Relaxation of the mouse isolated aorta and carotid artery in response to adenosine analogues in genetically-modified mice lacking the adenosine A(2A) receptor.

The aim of this study was to characterise the receptor(s) mediating responses to adenosine and/or adenosine analogues in mouse isolated aorta and carotid artery. In addition, since mice lacking the A(2A) adenosine receptor are reported to be hypertensive, the possibility that this gene deletion or the altered phenotype results in alteration of responses mediated via adenosine analogues was investigated. This was achieved by comparing results obtained in parallel within single experiments using tissues from A(2A) knock-out animals and their wild-type littermates.In aortic rings, adenosine and 5'- N-ethylcarboxamidoadenosine (NECA) caused relaxations above 10 microM and 30 microM, respectively, which were unaffected by either 8-sulphophenyltheophylline (8-SPT, 100 microM) or A(2A) receptor knockout. 2-[ p-(2-Carbonylethyl)phenylethylamino]-5'- N-ethylcarboxamidoadenosine (CGS 21680) was virtually inactive. R- N(6)-Phenylisopropyladenosine (R-PIA) induced relaxations which were not inhibited by 8-SPT (100 microM) or altered by A(2A) receptor knockout. No A(1)-mediated contractile responses were observed in wild-type or knock-out tissues in contrast with results in mice of the same strain obtained commercially rather than from our breeding programme. In carotid artery rings NECA contracted at low concentrations (0.1-1 microM) and relaxed at higher concentrations. Curves to NECA were not different in tissues from wild-type and A(2A) receptor knock-out mice and both the contractile and relaxant phases were right-shifted by 8-SPT (100 microM) in tissues from animals of both genotype. 1,3-Dipropyl-8-cyclopentylxanthine (DPCPX, 3 nM) attenuated contractile NECA responses but did not affect relaxant responses. CGS 21680 was inactive in carotid artery rings from both wild-type and A(2A) receptor knock-out mice. In the presence of DPCPX (30 nM) to abolish contractions, R-PIA induced relaxant curves which were not different in tissues from wild-type and A(2A) knock-out mice and were not inhibited by 8-SPT (100 microM). These results confirm the absence of A(2A) or A(2B) receptors in murine aorta and suggest that relaxations to NECA in carotid artery are A(2B) receptor-mediated whilst contractions are A(1) receptor-mediated. They also indicate the presence of an antagonist-resistant site activated by R-PIA in both vascular preparations. There is no evidence for compensatory changes in responses mediated by adenosine and its analogues due to the gene deletion or the reported resulting hypertensive phenotype in either aortic or carotid arterial rings obtained from A(2A) knock-out mice.

Role of cyclic nucleotides in vasodilations of the rat thoracic aorta induced by adenosine analogues.

Although adenosine analogues such as 5'-N-ethylcarboxamidoadenosine (NECA) relax the rat thoracic aorta in a partially endothelium-dependent manner via adenosine A(2A) receptors, others such as N(6)-R-phenylisopropyladenosine (R-PIA) act via an endothelium-independent, antagonist-insensitive mechanism. The role of cyclic nucleotides in these relaxations was investigated in isolated aortic rings using inhibitors of adenylate and guanylate cyclases as well as subtype-selective phosphodiesterase inhibitors. The adenylate cyclase inhibitor 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ 22536; 100 microM) significantly inhibited responses to NECA, but not responses to R-PIA. The type IV (cyclic AMP-selective) phosphodiesterase inhibitor 4-[(3-butoxy-4-methoxyphenyl)methyl]-2-imidazolidinone (RO 20-1724; 30 microM) significantly enhanced responses to NECA and to a lesser extent those to R-PIA. The guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3a]quinoxalin-1-one (ODQ; 100 microM) significantly inhibited responses to NECA and acetylcholine but not responses to R-PIA. The selective phosphodiesterase V (cyclic GMP-selective) inhibitors, zaprinast (10 microM) and 4-[[3',4'-(methylenedioxy)benzyl]amino]-6-methoxyquinazoline (MMQ; 1 microM), had no significant effect on responses to either NECA or R-PIA, but enhanced responses to acetylcholine. These results are consistent with the effects of NECA being via activation of endothelial receptors to release NO which stimulates guanylate cyclase, as well as smooth muscle receptors coupled to stimulation of adenylate cyclase. The lack of effect of zaprinast and MMQ on responses to NECA are likely to be due to simultaneous activation of both adenylate and guanylate cyclases in the smooth muscle, as cyclic AMP reduces the sensitivity of phosphodiesterase V to inhibitors. These results also suggest that the effects of R-PIA are via neither of these mechanisms.

Characterisation of adenosine receptors mediating relaxation in hamster isolated aorta.

The aim of this study was to characterise the receptor(s) mediating relaxations to adenosine and its analogues in the hamster isolated aorta. Adenosine relaxed the aorta but there was no significant difference between pIC20 values in the absence and presence of 8-sulphophenyltheophylline (8-SPT, 50 microM), although there was a small right-shift (approximately threefold) of the lower portion of the curve in the presence of 8-SPT. However, in the presence of the adenosine uptake inhibitor nitrobenzylthioinosine (NBTI, 1 microM), curves to adenosine were left-shifted by approximately 100-fold and an apparent pK(B) for 8-SPT of 5.79+/-0.05 was obtained. Likewise, 5'-N-ethylcarboxamidoadenosine (NECA) relaxed the aorta but curves were biphasic. The first phase of the curve was blocked by 8-SPT (10-100 microM, pA2 = 5.75+/-0.14) and the A2A-selective antagonist 4-(2-[7-amino-2-(2-furyl) [1,2,4]-triazolo[2,3-a][1,3,5]triazin-5-ylaminolethyl) phenol (ZM 241385, 3 nM-1 microM, pK(B)=9.17+/-0.10). Similarly, the A2A-selective agonist 2-[p)-(2-carbonylethyl)-phenylethylamino]-5'-N-ethylcarboxam idoadenosine (CGS 21680) relaxed the tissues but curves were biphasic and the first phase was again blocked by ZM 241385 (10 nM, apparent pK(B)=9.06+/-0.34). In contrast, relaxations to N6-R-phenylisopropyladenosine (R-PIA), N6-cyclopentyladenosine (CPA), 2-chloroadenosine (2-CADO) and N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB-MECA) were not blocked by 8-SPT (50 microM). Responses to IB-MECA were also not blocked by the A3 receptor antagonist 3-ethyl-5-benzyl-2-methyl-6-phenyl-4-phenylethynyl-1,4-(+/-)-dihyd ropyridine-3,5-dicarboxylate (MRS 1191, 30 microM). The asymptote of the first phase of curves to NECA was markedly reduced (and in some preparations the first phase was completely abolished) both in the presence of N(G)-nitro-L-arginine methyl ester (L-NAME, 0.1 mM), and in the absence of endothelium. Likewise, the first phase of curves to CGS 21680 was abolished both in the presence of L-NAME (0.1 mM) and in the absence of endothelium. In contrast, there were only relatively small shifts to the right of curves to adenosine and the other analogues in the presence of L-NAME or the absence of endothelium (between three- and fivefold). The data suggest the presence of A2A receptors which are located on the endothelium and mediate release of nitric oxide. These receptors are activated by NECA, CGS 21680 and adenosine (in the presence of uptake blockade). The resistance to blockade of relaxations to adenosine (in the absence of uptake inhibitor), CPA, R-PIA, 2-CADO, IB-MECA and high concentrations of NECA and CGS 21680 by 8-SPT or ZM 241385 suggests the presence of an additional mechanism(s). Data obtained with adenosine in the absence and presence of NBTI suggest that the endogenous ligand may cause relaxation via an intracellular mechanism.

Receptor specificity and trigemino-vascular inhibitory actions of a novel 5-HT1B/1D receptor partial agonist, 311C90 (zolmitriptan).

1. 311C90 (zolmitriptan zomig: (S)-4[[3-[2-(dimethylamino)ethyl]-1H-indol-5-yl]methyl]-2-oxazolidinone) is a novel 5-HT1B/1D receptor agonist with proven efficacy in the acute treatment of migraine. Here, we describe the receptor specificity of the drug and its actions on trigeminal-evoked plasma protein extravasation into the dura mater of the anaesthetized guinea-pig. 2. At the "5-HT1B-like' receptor mediating vascular contraction (rabbit saphenous vein), the compound was a potent (p[A50] = 6.79 +/- 0.06) partial agonist achieving 77 +/- 4% of the maximum effect to 5-hydroxytryptamine (5-HT). In the same experiments, sumatriptan (p[A50] = 6.48 +/- 0.04) was half as potent as 311C90 and produced 97 +/- 2% of the 5-HT maximum effect. Studies in which receptor inactivation methods were used to estimate the affinity (pKA) and efficacy relative to 5-HT (tau rel) for each agonist confirmed that 311C90 exhibits higher affinity than sumatriptan (pKA = 6.63 +/- 0.04 and 6.16 +/- 0.03, respectively) and that both drugs are partial agonists relative to 5-HT (tau rel = 0.61 +/- 0.03 and 0.63 +/- 0.10, respectively, compared to 5-HT = 1.0). 3. Consistent with its effects in rabbit saphenous vein, 311C90 also produced concentration-dependent contractions of primate basilar artery and human epicardial coronary artery rings. In basilar artery, agonist potency (p[A50] = 6.92 +/- 0.07) was similar to that demonstrated in rabbit saphenous vein, again being 2-3 fold higher than for sumatriptan (p[A50] = 6.46 +/- 0.03). Both agonists produced about 50% of the maximum response obtained with 5-HT in the same preparations. In rings of human coronary artery, the absolute potency of 311C90 and sumatriptan was higher than in primate basilar artery (p[A50] = 7.3 +/- 0.1 and 6.7 +/- 0.1, respectively), but maximum effects relative to 5-HT were lower (37 +/- 8% and 35 +/- 7%, respectively). In both types of vessel, the inability of 5-HT1B/1D agonists to achieve the same maximum as the endogenous agonist 5-HT is explained by the additional presence of 5-HT2A receptors. 4. 311C90 displayed high affinity at human recombinant 5-HT1D (formerly 5-HT1D alpha) and 5-HT1B (formerly 5-HT1D beta) receptors in transfected CHO-K1 cell membranes (pIC50 values = 9.16 +/- 0.12 and 8.32 +/- 0.09, respectively). In intact cells, the drug produced concentration-dependent inhibition of forskolin-stimulated adenylyl cyclase (p[A50] = 9.9 and 9.5, respectively) achieving the same maximum effect as 5-HT. Excepting human recombinant 5-HT1A and 5-ht1F receptors at which the drug behaved as an agonist with modest affinity (pIC50 = 6.45 +/- 0.11 and 7.22 +/- 0.12, respectively), 311C90 exhibited low, or no detectable affinity (pKi or pKB < or = 5.5) at numerous other monoamine receptors, including other 5-HT receptor subtypes. 5. When administered to anaesthetized guinea-pigs ten minutes before unilateral electrical stimulation of the trigeminal ganglion (1.2 mA, 5 Hz, 5 ms, 5 min), 311C90 (3-30 micrograms kg-1, i.v.) caused a dose-dependent inhibition of [125I]-albumin extravasation within the ipsilateral dura mater. At the same doses, the drug also produced dose-dependent falls in cranial vascular conductance (32.3 +/- 7.5% at 30 micrograms kg-1), as measured in the ear by laser doppler flowmetry. 6. These results show that 311C90, a novel member of the 5-HT1B/1D agonist drug class, exhibits a high degree of pharmacological specificity. Its potent partial agonist action at "5-HT1B-like' receptors in intracranial arteries, coupled with potent agonism at 5-HT1D and 5-HT1B receptors and an ability to inhibit neurogenic plasma protein extravasation in the dura, are consistent with its utility as an effective acute treatment for migraine.

Adenosine analogues relax guinea-pig taenia caeci via an adenosine A2B receptor and a xanthine-resistant site.

In this study we have sub-classified the adenosine A2 receptor mediating relaxation in the guinea-pig taenia caecum using the adenosine A2A receptor-selective agonist CGS 21680 (2-[p-(2-carboxyethyl)phenylamino]-5'-N-ethylcarboxamidoadenosine) and the adenosine A2A receptor-selective antagonist 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). CGS 21680 did not elicit relaxations, and a pKB value of 7.80 was obtained for ZM 241385 against 5'-N-ethylcarboxamidoadenosine suggesting the presence of adenosine A2B receptors. Relaxations are also mediated via a xanthine-resistant site. In this study relaxations to the adenosine A3 receptor agonist IB-MECA (N6-(3-iodo-benzyl)adenosine-5'-N-methyluronamide) were blocked by neither 8-sulphophenyltheophylline (100 microM) nor the adenosine A3 receptor antagonist BW-A1433 (1,3-dipropyl-8-(4-acrylate)phenylxanthine, 100 microM), suggesting that this site is not an adenosine A3 receptor.

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-ethylcarboxami doadenosine (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 microM) 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 microM and IB-MECA elicited relaxant responses which were resistant to blockade by 8-SPT and BWA1433 (100 microM). 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 microM) 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 microM) 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 microM), 8-SPT (100 microM) gave significant rightward shifts of E/[A] curves to adenosine. 6. ZM 241385 (0.1-1 microM) 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 microM ZM 241385. In the presence of NBTI E/[A] curves to adenosine were significantly rightward shifted by ZM 241385 (0.1 microM, 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 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

Pharmacological analysis of the interaction between purinoceptor agonists and antagonists in the guinea-pig taenia caecum.

1. In the absence of adenosine uptake inhibition, adenosine produced a concentration-dependent (threshold 30 microM) relaxation of the 5-methylfurmethide pre-contracted guinea-pig taenia caecum. The relaxation was not blocked by 8-phenyltheophylline (8-PT, 3 microM) or 1,3-dipropyl, 8-cyclopentylxanthine (DPCPX, 30 microM). 2. In the presence of the adenosine uptake inhibitor, dipyridamole (Dip, 3 microM), a biphasic adenosine concentration-effect curve was obtained (threshold 0.3 microM). The time course of the responses to adenosine in the absence of Dip was similar to that of the second phase responses in the presence of Dip and occurred over the same adenosine concentration-range. 5'-(N-ethyl) carboxamido-adenosine (NECA) concentration-effect curves (in the absence of Dip) were also biphasic. Only the first phases of the concentration-effect curves obtained with NECA and adenosine (plus Dip) were inhibited by 8-PT. The pA2 values for 8-PT of 6.7 and 7.0 versus adenosine and NECA, respectively, were consistent with actions at P1-purinoceptors. There was a trend towards an increase in the upper asymptote of the first phase of the NECA curve in the presence of increasing concentrations of 8-PT. The A1-purinoceptor selective antagonist, DPCPX, also blocked only the first phase of the NECA concentration-effect curve and produced a significant increase in the upper asymptote. The pA2 value (6.8) obtained was consistent with activation of A2-subtype P1-purinoceptors by the low concentrations of NECA. 3. There was no correlation between A1-purinoceptor affinity and the propensity to cause the increase in the upper asymptote of the first phase of the NECA concentration-effect curves amongst a series of 9-methyl adenine analogues, suggesting that the amplification was not due to inhibition of an underlying A1-purinoceptor-mediated contractile response.4. DPCPX (10 microM) produced a significant increase in the upper asymptote of the NECA concentration effect curve, but had no effect on isoprenaline curves whereas the phosphodiesterase inhibitor Ro20-1724 (30 microM) produced a significant increase in the upper asymptote of both NECA and isoprenaline concentration-effect curves. Therefore the amplification of the first phase responses by DPCPX did not appear to be due to phosphodiesterase inhibition.5. It was not possible to conclude whether second phase responses to adenosine and NECA were mediated by intracellular or extracellular sites of action. However, if intracellular sites of action were involved then adenosine did not apparently gain access by the Dip-sensitive uptake system.

Estimation of agonist affinity and efficacy by direct, operational model-fitting.

A few years ago Black and Leff (1983) presented a theoretical model for agonist action that can be fitted directly to experimental agonist concentration-effect data in order to estimate affinities and relative efficacies of agonists. The model has been successfully applied to data obtained using experimental designs commonly employed in pharmacologic experiments. This article describes the methodology used to fit the model equations to data for full and partial agonists, shows how affinity and efficacy estimates are calculated for these values, and shows how experimental design influences these calculations.