Vasodilatory effects of adenosine A2 receptor agonists CGS 21680 and CGS 22492 in human vasculature

The vasodilatory effects of the adenosine analogs, 5'-N-ethylcarboxamidoadenosine (NECA), 2-[p-(2-carboxyethyl)phenethyl amino]-5'-N-ethylcarboxamidoadenosine (CGS 21680) and 2-[(2-cyclohexylethyl)amino]adenosine (CGS 22492) in human coronary, internal mammary artery and saphenous vein were examined in vitro. All produced concentration-dependent relaxations in arterial as well as venous rings contracted with 35 mM KCl. The concentration-response curves for NECA and CGS 21680 were parallel in the coronary. The adenosine A2 receptor antagonist, 9-chloro-2-(2-furyl)[1,2,4]triazolo[1,5-c]quinazolin-5-amine (CGS 15943A) significantly attenuated the relaxing response to the adenosine analogs in coronary artery. Although NECA and CGS 22492 were equally as effective at the highest concentration administered (both achieving approximately 70% relaxation at 10(-4) M) NECA (EC50 = 1.25 +/- 0.11 microM) induced greater vasodilation at lower concentrations than CGS 22492 (EC50 = 11.27 +/- 1.53 microM). CGS 21680 was the least potent of the agents tested achieving only 44% relaxation at 10(-4) M (EC50 = 4.71 +/- 0.46 microM). Coronary artery appeared to be more responsive than internal mammary artery or saphenous vein which displayed only marginal relaxation to these agents.     

A1 adenosine receptors modulate respiratory activity of the neonatal mouse via the cAMP-mediated signaling pathway

The effects of adenosine and its analogs on the function of the respiratory center were studied in the spontaneously active rhythmic slice of neonatal and juvenile mice (4-14 days old). Whole cell, spontaneous postsynaptic currents (sPSCs) and single channel KATP currents were recorded in inspiratory neurons of the pre-B?tzinger complex. Adenosine (50-600 microM) inhibited the respiratory rhythm. This was accompanied by increase in the activity of KATP channels in cell-attached patches. The A1 adenosine receptor agonist, 2-chloro-N6-cyclopentyladenosine (CCPA, 0.3-2 microM), inhibited the respiratory rhythm, sPSCs, and enhanced activity of KATP channels. The A1 adenosine receptor antagonist, 8-cyclopentyl-1, 3-dipropylxanthine (DPCPX, 1-3 microM), showed opposite effects and occluded the CCPA actions. Agents specific for A2 adenosine receptors (CGS 21860 and NECA, both applied at 1-10 microM) were without effect. Elevation of intracellular cAMP concentration ([cAMP]i) by 8-Br-cAMP (200-500 microM), forskolin (0.5-2 microM), or isobutylmethylxantine (IBMX, 30-90 microM) reinforced the rhythm, whereas NaF (100-800 microM) depressed it. The open probability of single KATP channels in cell-attached patches decreased after application of forskolin and increased in the presence of NaF. [cAMP]i elevation reversed the effects of A1 receptors both on the respiratory rhythm and KATP channels. A1 receptors and [cAMP]i modified the hypoxic respiratory response. In the presence of A1 agonists the duration of hypoxic augmentation shortened, and depression of the respiratory rhythm occurred earlier. Elevation of [cAMP]i prolonged augmentation and delayed the development of the depression. We conclude that A1 adenosine receptors modulate the respiratory rhythm via inhibition of intracellular cAMP production and concomitant activation of KATP channels.     

Involvement of P1 receptors in the effect of forskolin on cyclic AMP accumulation and export in PC12 cells

In PC12 cells, forskolin as well as the adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA) increased intracellular adenosine-3',5'-cyclic monophosphate (cyclic AMP) levels, which peaked at 45-60 minutes and declined thereafter. Maximum levels were 3000 and 1700 pmol/10(6) cells during treatment with 10 microM forskolin or 0.1 microM NECA, respectively. Extracellular cyclic AMP rose with time, at mean rates of 24.7 (forskolin) and 11.3 (NECA) pmol/min/10(6) cells. With either drug, a linear correlation was obtained between the calculated time integral of intracellular cyclic AMP and the measured extracellular cyclic AMP levels, indicating that the outflow of cyclic AMP was sustained by a nonsaturated transport system. The ability of forskolin to increase intracellular and extracellular cyclic AMP levels was hindered in a concentration-dependent manner by 8-(p-sulfophenyl)theophylline (8-SPT). A similar inhibition was exerted by other two adenosine receptor antagonists, 8-cyclopentyl-1,3-dipropylxanthine and 3,7-dimethyl-1-propargylxanthine. The concentration-response curve to adenosine was shifted to the right by 25 microM 8-SPT, whereas that of forskolin was shifted downwards. Adenosine deaminase (ADA, EC 3.5.44, 1 U/mL) reduced the intracellular cyclic AMP response to forskolin by 68%, whereas the adenosine transport inhibitor, dipyridamole (10 microM), significantly increased 1 and 10 microM forskolin-dependent cyclic AMP accumulation. Erythro-9-(2-hydroxy-3-nonyl)adenine (10 microM), an inhibitor of ADA, and alpha,beta-methyleneadenosine 5'-diphosphate (100 microM), an inhibitor of ecto-5'-nucleotidase, did not alter forskolin activity. These results demonstrate that a cyclic AMP extrusion system operates in PC12 cells during adenylyl cyclase stimulation by forskolin and that this stimulation involves a synergistic interaction with endogenous adenosine. However, extruded cyclic AMP does not appear to significantly contribute to the formation of the endogenous adenosine pool.     

The foot in hereditary motor and sensory neuropathies in children

We investigated the regulation of COX-2 expression and activity by adenosine receptors in rat microglial cells. The selective adenosine A2a-receptor agonist CGS21680 and the non-selective adenosine A1- and A2-receptor agonist 5'-N-ethylcarboxiamidoadenosine (NECA) induced an increase in COX-2 mRNA levels and the synthesis of prostaglandin E2 (PGE2). The adenosine A1-receptor agonist cyclopentyladenosine (CPA) was less potent, and the adenosine A1-receptor-specific agonist N6-2-(-aminophenylo)ethyladenosine (APNEA) showed only marginal effects. Microglia expressed adenosine A1-, A2a-, and A3-, but not A2b-receptor mRNAs, whereas astroglial cells expressed adenosine A2b- but not A2a-receptor mRNA. The adenosine A2a-receptor selective antagonist (E)-8-(3,4-dimethoxystyryl)-1,3-dipropyl-7-methylxanthine (KF17837) inhibited both CGS21680-induced COX-2 expression and PGE2 release. CGS21680-increased PGE2 levels were inhibited by dexamethasone, by the nonsteroidal antiinflammatory drug meloxicam, and by the adenylyl cyclase inhibitor 9-(tetrahydro-2-furanyl)-9H-purine-6-amine (SQ22536). CGS21680 and NECA both increased intracellular cAMP levels in microglial cells. Dibutyryl cAMP as well as forskolin induced the release of PGE2. The results strongly suggest that adenosine A2a-receptor-induced intracellular signaling events cause an up-regulation of the COX-2 gene and the release of PGE2. Apparently, the cAMP second messenger system plays a crucial role in COX-2 gene regulation in rat microglial cells. The results are discussed with respect to neurodegenerative disorders of the CNS such as Alzheimer's disease, in which activated microglia are critically involved and COX inhibitors may be of therapeutic benefit.     

Further investigations into adenosine A1 receptor-mediated contraction in rat colonic muscularis mucosae and its augmentation by certain alkylxanthine antagonists

1. The alkylxanthine antagonists, 8-phenyltheophylline (8-PT), 8-p-sulphophenyltheophylline (8-SPT) and 1,3,7-trimethylxanthine (caffeine) produced rightward displacements of contractile concentration-effect curves to 5'-N-ethylcarboxamidoadenosine (NECA) in rat isolated colonic muscularis mucosae (RCMM) with concentration-ratios consistent with adenosine receptor blockade. The non-xanthine antagonist, 9 fluro-2-(2-furyl)-5,6-dihydro [1,2,4] triazo to [1,5-c]-quinazin-imine (CGS15943A) also antagonized contractions to NECA with an affinity (pKB8.1-8.5) consistent with adenosine A1 receptor blockade. 2. In addition to producing rightward shifts of the concentration-response curves, the maximum contractions to 5'-N-ethylcarboxamidoadenosine (NECA) were also markedly increased in the presence of 8-PT (by 83 +/- 16% at 1 microM), 8-SPT (by 37 +/- 7% at 10 microM) and caffeine (by 45 +/- 5% at 100 microM) but were unaffected by CGS15943A (at 0.01 and 0.03 microM). 3. As with NECA, the maximum contractions to the adenosine A1 receptor agonists R-phenylisopropyladenosine (R-PIA) and N-[(1S, trans)-2-hydroxyclopentyl] adenosine (GR79236) were both antagonized and augmented by 8-PT. In addition, the contractions to NECA in the presence of 8-PT (1 microM) were inhibited by nanomolar concentrations of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). 4. The non-selective phosphodiesterase (PDE) inhibitor, 3-isobutyl-1-methylxanthine (1 microM) produced a marked increase in the NECA maximum without producing a rightward shift in the NECA curve, whereas a higher concentration (10 microM) virtually abolished responses. The PDE type III inhibitor,milrinone (1 microM), the type IV inhibitor, rolipram (10 microM), and the type V PDE inhibitor, zaprinast(3 microM), were all without effect on NECA responses in RCMM.5. Partial inhibitions of contractions to NECA were produced by indomethacin (at 3 or 10 micro M) or piroxicam (at 3 microM). Responses to GR79236 were also partially inhibited by indomethacin. In the presence of indomethacin, 8-PT was still able to enhance markedly the maximum contractions obtained to NECA in RCMM.6. The present study has shown that certain alkylxanthine antagonists (but not the non-xanthineCGS15943A) produced a marked augmentation of adenosine Al receptor-mediated contractions inRCMM. The mechanism of this augmentation is, as yet, not known but is unlikely to result from inhibition of PDE. This study has also shown that adenosine Al receptor-induced contractions inRCMM are mediated, in part, via products of the cyclo-oxygenase pathway.     

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     

Binding of the novel nonxanthine A2A adenosine receptor antagonist [3H]SCH58261 to coronary artery membranes

This study demonstrates quantification of A2A adenosine receptors (A2AAdoRs) in membranes prepared from porcine coronary arteries, porcine striatum, and PC12 cells. Radioligand binding assays were performed using the new selective A2AAdoR antagonist radioligand [3H]-5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo [4,3-epsilon]-1,2,4-triazolo[1,5-c)pyrimidine ([3H]SCH58261). Binding of the radioligand to membranes was rapid, reversible, and saturable. The densities of A2AAdoRs in membranes prepared from porcine coronary arteries, porcine striatum, and PC12 cells were 900 +/- 61, 892 +/- 35, and 959 +/- 76 fmol/mg protein, respectively. Equilibrium dissociation constants (Kd values) calculated from results of saturation binding assays were 2.19, 1.20, and 0.81 nmol/L, and Kd values calculated from results of association and dissociation assays were 2.42, 1.01, and 0.40 nmol/L for [3H]SCH58261 binding to membranes prepared from porcine coronary arteries, porcine striatum, and PC12 cells, respectively. The specific binding of [3H]SCH58261 as a percentage of total binding at a radioligand concentration equal to the Kd value was 65% to 90% in the three membrane preparations. The order of ligand potencies determined by assay of competition binding to sites in porcine coronary membranes using [3H]SCH58261, unlabeled antagonists (SCH58261, 8-(3-chlorostyryl)caffeine [CSC], and xanthine amine congener [XAC]), and unlabeled agonists ([3H]2-p-(2-carboxyethyl)-phenethylamino-5'-N-ethylcarboxamidoaden osine [CGS 21680], 2-hexynyl-5'-N-ethylcarboxamidoadenosine [HE-NECA], [3H]5'-N-ethylcarboxamidoadenosine [NECA], and R(-)N6-(2-phenylisopropyl)adenosine [R-PIA]) was SCH58261 > HE-NECA = CSC = CGS 21680 = XAC > NECA = R-PIA. The Hill coefficients of displacement by A2AAdoR ligands of [3H]SCH58261 binding were not significantly different from unity, indicating that [3H]SCH58261 bound to a group of homogeneous noninteracting sites in all membrane preparations. The order of ligand potencies to compete for [3H]SCH58261 binding sites in porcine striatal and PC12 cell membranes was, in part, different from that for porcine coronary arterial membranes. The different rank orders of potencies for agonists and antagonists at A2A receptors of porcine coronary arteries, striatum, and PC12 cells and significant differences in absolute values of potency of ligands in the three preparations may indicate the existence of different subtypes of A2AAdoRs. The antagonist radio-ligand [3H]SCH58261 should be of value for pharmacological characterization of A2A adenosine receptors in other preparations.     

Effect of adenosine receptor agonists on release of the nucleoside analogue [3H]formycin B from cultured smooth muscle DDT1 MF-2 cells

Adenosine has receptor-mediated effects in a variety of cell types and is predominantly formed from ATP by a series of nucleotidase reactions. Adenosine formed intracellularly can be released by bidirectional nucleoside transport processes to activate cell surface receptors. We examined whether stimulation of adenosine receptors has a regulatory effect on transporter-mediated nucleoside release. DDT1 MF-2 smooth muscle cells, which possess nitrobenzylthioinosine-sensitive (ES) transporters as well as both adenosine A1 and A2 receptors, were loaded with the metabolically stable nucleoside analogue [3H]formycin B. N6-cyclohexyladenosine (CHA), a selective adenosine A1 receptor agonist, produced a concentration-dependent inhibition of [3H]formycin B release with an IC50 value of 2.7 microM. Further investigation revealed CHA interacts directly with nucleoside transporters with a Ki value of 3.3 microM. Neither 5'-N-ethylcarboxamidoadenosine (NECA), a mixed adenosine A1 and A2 receptor agonist, nor CGS 21680, a selective adenosine A2A receptor agonist, affected nucleoside release. We conclude that release of the nucleoside formycin B from DDT1 MF-2 cells is not regulated by adenosine A1 or A2 receptor activation.     

Induction of apoptosis in HL-60 human promyelocytic leukemia cells by adenosine A(3) receptor agonists

The effects of adenosine (ADO) analogs on cells of the human promyelocytic HL-60 line were examined. ADO A(3) receptor agonists, N(6)-(3-iodobenzyl)adenosine-5'-N-methylcarboxamide (IB-MECA, 30-60 microM) and 2-chloro-N(6)-(3-iodobenzyl)adenosine-5'-N-methyluronamide (CI-IB-MECA, 10-30 microM) induced apoptotic cell death. In contrast, neither an A(1)/A(2) antagonist (XAC) nor other selective ADO receptor agonists (CPA, NECA and CGS21680) induced apoptosis at concentrations of <30 microM. Both IB-MECA and CI-IB-MECA significantly induced Ca(2+) release from intracellular Ca(2+) pools followed by Ca(2+) influx, suggesting the presence of phospholipase C-coupled ADO A(3) receptors on HL-60 cells. This was further supported by the presence of mRNA of ADO A3 receptor in the cells. These results suggest that activation of ADO A(3) receptors is responsible for the ADO-induced apoptosis in HL-60 cells and could be of potential therapeutic value in the treatment of leukemia.     

Adenosine A2A receptors modulate the binding characteristics of dopamine D2 receptors in stably cotransfected fibroblast cells

In membrane preparations from rat striatum, where adenosine A2A and dopamine D2 receptors are coexpressed, stimulation of adenosine A2A receptors was found to decrease the affinity of dopamine D2 receptors for dopamine agonists. We now demonstrate the existence of this antagonistic interaction in a fibroblast cell line (Ltk-) stably transfected with the human dopamine D2 (long-form) receptor and the dog adenosine A2A receptor cDNAs (A2A-D2 cells). In A2A-D2 cells, but not in control cells only containing dopamine D2 receptors (D2 cells), the selective adenosine A2A agonist 2-[p-(2-carboxyethyl)-phenethylamino]-5'-N-ethyl-carboxamido adenosine (CGS 21680) induced a 2-3-fold decrease in the affinity of dopamine D2 receptors for dopamine, as shown in competition experiments with dopamine versus the selective dopamine D2 antagonist [3H]raclopride. By contrast, activation of the constitutively expressed adenosine A2B receptors with 5'-N-ethyl-carboxamidoadenosine (NECA) did not modify dopamine D2 receptor binding. In A2A-D2 cells CGS 21680 failed to induce or induced only a small increase in adenosine-3',5'-cyclic-monophosphate (cAMP) accumulation. In D2 cells NECA- or forskolin-induced adenylyl cyclase activation was not associated with any change in dopamine D2 receptor binding. These results indicate that adenylyl cyclase activation is not involved in the adenosine A2A receptor-mediated modulation of the binding characteristics of the dopamine D2 (long-form) receptor.     

Adenosine receptor-mediated relaxation of guinea-pig precontracted, isolated trachea

1. We have investigated the pharmacological profile of the adenosine receptor mediating relaxation of the carbachol pre-contracted guinea-pig trachea. 2. 5'-N-Ethylcarboxamidoadenosine (NECA) and 2-chloroadenosine elicited concentration-dependent relaxations with pD2 (-log10 half-maximal values) of 6.37 +/- 0.04 and 5.25 +/- 0.09, with maximal relaxations of 73 +/- 7 and 208 +/- 38%, respectively. In the presence of 10 microM NECA, 2-chloroadenosine was able to relax the tissue further with a pD2 value of 4.74 +/- 0.11 and a maximal response of 252 +/- 68%. 3. CGS 21680, APEC and adenosine failed to elicit significant relaxations of precontracted tracheal rings at concentrations below 10 microM. At 10 microM, adenosine analogues elicited relaxations with the following order of magnitude (% relaxation): 2-chloroadenosine (75 +/- 16%) = NECA (69 +/- 16%) > APEC (25 +/- 8%) > CGS 21680 (11 +/- 2%) > adenosine (6 +/- 4%). 4. NECA-induced relaxation of precontracted trachea was antagonized by adenosine receptor antagonists with the rank order of apparent affinity (Ki, nM): PD 115,199 (27 +/- 8) = XAC (43 +/- 11) > CP 66,713(285 +/- 89) = DPCPX (316 +/- 114). 5. We conclude that the adenosine analogue-induced relaxation of guinea-pig tracheal rings fails to fit into the current classification of A2 adenosine receptors.     

Binding of the radioligand [3H]-SCH 58261, a new non-xanthine A2A adenosine receptor antagonist, to rat striatal membranes

1. The present study describes the binding to rat striatal A2A adenosine receptors of the new potent and selective antagonist radioligand, [3H]-5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazol o [1,5-c] pyrimidine, [3H]-SCH 58261. 2. [3H]-SCH 58261 specific binding to rat striatal membranes ( > 90%) was saturable, reversible and dependent upon protein concentration. Saturation experiments revealed that [3H]-SCH 58261 labelled a single class of recognition sites with high affinity (Kd = 0.70 nM) and limited capacity (apparent Bmax = 971 fmol mg-1 of protein). The presence of 100 microM GTP in the incubation mixture did not modify [3H]-SCH 58261 binding parameters. 3. Competition experiments showed that [3H]-SCH 58261 binding is consistent with the labelling of A2A striatal receptors. Adenosine receptor agonists competed with the binding of 0.2 nM [3H]-SCH 58261 with the following order of potency: 2-hexynyl-5'-N-ethyl carboxamidoadenosine (2HE-NECA) > 5'-N-ethylcarboxamidoadenosine (NECA) > 2-[4-(2-carboxyethyl)-phenethylamino]-5'-N-ethylcarboxamidoadenosi ne (CGS 21680) > 2-phenylaminoadenosine (CV 1808) > R-N6-phenylisopropyladenosine (R-PIA) > N6-cyclohexyladenosine (CHA) = 2-chloro-N6-cyclopentyladenosine (CCPA) > S-N6-phenylisopropyladenosine (S-PIA). 4. Adenosine antagonists inhibited [3H]-SCH 58261 binding with the following order: 5-amino-9-chloro-2-(2-furyl)-[1,2,4]-triazolo[1,5-c] quinazoline (CGS 15943) > 5-amino-8-(4-fluorobenzyl)-2-(2-furyl)-pyrazolo [4,3-e]-1,2,4-triazolo [1,5-c] pyrimidine (8FB-PTP) = SCH 58261 > xanthine amine congener (XAC) = (E,18%-Z,82%)7-methyl-8-(3,4-dimethoxystyryl)-1,3-dipropylxanthine (KF 17837S) > 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) > or = 8-phenyltheophylline (8-PT). 5. The Ki values for adenosine antagonists were similar to those labelled with the A2A agonist [3H]-CGS 21680. Affinities of agonists were generally lower. The A1-selective agonist, R-PIA, was found to be about 9 fold more potent than its stereoisomer, S-PIA, thus showing the stereoselectivity of [3H]-SCH 58261 binding. Except for 8-PT, the adenosine agonists and antagonists examined inhibited [3H]-SCH 58261 binding with Hill coefficients not significantly different from unity. 6. The present results indicate that [3H]-SCH 58261 is the first non-xanthine adenosine antagonist radioligand which directly labels A2A striatal receptors. High receptor affinity, good selectivity and very low non-specific binding make [3H]-SCH 58261 an excellent probe for studying the A2A adenosine receptor subtype in mammalian brain.     

Functional characterization of adenosine receptors in the nucleus tractus solitarius mediating hypotensive responses in the rat

1. The aim of this study was to characterize adenosine receptors located in the nucleus tractus solitarius (NTS) that mediate decreases in blood pressure in the anaesthetized rat. To determine the adenosine receptor subtype involved, a range of selective agonists and antagonists were studied and their relative potencies evaluated. 2. The rank order of agonist potency in inducing decreases in diastolic blood pressure was N6-cyclopentyladenosine (CPA) > N6-cyclohexyladenosine (CHA) > N-ethyl-carboxamidoadenosine (NECA) > or = 2-phenylaminoadenosine (CV1808) > 2-p-(carboxyethyl)phenethylamino-5' N-ethylcarboxamidoadenosine (CGS 21680) > N6-(2-(4-aminophenyl)ethyl)-adenosine (APNEA). 3. The hypotensive action of CPA following microinjection into the NTS was antagonized by i.v. infusions (50 micrograms kg-1 min-1) of adenosine receptor antagonists, 8-cyclopentyl-1,3 dipropylxanthine (DPCPX), 8-phenyltheophylline (8-PT), 8-(p-sulphophenyl)theophylline (8-SPT), and 1,3-dipropyl-8-N-(2-diethylamino)ethyl)-N methyl-4-(2,3,6,7-tetrahydro-2,6-dioxo) benzenesulphonamidexanthine (PD 115199). The antagonist potency order was DPCPX > PD115199 > or = 8-PT. Intravenous infusion of 8-SPT had no effect on blood pressure responses to microinjection of CPA into the NTS. 4. The results suggest that adenosine A1 receptors in the NTS mediate hypotensive responses in the anaesthetized rat preparation.     

Characterization of ocular hypertension induced by adenosine agonists

PURPOSE: Previous studies have shown that adenosine agonists may induce a rise in intraocular pressure (IOP), a reduction in IOP, or both. Although the reduction in IOP results from the activation of adenosine A1 receptors, the mechanisms responsible for the rise in IOP have not been investigated. This study examines the receptors and mechanisms responsible for the adenosine agonist-induced rise in IOP. METHODS: The ocular effects of the nonselective adenosine agonist NECA, the relatively selective adenosine A2 agonist CV-1808, the A2a agonist CGS-21680, and the A1 agonist R-PIA were evaluated. RESULTS: The topical administration of CV-1808 produced a rapid rise in IOP, with a maximum increase of 15.6 +/- 1.6 mm Hg. Dose-response curves demonstrated that each agonist produced a dose-related rise in IOP with the following rank order of potency: NECA > CV-1808 > > R-PIA = CGS-21680. At times corresponding to the rise in IOP, the administration of high doses of CV-1808 (165 micrograms) produced a significant increase in aqueous humor flow and protein concentration. Increases in IOP and aqueous humor protein levels induced by CV-1808 were blocked by pretreatment with the adenosine A2 antagonist DMPX. In vitro studies demonstrated that CV-1808 did not alter cyclic adenosine monophosphate production in the rabbit iris-ciliary body. In cats, topical administration of CV-1808 produced a rapid rise in IOP, with a maximum increase of 8.1 +/- 2.4 mm Hg and an ED50 of 73 +/- 2.9 micrograms. This rise in IOP was blocked by DMPX pretreatment. CONCLUSIONS: These data demonstrate that adenosine receptor agonists can induce an acute rise in IOP in rabbits and cats. On the basis of pharmacologic characteristics, the rise in IOP is consistent with the activation of ocular adenosine A2 receptors. Functional studies indicate that at high doses, this rise in IOP involves an increase in aqueous flow and the breakdown of the blood-aqueous barrier.     

A1 adenosine receptor modulation of electrically-evoked contractions in the bisected vas deferens and cauda epididymis of the guinea-pig

1. The effects of adenosine receptor agonists upon both electrically-evoked and phenylephrine-induced contractile responses were investigated in the bisected vas deferens and the cauda epididymis of the guinea-pig. Electrical field-stimulation (10 s trains of pulses at 9 Hz, 0.1 ms duration, supramaximal voltage) elicited biphasic and monophasic contractile responses from preparations of bisected vas deferens and cauda epididymis, respectively; these responses were abolished by tetrodotoxin (300 nM). 2. In the prostatic half of the vas deferens the A1 selective adenosine receptor agonists, N6-cyclopentyladenosine (CPA) and (2S)-N6-[2-endo-norbornyl]adenosine ((S)-ENBA) and the non-selective A1/A2 adenosine receptor agonist, 5'-N-ethylcarboxamidoadenosine (NECA) inhibited electrically-evoked contractions (pIC50+/-s.e.mean values 6.15+/-0.24, 5.99+/-0.26 and 5.51+/-0.24, respectively). The responses to CPA were blocked by the A1 adenosine receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine, DPCPX (100 nM). 3. In the epididymal half of the vas deferens NECA potentiated (at < or = 100 nM) and inhibited (at > or = 1 microM) electrically-evoked contractions. In the presence of the non-selective alpha-adrenoceptor antagonist phentolamine (3 microM), the alpha1-adrenoceptor antagonist, prazosin (100 nM), or at a reduced train length (3 s) NECA inhibited electrically-evoked contractions (pIC50 values 6.05+/-0.25, 5.97+/-0.29 and 5.71 +/-0.27, respectively). CPA (at 10 microM) also inhibited electrically-evoked contractions in this half of the vas deferens. In the presence of prazosin (100 nM), CPA also inhibited electrically-evoked contractions (pIC50 6.14+/-0.67); this effect was antagonized by DPCPX (30 nM, apparent pK(B) 8.26+/-0.88). In the presence of the P2 purinoceptor antagonist, suramin (300 microM), CPA (up to 1 microM) potentiated electrically-evoked contractions. 4. NECA, CPA and APNEA potentiated electrically-evoked contractions in preparations of cauda epididymis (pEC50 values 7.49+/-0.62, 7.65+/-0.74 and 5.84+/-0.86, respectively), the response to CPA was competitively antagonized by DPCPX (100 nM) with an apparent pK(B) value of 7.64+/-0.64. 5. The alpha1-adrenoceptor agonist phenylephrine elicited concentration-dependent contractile responses from preparations of bisected vas deferens and cauda epididymis. NECA (1 microM) potentiated responses to phenylephrine (< or = 1 microM) in the epididymal, but not in the prostatic half of the vas deferens. In preparations of epididymis NECA (1 microM) shifted phenylephrine concentration response curves to the left (4.6 fold). In the presence of a fixed concentration of phenylephrine (1 microM), NECA elicited concentration-dependent contractions of preparations of the epididymal half of the vas deferens and of the epididymis (pEC50 values 7.57+/-0.54 and 8.08+/-0.18, respectively). NECA did not potentiate responses to ATP in either the epididymal half of the vas deferens or the epididymis. 6. These studies are consistent with the action of stable adenosine analogues at prejunctional A1 and postjunctional A1-like adenosine receptors. The prejunctional A1 adenosine receptors only inhibit the electrically-evoked contractions of purinergic origin (an effect predominant in the prostatic half of the vas deferens). At the epididymis, where electrically-evoked contractions are entirely adrenergic, the predominant adenosine receptor agonist effect is a potentiation of alpha1-adrenoceptor-, but not of ATP-induced contractility.     

Effects of pH on responses to adenosine, CGS 21680, carbachol and nitroprusside in the isolated perfused superior mesenteric arterial bed of the rat

1. The receptors mediating the vasodilator responses to adenosine in the isolated mesenteric arterial bed of the rat were identified by use of selective agonists and antagonists and the involvement of the endothelium was examined. 2. Adenosine-mediated dilatation of the mesentery was potentiated by the nitric oxide synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME, 100 microM), but in contrast, removal of the endothelium substantially reduced the responses to adenosine. 3. The order of potency of adenosine receptor agonists was: 5'-N-ethylcarboxamidoadenosine (NECA) > 2-p-(-2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS 21680) > 2-chloro-N6-cyclopentyl-adenosine (CCPA) > or = adenosine, suggesting the presence of A2A receptors. 4. Adenosine-mediated dilatation was inhibited by the non-selective adenosine receptor antagonist, 8-phenyltheophylline (3 microM) and by the A2A receptor antagonist 8-(3-chlorostyryl)caffeine (500 nM), but was unaffected by the A1 receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 10 nM). 5. Reducing the pH of the perfusate to 6.8 potentiated the actions of both CGS 21680 and adenosine, but the vasodilator effects of carbachol were the same at both pH values. The adenosine response at the lower pH as at pH 7.4, was unaffected by DPCPX. The actions of the nitrovasodilator, sodium nitroprusside, were also potentiated at pH 6.8 relative to those at the higher pH value but smaller responses were obtained at the lower pH value with forskolin, a stimulator of adenylyl cyclase, than at pH 7.4. 6. It is concluded that the adenosine receptor mediating dilatation of the rat mesenteric arterial bed is of the A2A subtype, that the response, under the conditions used, is apparently partly dependent on the endothelium (but not due to the release of nitric oxide), and that the response to activation of this receptor is potentiated by a reduction in pH which is similar to that seen in ischaemic conditions.     

Adenosine analogues with specificity for A2 receptors bind to mouse spermatozoa and stimulate adenylate cyclase activity in uncapacitated suspensions

Adenosine and its analogues, known to stimulate adenylate cyclase activity in somatic cells via A2 receptors, can accelerate capacitation in mouse spermatozoa and thereby enhance fertilizing ability in vitro. Indirect evidence has suggested that adenosine can modulate mouse sperm adenylate cyclase, implicating this enzyme and cAMP in the observed functional responses. In the present study we provide evidence that [3H]5'-N-ethylcarboxamidoadenosine (NECA), an adenosine analogue with specificity for stimulatory A2 adenosine receptors, can bind to mouse spermatozoa. This binding can be displaced by both unlabelled NECA and 2-chloroadenosine, another A2 receptor agonist, but not by cyclopentyladenosine, an inhibitory A1 receptor agonist, suggesting that the NECA binding is specific for A2 receptors. The presence of S-(p-nitrobenzyl)-6-thioinosine, an adenosine transport inhibitor, did not affect binding, indicating an external site for interaction with sperm cells. Saturable specific binding of [3H]NECA to mouse spermatozoa incubated at 37 degrees C was observed, with a Bmax of 5.17 pmol mg-1 protein and a Kd value of 930 nmol l-1. Binding data were consistent with the presence of a single major class of receptor. In addition to demonstrable binding of [3H]NECA, both NECA and 2-chloroadenosine significantly stimulated adenylate cyclase activity in a concentration-dependent manner, with NECA being effective at a lower concentration. Furthermore, the hydrolysis-resistant GTP analogue Gpp(NH)p, alone and in the presence of either NECA or 2-chloroadenosine, also significantly stimulated enzyme activity. In somatic cells, expression of responses to adenosine usually requires GTP and G proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of exogenous phytohormones on plastid tRNA modifications in ragi coleoptiles

The influence of adenosine and selective A1 and A2 agonists and antagonists was investigated on the cholinergic and the excitatory non-cholinergic (e-NC) contractions induced by electrical field stimulation in the guinea-pig bronchi. Adenosine (10 nM-1 mM) induced a concentration-dependent inhibition of the e-NC contraction (EC50 = 90 +/- 14 microM), whereas the cholinergic peak was only slightly affected. Preincubation of the tissue with the adenosine uptake blocker dipyridamole (10 microM) significantly shifted the concentration-inhibition curve to adenosine to the left (EC50 = 10 +/- 1 microM), suggesting an interaction with extracellular adenosine receptors of A1 and/or A2 subtype. To characterize the receptor type involved in this effect, selective adenosine derivatives were studied. The agonist to both A1 and A2 adenosine receptors, 5'-N-ethylcarboxamidoadenosine (NECA) was more potent than the selective A1 agonist, (-)-R-6-phenylisopropyladenosine (R-PIA), in inhibiting the e-NC contraction (EC50 = 0.10 +/- 0.04 and 0.60 +/- 0.12 microM, respectively, with a maximal inhibition of 70 and 45%, respectively). The concentration-response curve to NECA was shifted to the right by the A2 receptor selective antagonist 3,7-dimethyl-1-propargylxanthine (DMPX) (10 microM) (EC50 = 1.4 +/- 0.5 microM) as well as by the specific A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) (10 microM) (EC50 = 0.7 +/- 0.3 microM). The inhibitory effect induced by the association of both antagonists, DPCPX and DMPX, was considerably potentiated (EC50 > 22 +/- 2.5 microM). The effect of R-PIA was also shifted to the right by DPCPX (EC50 = 8.2 +/- 1.6 microM) but was not modified by DMPX. The contractile response to exogenous substance P was unaffected by NECA pretreatment (0.3 microM). Altogether, these results suggest that adenosine-induced inhibition of e-NC contraction of guinea-pig bronchi is mediated through activation of both A1 and A2 adenosine receptors linked to inhibition of the release of neuropeptides from C-fibre nerve endings.     

3H]SCH 58261, a selective adenosine A2A receptor antagonist, is a useful ligand in autoradiographic studies

We have characterized the new potent and selective nonxanthine adenosine A2A receptor antagonist SCH 58261 as a new radioligand for receptor autoradiography. In autoradiographic studies using agonist radioligands for A2A receptors ([3H]CGS 21680) or A1 receptors (N6-[3H]cyclohexyladenosine), it was found that SCH 58261 is close to 800-fold selective for rat brain A2A versus A1 receptors (Ki values of 1.2 nM versus 0.8 microM). Moreover, receptor autoradiography showed that [3H]SCH 58261, in concentrations below 2 nM, binds only to the dopamine-rich regions of the rat brain, with a K(D) value of 1.4 (0.8-1.8) nM. The maximal number of binding sites was 310 fmol/mg of protein in the striatum. Below concentrations of 3 nM, the nonspecific binding was <15%. Three adenosine analogues displaced all specific binding of [3H] SCH 58261 with the following estimated Ki values (nM): 2-hex-1-ynyl-5'-N-ethylcarboxamidoadenosine, 3.9 (1.8-8.4); CGS 21680, 130 (42-405); N6-cyclohexyladenosine, 9,985 (3,169-31,462). The binding of low concentrations of SCH 58261 was not influenced by either GTP (100 microM) or Mg2+ (10 mM). The present results show that in its tritium-labeled form, SCH 58261 appears to be a good radioligand for autoradiographic studies, because it does not suffer from some of the problems encountered with the currently used agonist radioligand [3H]CGS 21680.