Adenosine A2A receptors in ventral striatum, hypothalamus and nociceptive circuitry implications for drug addiction, sleep and pain.

Adenosine A2A receptors localized in the dorsal striatum are considered as a new target for the development of antiparkinsonian drugs. Co-administration of A2A receptor antagonists has shown a significant improvement of the effects of l-DOPA. The present review emphasizes the possible application of A2A receptor antagonists in pathological conditions other than parkinsonism, including drug addiction, sleep disorders and pain. In addition to the dorsal striatum, the ventral striatum (nucleus accumbens) contains a high density of A2A receptors, which presynaptically and postsynaptically regulate glutamatergic transmission in the cortical glutamatergic projections to the nucleus accumbens. It is currently believed that molecular adaptations of the cortico-accumbens glutamatergic synapses are involved in compulsive drug seeking and relapse. Here we review recent experimental evidence suggesting that A2A antagonists could become new therapeutic agents for drug addiction. Morphological and functional studies have identified lower levels of A2A receptors in brain areas other than the striatum, such as the ventrolateral preoptic area of the hypothalamus, where adenosine plays an important role in sleep regulation. Although initially believed to be mostly dependent on A1 receptors, here we review recent studies that demonstrate that the somnogenic effects of adenosine are largely mediated by hypothalamic A2A receptors. A2A)receptor antagonists could therefore be considered as a possible treatment for narcolepsy and other sleep-related disorders. Finally, nociception is another adenosine-regulated neural function previously thought to mostly involve A1 receptors. Although there is some conflicting literature on the effects of agonists and antagonists, which may partly be due to the lack of selectivity of available drugs, the studies in A2A receptor knockout mice suggest that A2A receptor antagonists might have some therapeutic potential in pain states, in particular where high intensity stimuli are prevalent.

Receptors for ADP on human blood platelets.

It is well established that ADP causes aggregation of human blood platelets, and indeed it was the first aggregating agent to be studied, but the ways in which platelets respond to ADP are still relatively obscure. Although it is apparent that increases in intracellular Ca2+ concentrations are of major importance in activating platelets, it is not clearly understood how ADP causes these increases and what other signal transduction mechanisms it uses. It is not even clear whether ADP causes its effects by interacting with only one receptor, or whether multiple receptors for ADP exist on platelets. In this review, Susanna Hourani and David Hall examine some of the conflicting evidence in this field, and draw some tentative conclusions about the number and nature of receptors for ADP on human platelets.

Adenosine receptor subtypes.

The numerous and widespread effects of adenosine provide both an opportunity for the development of novel therapeutic agents acting via adenosine receptors and the challenge of achieving selectivity of action. The feasibility of achieving selectivity is enhanced if receptor subtypes can be identified. Biochemical, functional and receptor-cloning studies are beginning to provide convergent data supporting the existence of A1, A2A, A2B and A3 receptors. However, studies of the functional significance of these receptors in intact tissues both in vitro and in vivo have lagged behind the biochemical studies. In this article, Michael Collis and Susanna Hourani review the current status of adenosine receptor classification and propose that ligands with greater selectivity need to be evaluated in a wide range of functional preparations if the therapeutic potential of this area is to be realized.

The regulation of vascular function by P2 receptors: multiple sites and multiple receptors.

Although the effects of nucleotides in the cardiovascular system have been known for almost 70 years, it is only in the past few years that some of the P2 receptors at which they act have been cloned and characterized. It is now clear that the control of cardiovascular function by nucleotides is complex, involving multiple receptors and multiple effects in the different cell types of importance. In this review Mike Boarder and Susanna Hourani summarize the P2 receptors that are present in endothelial cells, platelets, smooth muscle and nerves, the signalling pathways that they activate and the responses that are produced. They also discuss the important role of nucleotides in the interactions between the different cell types, and the implications of this in vascular disease.

Desensitization of the guinea-pig urinary bladder by the enantiomers of adenylyl 5'-(beta, gamma-methylene)-diphosphonate and by substance P.

Adenosine 5'-triphosphate (ATP), substance P (SP) and non-cholinergic nerve stimulation contracted the guinea-pig urinary bladder. SP and two poorly-degradable analogues of ATP, the enantiomers of adenylyl 5'-(beta, gamma-methylene)-diphosphonate (AMP-PCP and L-AMP-PCP), were used to desensitize the guinea-pig bladder. Desensitization of the bladder by AMP-PCP (50 microM) or by L-AMP-PCP (50 microM) abolished the responses to ATP, and inhibited the responses to non-cholinergic nerve stimulation and to SP. The responses to histamine were unaffected. Desensitization by SP (1 microM) inhibited the responses to SP itself, but not the responses to ATP, L-AMP-PCP or non-cholinergic nerve stimulation. These results suggest that SP may act partly by releasing ATP, and support the suggestion that ATP rather than SP is the non-cholinergic stimulatory transmitter.

Some pharmacological and biochemical interactions of the enantiomers of adenylyl 5'-(beta, gamma-methylene)-diphosphonate with the guinea-pig urinary bladder.

Adenosine 5'-triphosphate (ATP) and adenylyl 5'-(beta, gamma-methylene)-diphosphonate (AMP-PCP) both contracted the guinea-pig urinary bladder, but the response to AMP-PCP was much greater. We synthesized the enantiomer of AMP-PCP, L-adenylyl 5'-(beta, gamma-methylene)-diphosphonate (L-AMP-PCP), and tested it on the guinea-pig bladder. L-AMP-PCP contracted the guinea-pig bladder, and was more potent than AMP-PCP and much more potent than ATP. The potential breakdown product of L-AMP-PCP, L-adenosine, unlike adenosine (the breakdown product of AMP-PCP), did not inhibit contractions of the guinea-pig bladder. ATP and its enantiomer L-adenosine 5'-triphosphate (L-ATP) were rapidly degraded by the muscle, and AMP-PCP was also degraded, but more slowly. L-AMP-PCP, however, was completely resistant to degradation. L-AMP-PCP would appear to be a useful ATP analogue, as it is potent and resistant to degradation, and its potential breakdown product, L-adenosine, is inactive.

Effects of purines on the longitudinal muscle of the rat colon.

1. Adenosine and adenosine 5'-triphosphate (ATP) have been reported to cause relaxation of the rat colon longitudinal muscle preparation; the purinoceptors mediating this effect were investigated by use of a series of agonists and antagonists. 2. The tissue was precontracted with carbachol (1 microM), and the purines induced reversible relaxations with a potency order of 5'-N-ethylcarboxamidoadenosine (NECA) greater than N6-cyclopentyladenosine (CPA) = adenosine 5'-(alpha, beta-methylene) triphosphonate (AMPCPP) greater than adenosine = adenylyl 5'-(beta, gamma-methylene) disphosphonate (AMPPCP) = ATP. The P1-selective antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) (3 microM) shifted to the right the log concentration-response curves of all these agonists except for AMPCPP, indicating that they all act via P1-purinoceptors. The order of potency of the adenosine analogues and the relatively high concentrations of the antagonist required indicated that these receptors are of the A2 subtype. The P2-selective antagonist suramin (300 microM) inhibited responses to AMPCPP, but not to the other agonists. 3. The dephosphorylation of the nucleotides was studied by high performance liquid chromatography following incubation with the longitudinal muscle preparation for up to 30 min. ATP was rapidly degraded, largely to adenosine, and AMPPCP and AMPCPP were also degraded, although more slowly, to adenosine and adenosine 5'-(alpha, beta-methylene) diphosphonate (AMPCP) respectively. AMPCP, like AMPCPP, caused relaxations by acting on P2-purinoceptors, as it was also inhibited by suramin (300 microM). Incubation of the tissue with adenosine deaminase abolished responses to adenosine, reduced those to ATP and AMPPCP, but had no effect on those to AMPCPP.ATP and AMPPCP therefore appear to be acting on the A2 receptors in this tissue largely via their degradation product adenosine.4. The longitudinal muscle of the rat colon therefore contains both P.- and P2-purinoceptors, which both mediate relaxation. The P,-purinoceptors are of the A2 subtype and the P2-purinoceptors are probably of the P2Y subtype, although the rapid degradation of the nucleotides means that it is difficult to classify them with certainty.

5'-N-ethylcarboxamidoadenosine: a potent inhibitor of human platelet aggregation.

1 5'-N-ethylcarboxamidoadenosine (NECA) is an adenosine analogue which is 22,900 times more potent than adenosine as a vasodilator. Adenosine and some of its analogues are also inhibitors of human platelet aggregation. NECA was tested for its effects on human platelets. 2 NECA (1 microM) inhibited human platelet aggregation induced by adenosine 5'-diphosphate (ADP), adrenaline, 5-hydroxytryptamine (5-HT) and thrombin more powerfully than adenosine. NECA was 5 to 10 times more potent than adenosine at inhibiting ADP- and adrenaline-induced aggregation. 3 NECA, like adenosine, caused dose-dependent increases in levels of platelet adenosine 3',5'-cyclic monophosphate (cyclic AMP), which were competitively inhibited by theophylline, an adenosine antagonist. 4 These effects of NECA, like those of adenosine, were completely stereospecific as the L-enantiomer of NECA was inactive. 5 NECA did not interfere with the inhibition by ADP of prostaglandin E1 (PGE1)-stimulated adenylate cyclase. 6 NECA is the most potent analogue of adenosine tested so far on human platelets, and is the first example of a 5' modification to retain affinity for the platelet adenosine receptor.

Partial agonist behaviour of adenosine 5'-O-(2-thiodiphosphate) on human platelets.

1 The effects of an adenosine diphosphate (ADP) analogue, adenosine 5'-O-(2-thiodiphosphate) (ADP-beta-S), in which a terminal phosphate oxygen has been replaced by sulphur, were studied on human platelets. 2 ADP-beta-S induced platelet aggregation and inhibited prostaglandin E1 (PGE1)-stimulated adenylate cyclase but in both cases was less potent than ADP and did not achieve the same maximal effects. 3 Both actions of ADP could be inhibited by the simultaneous addition of ADP-beta-S (50 microM). 4 Aggregation induced by 11 alpha, 9 alpha-epoxymethano prostaglandin H2 (a stable endoperoxide analogue) was not inhibited by simultaneous addition of ADP-beta-S (50 microM). 5 The behaviour of ADP-beta-S towards human platelets was consistent with it being a partial agonist.

Effects of RP and SP diastereoisomers of adenosine 5'-O-(1-thiodiphosphate) on human platelets.

1 RP and SP diastereoisomers of adenosine 5'-O-(1-thiodiphosphate) ((R)-ADP-alpha-S and (S)-ADP-alpha-S), an adenosine 5'-diphosphate (ADP) analogue, were tested on intact human platelets. 2 Each diastereoisomer induced aggregation, (S)-ADP-alpha-S being 5 times more potent than (R)-ADP-alpha-S but they achieved only 75% of the maximal effect of ADP. 3 Aggregation induced by each diastereoisomer was competitively inhibited by ATP (50 microM). 4 Simultaneous addition of each diastereoisomer inhibited aggregation induced by ADP but not by 11 alpha, 9 alpha-epoxymethano prostaglandin H2, a stable endoperoxide analogue. Both diastereoisomers are therefore partial agonists at the ADP receptor mediating aggregation. 5 Unlike ADP, neither diastereoisomer inhibited prostaglandin E1 (PGE1)-stimulated adenylate cyclase, but each competitively inhibited the effect of aDP, with (S)-ADP-alpha-S again being 5 times more potent than (R)-ADP-alpha-S. 6 These are the first reported examples of ADP analogues to induce platelet aggregation without inhibiting PGE1-stimulated adenylate cyclase.

Competitive inhibition by adenosine 5'-triphosphate of the actions on human platelets of 2-chloroadenosine 5'-diphosphate, 2-azidoadenosine 5'-diphosphate and 2-methylthioadenosine 5'-diphosphate.

1 Adenosine 5'-diphosphate (ADP) induces human platelet aggregation and noncompetitively inhibits stimulated human platelet adenylate cyclase; these two effects are mediated by the same ADP receptor, at which adenosine 5'-triphosphate (ATP) is a competitive antagonist. 2 Two ADP analogues, 2-azidoadenosine 5'-diphosphate (2-azido-ADP) and 2-methylthioadenosine 5'-diphosphate (2-methylthio-ADP) have been reported to be more potent as inhibitors of adenylate cyclase than they are as aggregating agents, but no evidence has been presented that these actions are mediated solely by the ADP receptor. 3 We therefore tested the ability of ATP to inhibit the actions of these compounds and of another ADP analogue, 2-chloroadenosine 5'-diphosphate (2-chloro-ADP). 4 2-Chloro-ADP, 2-azido-ADP and 2-methylthio-ADP each induced aggregation and inhibited stimulated adenylate cyclase. Both of these actions were competitively inhibited by ATP (50 microM) with pA2 values similar to those previously found for inhibition by ATP of these effects of ADP. 5 The reported greater potency of 2-azido-ADP and of 2-methylthio-ADP as inhibitors of adenylate cyclase than as aggregating agents is therefore due only to their greater efficacy for this effect, not to some extra actions elsewhere.

Specific but noncompetitive inhibition by 2-alkylthio analogues of adenosine 5'-monophosphate and adenosine 5'-triphosphate of human platelet aggregation induced by adenosine 5'-diphosphate.

Some 2-alkylthio derivatives of adenosine 5'-monophosphate (AMP), adenosine 5'-monophosphorothioate (AMPS) and adenosine 5'-triphosphate (ATP) were examined as inhibitors of human platelet aggregation. 2-Methylthio-AMP, 2-ethylthio-AMP, 2-(pentan-l-yl)thio-AMP, 2-ethylthio-AMPS, 2-methylthio-ATP and 2-ethylthio-ATP (100 microM) each inhibited aggregation induced by adenosine 5'-diphosphate (ADP) but not by 11 alpha, 9 alpha-epoxymethano prostaglandin H2, a stable endoperoxide analogue. Log dose-response curves to ADP in the absence and presence of each inhibitor were not parallel and the inhibition could not be overcome by high concentrations of ADP. The ATP analogues achieved greater inhibition of aggregation induced by ADP (5 microM) than did the AMP analogues. The order of potency of the AMP analogues was 2-ethylthio-AMPS greater than 2-ethylthio-AMP greater than 2-(pentan-l-yl)thio-AMP greater than 2-methylthio-AMP, and 2-methylthio-ATP was more potent than 2-ethylthio-ATP. These 2-alkylthio substituted analogues of AMP and ATP are specific but noncompetitive inhibitors of ADP-induced human platelet aggregation.

Differential effects of suramin on P2-purinoceptors mediating contraction of the guinea-pig vas deferens and urinary bladder.

1. The effect of the P2-purinoceptor antagonist, suramin, was investigated on contractions of the guinea-pig vas deferens and urinary bladder induced by adenosine 5'-triphosphate (ATP) and by the other naturally occurring nucleoside triphosphates. 2. ATP, guanosine 5'-triphosphate (GTP), cytidine 5'-triphosphate (CTP), inosine 5'-triphosphate (ITP) and uridine 5'-triphosphate (UTP) (0.1-500 microM) each contracted both the guinea-pig bladder and the guinea-pig vas deferens. In the vas deferens the order of potency of the nucleotides was ATP >> CTP > GTP > or = UTP = ITP, and in the bladder it was ATP >> CTP = GTP > UTP = ITP, although maximal responses to these agonists were not achieved in either tissue. 3. Suramin (30 microM-1 mM) dose-dependently inhibited ATP-induced contractions of the bladder in an apparently non-competitive manner, causing a reduction in the slope of the concentration-response curve to ATP. In contrast, suramin (5 microM-1 mM) had little inhibitory effect on ATP-induced contractions of the vas deferens, and indeed at concentrations of 100 microM and above markedly potentiated high concentrations of ATP (100-500 microM). The contractions induced by CTP, GTP, UTP and ITP (1-500 microM) were, however, abolished by suramin (1 mM) in each tissue. 4. Desensitization of the P2X purinoceptors in the guinea-pig vas deferens with adenosine 5'-alpha,beta-methylenetriphosphonate (AMPCPP) (300 microM) abolished contractions induced by ATP (1 microM-1 mM) in the absence of suramin. However, the contractions induced in the presence of suramin were unaffected by prior desensitization, indicating that they were not mediated by P2X-purinoceptors.5. ATP (100 MicroM) was dephosphorylated by both isolated tissue preparations under the conditions of these experiments, breakdown products being detectable after 2 min, with the major breakdown product in the bladder being inosine whereas that in the vas deferens was adenosine. Approximately 35% of the ATP remained intact after incubation for 30 min with the bladder, and approximately 45% remained after incubation for 30 min with the vas deferens. In each tissue this degradation was inhibited by suramin (1 mM), so that after incubation of ATP (100 MicroM) in the presence of suramin for 30 min,approximately 50% remained in the case of the bladder and approximately 65% remained in the vas deferens. However, inhibition of the production of the inhibitory agonist, adenosine by suramin did not appear to be responsible for the potentiation observed in the vas deferens, as the PI-purinoceptor antagonist 8-sulphophenyltheophylline (100 MicroM) did not reduce this potentiation.6. Chelation of divalent cations did not appear to account for the enhancement by suramin of ATP-induced contractions of the vas deferens, as the enhancement was still observed when Mg2+ was omitted from the buffer or when its concentration (normally 1.2 mM) was increased ten fold to 12 mM,or when the concentration of Ca2+ (normally 2.5 mM) was reduced to 0.83 mM. Even in the absence of Mg2+ and with the Ca2+ concentration reduced to 0.83 mM, no inhibition by suramin (1 mM) of ATP-induced contractions was observed.7. The most likely explanation for the potentiation by suramin of the ATP-induced contractions of the vas deferens is the co-existence of inhibitory P2Y-purinoceptors. However, no consistent relaxations to ATP (1-100 MicroM) or to the more potent P2Y-purinoceptor agonist 2-methylthioadenosine 5'-triphosphate(2-MeSATP) (0.01-100 MicroM) could be detected in the vas deferens precontracted with KCl (35 mM), even after desensitization of P2x-purinoceptors with AMPCPP (300 MicroM). Similarly, ATP (1-100 MicroM) or 2-MeSATP (0.01-1100 MicroM) added before KCI (35 mM), carbachol (10 JM) or noradrenaline (10 MicroM) did not reduce subsequent contractions to these agents.8. The differential effect of suramin on the contractions induced by ATP in the bladder and the vas deferens was unexpected, and shows that the receptor populations by which ATP acts in these tissues may not be identical. The failure of suramin to inhibit responses to ATP in the vas deferens suggests that this tissue, in addition to possessing P2x-purinoceptors may also possess a suramin-insensitive contractile ATP receptor revealed in the presence of suramin.

Differential effects of adenine nucleotide analogues on shape change and aggregation induced by adnosine 5-diphosphate (ADP) in human platelets.

Adenosine 5'-diphosphate (ADP) induces human blood platelets to aggregate and change shape, and it has been suggested that these two responses are mediated by more than one subtype of ADP receptor. The structure-activity relationships for several analogues of adenine nucleotides in causing aggregation and shape change were measured and compared in washed platelets using an aggregometer. ADP and its analogues 2-methylthioadenosine 5'-diphosphate (2-methylthio-ADP), adenosine 5'(alpha,beta-methylene)diphosphonate (AMPCP), S(P)-adenosine 5'-O-(1-thiodiphosphate) (AD-P alphaS) and adenosine 5'-O-(2-thiodiphosphate) (ADPbetaS) were used as agonists. Adenosine 5'-triphosphate (ATP) and its analogues, P1, P5-diadenosine pentaphosphate (ApsA), adenosine (5'-(alpha,beta-methylene)triphosphonate (AMPCPP), 2-methylthioadenosine 5'-triphosphate (2-methylthio-ATP) and uridine 5'-triphosphate (UTP), as well as the trypanocidal drug suramin, were used as antagonists. In general, the structure-activity relationships for both responses were similar, but for some analogues differences were observed. ADPalphaS and ADPbetaS were much more potent agonists relative to ADP for shape change than for aggregation and indeed ADPalphaS antagonized ADP-induced aggregation with an apparent pK(B) value of 5.5+/-0.1. 2-Methylthio-ATP also had different effects in aggregation and shape change, being a much higher affinity antagonist of aggregation than of shape change with an apparent pK(B) value of 7.0+/-0.2 for aggregation and 5.2+/-0.2 for shape change. These results support the suggestion that these two responses are mediated by multiple ADP receptors on human platelets, and are consistent with shape change being mediated via one receptor (the P2Y1 receptor) with aggregation requiring the activation of two receptors (the P2Y1 and another P2Y receptor).

Effects of suramin on contractions of the guinea-pig vas deferens induced by analogues of adenosine 5'-triphosphate.

1. Adenosine 5'-triphosphate (ATP) and some of its analogues contract the guinea-pig vas deferens, acting via receptors which have been classified as P2X-purinoceptors. We have recently shown, however, that the effects of ATP are enhanced, rather than inhibited, by the non-selective P2 antagonist, suramin, and that this enhancement could not easily be explained in terms of inhibition by suramin of the breakdown of ATP. We therefore investigated the effects of suramin on contractions induced by ATP analogues, to define the structure-activity relationships of the suramin-resistant response. 2. In the absence of suramin, the order of potency for ATP analogues was adenosine 5'-(alpha,beta-methylene)triphosphonate (AMPCPP) = P1,P5-diadenosine pentaphosphate (Ap5A) = adenosine 5'-tetraphosphate (Ap4) > adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) = adenylyl 5'-(beta,gamma-methylene) diphosphonate (AMPPCP) > P1,P5-diadenosine tetraphosphate (Ap4A) > adenosine 5'-O-(2- thiodiphosphate) (ADP beta S) > 2-methylthioadenosine 5'-triphosphate (MeSATP) > or = ATP > adenosine 5'-diphosphate (ADP). This is generally in agreement with previously reported structure-activity relationships in this tissue. 3. In the presence of suramin (1 mM), responses to Ap5A, Ap4A, AMPPCP, ADP beta S and ADP were abolished or greatly reduced, and contractions induced by AMPCPP, Ap4 and ATP gamma S were inhibited. Contractions induced by MeSATP however, like those induced by ATP itself, were not reduced, but at concentrations above 100 microM were enhanced. In the presence of suramin (1 mM) the order of potency of analogues was therefore AMPCPP = Ap4> ATP = MeSATP> ATP gamma S, with all other analogues tested being essentially inactive at concentrations up to 500 microM.4. Contractile responses of the vas deferens to transmural nerve stimulation (1-50 Hz) in the presence of the alpha-adrenoceptor antagonist, phentolamine (10 microM), were abolished by suramin (1 mM). This is in agreement with previous reports that suramin inhibits the excitatory junction potential, a response thought to be mediated by P2 purinoceptors. It is however hard to reconcile the evidence implicating ATP as the non-adrenergic transmitter responsible for this response with the failure of suramin to inhibit the contractions induced by ATP itself while abolishing nerve-mediated contractions.5. In conclusion, these results confirm our previous findings of a suramin-resistant component to the ATP-induced contraction in the guinea-pig vas deferens, and show that the structure-activity relationships of this response are not identical to those of any known P2-purinoceptor subclass. Although the inhibition by suramin of the breakdown of ATP may contribute to the suramin-resistance of some of the ATP analogues, it does not appear to provide the full explanation.

Activation of multiple sites by adenosine analogues in the rat isolated aorta.

1. The presence of A2 receptors mediating relaxation in the rat isolated aorta has been previously demonstrated. However, agonist dependency of the degree of rightward shift elicited by 8-sulphophenyltheophylline (8-SPT) led to the suggestion that the population of receptors in this tissue is not a homogeneous one. In this study we have re-examined the effects of 8-SPT in the absence and presence of the NO synthase inhibitor L-NAME (NG-nitro-L-arginine methyl ester) and investigated antagonism of responses by the potent A2a receptor ligands PD 115,199 (N-[2-dimethylamino)ethyl]-N-methyl-4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3 dipropyl-1H-purin-8-yl)) benzene sulphonamidexanthine), ZM 241385 (4-(2-[7-amino-2-(2-furyl) [1,2,4]-triazolo[2,3-a][1,3,5]triazin-5-yl amino]ethyl)phenol), and CGS 21680 (2-[p-(2-carboxyethyl)phenylamino]-5'-N-ethylcarboxamidoadenosine). We have also investigated the antagonist effects of BWA1433 (1,3-dipropyl-8-(4-acrylate)phenylxanthine) which has been shown to have affinity at rat A3 receptors. 2. Adenosine, R-PIA (N6-R-phenylisopropyl adenosine), CPA (N6-cyclopentyladenosine) and NECA (5'-N-ethylcarboxamidoadenosine) all elicited relaxant responses in the phenylephrine pre-contracted rat isolated aorta with the following potency order (p[A50] values in parentheses): NECA (7.07 +/- 0.11) > R-PIA (5.65 +/- 0.10) > CPA (5.05 +/- 0.12) > adenosine (4.44 +/- 0.12). 3. 8-SPT (10-100 microM) caused parallel rightward shifts of the E/[A] curves to NECA (pKB = 5.23 +/- 0.16). A smaller rightward shift of E/[A] curves to CPA was observed (pA2 = 4.85 +/- 0.17). However, no significant shifts of E/[A] curves to either adenosine or R-PIA were observed. 4. In the absence of endothelium E/[A] curves to NECA and CPA were right-shifted compared to controls. However, removal of the endothelium did not produce a substantial shift of adenosine E/[A] curves, and E/[A] curves to R-PIA were unaffected by removal of the endothelium. 5. In the presence of L-NAME (100 microM) E/[A] curves to NECA and CPA were right-shifted. However, no further shift of the CPA E/[A] curve was obtained when 8-SPT (50 microM) was administered concomitantly. The locations of curves to R-PIA and adenosine were unaffected by L-NAME (100 microM). 6. In the presence of PD 115,199 (0.1 microM) a parallel rightward shift of NECA E/[A] curves was observed (pA2 = 7.50 +/- 0.19). PD 115,199 (0.1 and 1 microM) gave smaller rightward shifts of E/[A] curves to R-PIA and CPA, but E/[A] curves to adenosine were not significantly shifted in the presence of PD 115,199 (0.1 or 1 microM). 7. The presence of ZM 241385 (3 nM-0.3 microM) caused parallel rightwad shifts of NECA E/[A] curves (pKB = 8.73 +/- 0.11). No significant shifts of E/[A] curves to adenosine, CPA or R-PIA were observed in the presence of 0.1 microM ZM 241385. 8. CGS 21680 (1 microM) elicited a relaxant response equivalent to approximately 40% of the NECA maximum response. In the presence of this concentration of CGS 21680, E/[A] curves to NECA were right-shifted in excess of 2-log units, whereas E/[A] curves to R-PIA were not significantly shifted. 9. BWA1433 (100 microM) caused a small but significant right-shift of the E/[A] curve to R-PIA yielding a pA2 estimate of 4.1 IB-MECA (N6-(3-iodo-benzyl)adenosine-5(1)-N-methyl uronamide) elicited relaxant responses which were resistant to blockade by 8-SPT (p[A]50 = 5.26 +/- 0.13). 10. The results suggest that whereas relaxations to NECA (10 nM-1 microM) are mediated via adenosine A2a receptors, which are located at least in part on the endothelium, R-PIA and CPA may activate A2b receptors on the endothelium and an additional, as yet undefined site, which is likely to be located on the smooth muscle and which is not susceptible to blockade by 8-SPT, PD 115,199 or ZM 241385. This site is unlikely to be an A3 receptor since the very small shift obtained in the presence of BWA1433 (100 microM), and the low potency of IB-MECA is not consistent with the affin

Effects of analogues of adenine nucleotides on increases in intracellular calcium mediated by P2T-purinoceptors on human blood platelets.

1. By use of a number of analogues of adenine nucleotides, the structure-activity relationships of the human platelet receptor for adenosine 5'-diphosphate (ADP) mediating increases in intracellular calcium were investigated, and compared with the known structure-activity relationships for induction by ADP of platelet aggregation. 2. ADP, 2-methylthioadenosine 5'-diphosphate (2-methylthio-ADP), adenosine 5'-O-(1-thiodiphosphate) (ADP-alpha-S) and adenosine 5'-O-(2-thiodiphosphate) (ADP-beta-S) each induced increases in intracellular calcium in a manner similar to their reported ability to induce human platelet aggregation. The effects of these agonists were antagonized by ATP, with a pA2 value in each case consistent with the inhibition by ATP of ADP-induced aggregation. In the case of ADP, the inhibition by ATP of increases in intracellular calcium was shown to be competitive by Schild analysis. 3. Of the analogues tested as inhibitors of the effect of ADP on intracellular calcium, 2-chloroadenosine 5'-triphosphate (2-chloro-ATP), adenosine 5'-O-(1-thiotriphosphate) (ATP-alpha-S), P1, P5-diadenosine pentaphosphate (Ap5A) and adenylyl 5'-(beta,gamma-methylene)diphosphonate (AMPPCP) were apparently competitive antagonists, although only one concentration of each antagonist was used. There was a good correlation between the pA2 values found here for these antagonists including ATP, and their pA2 values reported for inhibition of ADP-induced aggregation. Adenosine 5'-(alpha, beta-methylene)triphosphate (AMPCPP) and uridine 5'-triphosphate (UTP) (100 microM) were only very weak inhibitors of the effect of ADP on intracellular calcium, and this is consistent with their weak actions as inhibitors of aggregation. 2-Methylthioadenosine 5'-triphosphate (2-methylthio-ATP) (50 microM) non-competitively inhibited the effect of ADP on intracellular calcium, in a very similar way to its inhibition of ADP-induced aggregation.4. The good correspondence found for these analogues between their effect on intracellular calcium and on aggregation confirms that there is a causal relationship between these actions of ADP, and that they are mediated by the same receptor on platelets. These findings cast further doubt on the use of the affinity reagent 5'-fluorosulphonylbenzoyladenosine (FSBA) as an antagonist and label for the ADP receptor, as this compound has been reported to inhibit aggregation but not ADP-induced increases in intracellular calcium.

Contractile effects of uridine 5'-triphosphate in the rat duodenum.

1. Previous studies have shown that the rat duodenum relaxes to adenosine and adenosine 5'-triphosphate (ATP) via P1 and P2Y purinoceptors respectively, but in preliminary studies uridine 5'-triphosphate (UTP) was found to contract this tissue. The non-selective P2 antagonist suramin and a number of nucleotides were therefore used to investigate this response further. 2. ATP, UTP, adenosine 5'-diphosphate (ADP), adenosine 5'-O-(3-thiotriphosphate) (ATP-gamma-S), guanosine 5'-triphosphate (GTP) and uridine 5'-diphosphate (UDP) each relaxed the duodenum, with an agonist potency order of ATP = ADP > ATP-gamma-S >> GTP >> UTP = UDP, consistent with the presence of a P2Y purinoceptor mediating relaxation. 3. ATP-gamma-S, UTP and UDP each contracted the duodenum with an agonist potency order of ATP-gamma-S > UTP > UDP, although maximal responses to these agonists were not obtained at a concentration of 267 microM (ATP-gamma-S) and 300 microM (UTP and UDP). No contractions were observed with any of the other agonists at concentrations up to 300 microM. 4. Indomethacin (25 microM) did not inhibit the contractions induced by UTP, indicating that they were not mediated via production of prostaglandins. 5. Suramin (100 microM and 1 mM) inhibited relaxations induced by ATP, shifting the concentration-response curve to the right, with the maximal response to ATP being decreased by the higher concentration of suramin (1 mM). Suramin (1 mM) inhibited relaxations induced by ATP-gamma-S, shifting the concentration-response curve to the right, and completely abolished contractions induced by ATP-gamma-S. In contrast, suramin (100 JAM and 1 mM) had no effect on contractions induced by UTP.Contractions induced by UTP were, however, less sustained in the presence of suramin, which also affected the basal tone of some tissues when precontracted with carbachol (0.1 microM). In the presence of suramin (I mM), no contractions to ATP were observed.6. These results confirm that in the rat duodenum there is a P2Y purinoceptor that mediates relaxation in response to a number of purine nucleotides, and at which the pyrimidine nucleotides UTP and UDP are almost inactive. There are also receptors at which UTP and ATP-y-S act to cause contraction.Suramin discriminates between the contractile effects of these two agonists, which may indicate the presence of a suramin-insensitive pyrimidinoceptor as well as a suramin-sensitive receptor for ATP-y-S.An alternative explanation is that the differential effects of suramin are via its actions as an antagonist in addition to its action as an ectonucleotidase inhibitor.

Differential inhibition by adenosine or by prostaglandin E1 of human platelet aggregation induced by adenosine 5'-O-(1-thiodiphosphate) and adenosine 5'-O-(2-thiodiphosphate).

Adenosine 5'-diphosphate (ADP) induces human platelet aggregation and inhibits stimulated adenylate cyclase. Adenosine 5'-O-(1-thiodiphosphate) (ADP-alpha-S) and adenosine 5'-O-(2-thiodiphosphate) (ADP-beta-S) act at the ADP receptor and achieve the same maximal rate of human platelet aggregation as each other. Adenosine and prostaglandin E1, which noncompetitively inhibit ADP-induced aggregation by stimulating adenylate cyclase, inhibit aggregation induced by ADP-x-S more than aggregation induced by ADP-beta-S. ADP-x-S, unlike ADP-beta-S and ADP itself, does not inhibit stimulated adenylate cyclase. This suggests that the inhibition of stimulated adenylate cyclase by ADP, although not a cause of aggregation, may be of physiological importance in reducing the effects of endogenous agents such as adenosine and prostaglandins (for example, prostacyclin) to which the platelet may be exposed.

Adenosine 5-diphosphate antagonists and human platelets: no evidence that aggregation and inhibition of stimulated adenylate cyclase are mediated by different receptors.

1 Adenosine 5'-diphosphate (ADP) induces human platelet aggregation and noncompetitively inhibits stimulated human platelet adenylate cyclase; it has been suggested that these two effects are mediated by separate ADP receptors on the platelet surface. 2 Adenosine 5'-triphosphate and seven adenine nucleotide analogues were tested as inhibitors of both effects of ADP on human platelets, and were found to be competitive. 3 pA2 values were calculated for each antagonist for inhibition of both effects of ADP, and a good correlation (correlation coefficient 0.87; p less than 0.01) was found between the pA2 values for inhibition of ADP-induced aggregation and the pA2 values for inhibition of the effect of ADP on stimulated adenylate cyclase. 4 Such a correlation does not support the suggestion that ADP-induced aggregation and the inhibition by ADP of stimulated adenylate cyclase are mediated by two separate receptors.