Accelerated resequestration of cytosolic calcium and suppression of the pro-inflammatory activities of human neutrophils by CGS 21680 in vitro

We have investigated the effects of the adenosine A(2A) receptor agonist CGS 21680 (0.01 - 1 microM) on reactive oxidant production by, and elastase release from FMLP-activated human neutrophils, as well as on cytosolic Ca(2+) fluxes and intracellular concentrations of cyclic AMP. Oxidant production, elastase release and cyclic AMP were assayed using lucigenin-enhanced chemiluminescence, colourimetric and radioimmunoassay procedures respectively, while cytosolic Ca(2+) fluxes were measured by fura-2 spectrofluorimetry in combination with radiometric procedures which distinguish between net efflux and influx of the cation. Treatment of neutrophils with CGS 21680 did not affect the FMLP-activated release of Ca(2+) from intracellular stores, but resulted in dose-related acceleration of the rate of decline in fura-2 fluorescence, as well as decreases in both efflux and store-operated influx of Ca(2+), compatible with enhancement of resequestration of the cation by the endo-membrane Ca(2+)-ATPase. These effects on neutrophil Ca(2+) handling were associated with increased intracellular cyclic AMP and with inhibition of oxidant production and release of elastase. In contrast, treatment of neutrophils with the selective A(2A) receptor antagonist, ZM 241385 (2.5 microM), prevented the transient increase in cyclic AMP in FMLP-activated neutrophils which was associated with delayed sequestration of incoming Ca(2+) during store-operated influx. The CGS 21680-mediated reduction of Ca(2+) efflux from FMLP-activated neutrophils was also antagonized by pretreatment of the cells with ZM 241385 (2.5 microM), as well as by thapsigargin (1 microM), an inhibitor of the endo-membrane Ca(2+)-ATPase. ZM 241385 also neutralized the cyclic AMP-elevating and anti-inflammatory interactions of CGS 21680 with neutrophils. We conclude that A(2A) receptors regulate the pro-inflammatory activities of human neutrophils by promoting cyclic AMP-dependent sequestration of cytosolic Ca(2+).     

Adenosine A3 receptors on human eosinophils mediate inhibition of degranulation and superoxide anion release.

The role of adenosine A3 receptors on human eosinophil degranulation and superoxide anion (O2-) release was studied in vitro using the complement fragment C5a as the main stimulus and employing a number of selective agonists and antagonists. In the presence of cytochalasin B (CB), C5a induced a dose-dependent release of the granular eosinophil peroxidase (EPO), but not O2-, whereas in the absence of CB O2- , but not EPO, was released. C5a-induced EPO release was inhibited dose-dependently by the selective A3 agonist N6-(3-iodobenzyl)-5'-N-methylcarbamoyladenosine (IB-MECA) and to a lesser extent by the less-selective N6-2-(4-amino-3-iodophenyl) ethyladenosine (APNEA). The IC50 (95% CI) for IB-MECA was 6.8 microM (3.1-12.0 microM). At concentrations up to 100 microM, neither adenosine nor the selective A1 agonist N-cyclopentyladenosine (CPA) and the selective A2 agonist 2-[[2-[4-(2-carboxyethyl)phenyl]ethyl]amino]-N-ethylcarboxamidoadenosine (CGS 21680) had any significant effect. The inhibitory effect of IB-MECA was almost completely abolished by pre-treatment with 1 microM of the selective A3 antagonist 9-chloro-2-(2-furyl)-5-phenylactylamino[1,2,4]triazolo[1,5-c]quina zoline (MRS 1220), but not the selective A1 antagonist 1,3-dipropyly-8-cyclopentylxanthine (DPCPX) or the selective A2 antagonist 3,7-dimethyl-1-propargylxanthine (DMPX). IB-MECA also significantly inhibited C5a-induced O2- release with IC50 (95% CI) of 9.5 microM (4.6-13.1 microM) whereas adenosine and the A1 agonist CPA potentiated this effect at low concentrations. The potentiation appeared to be a result of their direct O2- release from these cells, probably mediated via A1 receptors. The inhibition by IB-MECA was selectively reversed by MRS 1220. These results show that the A3 receptors on human eosinophils mediate inhibition of both degranulation and O2- release and suggest a therapeutic potential for A3 agonists in diseases such as asthma in which activated eosinophils are involved.     

The anti-inflammatory interactions of epinephrine with human neutrophils in vitro are achieved by cyclic AMP-mediated accelerated resequestration of cytosolic calcium

This study was designed to evaluate the effects of epinephrine (0.01-1 microM) on superoxide production by, and release of elastase from human neutrophils activated with the chemotactic tripeptide, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) (1 microM) in vitro, and to relate alterations in these responses to changes in adenosine 3,5' cyclic monophosphate (cAMP) and cytosolic free Ca(2+). Cyclic AMP, superoxide production and elastase release were measured by radioimmunoassay, lucigenin-enhanced chemiluminescence, and a colorimetric procedure respectively. Cytosolic Ca(2+) fluxes were measured by fura-2 spectrofluorimetry in combination with radiometric procedures that enable distinction between net efflux and influx of the cation. Epinephrine treatment of neutrophils resulted in increased cAMP and dose-related inhibition of both superoxide production and elastase release, which was potentiated by the type 4 phosphodiesterase inhibitor, rolipram, and attenuated by propranolol, but not by selective beta(1)-, alpha(1)- or alpha(2)-adrenoreceptor antagonists. Although epinephrine did not affect the FMLP-activated abruptly-occurring increase in fura-2 fluorescence intensity, indicating no effects on the release of Ca(2+) from neutrophil intracellular stores, this agent accelerated the rate of decline in fluorescence in the setting of decreased efflux and a reduction in store-operated influx of Ca(2+). These effects of epinephrine on the clearance of Ca(2+) from the cytosol of FMLP-activated neutrophils were attenuated by propranolol, and are compatible with enhancement of the activity of the cAMP-dependent Ca(2+) sequestering/resequestering endo-membrane Ca(2+)-ATPase. We conclude that epinephrine down-regulates the pro-inflammatory activities of neutrophils by cAMP-mediated enhancement of the clearance of cytosolic Ca(2+).     

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.     

Effects of adenosine receptor agonists on guinea-pig isolated working hearts and the role of endothelium and NO

The hypothesis that the coronary vasodilator effects of adenosine receptor agonists are independent of the vascular endothelium or mediators derived therefrom was examined in guinea-pig isolated working hearts. Adenosine receptor agonists, 5'-(N-ethylcarboxamido)-adenosine (NECA; two-fold selective for A2 over A1 receptors), 2-[p-(2-carboxyethyl)phenylethylamino]-5'-N-ethylcarboxamidoadenosine (CGS21680; A2A selective), N6-cyclopentyl-adenosine (CPA; A1 selective) and N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide (IB-MECA; A3 selective), were infused (3 x 10(-7) M) after endothelium removal by passing oxygen through the coronary circulation. In spontaneously beating hearts, CGS21680 and NECA increased, while CPA decreased, coronary flow. NECA and CPA reduced heart rate, left ventricular pressure and aortic output. The nitric oxide synthase (NOS) inhibitor, N(G)-nitro-L-arginine (L-NOARG; 3 x 10(-5) M) abolished the vasodilatation by NECA but not CGS21680, indicating that nitric oxide (NO) of a non-endothelial source mediated the NECA response. Coronary vasodilatation by CGS21680 was inhibited bythe A2A receptor antagonist, 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo [2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM241385). Indometacin (10(-6) M) attenuated the coronary vasodilatation to CGS21680, suggesting a partial role for cyclooxygenase products. IB-MECA had no effect, indicating no A3 receptor involvement. In paced working hearts, the responses were similar except CPA had no effect on coronary flow or aortic output and CGS21680 increased left ventricular pressure and the maximum rate of ventricular pressure rise. This study has demonstrated functionally effective removal of the endothelium by a novel method of passing oxygen through the coronary vasculature. A coronary vasodilator action of adenosine receptor agonists mediated via A2A receptors is endothelium- and NO-independent, but partially involves cyclooxygenase products.     

Release inhibitory receptors activation favours the A2A-adenosine receptor-mediated facilitation of noradrenaline release in isolated rat tail artery

1. Interactions between A(2A)-adenosine receptors and alpha(2)-, A(1)- and P2- release-inhibitory receptors, on the modulation of noradrenaline release were studied in isolated rat tail artery. Preparations were labelled with [(3)H]-noradrenaline, superfused with desipramine-containing medium, and stimulated electrically (100 pulses at 5 Hz or 20 pulses at 50 Hz). 2. Blockade of alpha(2)-autoreceptors with yohimbine (1 microM) increased tritium overflow elicited by 100 pulses at 5 Hz but not by 20 pulses at 50 Hz. 3. The selective A(2A)-receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS 21680; 1-100 nM) enhanced tritium overflow elicited by 100 pulses at 5 Hz. Yohimbine prevented the effect of CGS 21680, which was restored by the A(1)-receptor agonist N(6)-cyclopentyladenosine (CPA; 100 nM) or by the P2-receptor agonist 2-methylthioadenosine triphosphate (2-MeSATP; 80 microM). 4. CGS 21680 (100 nM) failed to increase tritium overflow elicited by 20 pulses at 50 Hz. The alpha(2)-adrenoceptor agonist 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14304; 30 nM), the A(1)-receptor agonist CPA (100 nM) or the P2-receptor agonist 2-MeSATP (80 microM) reduced tritium overflow. In the presence of these agonists CGS 21680 elicited a facilitation of tritium overflow. 5. Blockade of potassium channels with tetraethylammonium (TEA; 5 mM) increased tritium overflow elicited by 100 pulses at 5 Hz to values similar to those obtained in the presence of yohimbine but did not prevent the effect of CGS 21680 (100 nM) on tritium overflow. 6. It is concluded that, in isolated rat tail artery, the facilitation of noradrenaline release mediated by A(2A)-adenosine receptors is favoured by activation of release inhibitory receptors.     

Effects of adenosine A1 and A2A receptor activation on the evoked release of glutamate from rat cerebrocortical synaptosomes

1. The effects of adenosine A2A and A1 receptor activation on the release of glutamate were studied in rat cerebral cortex synaptosomes exposed in superfusion to adenosine receptor ligands. 2. Adenosine (0.1 microM) produced a significant potentiation of the Ca2+-dependent K+ (15 mM)-evoked [3H]-D-aspartate overflow (20.4+/-3.5%), which was blocked by A2A blocker SCH58261 (0.1 microM). At higher concentrations (10 - 1000 microM) adenosine inhibited in a DPCPX-sensitive manner the Ca2+-dependent K+-evoked [3H]-D-aspartate overflow. The inhibitory effect of adenosine at 1000 microM was significantly increased by SCH58261. This inhibition was antagonized by 1 microM DPCPX. Adenosine did not produce any effect on basal release. 3. The A2A receptor agonist CGS 21680 was ineffective on basal release, but stimulated the Ca2+-dependent K+-evoked overflow of [3H]-D-aspartate (EC50 approximately 1 pM). The effect of 0.01 nM CGS 21680 was totally sensitive to the A2A receptor antagonist SCH58261 (IC50 approximately 5 nM). 4. The A1 receptor agonist CCPA inhibited the Ca2+-dependent K+-evoked [3H]-D-aspartate overflow (EC50 approximately 20 nM). The effect of 100 nM CCPA was abolished by 100 nM of the A1 receptor antagonist DPCPX. 5. The K+ (15 mM)-evoked overflow of endogenous glutamate was enhanced by CGS 21680 (0.01 nM) and inhibited by CCPA (0.1 microM). The effect of CGS 21680 was abolished by SCH58261 (0.1 microM) and that of CCPA by DPCPX (0.1 microM). 6. It is concluded that adenosine and adenosine receptor agonists modulate glutamate release by activating inhibitory A1 and excitatory A2A receptors present on glutamatergic terminals of the rat cerebral cortex.     

Experimental hypothyroidism modifies specific binding of A1 and A2A analogues to adenosine receptors in the rat kidney

1 Binding kinetic studies with the adenosine analogues [3H]CPA (0.250-50 nm) and [3H]CGS21680 (0.1-100 nm) were performed in renal tissue from control (NL) and thyroidectomised (HTX) rats. We propose that the low renal adenosine content reported in hypothyroid rats may induce changes in the density and/or affinity of adenosine receptor, distributed in the cortex (C), outer medulla (OM), and inner medulla (IM) of the kidney. 2 [3H]CPA and [3H]CGS21680 binding saturation isotherms were fitted by nonlinear regression analysis and evaluated by Furchgott's method. These results revealed high (KH) and low (KL) affinity (KD) sites for both compounds. As expected, a heterogeneous pattern was observed for Bmax and KD values. 3 Bound [3H]CPA and [3H]CGS21680 were displaced by increasing concentrations of nonlabelled DPCPX and NECA, respectively, indicating the presence of A1 and A2A adenosine receptors distributed in the renal segments studied. 4 The relative intrinsic efficacy (epsilon) for [3H]CPA and [3H]CGS21680 showed extreme values (far from 1.0), 0.5 in IM NL and 2.70 in IM HTX for [3H]CGS21680. 5 Our results indicate that A2A adenosine receptor is predominant in IM from HTX, but A1 receptors are expressed preferentially in C in NL. 6 We conclude that the changes observed in number, affinity, and epsilon for the A2A receptor in IM from HTX might be responsible from alterations in medullary function, that is, incapacity for urine concentration as observed in the hypothyroid kidney.     

Characterization of human A(2B) adenosine receptors: radioligand binding, western blotting, and coupling to G(q) in human embryonic kidney 293 cells and HMC-1 mast cells.

Recombinant human A(2B) adenosine receptors (A(2B)ARs) and receptors extended on the amino terminus with hexahistidine and the FLAG epitope, DYKDDDDK (H/F-A(2B)) were stably overexpressed (to >20,000 fmol/mg protein) in human embryonic kidney 293 cells (HEK-A(2B)). By Western blotting, the H/F-A(2B) receptor runs as a 34.8-kDa glycoprotein. Pharmacological properties of A(2B)ARs were characterized with (125)I-3-aminobenzyl-8-phenyl-(4-oxyacetic acid)-1-propylxanthine (K(D), 36 nM). In competition binding assays, the affinity of agonists is reduced by substitution on either the N(6)- or the C-2 position of the adenine ring, whereas 5'-substitutions increase affinity, resulting in the potency order: 5'-N-ethylcarboxamidoadenosine (NECA) > N(6)-aminobenzyl-NECA approximately 2-chloroadenosine > 2-[4-(2-carboxyethyl)phenethylamino]-NECA (CGS21680) > N(6)-aminobenzyladenosine. The A(2B)AR is potently blocked by the A(2A)-selective antagonist 4-(2-[7-amino-2-[2-furyl][1,2, 4]triazolo-[2,3-a][1,3,5] triazin-5-yl-amino]ethyl)phenol (ZM241385; K(I), 32 nM for A(2B), 1.4 nM for A(2A)) and the A(1) selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (K(I), 50.5 nM for A(2B); 2.5 nM for A(1)). The K(I) values for the antiasthmatic xanthines, theophylline (7.8 microM) and enprofylline (6.4 microM), are below their therapeutic plasma concentrations (20 to 50 microM), and agree with K(I) determinations for inhibition of NECA-stimulated cAMP accumulation in HEK-A(2B) cells. NECA or N(6)-(2-iodo)benzyl-5'-N-methylcarboxamidodoadenosine (IB-MECA) stimulate inositol trisphosphates and calcium accumulation in HEK-A(2B) or HEK-A(3) cells, respectively, but only the A(3) response is prevented by pertussis toxin. In human HMC-1 mast cells, A(2B)AR activation stimulates calcium mobilization and cAMP accumulation. We conclude that HEK-A(2B) cells and HMC-1 mast cells possess A(2B)AR glycoproteins that are coupled to both G(q/11) and G(s).     

Relaxation of mouse isolated aorta to adenosine and its analogues does not involve adenosine A(1), A(2) or A(3) receptors

Relaxations to adenosine and analogues were investigated in the mouse aorta in the presence of the adenosine A(1) receptor-selective antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 30 nM), which did not affect relaxations to adenosine or its analogue N(6)-R-phenylisopropyladenosine (R-PIA) but abolished contractile adenosine A(1) receptor-mediated responses to these agonists. Relaxations to adenosine, 5'-N-ethylcarboxamidoadenosine, R-PIA, 2-[p-(2-carbonylethyl)-phenylethylamino]-5'-N-ethylcarboxamidoadenosine (CGS 21680), and N(6)-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB-MECA) were unaffected by the adenosine A(1)/A(2) receptor antagonist 8-sulphophenyltheophylline (100 microM). IB-MECA relaxations were unaffected by the adenosine A(3) receptor-selective antagonist 3-ethyl-5-benzyl-2-methyl-6-phenyl-4-phenylethynyl-1,4-(+/-)-dihydropyridine-3,5-dicarboxylate (MRS1191, 30 microM) and R-PIA relaxations were unaffected by N(G)-nitro-L-arginine methyl ester (100 microM) and endothelium removal. In conclusion, relaxant responses to adenosine and analogues do not involve adenosine A(1), A(2) or A(3) receptors and are endothelium- and nitric oxide-independent.     

Characterization of the adenosine receptor mediating contraction in rat colonic muscularis mucosae.

1. The objective of this study was to characterize the adenosine receptor mediating contraction in rat isolated colonic muscularis mucosae (RCMM). 2. Sequential additions of the adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA; 0.01-10 microM) elicited reproducible, concentration-related contractions in RCMM. The effects of NECA were mimicked by the adenosine A1 receptor-selective agonists cyclopentyladenosine (CPA), R-phenylisopropyladenosine (R-PIA) and N-[1S, trans)2-hydroxycyclopentyl] adenosine (GR79236) and by S-PIA (the stereoisomer of R-PIA). The adenosine A2 agonists N-[(2-methylphenyl)methyl] adenosine (metrifudil) and 2-[p-(2-carboxyethyl)phenethylamine]-5'-N-ethylcarboxamidoadenosine (CGS21680) also produced contractions in RCMM but were 54 and 165 times less potent respectively than NECA. The rank order of agonist potency for contraction of RCMM was CPA > or = GR79236 = R-PIA > or = NECA > > S-PIA = metrifudil > CGS21680, which is identical to that reported for the inhibition of spontaneous rate in rat isolated right atria and inhibition of lipolysis in rat isolated adipocytes by these same agonists. 3. R-PIA, S-PIA and metrifudil behaved as partial agonists in RCMM. 4. The adenosine A1 receptor-selective antagonist 8-cyclopentyl-1,3- dipropylxanthine (DPCPX) inhibited the contractions produced by all the adenosine agonists tested, with pKB values between 9.2 and 9.5. The non-selective adenosine antagonist 8-phenyltheophylline (8-PT) antagonized the effects of NECA but also markedly potentiated (by 93.0 +/- 10.2% at 3 microM) the maximum contractile response to NECA in RCMM. Neither 8-PT (3 microM) nor DPCPX (0.1 microM) had any effect on the contractions produced by carbachol.(ABSTRACT TRUNCATED AT 250 WORDS)     

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'-(beta,gamma-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 greater than or equal to CPA greater than AMPPCP = adenosine greater than 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 microM. 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 much greater than adenosine greater than CPA = CGS 21680, and a concentration of DPCPX of 1 microM was required to antagonize responses to NECA and adenosine. This suggests the presence of A2 receptors in this tissue.(ABSTRACT TRUNCATED AT 250 WORDS)     

Presynaptic adenosine A2A receptors enhance GABAergic synaptic transmission via a cyclic AMP dependent mechanism in the rat globus pallidus

1. We previously reported a presynaptic facilitatory action of A(2A) receptors on GABAergic synaptic transmission in the rat globus pallidus (GP). In the present study we identify the intracellular signalling mechanisms responsible for this facilitatory action of A(2A) receptors, using biochemical and patch-clamp methods in rat GP slices. 2. The adenosine A(2A) receptor selective agonist CGS21680 (1, 10 microM) and the adenylyl cyclase activator forskolin (1, 10 microM) both significantly increased cyclic AMP accumulation in GP slices. The CGS21680 (1 microM)-mediated increase in cyclic AMP was inhibited by the A(2A) receptor selective antagonist KF17837 (10 microM). 3. In an analysis of miniature inhibitory postsynaptic currents (mIPSCs), forskolin (10 microM) increased the mIPSC frequency without affecting their amplitude distribution, a result similar to that previously reported with CGS21680. 4. The adenylyl cyclase inhibitor 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ22,536, 300 microM) abolished the CGS21680-induced enhancement in the frequency of mIPSCs. 5. H-89 (10 microM), a selective inhibitor for cyclic AMP-dependent protein kinase (PKA), blocked the CGS21680-induced enhancement of the mIPSC frequency. 6. The calcium channel blocker CdCl(2) (100 microM) did not prevent CGS21680 from increasing the frequency of mIPSCs. 7. These results indicate that A(2A) receptor-mediated potentiation of mIPSCs in the GP involves the sequential activation of the A(2A) receptor, adenylyl cyclase, and then PKA, and that this facilitatory modulation could occur independently of presynaptic Ca(2+) influx.     

A3 adenosine receptor activation triggers phosphorylation of protein kinase B and protects rat basophilic leukemia 2H3 mast cells from apoptosis

Adenosine accumulates to high levels in inflamed or ischemic tissues and activates A3 adenosine receptors (ARs) on mast cells to trigger degranulation. Here we show that stimulation of rat basophilic leukemia (RBL)-2H3 mast-like cells with the A3 AR agonists N6-(3-iodo)benzyl-5'-N-methylcarboxamidodoadenosine (IB-MECA; 10 nM) or inosine (10 microM) stimulates phosphorylation of protein kinase B (Akt). IB-MECA (1 microM) also causes a >50% reduction in apoptosis caused by exposure of RBL-2H3 cells to UV light. Akt phosphorylation is not stimulated by 100 nM N6-cyclopentyladenosine (A1-selective) or CGS21680 (A2A-selective) and is absent in cells pretreated with wortmannin or pertussis toxin. The KI values of the AR antagonists BW-1433 and 8-sulfophenyltheophylline (8-SPT) were determined in radioligand binding assays for all four subtypes of rat ARs: BW-1433 (A1, 5.8 +/- 1.0 nM; A2A, 240 +/- 37; A2B, 30 +/- 10; A3, 12,300 +/- 3, 700); 8-SPT (A1, 3.2 +/- 1.2 microM; A2A, 57 +/- 4; A2), 2.2 +/- 0.8; A3, >100). BW-1433 and the A3-selective antagonist MRS1523 (5 microM), but not 8-SPT (100 microM), block IB-MECA-induced protection from apoptosis, confirming the A3 AR as the mediator of the antiapoptotic response. The data suggest that adenosine and inosine activate Gi-coupled A3 ARs to protect mast cells from apoptosis by a pathway involving the betagamma subunits of Gi, phosphatidylinositol 3-kinase beta, and Akt. We speculate that activation of A3 ARs on mast cells or other cells that express A3 ARs (e.g., eosinophils) may facilitate their survival and accumulation in inflamed tissues.     

Characterization of P1-purinoceptors on rat isolated duodenum longitudinal muscle and muscularis mucosae.

1. P1-purinoceptors mediating relaxation of the rat duodenum longitudinal muscle and contraction of the rat duodenum muscularis mucosae were characterized by the use of adenosine and its analogues, 5'-N-ethylcarboxamidoadenosine (NECA), N6-cyclopentyl-adenosine (CPA), N6-(phenylisopropyl)adenosine (R-PIA), 2-chloroadenosine (2-CADO) and 2-p-((carboxyethyl)phenethylamino)-5'-carboxamidoadenosine (CGS21680), as well as the P1-purinoceptor antagonist 8-phenyltheophylline (8-PT) and the A1-selective antagonist, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX). 2. In the rat duodenum longitudinal muscle, the order of potency of the adenosine agonists was CPA > NECA > adenosine > CGS21680. DPCPX antagonized responses to CPA and NECA at a concentration of 1 nM suggesting that they are acting at A1 receptors. A Schild plot versus CPA gave a slope near to unity (slope = 0.955) and a pA2 of 9.8 confirming that CPA was acting via A1 receptors. Schild analysis for DPCPX versus NECA, however, gave a slope of 0.674 suggesting that NECA was acting on both A1 and A2 receptors. CGS21680, a selective A2a agonist, was much less potent than adenosine suggesting that the A2 receptors are of the A2b subtype. 3. In the rat duodenum muscularis mucosae, the order of potency of the adenosine agonists was NECA > or = R-PIA = CPA > 2-CADO > adenosine, and DPCPX antagonized responses to CPA and NECA at a concentration of 1 microM. CGS21680, at a concentration of 10 microM, had no effect on this tissue. This suggests the presence of A2 receptors in this tissue and that they are of the A2b subtype. 4. These results are in agreement with previous studies in the whole duodenum showing the presence of A1 and A2b receptors causing relaxation, and this shows that the longitudinal muscle dominates the response of the whole tissue. In addition, a contractile A2b receptor has been revealed on the muscularis mucosae, the first time this subtype has been reported to elicit an excitatory response in a smooth muscle preparation.     

Pharmacological characterisation of the adenosine receptor mediating increased ion transport in the mouse isolated trachea and the effect of allergen challenge

The effect of adenosine on transepithelial ion transport was investigated in isolated preparations of murine trachea mounted in Ussing chambers. The possible regulation of adenosine receptors in an established model of allergic airway inflammation was also investigated. Mucosally applied adenosine caused increases in short-circuit current (I(SC)) that corresponded to approximately 50% of the response to the most efficacious secretogogue, ATP (delta I(SC) 69.5 +/- 6.7 microA cm2). In contrast, submucosally applied adenosine caused only small (<20%) increases in I(SC), which were not investigated further. The A1-selective (N6-cyclopentyladenosine, CPA, 1 nM-10 microM), A2A-selective (2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxoamido adenosine; CGS 21680; 0.1-100 microM) and A3-selective (1-deoxy-1-[6-[[(3-iodophenyl)-methyl]amino]-9H-purin-9-yl]-N-methyl-beta-D-ribofuranuronamide; IB-MECA; 30 nM-100 microM) adenosine receptor agonists were either equipotent or less potent than adenosine, suggesting that these receptors do not mediate the response to adenosine. The A1 receptor selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 10 nM-1 microM) caused a rightward shift of the adenosine concentration-effect curve only at 1 microM. The mixed A2A/A2B receptor antagonist 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385) also caused rightward shift of the adenosine concentration-effect curve, again only at micromolar concentrations, suggestive of the involvement of A2B receptors. In preparations from animals sensitised to ovalbumin and challenged over 3 days with aerosol ovalbumin, a decrease in baseline I(SC) was observed and responses to ATP were diminished. Similarly, the amplitude of responses to adenosine were attenuated although there was no change in potency. These results suggest that the A2B receptor mediates the I(SC) response to adenosine in the mouse trachea. This receptor does not appear to be regulated in a standard asthma model.     

Metabolism of methylarginines by human vasculature; implications for the regulation of nitric oxide synthesis.

1. The effect of the A2A adenosine receptor agonist, 2-p-(2-carboxyethyl)phenethyl-amino-5'-N-ethylcarboxamidoadenosine (CGS 21680) on the potassium evoked release of [3H]-gamma-aminobutyric acid ([3H]-GABA) from nerve terminals derived from the caudate-putamen and the globus pallidus of the rat was compared. In both preparations CGS 21680 (1 nM) inhibited the [3H]-GABA release evoked by 15 mM KCl but had no effect on that evoked by 30 mM KCl. 2. The ability of CGS 21680 (1 nM) to inhibit the release of [3H]-GABA from striatal nerve terminals was unaffected by the presence of the GABA receptor antagonists, bicuculline (10 microM), phaclofen (100 microM) and 2-hydroxysaclofen (100 microM). Similarly the opioid receptor antagonist, naloxone (10 microM), the adenosine A1 receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 40 nM), and the cholinoceptor antagonists, mecamylamine (10 microM) and atropine (100 nM) had no effect on this inhibition. 3. The ability of CGS 21680 (0.1 nM) to stimulate the release of [3H]-acetylcholine ([3H]-ACh) from striatal nerve terminals was unaffected by the presence of bicuculline (10 microM), 2-hydroxysaclofen (100 microM), phaclofen (100 microM), naloxone (10 microM) and DPCPX (4 nM). 4. The novel A2A receptor antagonist, (E)-8-(3,4-dimethoxystyryl)-1,3-dipropyl-7-methylxanthine (KF 17837), blocked the CGS 21680 (1 nM)-induced inhibition of [3H]-GABA efflux with an EC50 of approximately 30 nM and also antagonized the CGS 21680 (0.1 nM)-induced stimulation of [3H]-ACh release with an EC50 of approximately 0.3 nM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative effects of a selective adenosine A2 receptor agonist, CGS 21680, and nitroprusside in vascular smooth muscle.

CGS 21680 (2-[p-(2-carboxyethyl)phenylethylamino]-5'-N-ethyolcarboxamidoa denosine) is an adenosine agonist that has been reported recently to bind selectively to adenosine A2 receptors in rat brain. This adenosine agonist, and the parent compound NECA (5'-N-ethylcarboxamidoadenosine), were found to be potent vasorelaxants of prostaglandin F2 alpha (PGF2 alpha) precontracted porcine coronary smooth muscle with EC50s of 4.5 and 9.7 nM, respectively. Schild analysis of the inhibition of CGS 21680, NECA and 2-chloroadenosine induced relaxation of the porcine coronary artery by CGS 15943 (9-chloro-2-(2-furanyl)[1,2,4]triazolo[1,5-C]quinazolin-5-amine), an A2 receptor antagonist, yielded identical pA2 values for the antagonist (approximately 9.3). This indicates that the same receptor mediates the effects of these three adenosine agonists. NECA and CGS 21680 were equipotent in most vascular preparations except in the canine coronary artery. Porcine coronary arterial rings contracted with PGF2 alpha were relaxed by NECA or CGS 21680 as well as by nitroprusside; those contracted with KCl (40 mM) were relaxed only by nitroprusside. In rabbit aorta, contractions induced by phenylephrine or PGF2 alpha were inhibited by nitroprusside but not by NECA or CGS 21680. Thus, the adenosine A2 receptor agonists, NECA and CGS 21680, are potent vasorelaxants that display regional vascular and species variations that differ from those of nitroprusside.     

Radioligand binding and functional responses of ligands for human recombinant adenosine A(3) receptors

The binding and functional properties of adenosine receptor ligands were compared in Chinese hamster ovary cells transfected with human adenosine A(3) receptors. Inhibition of [(125)I]-aminobenzyl-5'-N-methylcarboamidoadenosine ([(125)I]-AB-MECA) binding by adenosine receptor ligands was examined in membrane preparations. Inhibition of forskolin-induced cAMP accumulation by agonists was measured using a cAMP enzyme immunoassay. The rank order of agonist potency for both assays was N(6)-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB-MECA) > 5'-N-ethylcarboxamidoadenosine (NECA) > (-)-N(6)-[(R)-phenylisopropyl] adenosine (R-PIA) > 4-aminobenzyl-5'-N-methylcarboxamidoadenosine (AB-MECA) > N(6)-cyclopentyl adenosine (CPA) > adenosine. The radioligand binding rank order of antagonist potency was N-[9-chloro-2-(2-furanyl)[1,2,4]-triazolo[1,5-c]quinazolin-5-benzeneacetamide (MRS1220) > 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) > 8-phenyltheophylline (8-PT) > 8-(p-sulfophenyl)-theophylline (8-SPT). MRS1220 competitively inhibited the effect of IB-MECA on cAMP production, with a K(B) value of 0.35 nm. These data are characteristic of adenosine A(3) receptors. The absence of Mg(2+) and presence of guanosine 5'-(gamma-thio)triphosphate (GTPgammaS) significantly reduced agonist binding inhibition potency, indicating binding to high- and low-affinity states. The IB-MECA, NECA and R-PIA IC(50) values were greater for the cAMP assay than for radioligand binding, suggesting an efficient stimulus-response transduction pathway.