details. (b) Arunlakshana-Schild plot from 0 ._z the data presented in (a). The slope of the plot was 1.07 ± 0.08. The estimated pKB was 7.6 ± 0.1 (n = 4 experiments). Data shown are the 4)Tn mean of triplicate determinations and the results shown are represen- tative of those obtained in four separate experiments. o

. * 0.0 0_ 100 * 5.0 4.5 0A -log [Suramin] M 75 0~0 Figure 1 Effect of suramin on P2y-purinoceptor-mediated inhibition of adenylyl cyclase activity in C6 glioma cells. Cyclic AMP 50 accumulation in [3H]-adenine-labelled C6 glioma cells was quan- titated as described in Methods. (a) The concentration dependence of 2MeSATP for inhibition of isoprenaline-stimulated cyclic AMP ~25- accumulation was determined in the absence (0), or in the presence of 3 (*), 10 (A), 20 (-), and 100LM (0) suramin. The maximal cyclic AMP accumulation stimulated by 10 jsM isoprenaline was 10 9 8 19000 ± 2000 c.p.m. Maximal effective concentrations of 2MeSATP -log [2MeSATP] M resulted in 40-50% inhibition of the response to isoprenaline. Data are presented as percentage of the isoprenaline-induced cyclic AMP Figure 3 Lack of effect of PPADS on P2y-purinoceptor-mediated accumulation inhibitable by a maximally effective concentration of inhibtion of adenylyl cyclase activity in C6 glioma cells. Cyclic AMP 2MeSATP observed in the absence of antagonist. (b) Arunlakshana- accumulation in [3H]-adenine-labelled C6 glioma cells was quan- Schild plot from the data presented in (a). The slope of the plot was titated as indicated under Methods. The concentration-dependence 0.94 and the estimated pKB obtained from three separate experiments of 2MeSATP in inhibiting isoprenaline-stimulated cyclic AMP was 5.4 ± 0.2. Data shown are the mean of triplicate determinations accumulation was determined in the absence (0) or in the presence and the results shown are representative of those obtained in three of 0.3 (*), 30 (A), and 100 gM (U) PPADS. See legend of Figure 1 separate experiments. for details. For abbreviations, see text. P2-PURINOCEPTOR ANTAGONISTS 617

a a 0 x 15 x 30CL 12- . 6 0)U)

0 U)0. -C 06 s° W 93 10 9 8 7 0. a 0.a 0 0 L-l

10 9 8 6 7 5 4 3 I -log [2MeSATPI,M b 2.0-

_~3 E 1.5- x

W- 00 6. *r- 1.0 6 0 0 10 0. 0.m 0 U)

0 7 65 4 3 C -log [PPADS], m mI Figure 4 Effect of PPADS on P2y-purinoceptor-mediated activation of phospholipase C in turkey erythrocyte membranes. Membranes -log [2MeSATP] M from [3H]-inositol-labelled erythrocytes were prepared as described in Methods. (a) Release of [3H]-inositol phosphates was measured in the Figure 5 Effects of suramin and reactive blue 2 on P2y- presence of 1 gM GTPVS and the indicated concentrations of purinoceptor-mediated activation of phospholipase C in turkey 2MeSATP in the absence (0), or in the presence of 1 (0), 3 (*), 5 erythrocyte membranes. Membranes from [3H]-inositol-labelled (b), 10 (A), 20 (A), or 30 tM PPADS (U). Data shown are the erythrocytes were prepared as described in Methods. (a) Release of mean of duplicate determinations, and the results shown are [3H]-inositol phosphates was measured in the presence of I tim representative of those obtained in three separate experiments. [3H]- GTPyS and the indicated concentrations of 2MeSATP in the absence inositol phosphates released by 1 gM GTP-yS in the absence of (@), or in the presence of 1 (0), 3 (*), 10 (O), 30 (A) or 100 gM 2MeSATP (500 ± 200 c.p.m.) were subtracted from the data. (b) The suramin (A). (b) Release of [3H]-inositol phosphates was measured corresponding Arunlakshana-Schild plot from the data presented in in the presence of I juM GTPyS and the indicated concentrations of (a). The slope of the plot was 1.1 ± 0.1 and the estimated pKB was 2MeSATP in the absence (0) or in the presence of 1 (0), 3 (*), 10 5.9 ± 0.1 (n = 3 experiments). For abbreviations, see text. (O), 30 (A), or 100 LM reactive blue 2 (A). Data shown are the mean of duplicate determinations and the results shown are represen- tative of those obtained in three different experiments.

C. The effects of PPADS, suramin and reactive blue 2 on the P2y-purinoceptor-stimulated inositol lipid response of turkey erythrocyte membranes were examined. In contrast to the 2MeSATP (Figure 5). These results indicate that the lack of effect of PPADS on the C6 cell P2Y-purinoceptor, inhibitory effects of suramin and reactive blue 2 on the PPADS produced a 30 to 45 fold shift to the right in the inositol phosphate response of 2MeSATP do not solely occur concentration-effect curve of 2MeSATP in turkey erythrocyte by competitive inhibition. membranes without producing any significant effect on the Based on the broad range of potential effects of the maximal response (Figure 4a). Arunlakshana-Schild analysis available P2-antagonists, it is not unlikely that these com- of these data indicated that PPADS is a competitive pounds produce non-specific effects, particularly at high con- antagonist of the phospholipase C-coupled P2Y-purinoceptor. centrations. This possibility is even more likely to occur in The pKB for PPADS was 5.9 ± 0.1, and the slope of the cell-free assays, as with the turkey erythrocyte membrane Schild regression was 1.1 ± 0.1 (Figure 4b). Higher concen- preparation used in this study. To determine whether the trations, i.e. greater than 50 pM PPADS, produced further decrease in the maximal response to 2MeSATP observed in shifts to the right in the concentration-effect curves for membranes treated with suramin and reactive blue 2 was 2MeSATP but also resulted in decreases in the maximal related to the inactivation of the P2y-purinoceptor, the effects inositol phosphate response (data not shown). of the antagonists on the P-adrenoceptor promoted activation Treatment of turkey erythrocyte membranes with suramin of phospholipase C (Rooney et al., 1991; Vaziri & Downes, or reactive blue 2 also produced a concentration-dependent 1992) were studied. In contrast to the marked shifts to the shift to the right of the concentration-effect curve of right of the concentration-effect curves of 2MeSATP (Figure 2MeSATP for activation of phospholipase C (Figure 5). For 5), no effect of suramin or reactive blue 2 on the EC50 for the example, the EC50 for 2MeSATP increased by 10 and 50 fold isoprenaline response was observed (Figure 6a). However, in the presence of 30 pM suramin and 30 pM reactive blue 2, both antagonists produced a concentration-dependent respectively. However, this shift in the concentration-effect decrease in the maximal inositol phosphate response to curve was accompanied by a significant decrease in the max- isoprenaline (Figure 6a, and data not shown). Consistent imal stimulation of inositol phosphates produced by with a specific antagonist effect, 30 JLM PPADS produced a 618 J.L. BOYER et al.

purinoceptor responses in turkey erythrocytes and C6 glioma °- 25 cells, have allowed us to determine the pharmacological prop- x erties of putative P2-purinoceptor antagonists at the most E 20 immediate response of the activation of the receptor, the d generation of the second messenger. The pyridoxal phosphate C) derivative, PPADS, exhibited selectivity for the phos- w.00. 15- pholipase C-coupled P2y-purinoceptor of the turkey eryth- sa rocyte, whereas no activity against the adenylyl cyclase- oU, 10 coupled P2Y-purinoceptor in C6 glioma cells was observed 0. (Figure 3). The effect of PPADS was competitive (Figure 4), and its selectivity was confirmed by a lack of effect on 0 activation of phospholipase C by P-adrenoceptors (Figure 0 6b). These results with PPADS strongly suggest that the C- 8 7 6 5 4 3 activation of phospholipase C and the inhibition of adenylyl I 10 9 cyclase are mediated by different P2Y-purinoceptor subtypes. Evidence for heterogeneity within the P2N-purinoceptor sub- 25 class also has been obtained recently in studies of the effects o6x of a large series of nucleotide analogues on the responses E 20 mediated by ATP receptors on the guinea-pig taenia coli, rabbit aorta, rabbit mesenteric artery and turkey erythrocytes (3 (Fischer et al., 1993; Burnstock et al., 1994). 0,( 15 Lambrecht et al. (1992) and Ziganshin et al. (1993) have 0. reported the antagonist properties of PPADS onW P2X- W 10 purinoceptor-mediated mechanical responses of the rabbit 0 vas deferens and urinary bladder detrusor muscle. The effects of PPADS were found to be selective, since mechanical res- 0. ponses induced by a,-adrenoceptor, M2- and M3-muscarinic, 0 H,-histamine, and A,-adenosine receptors were not affected. O I 8 7 6 5 4 3 The binding of [3H]-a,p-methylene ATP to urinary bladder 10 9 membranes was also blocked by PPADS in the same concen- -log [Isoprenaline], M tration-range (Ziganshin et al., 1993). It is clear from our data that PPADS also interacts with P2Y-purinoceptors, and Figure 6 Effects of suramin, reactive blue 2, and PPADS on P- adrenoceptor-mediated activation of phospholipase C in turkey therefore, a more extensive pharmacological analysis of the erythrocyte membranes. Membranes from [3H]-inositol-labelled effects of PPADS on other P2-purinoceptor systems is neces- erythrocytes were prepared as described in Methods. Release of sary to establish its overall specificity towards ATP-mediated [3H]-inositol phosphates was measured in the presence of I jtM responses. GTPyS and the indicated concentrations of isoprenaline in the Suramin and reactive blue 2 have been shown to produce absence (0), or in the presence of 30 IM suramin (b), 30 jM multiple effects on a variety of biological systems (Fedan & reactive blue 2 (*) (a), or 30 tiM PPADS (0) (b). Data shown are Lamport, 1990; Voogd et al., 1993). In spite of this broad the mean of duplicate determinations and the results shown are range of effects, these compounds have proved useful as representative of those obtained in three separate experiments. functional antagonists of ATP-stimulated responses in some tissues (Dunn & Blakely, 1978; Choo, 1980; Manzini et al., 1986; Reilly et al., 1987; Rice & Singleton, 1989; Hoyle et al., 30 fold increase in the EC50 for 2MeSATP (Figure 4a) with- 1990; Leff et al., 1990). Within a defined concentration-range out modifying significatively the concentration-effect curve of both suramin and reactive blue 2 behaved as competitive isoprenaline (Figure 6b). The decrease in the maximal antagonists of the adenylyl cyclase-coupled P2Y-purinoceptor inositol phosphate response to isoprenaline produced by of C6 cells. The apparent affinity of reactive blue 2 30 gLM antagonist was 6%, 23% and 46% for PPADS, (pKB = 7.6 ± 0.1) is the highest that to our knowledge has suramin, and reactive blue 2, respectively. These data indicate been reported for this compound in any ATP response. that the inhibitory effect of suramin, reactive blue 2, and that Suramin and reactive blue 2 also shifted to the right the observed with higher concentrations of PPADS on the concentration-effect curve of 2MeSATP for the stimulation inositol phosphate response to isoprenaline are non- of phospholipase C in the turkey erythrocyte preparation competitive, and probably occur as a result of a non-specific (Figure 5). In contrast to the apparently competitive inhibi- interaction with the G-protein involved or phospholipase C. tion by suramin and reactive blue 2 of P2Y-purinoceptors on Consistent with these results, reactive blue 2 and high con- C6 cells (Figures 1 and 2), the inhibition of the turkey centrations of suramin also inhibited the activation of phos- erythrocyte P2 -purinoceptor was more complex; significant pholipase C produced by A1F4- whereas PPADS at concen- decreases in the maximal response of phospholipase C occur- trations up to 100 jAM had no effect on the response to A1F4 red coincidently with the rightward shifts in the (data not shown). Taken together, these data indicate that in concentration-effect curve for 2MeSATP (Figure 5). Suramin the turkey erythrocyte preparation, the effects of suramin and decreased the maximal response of phospholipase C to reactive blue 2 on the response of 2MeSATP are produced by isoprenaline without affecting the EC;0 for this p- at least two independent effects: a specific effect at the P2Y- adrenoceptor agonist. These data suggest that in the turkey purinoceptor (as evidenced by the shift to the right of the erythrocyte preparation, suramin interacts with the P2y- concentration-effect curve of 2MeSATP (Figure 5)) and a purinoceptor but not with the P-adrenoceptor and that the non-specific effect at the level of G-protein/phospholipase C decrease in the maximal responses to P2Y-purinoceptor- and (as evidenced by their effects on the response to A1F4- and P-adrenoceptor-stimulation results from effects at sites other by the decrease in the maximal phospholipase C response to than the receptor, probably at the level of the G-protein or isoprenaline without a change in the EC50 value). phospholipase C. Consistent with this idea, the effects of suramin on the maximal responses to P2y-purinoceptor- and Discussion P-adrenoceptor-stimulation were similar. Reactive blue 2 also decreased the maximal response of The relative simplicity of the model systems studied here, phospholipase C to isoprenaline without affecting the EC50. together with the exceptional sensitivity of the P2y- However, in contrast with suramin, the depression of the P2-PURINOCEPTOR ANTAGONISTS 619 response of phospholipase C to 2MeSATP produced by reac- In summary, our results show that PPADS selectively tive blue 2 was larger than that of. isoprenaline, consistent blocks the phospholipase C coupled P2y-purinoceptor of with a non competitive effect of reactive blue 2 on the turkey erythrocytes. These results support the hypothesis that P2Y-purinoceptor. activation of phospholipase C and inhibition of adenylyl The overall affinities of PPADS and suramin observed in cyclase are mediated by different P2y-purinoceptors. The these model systems were in a good agreement with the definitive answer to this question should come from values reported in most previous studies with mammalian molecular cloning of the receptors involved. The predicted tissue preparations (Leff et al., 1990; Lambrecht et al., 1992; amino acid sequence of a P2y-purinoceptor subtype has been Wilkinson et al., 1993). However, the affinity of reactive blue reported recently (Webb et al., 1993), and we have shown 2 for the adenylyl cyclase-coupled P2Y-purinoceptor of C6 that this receptor couples to phospholipase C but not to glioma cells (; 30 nM) was at least 50 fold higher than the adenylyl cyclase (Filtz et al., 1994). Availability of the affinities reported for P2y-purinoceptors in other tissues nucleotide sequence of the P2y-purinoceptor should facilitate (Choo, 1981; Crema et al., 1983; Reilly et al., 1987; Rice & the identification of new members of this family of receptors Singleton, 1989; Soltoff et al., 1989). As was observed here, and the identification of their signalling mechanisms. the selectivity and specificity of the available P2-purinoceptor antagonists is frequently limited to a set of particular experi- mental conditions and to a narrow range of concentrations. More selective antagonists obviously are needed. However, all three compounds studied here can be used as a starting This work was supported by United States Public Health Service point for the development of more potent and selective Grants GM 38213, GM 29563, and HL 32322. We gratefully ack- drugs. nowledge the help of Seon Kyung Judy Kim and John O'Tuel.

References ARUNLAKSHANA, 0. & SCHILD, H.O. (1959). Some quantitative uses FLITZ, T.A., LI, Q., BOYER, J.L., NICHOLAS, R.A. & HRDEN, T.K. of drug antagonists. Br. J. Pharmacol. Chemother., 14, 48-58. (1994). Expression of a cloned P2y- that BERRIE, C.P., HAWKINS, P.T., STEPHENS, L.R., HARDEN, T.K. & couples to phospholipase C. Mol. Pharmacol., 46, 8-14. DOWNES, C.P. (1989). Phosphatidylinositol 4,5-bisphosphate FINE, J., COLE, P. & DAVIDSON, J.S. (1989). Extracellular nucleotides hydrolysis in turkey erythrocytes is regulated by P2y- stimulate receptor-mediated calcium mobilization and inositol purinoceptors. Mol. Pharmacol., 35, 526-532. phosphate production in human fibroblasts. Biochem. J., 263, BOYER, J.L., DOWNES, C.P. & HARDEN, T.K. (1989). Kinetics of 371 -376. activation of phospholipase C by P2-purinergic receptor agonists FISCHER, B., BOYER, J.L., HOYLE, C.H.V., ZIGANSHIN, A.U., BRIZ- and guanine nucleotides. J. Biol. Chem., 264, 884-890. ZOLARA, A.L., KNIGHT, G.E., ZIMMET, J., BURNSTOCK, G., BOYER, J.L., LAZAROWSKI, E.R., CHEN, X.-H. & HARDEN, T.K. HARDEN, T.K. & JACOBSON, K.A. (1993). Identification of (1993). Identification of a P2Y-purinergic receptor that inhibits potent, selective P2y-purinoceptor agonists: structure-activity rela- adenylyl cyclase. J. Pharmacol. Exp. Ther., 267, 1140-1146. tionships for 2-thioether derivatives of adenosine 5'-triphosphate. BROWN, H.A., LAZAROWSKI, E.R., BOUCHER, R.C. & HARDEN, T.K. J. Med. Chem., 36, 3937-3946. (1991). Evidence that UTP and ATP regulate phospholipase C FORSBERG, E.J., FEBERSTEIN, G., SHOHAMI, E. & POLLARD, H.B. through a common extracellular 5' nucleotide receptor in airway (1987). stimulates inositol phospholipid epithelial cells. Mol. Pharmacol., 40, 648-655. metabolism and prostacyclin formation in adrenal medullary BURNSTOCK, G. (1978). A basis for distinguishing two types of endothelial cells by means of P2-purinergic receptors. Proc. Natl. purinergic receptors. In Cell Membrane Receptors for Drugs and Acad. Sci. U.S.A., 84, 5630-5634. Hormones, a Multidisciplinary Approach. ed. Staub R.W. & Bolis FREDERICH, R.M., WALDO, G.L., HARDEN, T.K. & PERKINS, J.P. L. pp. 107-118. New York: Raven Press. (1983). Characterization of agonist-induced P-adrenergic receptor- BURNSTOCK, G. & KENNEDY, C. (1985). Is there a basis for distin- specific desensitization in C6-2B glioma cells. J. Cyclic Nucleotide guishing two types of P2-purinoceptors? Gen. Pharmacol., 16, Res., 9, 103-118. 433-440. GORDON, J.L. (1986). Extracellular ATP: effects, sources and fate. BURNSTOCK, G.. FISCHER, B., HOYLE, C.H.V., MAILLARD, M., ZIN- Biochem. J., 233, 309-319. GASHIN, A.U., BRIZZOLARA, A.L., VON ISAKOVICS, A., BOYER, HARDEN, T.K., SCHEER, A.G. & SMITH, M.M. (1982). Differential J.L., HARDEN, T.K. & JACOBSON, K.A. (1994). Structure activity modification of the interaction of cardiac muscarinic cholinergic relationship for derivatives of adenosine-5'-triphosphate as and P-adrenergic receptors with guanine nucleotide binding agonists at P2 purinoceptors: heterogeneity within P2X and P2y site(s). Mol. Pharmacol., 21, 570-580. subtypes. Drug Dev. Res., 31, 206-219. HARDEN, T.K., HAWKINS, P.T., STEPHENS, L., BOYER, J.L. & CHAREST, R., BLACKMORE, P.F. & EXTON, J.H. (1985). Charac- DOWNES, C.P. (1988). Phosphoinositide hydrolysis of terization of responses of isolated hepatocytes to ATP and ADP. 5'-[y-thio]triphosphate-activated phospholipase C of turkey eryth- J. Biol. Chem., 260, 15789-15794. rocyte membranes. Biochem. J., 252, 583-593. CHOO, L.K. (1981). The effect of reactive blue, an antagonist of ATP, HOYLE, C.H.V., KNIGHT, G.E. & BURNSTOCK, G. (1990). Suramin on the isolated urinary bladders of guinea-pig and rat. J. Pharm. antagonizes responses to P2-purinoceptor agonists and purinergic Pharmacol., 33, 248-250. nerve stimulation in the guinea-pig urinary bladder and taenia CREMA, A., FRIGO, G.M., LECCHINI, S., MANZ, L., ONORI, L. & coli. Br. J. Pharmacol., 99, 617-621. TONINI, M. (1983). receptors in the guinea-pig internal LAMBRECHT, G., FRIEBE, T., GRIMM, U., WINDSCHEIF, U., BUN- anal sphincter. Br. J. Pharmacol., 78, 599-603. GARDT, E., HILDERBRANDT. C., BAUMERT, H., SPATZ- DUBYAK, G.R., COWEN, D.S. & MUELLER, L.M. (1988). Activation KUMBEL, G. & MUTSCHLER, E. (1992). PPADS, a novel func- of inositol phospholipid breakdown in HL60 cells by P2- tionally selective antagonist of P2 purinoceptor-mediated res- purinergic receptors for extracellular ATP. Evidence for media- ponses. Eur. J. Pharmacol., 271, 217-219. tion by both pertussis toxin-sensitive and pertussis toxin- LEFF, P., WOOD, B.E. & O'CONNOR, S.E. (1990). Suramin is a slowly- insensitive mechanisms. J. Biol. Chem., 263, 18108-18117. equilibrating but competitive antagonist at P2x-receptors in the DUBYAK, G.R. & EL-MOTASSIM, C. (1993). Signal transduction via rabbit isolated ear artery. Br. J. Pharmacol., 101, 645-649. P2-purinergic receptors for extracellular ATP and other LIN, W.-W. & CHUANG, D.-M. (1993). Endothelin- and ATP-induced nucleotides. Am. J. Physiol., 34, C577-C606. inhibition of adenylyl cyclase activity in C6 glioma cells: role of DUNN, P. & BLAKELY, A.G.H. (1988). Suramin: a reversible P2- GQ and calcium. Mol. Pharmacol., 44, 158-165. purinoceptor antagonist in the mouse vas deferens. Br. J. Phar- LONDOS, C., COOPER, D.M.F. & WOLFF, J. (1980). Subclasses of macol., 93, 243-245. adenosine receptors. Proc. Natl. Acad. Sci. U.S.A., 77, FEDAN, J.S. & LAMPORT, S.J. (1990). P2-purinoceptor antagonists. 2551 -2554. Ann. N.Y. Acad. Sci., 603, 182-197. 620 J.L. BOYER et al.

MANZINI, S., HOYLE, C.H.V. & BURNSTOCK, G. (1986). An elect- SATO, K., OKAJIMA, F. & KONDO, Y. (1992). Extracellular ATP rophysiological analysis of the effect of reactive blue 2, a putative stimulates three different receptor-signal transduction systems in P2-purinergic receptor antagonist, on inhibitory junction poten- FRTL-5 thyroid cells. Biochem. J., 23, 281-287. tials of rat cecum. Eur. J. Pharmacol., 127, 197-204. SOLTOFF, S.P., MCMILLAN, M.K. & TALAMO, B.R. (1989). Coomas- MEEKER, R.B. & HARDEN. T.K. (1982). Muscarinic receptor- sie brilliant blue G is a more potent antagonist of P2 purinergic mediated activation of phosphodiesterase. Mol. Pharmacol., 22, responses than reactive blue 2 (cibacron blue 3GA) in rat parotid 310-319. acinar cells. Biochem. Biophys. Res. Commun., 165, 1279-1285. MEYERHOF, W., MOLLER-BRECHLIN, R. & RICHTER, D. (1991). STUTCHFIELD, J. & COCKCROFT, S. (1990). Undifferentiated HL-60 Molecular cloning of a novel putative G-protein coupled receptor cells respond to extracellular ATP and UTP by stimulating phos- expressed during rat spermiogenesis. FEBS Lett., 284, 155-160. pholipase C activation and exocytosis. FEBS Lett., 262, 256-258. O'CONNOR, S.E., DAINTY, I.A. & LEFF, P. (1991). Further sub- VALEINS, H., MERLE, M. & LABOUESSE, J. (1992). Pre-steady state classification of ATP receptors based on agonist studies. Trends study of the P-adrenergic and purinergic receptor interaction in Pharmacol. Sci., 12, 137-141. C6 cell membranes. Mol. Pharmacol., 42, 1033-1041. OKAJIMA, F., KIOICHI, J.S., NAZAREA, M., SHO, K. & KONDO, Y. VAN CALKER, D., MULLER, M. & HAMPRECHT, B. (1979). Adenosine (1989). A permissive role of pertussis toxin substrate G-protein in regulates via two different types of receptors the accumulation of P2-purinergic stimulation of PI turnover and arachidonic acid cyclic AMP in cultured brain cells. J. Neurochem., 33, 999-1005. release in FRTL-5 thyroid cells. J. Biol. Chem., 264, VAZIRI, C. & DOWNES, C.P. (1992). G-protein-mediated activation of 13029-13037. turkey erythrocyte phospholipase C by P-adrenergic and P2y- OKAJIMA, F., TOKUMITSU, Y., KONDO, Y. & UI, M. (1987). P2- purinergic receptors. Biochem. J., 284, 917-922. purinergic receptors are coupled to two signal transduction VOOGD, T.E., VANSTERKENBURG, E.L.M., WILTING, J. & JANSSEN, systems leading to inhibition of cAMP generation and to produc- L.H.M. (1993). Recent research on the biological activity of tion of inositol triphosphate in rat hepatocytes. J. Biol. Chem., suramin. Pharmacol. Rev., 45, 177-203. 262, 13483-13490. WEBB, T.E., SIMON, J., KRISHEK, B.J., BATESON, A.N., SMART, T.G., PIANET, I., MERLE, M. & LABOUESSE, J. (1989). ADP and, KING, B.F., BURNSTOCK, G. & BARNARD, E.A. (1993). Cloning indirectly, ATP are potent inhibitors of cAMP production in and functional expression of a brain G-protein-coupled ATP intact isoproterenol-stimulated C6 glioma cells. Biochem. Biophys. receptor. FEBS Lett., 324, 219-225. Res. Commun., 163, 1150-1157. WILKINSON, G., PURKISS, J.R. & BOARDER, M.R. (1993). The PIROTTON, S., RASPE, E., DEMOLLE, D., ERNEUX, C. & regulation of aortic endothelial cells by and BOEYNAEMS, J.M. (1987). Involvement of inositol 1,4,5- involves co-existing P2y-purinoceptors and nucleotide receptors triphosphate and calcium in the action of adenine nucleotides on linked to phospholipase C. Br. J. Pharmacol., 108, 689-693. aortic endothelial cells. J. Biol. Chem., 262, 17461-17466. ZHOU, Q.-Y., LI, C., OLAH, M.E., JOHNSON, R.A., STILES, G.A. & REILLY, W.M., SAVILLE, V.L. & BURNSTOCK, G. (1987). An assess- CIVELLI, 0. (1992). Molecular cloning and characterization of an ment of the antagonistic activity of reactive blue 2 at PI- and adenosine receptor: the A3 adenosine receptor. Proc. Natl. Acad. P2-purinoceptors: supporting evidence for purinergic innervation Sci. U.S.A., 89, 7432-7436. of the rabbit portal vein. Eur. J. Pharmacol., 140, 47-53. ZIGANSHIN, A.U., HOYLE, C.H.V., BO, H., LAMBRECHT, G., MUTS- RICE, W.A. & SINGLETON, F.M. (1989). Reactive blue 2 selectively CHLER, E., BAUMERT, H.G. & BURNSTOCK, G. (1993). PPADS inhibits P2y-purinoceptor-stimulated surfactant phospholipid selectively antagonizes P2x-purinoceptor-mediated responses in secretion from isolated alveolar type II cells. Br. J. Pharmacol., the rabbit urinary bladder. Br. J. Pharmacol., 110, 1491-1495. 97, 158-162. ROONEY, T.A., HAGER, R. & THOMAS, A.P. (1991). P-Adrenergic (Received March 18, 1994 receptor-mediated phospholipase C activation independent of Revised May 27, 1994 cAMP formation in turkey erythrocyte membranes. J. Biol. Accepted June 6, 1994) Chem., 266, 15068-15074.