Br. J. Pharmacol. (1991), 104, 685-690
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Direct effects of adenylyl 5'-(,y-methylene)diphosphonate, a stable ATP analogue, on relaxant P1-purinoceptors in smooth muscle S.M.O. Hourani, S.J. Bailey, J. Nicholls & I. Kitchen Receptors and Cellular Regulation Research Group, School of Biological Sciences, University of Surrey, Guildford, Surrey, GU2 5XH 1 Previous results obtained with the rat colon muscularis mucosae, which contracts in response to adenosine and adenosine 5'-triphosphate (ATP), had suggested that adenylyl 5'4fy-methylene)diphosphonate (AMPPCP), a stable ATP analogue, acted on Pl-purinoceptors rather than, as expected, on P2-purinoceptors. This possibility has been examined in two tissues in which adenosine and ATP both cause relaxation, the guinea-pig taenia caeci and the rat duodenum. 2 ATP, 2-methylthio-ATP (2-MeSATP), AMPPCP, adenosine 5'-(a4,-methylene)triphosphonate (AMPCPP) and adenosine each relaxed the taenia caeci and the duodenum, and the order of potency of the nucleotides in each tissue was 2-MeSATP > ATP > AMPCPP > AMPPCP, indicating that these effects were mediated by P2y-purinoceptors. 3 The P, antagonist 8-(p-sulphophenyl)theophyuine (8-SPT) (100pM) did not affect the responses to ATP, 2-MeSATP or AMPCPP in either tissue, but inhibited the responses of adenosine and of AMPPCP in both tissues. In the duodenum a lower concentration of 8-SPT caused a parallel shift to the right of the concentration-response curve to adenosine and to AMPPCP but to different extents, with AMPPCP being inhibited more powerfully than adenosine. A dose-ratio of around 5 was observed for adenosine and AMPPCP at concentrations of 8-SPT of 20gM and 2pM respectively, but Schild analysis resulted in plots with slopes greater than unity. In the taenia caeci, however, 8-SPT inhibited adenosine more powerfully than AMPPCP, and a range of concentrations (10-20gM) only caused a two fold shift in the concentration-response curve for AMPPCP, although the concentration-response curve to adenosine was shifted in a concentration-dependent manner and Schild analysis gave a pA2 value of 5.13 with a slope of 0.90. 4 As has been shown in other tissues, including the guinea-pig taenia caeci, ATP (100pMm) was rapidly dephosphorylated by enzymes present in the rat duodenum, with less than 10% remaining after 20min incubation, whereas AMPPCP (100pMm) was resistant to degradation, with greater than 90% remaining at the same time point. 5 AMPPCP therefore has pronounced but variable agonist actions on P,-purinoceptors, and appears to act entirely via these receptors on the rat duodenum although in the guinea-pig taenia caeci this action is less important and it acts largely via P2y-purinoceptors. These Pl-purinoceptor effects of AMPPCP are direct and are not due to its degradation to adenosine. Keywords: Purines; adenosine; ATP; smooth muscle; analogues of ATP; receptors
Introduction Extracellular adenosine and adenosine 5'-triphosphate (ATP) are thought to cause their pharmacological effects by acting at two distinct receptor types called Pj- and P2-purinoceptors respectively (Burnstock, 1978). Each of these receptors has been subdivided into at least two major subtypes, the Pl-purinoceptors into Al and A2 (Williams, 1987) and the P2-purinoceptors into P2x and P2Y (Burnstock & Kennedy, 1985) with additional P2-purinoceptor subtypes existing on platelets (P2T) and some other cell types such as mast cells (P2Z) (Gordon, 1985). In smooth muscle, adenosine usually causes relaxation probably via A2 receptors but contracts some tissues via Al receptors (Kenakin & Pike, 1987; Smith et al., 1988), whereas ATP can cause either relaxation via P2Y or contraction via P2x depending on the tissue (White, 1988; Burnstock, 1989). Adenosine also presynaptically inhibits neurotransmitter release in many preparations, an effect which is generally thought to be mediated by Al receptors (Williams, 1987; Burnstock, 1989) although a separate subtype, A3, has also been proposed to mediate this effect (Ribeiro & Sebastiao, 1986). As well as acting directly on P2-purinoceptors, ATP is rapidly dephosphorylated ultimately to adenosine by ectonucleotidases present on smooth muscle, and this breakdown can complicate studies of the actions of ATP and its ana-
logues. For example, the resistance to dephosphorylation of ATP analogues correlates well with their potency in contracting the guinea-pig bladder via P2x-purinoceptors, but not with their potency in relaxing the guinea-pig taenia caeci via P2y-purinoceptors, implying that degradation does greatly affect potency on some tissues (Welford et al., 1986; 1987). Although no selective inhibitors of the breakdown of ATP are known (Hourani & Chown, 1989), analogues of ATP in which a bridging oxygen between the phosphate groups has been replaced by a methylene group, such as adenosine 5'-(4methylene)triphosphonate (AMPCPP) and adenylyl 5'fiymethylene)diphosphonate (AMPPCP) are much more resistant to degradation (Moody & Burnstock, 1982; Welford et al., 1986; 1987; Welford & Anderson, 1988; Bailey & Hourani, 1990). These compounds together with 2-substituted ATP analogues such as 2-methylthio-ATP (2-MeSATP), which is broken down as fast as ATP (Welford et al., 1986; 1987), are used to classify P2-purinoceptors, the order of potency on P2X receptors being AMPCPP, AMPPCP > ATP = 2-MeSATP and on P2y receptors being 2-MeSATP > ATP > AMPCPP, AMPPCP (Burnstock & Kennedy, 1985). Although the rapid degradation of ATP and of 2-MeSATP complicates the use of these agonists in classifying receptors, P2X and P2y receptors do possess different structure-activity relationships even for non-degradable analogues (Cusack et al., 1987) and selective
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agonists for these receptors have been found (Hourani et al., 1985; 1986; 1988). In some tissues ATP has effects which appear to be mediated by P1 receptors rather than P2 receptors, although it is not clear whether ATP is acting directly or via adenosine formed by dephosphorylation (Dahlen & Hedqvist, 1980; Collis & Pettinger, 1982; Moody & Burnstock, 1982; Moody et al., 1984; Wiklund et al., 1985; Shinozuka et al., 1988). The finding that the more resistant analogue, AMPPCP, also has actions that appear to be mediated by P1-purinoceptors has been taken by some authors as evidence that ATP acts directly on P1 receptors (Dahlen & Hedqvist, 1980; Collis & Pettinger, 1982; Wiklund et al., 1985) although Shinozuka et al. (1988) suggested that these receptors, since they respond to adenosine and to nucleotides, represent a unique class which they called P3. Moody & Burnstock (1982) attributed the P1 effects of AMPPCP to its breakdown to adenosine, which was, however, much slower than that of ATP. They found no P1 effect of AMPCPP which they believed to be due to a greater resistance to degradation of this analogue although after 10min it had been broken down to the same extent as AMPPCP. We have recently demonstrated (Bailey & Hourani, 1990) that in the rat colon muscularis mucosae, AMPPCP acts via P1 receptors as its effects are inhibited by the adenosine antagonist 8-(p-sulphophenyl)theophylline (8-SPT), whereas ATP, 2-MeSATP and AMPCPP act, as one would expect, on P2 receptors and are not inhibited by 8-SPT. This action of AMPPCP is almost certainly not due to its degradation to adenosine, as AMPPCP was highly resistant to degradation in this tissue whereas ATP was more readily broken down but nevertheless had no effect via P1-purinoceptors. It appears therefore that AMPPCP may have an unexpected direct P1 agonist Action which could explain some of the findings referred to above. However, the rat colon muscularis mucosae has rather unusual responses to purines, as it contracts rather than relaxes to adenosine, and also contracts to ATP via a P2y receptor rather than the expected P2x receptor (Bailey & Hourani, 1990). In the present study we therefore investigated whether the responses of AMPPCP were also mediated by P1-purinoceptors rather than by P2y-purinoceptors in two preparations which relax to adenosine and to ATP, the rat duodenum and the guinea-pig taenia caeci.
Methods Isolated tissue studies Male Wistar rats (200-250 g) were killed by cervical dislocation and the duodenum dissected out by cutting at the base of the pyloris and 1.5cm from this point, mounted in 1O ml organ baths by threads tied around each end so that the lumen was closed and maintained in Krebs solution of the following composition (mM): NaCl 118, KCI 4.8, MgSO4 1.2, CaCl2 2.5, KH2PO4 1.2, NaHCO3 25 and glucose 11. Male albino guinea-pigs (250450 g) were killed by cervical dislocation and the taenia caeci (10mm lengths) dissected out, mounted in 3ml organ baths and maintained in modified Krebs solution of the following composition (mM): NaCl 120, KCI 5.9, MgCl2 1.2, CaCl2 2.5, KH2PO4 1.2, NaHCO3 15.4 and glucose 11. For both preparations the Krebs solution was bubbled with 95% 2 :5% CO2 and warmed to 35°C, and the tissues were equilibrated for at least 45 min before addition of drugs. A resting tension of 1 g was applied to the tissues and force was recorded isometrically with Grass FT03 transducers and displayed on Grass 79D polygraphs. Concentration-response curves were obtained noncumulatively, and the inhibitory responses were quantified by precontracting the tissues with carbachol (40 nm for the taenia caeci, 100 nm for the duodenum) before challenge with purines, and expressed as % inhibition of carbachol contraction. In the taenia caeci, the concentration of carbachol used induced a
sustained contraction of 50-70% of the maximal response, whereas in the duodenum the concentration of carbachol used increased the spontaneous contractions of the tissue to around 50% of the maximum, and made them more regular (see Nicholls et al., 1990 for further details and representative traces for the duodenum). In each tissue, doses of carbachol were applied at approximately 10min intervals, and no tachyphylaxis was observed to either carbachol or any of the purines used. After control concentration-response curves to purines had been obtained, the tissues were incubated for 30 min with 8-SPT and the concentration-response curves were repeated in the presence of this antagonist. Responses to agonists returned to the control levels after no more than 40 min washout.
Degradation studies The rat duodenum was dissected out as above and incubated in gassed Krebs solution at 35°C for 3 h with the buffer being changed every 15 min. The tissues were then placed in aerated Krebs solution (2.5 ml) at 35°C, containing ATP or AMPPCP (100pUM), and 100,a aliquots were taken at various times and frozen for later analysis by high performance liquid chromatography (h.p.l.c.). To check for leakage of enzymes, tissues which had been washed as above were incubated in Krebs solution (2.5 ml) alone for 45 min, the tissues were then removed, the purines added to the buffer which had contained the tissue and samples were taken for analysis at 0 and 20 min. Samples were analysed with a Waters h.p.l.c. with a Techsphere 5pum ODS C18 column, eluted with 0.1 M KH2PO4/8mM tetrabutylammonium hydrogen sulphate (pH 6.0) (Solvent A) and a 60:40 mixture of Solvent A and acetonitrile (pH 6.73) (Solvent B), using a non-linear gradient (0-2.5 min 0% B, 2.55min 0-20% B, 5-10min 2040% B, 10-13 min 40-100% B, 13-18min 100% B), at a flow rate of 1.3mlmin-1. The purines were detected by u.v. absorbance at 259 nm, and quantified from the height of their absorbance peaks, which was linearly related to the concentration in the incubation mixture.
Materials Adenosine, ATP, AMPPCP, AMPCPP, tetrabutylammonium hydrogen sulphate and carbachol were obtained from Sigma Chemical Co., U.K., 2-MeSATP and 8-SPT were obtained from Research Biochemicals Inc., and buffer salts and solvents were of analytical or h.p.l.c. grade and were obtained from BDH.
Results
Guinea-pig taenia caeci ATP, adenosine, AMPPCP, AMPCPP and 2-MeSATP each relaxed the carbachol-contracted taenia caeci, and the order of potency was 2-MeSATP > ATP > AMPCPP > AMPPCP > adenosine (Figure 1). The responses to ATP, 2-MeSATP and AMPCPP were not inhibited by 8-SPT (100puM) (Figure la), whereas those to adenosine (Figure lb) and, to a lesser extent, those to AMPPCP were inhibited (Figure ic). The inhibition of adenosine by 8-SPT was concentrationdependent, with increasing shifts in the concentrationresponse curve as the concentration of antagonist was increased from 10-200pM (Figure 2a). In contrast, the same shift, approximately 2 fold, was observed in the concentrationresponse curve to AMPPCP for concentrations of antagonist from 10-200,UM (Figure 2c). The Schild plot for 8-SPT as an inhibitor of the effects of adenosine, derived from the data in Figure 2a by calculating dose-ratios for 25% inhibition of the carbachol-induced contraction in the presence and absence of antagonist, was linear (r = 0.993) with a slope of 0.90 and gave a pA2 value of 5.13 (Figure 2b).
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Figure 2 Relaxations of the guinea-pig taenia caeci induced by (a) adenosine or (c) AMPPCP in the absence (0) or presence of 8-SPT at 10pM (0), 20OuM (A), 50,oM (U), 100pM (V) or 200paM (*). Each point is the mean of at least 4 determinations and the vertical bars show s.e.mean. The Schild plot derived from the data in (a) is shown in (b). The line shown is that calculated by least squares linear regression analysis, has a slope of 0.90 and gives a pA2 value of 5.13 taken from the intercept on the horizontal axis. Abbreviations as in text.
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Figure 3 Effects of purines on rat duodenum in the absence (open symbols) or presence (closed symbols) of 8-SPT (10O1UM): (a) ATP
(O. 0); AMPCPP (A, A) and 2-MeSATP (EO, *); (b) adenosine (V, V); (c) AMPPCP (O, *). Each point is the mean of at least S determinations and the vertical bars show s.e.mean. Abbreviations as in text.
Rat duodenum ATP, adenosine, AMPCPP, AMPPCP and 2-MeSATP all relaxed the carbachol-contracted rat duodenum, and the order of was 2-MeSATP > ATP > AMPCPP > potency AMPPCP = adenosine (Figure 3). The responses to ATP, 2MeSATP and AMPCPP were not inhibited by 8-SPT (100pM) (Figure 3a), whereas those to adenosine (Figure 3b) and AMPPCP (Figure 3c) were abolished. Lower concentrations of 8-SPT shifted the concentrationresponse curves for adenosine and for AMPPCP to the right but to different extents, with a dose-ratio of around 5 being observed for adenosine and AMPPCP at concentrations of 8-SPT of 20pM and 2pM respectively (Figure 4). Schild analysis in this tissue using either agonist resulted in Schild plots with slopes greater than I (results not shown).
Degradation studies ATP (100pM) was rapidly degraded by the rat duodenum with only 9% remaining after 20min, and was mainly converted to AMP (33%) and inosine (45%) during this time (Figure 5a). AMPPCP (100pM) was resistant to degradation, with 92% remaining after 20min incubation (Figure Sb). ATP (100pM) added to buffer from which the tissue had been removed after incubation alone for 45min was degraded with 29% remaining after 20min, and the main degradation products were ADP (32%) and AMP (35%), with only 3% inosine being formed (Figure Sc). When AMPPCP was added to buffer from which the tissue had been removed, 97% remained after
20 min incubation (Figure 5d). No degradation of either purine was observed after incubation with buffer in which no tissue had been preincubated.
Discussion These results show that AMPPCP acts directly via Pl-purinoceptors in two tissues which relax to ATP and adenosine, as well as in the rat colon muscularis mucosae which contracts to the purines (Bailey & Hourani, 1990). The extent to which the actions of AMPPCP are mediated by P,-purinoceptors is not, however, the same in all tissues, as in the taenia caeci no more than a two fold shift in the doseresponse curve could be achieved even with 200pM 8-SPT, whereas in the rat duodenum, as in the rat colon, the doseresponse curve to AMPPCP was shifted at least as much as that to adenosine, indicating that in these tissues AMPPCP acts entirely via P1-purinoceptors. These actions of AMPPCP are unlikely to be due to adenosine formed as a result of degradation, as AMPPCP is known to be resistant to degradation in the guinea-pig taenia caeci (Welford et al., 1986) and the rat colon (Bailey & Hourani, 1990) and was also degraded to a negligible extent by the rat duodenum (Figure 5). Also, the actions of ATP, which is degraded much faster than AMPPCP in these tissues, were not inhibited by 8-SPT, showing that the degradation products do not influence activity. Indeed, although AMP and ADP were formed, the main product of degradation of ATP was the inactive inosine, and no adenosine was detected (Figure Sa), confirming the work of Franco et al. (1988), who also found the activity of adenosine
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inosine (V) by enzymes present in the rat duodenum (a and b) or released into buffer from the rat duodenum (c and d). Each point is the mean of at least 4 determinations and the vertical bars show
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deaminase to be relatively high in the rat duodenum. Some leakage of enzymes was detected, as ATP was also degraded by buffer in which tissue had been incubated, although in this case. the main degradation products were ADP and AMP, suggesting that 5'-nucleotidase and adenosine deaminase had remained bound to the tissue (Figure 5c). The order of potency of the ATP analogues in both tissues confirms that the P2-purinoceptor is of the P2Y subtype, with 2-MeSATP being much more potent than ATP and AMPCPP being somewhat less potent, with none of these compounds having any effect on Pl-purinoceptors which could interfere with this analysis. Indeed, the concentrationresponse curves for these compounds on the two tissues looked almost identical, with the potencies on each tissue being very similar and the curve for AMPCPP being steeper than those for ATP or 2-MeSATP, possibly because of its resistance to degradation, and therefore crossing the top of the ATP curve. This supports the idea that the receptors in these tissues are the same, although agonist potencies and the shapes of concentration-response curves do not provide conclusive information on receptor subtypes, and definite proof must await the development of true subtype-selective, competitive ATP antagonists. The results described here show that AMPPCP is of little value for the definition of P2-purinoceptor subtypes, as although on both tissues it is indeed less potent than ATP as expected for a
P2y-purinoceptor (Burnstock & Kennedy, 1985), it has a significant but variable effect on Pl-purinoceptors. In the taenia caeci its true potency at P2y-purinoceptors is presumably that observed in the presence of 10-200,uM 8-SPT and is thus roughly half that observed in the absence of antagonist, whereas in the duodenum no P2y effect is detectable at the concentrations tested and AMPPCP appears to act entirely as a P1 agonist in this tissue. Whether the observed differences in the effects of AMPPCP between the tissues reflect qualitative differences between the P2y receptor populations in these tissues is hard to investigate with the pharmacological tools available, but there seems no compelling reason to propose P2Y subtypes on this evidence. There are however some hints that the P1-purinoceptors may be different, although the small differences observed in the inhibitory potency of 8-SPT are hard to interpret with certainty, particularly in the absence of convincing Schild plots in the duodenum and in view of reports that P1 agonists and antagonists may in general have a rather higher affinity for rat tissues than for guinea-pig tissues (Collis et al., 1989). Why the Schild plots in the rat duodenum gave slopes greater than unity is not clear, but problems were also encountered with the dose-dependence of the antagonist in the rat colon (Bailey & Hourani, unpublished observations), and this could imply that 8-SPT is not acting in a purely competitive fashion in these tissues. In the guinea-pig taenia caeci the Schild plot against adenosine was satisfactory and no problems were reported by Collis et al. (1987) on the guinea-pig atria and aorta, showing that in these tissues 8-SPT is acting competitively, and that again there may be differences between rat and guinea-pig tissues. 8-SPT did not cause dose-dependent shifts in the concentration-response curve for AMPPCP in the taenia caeci where it was acting as a competitive adenosine antagonist, showing that the P1 action of AMPPCP is only a component of its overall effect in this tissue. In conclusion, it is now clear that AMPPCP cannot simply be regarded as a P2 agonist but also has a pronounced direct P1 agonist action as well, and the extent of this varies depending on the tissue chosen. This direct P1 effect complicates the use of AMPPCP as a stable ATP analogue, and may explain some earlier findings that AMPPCP (and ATP) acted like adenosine in some tissues (Dahlen & Hedqvist, 1980; Collis &
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Pettinger, 1982; Moody & Burnstock, 1982; Moody et al., 1984; Wiklund et al., 1985; Shinozuka et al., 1988). ATP could well be acting in these cases via its breakdown product adenosine, although we have found no evidence for a direct or indirect P1 effect, but it is likely that AMPPCP is acting directly on Pl-purinoceptors as in the results described here. The related stable analogue, AMPCPP, would appear to be a more suitable analogue to use in studies of P2-purinoceptors as it lacks this anomalous P1 effect, although it is not as selective as AMPPCP for P2x receptors and has a higher potency on P2y receptors, being almost as potent as ATP itself in the taenia caeci and rat duodenum, and furthermore has a greater effect than ATP at high concentrations. The potent, specific
P2x agonist L-AMPPCP, the enantiomer of AMPPCP, is probably the best compound to use, as it has little or no P2Y or P1 effects in the guinea-pig taenia caeci, aorta or ileum (Hourani et al., 1985; 1986) or the rat colon muscularis mucosae (Bailey & Hourani, 1990). Our results with AMPPCP show that it is possible for an ATP analogue to act directly on P,-purinoceptors however unlikely this may seem from the known structure-activity relationships, and emphasise the danger of making assumptions about the receptor selectivity of an agonist based solely on its structure. We thank the MRC for support for SJ.B. (98723473N), and the SERC for a studentship for J.N.
References BAILEY, S.J. & HOURANI, S.M.O. (1990). A study of the purinoceptors mediating contraction in the rat colon. Br. J. Pharmacol., 100, 753-756. BRUNS, R.F., LU, G.H. & PUGSLEY, T.A. (1986). Characterization of the A2 adenosine receptor labeled by [3H]NECA in rat striatal membranes. Mol. Pharmacol., 29, 331-346. BURNSTOCK, G. (1978). A basis for distinguishing two types of purinergic receptor. In Cell and Membrane Receptorsfor Drugs and Hormones: a Multidisciplinary Approach. ed. Bolis, L. & Straub, R.W. pp. 107-118. New York: Raven. BURNSTOCK, G. (1989). Purine receptors. In Adenosine Receptors in the Nervous System. ed. Ribeiro, J.A. pp. 1-14. London: Taylor & Francis. BURNSTOCK, G. & KENNEDY, C. (1985). Is there a basis for distinguishing two types of P2-purinoceptor? Gen. Pharmacol., 16, 433-440. COLLIS, M.G., JACOBSON, K.A. & TOMKINS, D.M. (1987). Apparent affinity of some 8-phenyl-substituted xanthines at adenosine receptors in guinea-pig aorta and atria. Br. J. Pharmacol., 92, 69-75. COLLIS, M.G. & PETTINGER, S.J. (1982). Can ATP stimulate P1-receptors in guinea-pig atrium without conversion to adenosine? Eur. J. Pharmacol., 81, 521-529. COLLIS, M.G., STOGGALL, S.M. & MARTIN, F.M. (1989). Apparent affinity of 1,3-dipropyl-8-cyclopentylxanthine for adenosine receptors in isolated tissues from guinea-pigs. Br. J. Pharmacol., 97, 1274-1278. CUSACK, N.J., HOURANI, S.M.O., LOIZOU, G.D. & WELFORD, L.A.
(1987). Pharmacological effects of isopolar phosphonate analogues of ATP on P2-purinoceptors in guinea-pig taenia coli and urinary bladder. Br. J. Pharmacol., 90, 791-795. DAHLEN, S.-E. & HEDQVIST, P. (1980). ATP, fy-methylene-ATP, and adenosine inhibit non-cholinergic non-adrenergic transmission in rat urinary bladder. Acta Physiol. Scand., 109, 137-142. FRANCO, R., HOYLE, C.H.V., CENTELLES, J.J. & BURNSTOCK, G.
(1988). Degradation of adenosine by extracellular adenosine deaminase in the rat duodenum. Gen. Pharmacol., 19, 679-681. GORDON, J.L. (1985). Extracellular ATP: Effects, sources and fate. Biochem. J., 233, 309-319. HOURANI, S.M.O. & CHOWN, J.A. (1989). The effects of some possible inhibitors of ectonucleotidases on the breakdown and pharmacological effects of ATP in the guinea-pig urinary bladder. Gen. Pharmacol., 20, 413-416. HOURANI, S.M.O., LOIZOU, G.D. & CUSACK, N.J. (1986). Pharmacological effects of L-AMPPCP on ATP receptors in smooth muscle. Eur. J. Pharmacol., 131, 99-103. HOURANI, S.M.O., WELFORD, L.A. & CUSACK, N.J. (1985). LAMPPCP, an ATP receptor agonist in guinea-pig bladder, is inactive on taenia coli. Eur. J. Pharmacol., 108, 197-200.
HOURANI, S.M.O., WELFORD, L.A., LOIZOU, G.D. & CUSACK, N.J.
(1988). Adenosine 5'(2-fluorodiphosphate) is a selective agonist at P2-purinoceptors mediating relaxation of smooth muscle. Eur. J. Pharmacol., 147, 131-136. KENAKIN, T.P. & PIKE, N.B. (1987). An in vitro analysis of purinemediated renal vasoconstriction in rat isolated kidney. Br. J. Pharmacol., 90, 373-381. MOODY, C.J. & BURNSTOCK, G. (1982). Evidence for the presence of P,-purinoceptors on cholinergic nerve terminals in the guinea-pig ileum. Eur. J. Pharmacol., 77, 1-9. MOODY, C.J., MEGHJI, P. & BURNSTOCK, G. (1984). Stimulation of P1-purinoceptors by ATP depends partly on its conversion to AMP and adenosine and partly on direct action. Eur. J. Pharmacol., 97, 47-54. NICHOLLS, J., HOURANI, S.M.O. & KITCHEN, I. (1990). The ontogeny of purinoceptors in rat urinary bladder and duodenum. Br. J. Pharmacol., 100, 874-878. RIBEIRO, J.A. & SEBASTIAO, A.M. (1986). Adenosine receptors and calcium: Basis for proposing a third (A3) adenosine receptor. Prog. Neurobiol., 26, 179-209. SHINOZUKA, K., BJUR, R.A. & WESTFALL, D.P. (1988). Characterisation of prejunctional purinoceptors on adrenergic nerves of the rat caudal artery. Naunyn-Schmiedebergs Arch. Pharmacol, 338, 221-227. SMITH, M.A., BUXTON, I.L.O. & WESTFALL, D.P. (1988). Pharmacological classification of receptors for adenyl purines in guinea-pig myometrium. J. Pharmacol. Exp. Ther., 247, 1059-1063. WELFORD, L.A. & ANDERSON, W.H. (1988). Purine receptors and guinea-pig trachea: evidence for a direct action of ATP. Br. J. Pharmacol., 95, 689-694. WELFORD, L.A., CUSACK, N.J. & HOURANI, S.M.O. (1986). ATP analogues and the guinea-pig taenia coli: a comparison of the structure activity relationships of ectonucleotidases with those of the P2-purinoceptor. Eur. J. Pharmacol., 129, 217-224. WELFORD, L.A., CUSACK, N.J. & HOURANI, S.M.O. (1987). The structure-activity relationships of ectonucleotidases and of excitatory P2-purinoceptors: evidence that dephosphorylation of ATP analogues reduces pharmacological potency. Eur. J. Pharmacol.,
141,123-130. WHITE, T.D. (1988). Role of adenine compounds in autonomic neurotransmission. Pharmacol. Ther., 38, 129-168. WIKLUND, N.P., GUSTAFSSON, L.E. & LUNDIN, J. (1985). Pre- and post-junctional modulation of cholinergic neuroeffector transmission by adenine nucleotides. Experiments with agonist and antagonist. Acta Physiol. Scand., 125, 681-691. WILLIAMS, M. (1987). Purine receptors in mammalian tissues: Pharmacology and functional significance. Annu. Rev. Pharmacol. Toxicol., 27, 315-345. (Received January 31, 1991 Revised July 30, 1991 Accepted August 8, 1991)
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Direct effects of adenylyl 5'-(,y-methylene)diphosphonate, a stable ATP analogue, on relaxant P1-purinoceptors in smooth muscle S.M.O. Hourani, S.J. Bailey, J. Nicholls & I. Kitchen Receptors and Cellular Regulation Research Group, School of Biological Sciences, University of Surrey, Guildford, Surrey, GU2 5XH 1 Previous results obtained with the rat colon muscularis mucosae, which contracts in response to adenosine and adenosine 5'-triphosphate (ATP), had suggested that adenylyl 5'4fy-methylene)diphosphonate (AMPPCP), a stable ATP analogue, acted on Pl-purinoceptors rather than, as expected, on P2-purinoceptors. This possibility has been examined in two tissues in which adenosine and ATP both cause relaxation, the guinea-pig taenia caeci and the rat duodenum. 2 ATP, 2-methylthio-ATP (2-MeSATP), AMPPCP, adenosine 5'-(a4,-methylene)triphosphonate (AMPCPP) and adenosine each relaxed the taenia caeci and the duodenum, and the order of potency of the nucleotides in each tissue was 2-MeSATP > ATP > AMPCPP > AMPPCP, indicating that these effects were mediated by P2y-purinoceptors. 3 The P, antagonist 8-(p-sulphophenyl)theophyuine (8-SPT) (100pM) did not affect the responses to ATP, 2-MeSATP or AMPCPP in either tissue, but inhibited the responses of adenosine and of AMPPCP in both tissues. In the duodenum a lower concentration of 8-SPT caused a parallel shift to the right of the concentration-response curve to adenosine and to AMPPCP but to different extents, with AMPPCP being inhibited more powerfully than adenosine. A dose-ratio of around 5 was observed for adenosine and AMPPCP at concentrations of 8-SPT of 20gM and 2pM respectively, but Schild analysis resulted in plots with slopes greater than unity. In the taenia caeci, however, 8-SPT inhibited adenosine more powerfully than AMPPCP, and a range of concentrations (10-20gM) only caused a two fold shift in the concentration-response curve for AMPPCP, although the concentration-response curve to adenosine was shifted in a concentration-dependent manner and Schild analysis gave a pA2 value of 5.13 with a slope of 0.90. 4 As has been shown in other tissues, including the guinea-pig taenia caeci, ATP (100pMm) was rapidly dephosphorylated by enzymes present in the rat duodenum, with less than 10% remaining after 20min incubation, whereas AMPPCP (100pMm) was resistant to degradation, with greater than 90% remaining at the same time point. 5 AMPPCP therefore has pronounced but variable agonist actions on P,-purinoceptors, and appears to act entirely via these receptors on the rat duodenum although in the guinea-pig taenia caeci this action is less important and it acts largely via P2y-purinoceptors. These Pl-purinoceptor effects of AMPPCP are direct and are not due to its degradation to adenosine. Keywords: Purines; adenosine; ATP; smooth muscle; analogues of ATP; receptors
Introduction Extracellular adenosine and adenosine 5'-triphosphate (ATP) are thought to cause their pharmacological effects by acting at two distinct receptor types called Pj- and P2-purinoceptors respectively (Burnstock, 1978). Each of these receptors has been subdivided into at least two major subtypes, the Pl-purinoceptors into Al and A2 (Williams, 1987) and the P2-purinoceptors into P2x and P2Y (Burnstock & Kennedy, 1985) with additional P2-purinoceptor subtypes existing on platelets (P2T) and some other cell types such as mast cells (P2Z) (Gordon, 1985). In smooth muscle, adenosine usually causes relaxation probably via A2 receptors but contracts some tissues via Al receptors (Kenakin & Pike, 1987; Smith et al., 1988), whereas ATP can cause either relaxation via P2Y or contraction via P2x depending on the tissue (White, 1988; Burnstock, 1989). Adenosine also presynaptically inhibits neurotransmitter release in many preparations, an effect which is generally thought to be mediated by Al receptors (Williams, 1987; Burnstock, 1989) although a separate subtype, A3, has also been proposed to mediate this effect (Ribeiro & Sebastiao, 1986). As well as acting directly on P2-purinoceptors, ATP is rapidly dephosphorylated ultimately to adenosine by ectonucleotidases present on smooth muscle, and this breakdown can complicate studies of the actions of ATP and its ana-
logues. For example, the resistance to dephosphorylation of ATP analogues correlates well with their potency in contracting the guinea-pig bladder via P2x-purinoceptors, but not with their potency in relaxing the guinea-pig taenia caeci via P2y-purinoceptors, implying that degradation does greatly affect potency on some tissues (Welford et al., 1986; 1987). Although no selective inhibitors of the breakdown of ATP are known (Hourani & Chown, 1989), analogues of ATP in which a bridging oxygen between the phosphate groups has been replaced by a methylene group, such as adenosine 5'-(4methylene)triphosphonate (AMPCPP) and adenylyl 5'fiymethylene)diphosphonate (AMPPCP) are much more resistant to degradation (Moody & Burnstock, 1982; Welford et al., 1986; 1987; Welford & Anderson, 1988; Bailey & Hourani, 1990). These compounds together with 2-substituted ATP analogues such as 2-methylthio-ATP (2-MeSATP), which is broken down as fast as ATP (Welford et al., 1986; 1987), are used to classify P2-purinoceptors, the order of potency on P2X receptors being AMPCPP, AMPPCP > ATP = 2-MeSATP and on P2y receptors being 2-MeSATP > ATP > AMPCPP, AMPPCP (Burnstock & Kennedy, 1985). Although the rapid degradation of ATP and of 2-MeSATP complicates the use of these agonists in classifying receptors, P2X and P2y receptors do possess different structure-activity relationships even for non-degradable analogues (Cusack et al., 1987) and selective
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agonists for these receptors have been found (Hourani et al., 1985; 1986; 1988). In some tissues ATP has effects which appear to be mediated by P1 receptors rather than P2 receptors, although it is not clear whether ATP is acting directly or via adenosine formed by dephosphorylation (Dahlen & Hedqvist, 1980; Collis & Pettinger, 1982; Moody & Burnstock, 1982; Moody et al., 1984; Wiklund et al., 1985; Shinozuka et al., 1988). The finding that the more resistant analogue, AMPPCP, also has actions that appear to be mediated by P1-purinoceptors has been taken by some authors as evidence that ATP acts directly on P1 receptors (Dahlen & Hedqvist, 1980; Collis & Pettinger, 1982; Wiklund et al., 1985) although Shinozuka et al. (1988) suggested that these receptors, since they respond to adenosine and to nucleotides, represent a unique class which they called P3. Moody & Burnstock (1982) attributed the P1 effects of AMPPCP to its breakdown to adenosine, which was, however, much slower than that of ATP. They found no P1 effect of AMPCPP which they believed to be due to a greater resistance to degradation of this analogue although after 10min it had been broken down to the same extent as AMPPCP. We have recently demonstrated (Bailey & Hourani, 1990) that in the rat colon muscularis mucosae, AMPPCP acts via P1 receptors as its effects are inhibited by the adenosine antagonist 8-(p-sulphophenyl)theophylline (8-SPT), whereas ATP, 2-MeSATP and AMPCPP act, as one would expect, on P2 receptors and are not inhibited by 8-SPT. This action of AMPPCP is almost certainly not due to its degradation to adenosine, as AMPPCP was highly resistant to degradation in this tissue whereas ATP was more readily broken down but nevertheless had no effect via P1-purinoceptors. It appears therefore that AMPPCP may have an unexpected direct P1 agonist Action which could explain some of the findings referred to above. However, the rat colon muscularis mucosae has rather unusual responses to purines, as it contracts rather than relaxes to adenosine, and also contracts to ATP via a P2y receptor rather than the expected P2x receptor (Bailey & Hourani, 1990). In the present study we therefore investigated whether the responses of AMPPCP were also mediated by P1-purinoceptors rather than by P2y-purinoceptors in two preparations which relax to adenosine and to ATP, the rat duodenum and the guinea-pig taenia caeci.
Methods Isolated tissue studies Male Wistar rats (200-250 g) were killed by cervical dislocation and the duodenum dissected out by cutting at the base of the pyloris and 1.5cm from this point, mounted in 1O ml organ baths by threads tied around each end so that the lumen was closed and maintained in Krebs solution of the following composition (mM): NaCl 118, KCI 4.8, MgSO4 1.2, CaCl2 2.5, KH2PO4 1.2, NaHCO3 25 and glucose 11. Male albino guinea-pigs (250450 g) were killed by cervical dislocation and the taenia caeci (10mm lengths) dissected out, mounted in 3ml organ baths and maintained in modified Krebs solution of the following composition (mM): NaCl 120, KCI 5.9, MgCl2 1.2, CaCl2 2.5, KH2PO4 1.2, NaHCO3 15.4 and glucose 11. For both preparations the Krebs solution was bubbled with 95% 2 :5% CO2 and warmed to 35°C, and the tissues were equilibrated for at least 45 min before addition of drugs. A resting tension of 1 g was applied to the tissues and force was recorded isometrically with Grass FT03 transducers and displayed on Grass 79D polygraphs. Concentration-response curves were obtained noncumulatively, and the inhibitory responses were quantified by precontracting the tissues with carbachol (40 nm for the taenia caeci, 100 nm for the duodenum) before challenge with purines, and expressed as % inhibition of carbachol contraction. In the taenia caeci, the concentration of carbachol used induced a
sustained contraction of 50-70% of the maximal response, whereas in the duodenum the concentration of carbachol used increased the spontaneous contractions of the tissue to around 50% of the maximum, and made them more regular (see Nicholls et al., 1990 for further details and representative traces for the duodenum). In each tissue, doses of carbachol were applied at approximately 10min intervals, and no tachyphylaxis was observed to either carbachol or any of the purines used. After control concentration-response curves to purines had been obtained, the tissues were incubated for 30 min with 8-SPT and the concentration-response curves were repeated in the presence of this antagonist. Responses to agonists returned to the control levels after no more than 40 min washout.
Degradation studies The rat duodenum was dissected out as above and incubated in gassed Krebs solution at 35°C for 3 h with the buffer being changed every 15 min. The tissues were then placed in aerated Krebs solution (2.5 ml) at 35°C, containing ATP or AMPPCP (100pUM), and 100,a aliquots were taken at various times and frozen for later analysis by high performance liquid chromatography (h.p.l.c.). To check for leakage of enzymes, tissues which had been washed as above were incubated in Krebs solution (2.5 ml) alone for 45 min, the tissues were then removed, the purines added to the buffer which had contained the tissue and samples were taken for analysis at 0 and 20 min. Samples were analysed with a Waters h.p.l.c. with a Techsphere 5pum ODS C18 column, eluted with 0.1 M KH2PO4/8mM tetrabutylammonium hydrogen sulphate (pH 6.0) (Solvent A) and a 60:40 mixture of Solvent A and acetonitrile (pH 6.73) (Solvent B), using a non-linear gradient (0-2.5 min 0% B, 2.55min 0-20% B, 5-10min 2040% B, 10-13 min 40-100% B, 13-18min 100% B), at a flow rate of 1.3mlmin-1. The purines were detected by u.v. absorbance at 259 nm, and quantified from the height of their absorbance peaks, which was linearly related to the concentration in the incubation mixture.
Materials Adenosine, ATP, AMPPCP, AMPCPP, tetrabutylammonium hydrogen sulphate and carbachol were obtained from Sigma Chemical Co., U.K., 2-MeSATP and 8-SPT were obtained from Research Biochemicals Inc., and buffer salts and solvents were of analytical or h.p.l.c. grade and were obtained from BDH.
Results
Guinea-pig taenia caeci ATP, adenosine, AMPPCP, AMPCPP and 2-MeSATP each relaxed the carbachol-contracted taenia caeci, and the order of potency was 2-MeSATP > ATP > AMPCPP > AMPPCP > adenosine (Figure 1). The responses to ATP, 2-MeSATP and AMPCPP were not inhibited by 8-SPT (100puM) (Figure la), whereas those to adenosine (Figure lb) and, to a lesser extent, those to AMPPCP were inhibited (Figure ic). The inhibition of adenosine by 8-SPT was concentrationdependent, with increasing shifts in the concentrationresponse curve as the concentration of antagonist was increased from 10-200pM (Figure 2a). In contrast, the same shift, approximately 2 fold, was observed in the concentrationresponse curve to AMPPCP for concentrations of antagonist from 10-200,UM (Figure 2c). The Schild plot for 8-SPT as an inhibitor of the effects of adenosine, derived from the data in Figure 2a by calculating dose-ratios for 25% inhibition of the carbachol-induced contraction in the presence and absence of antagonist, was linear (r = 0.993) with a slope of 0.90 and gave a pA2 value of 5.13 (Figure 2b).
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Figure 2 Relaxations of the guinea-pig taenia caeci induced by (a) adenosine or (c) AMPPCP in the absence (0) or presence of 8-SPT at 10pM (0), 20OuM (A), 50,oM (U), 100pM (V) or 200paM (*). Each point is the mean of at least 4 determinations and the vertical bars show s.e.mean. The Schild plot derived from the data in (a) is shown in (b). The line shown is that calculated by least squares linear regression analysis, has a slope of 0.90 and gives a pA2 value of 5.13 taken from the intercept on the horizontal axis. Abbreviations as in text.
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Figure 3 Effects of purines on rat duodenum in the absence (open symbols) or presence (closed symbols) of 8-SPT (10O1UM): (a) ATP
(O. 0); AMPCPP (A, A) and 2-MeSATP (EO, *); (b) adenosine (V, V); (c) AMPPCP (O, *). Each point is the mean of at least S determinations and the vertical bars show s.e.mean. Abbreviations as in text.
Rat duodenum ATP, adenosine, AMPCPP, AMPPCP and 2-MeSATP all relaxed the carbachol-contracted rat duodenum, and the order of was 2-MeSATP > ATP > AMPCPP > potency AMPPCP = adenosine (Figure 3). The responses to ATP, 2MeSATP and AMPCPP were not inhibited by 8-SPT (100pM) (Figure 3a), whereas those to adenosine (Figure 3b) and AMPPCP (Figure 3c) were abolished. Lower concentrations of 8-SPT shifted the concentrationresponse curves for adenosine and for AMPPCP to the right but to different extents, with a dose-ratio of around 5 being observed for adenosine and AMPPCP at concentrations of 8-SPT of 20pM and 2pM respectively (Figure 4). Schild analysis in this tissue using either agonist resulted in Schild plots with slopes greater than I (results not shown).
Degradation studies ATP (100pM) was rapidly degraded by the rat duodenum with only 9% remaining after 20min, and was mainly converted to AMP (33%) and inosine (45%) during this time (Figure 5a). AMPPCP (100pM) was resistant to degradation, with 92% remaining after 20min incubation (Figure Sb). ATP (100pM) added to buffer from which the tissue had been removed after incubation alone for 45min was degraded with 29% remaining after 20min, and the main degradation products were ADP (32%) and AMP (35%), with only 3% inosine being formed (Figure Sc). When AMPPCP was added to buffer from which the tissue had been removed, 97% remained after
20 min incubation (Figure 5d). No degradation of either purine was observed after incubation with buffer in which no tissue had been preincubated.
Discussion These results show that AMPPCP acts directly via Pl-purinoceptors in two tissues which relax to ATP and adenosine, as well as in the rat colon muscularis mucosae which contracts to the purines (Bailey & Hourani, 1990). The extent to which the actions of AMPPCP are mediated by P,-purinoceptors is not, however, the same in all tissues, as in the taenia caeci no more than a two fold shift in the doseresponse curve could be achieved even with 200pM 8-SPT, whereas in the rat duodenum, as in the rat colon, the doseresponse curve to AMPPCP was shifted at least as much as that to adenosine, indicating that in these tissues AMPPCP acts entirely via P1-purinoceptors. These actions of AMPPCP are unlikely to be due to adenosine formed as a result of degradation, as AMPPCP is known to be resistant to degradation in the guinea-pig taenia caeci (Welford et al., 1986) and the rat colon (Bailey & Hourani, 1990) and was also degraded to a negligible extent by the rat duodenum (Figure 5). Also, the actions of ATP, which is degraded much faster than AMPPCP in these tissues, were not inhibited by 8-SPT, showing that the degradation products do not influence activity. Indeed, although AMP and ADP were formed, the main product of degradation of ATP was the inactive inosine, and no adenosine was detected (Figure Sa), confirming the work of Franco et al. (1988), who also found the activity of adenosine
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deaminase to be relatively high in the rat duodenum. Some leakage of enzymes was detected, as ATP was also degraded by buffer in which tissue had been incubated, although in this case. the main degradation products were ADP and AMP, suggesting that 5'-nucleotidase and adenosine deaminase had remained bound to the tissue (Figure 5c). The order of potency of the ATP analogues in both tissues confirms that the P2-purinoceptor is of the P2Y subtype, with 2-MeSATP being much more potent than ATP and AMPCPP being somewhat less potent, with none of these compounds having any effect on Pl-purinoceptors which could interfere with this analysis. Indeed, the concentrationresponse curves for these compounds on the two tissues looked almost identical, with the potencies on each tissue being very similar and the curve for AMPCPP being steeper than those for ATP or 2-MeSATP, possibly because of its resistance to degradation, and therefore crossing the top of the ATP curve. This supports the idea that the receptors in these tissues are the same, although agonist potencies and the shapes of concentration-response curves do not provide conclusive information on receptor subtypes, and definite proof must await the development of true subtype-selective, competitive ATP antagonists. The results described here show that AMPPCP is of little value for the definition of P2-purinoceptor subtypes, as although on both tissues it is indeed less potent than ATP as expected for a
P2y-purinoceptor (Burnstock & Kennedy, 1985), it has a significant but variable effect on Pl-purinoceptors. In the taenia caeci its true potency at P2y-purinoceptors is presumably that observed in the presence of 10-200,uM 8-SPT and is thus roughly half that observed in the absence of antagonist, whereas in the duodenum no P2y effect is detectable at the concentrations tested and AMPPCP appears to act entirely as a P1 agonist in this tissue. Whether the observed differences in the effects of AMPPCP between the tissues reflect qualitative differences between the P2y receptor populations in these tissues is hard to investigate with the pharmacological tools available, but there seems no compelling reason to propose P2Y subtypes on this evidence. There are however some hints that the P1-purinoceptors may be different, although the small differences observed in the inhibitory potency of 8-SPT are hard to interpret with certainty, particularly in the absence of convincing Schild plots in the duodenum and in view of reports that P1 agonists and antagonists may in general have a rather higher affinity for rat tissues than for guinea-pig tissues (Collis et al., 1989). Why the Schild plots in the rat duodenum gave slopes greater than unity is not clear, but problems were also encountered with the dose-dependence of the antagonist in the rat colon (Bailey & Hourani, unpublished observations), and this could imply that 8-SPT is not acting in a purely competitive fashion in these tissues. In the guinea-pig taenia caeci the Schild plot against adenosine was satisfactory and no problems were reported by Collis et al. (1987) on the guinea-pig atria and aorta, showing that in these tissues 8-SPT is acting competitively, and that again there may be differences between rat and guinea-pig tissues. 8-SPT did not cause dose-dependent shifts in the concentration-response curve for AMPPCP in the taenia caeci where it was acting as a competitive adenosine antagonist, showing that the P1 action of AMPPCP is only a component of its overall effect in this tissue. In conclusion, it is now clear that AMPPCP cannot simply be regarded as a P2 agonist but also has a pronounced direct P1 agonist action as well, and the extent of this varies depending on the tissue chosen. This direct P1 effect complicates the use of AMPPCP as a stable ATP analogue, and may explain some earlier findings that AMPPCP (and ATP) acted like adenosine in some tissues (Dahlen & Hedqvist, 1980; Collis &
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Pettinger, 1982; Moody & Burnstock, 1982; Moody et al., 1984; Wiklund et al., 1985; Shinozuka et al., 1988). ATP could well be acting in these cases via its breakdown product adenosine, although we have found no evidence for a direct or indirect P1 effect, but it is likely that AMPPCP is acting directly on Pl-purinoceptors as in the results described here. The related stable analogue, AMPCPP, would appear to be a more suitable analogue to use in studies of P2-purinoceptors as it lacks this anomalous P1 effect, although it is not as selective as AMPPCP for P2x receptors and has a higher potency on P2y receptors, being almost as potent as ATP itself in the taenia caeci and rat duodenum, and furthermore has a greater effect than ATP at high concentrations. The potent, specific
P2x agonist L-AMPPCP, the enantiomer of AMPPCP, is probably the best compound to use, as it has little or no P2Y or P1 effects in the guinea-pig taenia caeci, aorta or ileum (Hourani et al., 1985; 1986) or the rat colon muscularis mucosae (Bailey & Hourani, 1990). Our results with AMPPCP show that it is possible for an ATP analogue to act directly on P,-purinoceptors however unlikely this may seem from the known structure-activity relationships, and emphasise the danger of making assumptions about the receptor selectivity of an agonist based solely on its structure. We thank the MRC for support for SJ.B. (98723473N), and the SERC for a studentship for J.N.
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