S.M.O. Hourani

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.

The structure-activity relationships of ectonucleotidases and of excitatory P2-purinoceptors: evidence that dephosphorylation of ATP analogues reduces pharmacological potency

The dephosphorylation of adenine nucleotides and their analogues by ectonucleotidases on the guinea-pig urinary bladder was studied using HPLC. The rate of dephosphorylation of each analogue was compared with its pharmacological potency at causing contraction. ATP, ADP and AMP were rapidly dephosphorylated, and substitution on the purine ring did not affect the rate of breakdown. The ectonucleotidases showed stereoselectivity towards the ribose moiety and towards the polyphosphate chain. In general, methylene isosteres of the nucleotides, and analogues in which one of the oxygen atoms on the terminal phosphate had been replaced, were resistant to degradation. None of the analogues that were readily dephosphorylated are more potent than ATP, and most but not all of the analogues resistant to degradation are more potent than ATP, suggesting that while resistance to degradation does not in itself confer high potency, susceptibility to degradation does limit the potency of ATP and its degradable analogues.

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.

ATP analogues and the guinea-pig taenia coli: a comparison of the structure-activity relationships of ectonucleotidases with those of the P2-purinoceptor

The dephosphorylation of adenine nucleotides and their analogues by ectonucleotidases on the guinea-pig taenia coli was studied using HPLC. The rate of dephosphorylation of each analogue was compared with its pharmacological potency relative to ATP. ATP, ADP and AMP were rapidly dephosphorylated, and substitution on the purine ring had no effect upon the rate of breakdown. The ectonucleotidases showed stereoselectivity towards the ribose, the unnatural L enantiomers of nucleotides being dephosphorylated more slowly. Analogues in which one of the oxygen atoms on the terminal phosphate had been replaced were resistant to degradation. Phosphorothioate analogues in which a sulphur was attached to the penultimate phosphorus were degraded stereoselectively. Methylene isosteres of ATP and ADP resisted degradation, except for homo-ATP which was dephosphorylated at the same rate as ATP. Overall, no correlation was found between the potency of an analogue and its rate of degradation.

Characterization of P1-purinoceptors on rat duodenum and urinary bladder.

1 The P1‐purinoceptors mediating relaxation of the rat duodenum and inhibition of contraction of the rat urinary bladder were characterized by use of adenosine and its analogues 5′‐N‐ethylcarboxamidoadenosine (NECA), N6‐cyclopentyladenosine (CPA) and 2‐p‐((carboxyethyl)phenethylamino)‐5′‐carboxamidoadenosine (CGS 21680), as well as the A1‐selective antagonist 1,3‐dipropyl‐8‐cyclopentylxanthine (DPCPX). The stable analogue of adenosine 5′‐triphosphate (ATP), adenylyl 5′‐(β,γ‐methylene)diphosphonate (AMPPCP), was also used as previous work had indicated that it has a direct action on some P1 receptors in addition to its P2‐purinoceptor activity. 2 In the rat duodenum, the order of potency of the adenosine agonists was NECA ≥ CPA > AMPPCP = adenosine > CGS 21680, and DPCPX antagonized CPA and AMPPCP at a concentration of 1 nm whereas equivalent antagonism of NECA and adenosine required a concentration of 1 μm. This suggests the presence of a mixture of A1 and A2 receptors in this tissue, with CPA and AMPPCP acting on the A1 and NECA and adenosine acting on the A2 receptors. 3 In the rat bladder, the order of potency of the adenosine agonists for inhibition of carbachol‐induced contractions was NECA ≫ adenosine > CPA = CGS 21680, and a concentration of DPCPX of 1 μm was required to antagonize responses to NECA and adenosine. This suggests the presence of A2 receptors in this tissue. ATP and AMPPCP each caused contractions which were not enhanced by DPCPX (1 μm) which suggests that in this tissue AMPPCP was acting only via P2 receptors and had no P1 agonist activity. That AMPPCP was active on the A1 receptors in the duodenum but inactive on the A2 receptors in the bladder implies that it has selectivity for the A1 subtype. 4 That CGS 21680, which has been reported to bind selectively to the high affinity A2a subclass of A2 receptors, had a very low potency on the A2 receptors in the duodenum and in the bladder suggests that these receptors are of the low affinity A2b subclass.

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 < 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.

Activation of two sites by adenosine receptor agonists to cause relaxation in rat isolated mesenteric artery

1 In this study we have characterized the receptor(s) in the rat mesenteric artery mediating relaxant responses to adenosine and a number of adenosine analogues, N6 ‐R‐phenylisopropyladenosine (R‐PIA), N6‐cyclopentyladenosine (CPA), N6‐(3‐iodo‐benzyl)‐adenosine‐5′‐N‐methyluronamide (IB‐MECA) and 5′‐N‐ethylcarboxamidoadenosine (NECA), by use of the non‐selective antagonist 8‐sulphophenyltheophylline (8‐SPT) and the A2A selective ligands 2‐[p‐(2‐carbonylethyl)‐phenylethylamino]‐5′‐N‐ethylcarboxamidoadenosine (CGS 21680) and 4‐(2‐[7‐amino‐2‐(2‐furyl)[1,2,4]‐triazolo[2,3‐a][1,3,5]‐triazin‐5‐ylamino]ethyl) phenol (ZM 241385). We have also studied the effects of endothelial removal and uptake inhibition by nitrobenzylthioinosine (NBTI) and the effects of the A3 receptor antagonist 1,3‐dipropyl‐8‐(4‐acrylate)phenylxanthine (BWA1433). 2 Adenosine, NECA, CPA and R‐PIA all elicited relaxant responses in tissues precontracted with phenylephrine (1 μM) with the following potency order: NECA>R‐PIA>adenosine=CPA. However, E/[A] curves to NECA were biphasic. CGS 21680 was inactive at concentrations up to 30 μM and IB‐MECA elicited relaxant responses which were resistant to blockade by 8‐SPT and BWA1433 (100 μM). 3 Removal of the endothelium produced a small but significant decrease in the asymptote of the high potency phase of E/[A] curves to NECA with no change in p[A]50. E/[A] curves to adenosine were not altered by removal of the endothelium. However, there were small rightward shifts of E/[A] curves to CPA and R‐PIA in the absence of endothelium. 4 Inhibition of uptake by NBTI (1 μM) had no effect on E/[A] curves to NECA, CPA or R‐PIA, but E/[A] curves to adenosine were significantly left‐shifted in the presence of NBTI. 5 8‐SPT (10–100 μM) caused significant rightward shifts of the high potency phase of the E/[A] curves to NECA (pA2=5.63±0.26). The second phase of the concentration‐response curve to NECA appeared to be resistant to blockade by 8‐SPT, as were E/[A] curves for adenosine, CPA or R‐PIA. However, in the presence of NBTI (1 μM), 8‐SPT (100 μM) gave significant rightward shifts of E/[A] curves to adenosine. 6 ZM 241385 (0.1–1 μM) produced significant rightward shifts of the high potency phase of NECA E/[A] curves (pA2=7.65±0.25 in the presence and 7.20±0.12 in the absence of endothelium), while curves to R‐PIA were not significantly shifted by 1 μM ZM 241385. In the presence of NBTI E/[A] curves to adenosine were significantly rightward shifted by ZM 241385 (0.1 μM, pA2=7.50±0.16). 7 In conclusion, the results suggest activation of A2B receptors located primarily on the smooth muscle by low concentrations of NECA and by adenosine under conditions of uptake blockade, and of another, as yet undefined site which may be intracellular, by higher concentrations of NECA, by CPA, R‐PIA and adenosine under conditions where uptake is operational.

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 μm) 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 μm) 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 μm) was administered concomitantly. The locations of curves to R‐PIA and adenosine were unaffected by L‐NAME (100 μm). 6 In the presence of PD 115,199 (0.1 μm) a parallel rightward shift of NECA E/[A] curves was observed (pA2 = 7.50 ± 0.19). PD 115,199 (0.1 and 1 μm) 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 μm). 7 The presence of ZM 241385 (3 nM‐0.3 μm) caused parallel rightward 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 μm ZM 241385. 8 CGS 21680 (1 μm) 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 μm) 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‐51‐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 μm) 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 μm), and the low potency of IB‐MECA is not consistent with the affinities of these compounds at the rat cloned A3 receptor. It is suggested that adenosine activates both the A2a and the undefined site, being most potent, but behaving as a partial agonist, at the A2a receptor.

Effects of the P2-purinoceptor antagonist, suramin, on human platelet aggregation induced by adenosine 5'-diphosphate.

1 The effects of suramin, a trypanocidal drug which has been reported to be a P2‐purinoceptor antagonist on smooth muscle, were investigated in human platelets, where adenosine 5′‐diphosphate (ADP) induces aggregation by acting on a subtype of purinoceptors which has been called P2T. 2 Suramin (100 μm) had no inhibitory effect on ADP‐induced platelet aggregation in plasma, even after 40 min incubation in the presence of bacitracin, a peptidase inhibitor, and did not affect the ability of adenosine 5′‐triphosphate (ATP) (40 μm) to inhibit competitively ADP‐induced aggregation. This lack of effect of suramin on platelets in plasma is probably due to its extensive binding to plasma proteins. 3 In washed platelets, suramin (50–400 μm) acted as an apparently competitive antagonist, causing parallel shifts to the right of the log concentration‐response curve to ADP. No depression of the maximal response to ADP was observed at concentrations of suramin (50–150 μm) for which full log concentration‐response curves to ADP could be obtained, but the slope of the Schild plot was around 2, indicating that this antagonism was not simply competitive. The apparent pA2 value for suramin, taken from this Schild plot, was 4.6. 4 Suramin (200–400 μm) also noncompetitively inhibited aggregation induced by U46619 (a thromboxane receptor agonist) or by 5‐hydroxytryptamine in the presence of adrenaline (100 μm), and caused a depression of the maximal response to these agonists. This nonspecific effect of suramin may explain the high Schild plot slope obtained against ADP. 5 These results provide evidence that the ADP receptor on human platelets is indeed similar to the P2‐purinoceptors responding to adenine nucleotides on smooth muscle and other tissues, and show that suramin cannot distinguish between the proposed subtypes of the P2‐purinoceptors.