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Immunoglobulins (IgG) from patients with Graves' disease increase inositol phosphate (IP) as well as cAMP production in rat thyroid FRTL-5 cells; IgGs from normal control subjects do not. Graves' IgG-and TSH-induced IP formation is inhibited by blocking TSH receptor (TSHR) antibodies from hypothyroid patients with primary myxedema, as is the cAMP response; this suggests that the Graves' IgG are acting through the TSHR to induce both the cAMP and phosphatidyl-inositol 4,5-biphosphate signal cascades in FRTL-5 thyroid cells as in cells with recombinant TSHR. Optimal conditions for measuring the Graves' IgG-induced IP increase include a NaCl-free Hanks' Balanced Salt Solution (HBSS) buffer system and a P1 purinergic receptor agonist; the action of each is additive. Optimization by NaCl-free HBSS is similar to that observed in cAMP assays and is specific for TSH or Graves' IgG; thus, NaCl-free HBSS did not affect ATP-induced, and actually inhibited norepinephrine-induced, IP production in FRTL-5 cells. The P1 purinergic receptor agonist acts via receptor cross-talk, which also allows further optimization of cAMP assays. Thus, adenosine deaminase improves Graves' IgG-induced cAMP production by removing adenosine from the medium. Although NaCl-free HBSS improved TSH- or Graves' IgG-induced IP and cAMP production in cells with recombinant TSHR; the modulatory action of phenylisopropyladenosine was lost.

1. Radioligand binding and functional studies were undertaken to investigate the P1-purinoceptors present in the separated myometrial layers and the endometrium of the guinea-pig uterus. 2. In preparations of endometrium-denuded circular myometrium, the A2-selective agonists (2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamido-adeno sin e (CGS 21680, 100 mumol/L) and N-ethylcarboxamido adenosine (NECA, 1-10 mumol/L) inhibited contractile responses to phenylephrine. In preparations of endometrium-intact circular myometrium, NECA (10 mumol/L) enhanced responses to phenylephrine. NECA did not modulate the spontaneous contractions of longitudinal myometrium. 3. Homogenate binding studies with circular myometrium, longitudinal myometrium and endometrium revealed saturable high affinity [3H]-NECA binding sites. The mean maximal densities of binding sites (Bmax) were 2.08, 14.7 and 15.5 fmol/mg protein, and pKD (neg. log dissociation constant) values were 9.82, 9.19 and 7.44, respectively. 4. (R-) and (S-)-N6-(2-phenylisopropyl)adenosine (R- and S-PIA) both competed for two [3H]-NECA binding sites in preparations of circular myometrium. CGS 21680 competed for two [3H]-NECA binding sites in preparations of endometrium and longitudinal myometrium. All other agonist competition was for one site only. The rank orders of potency of high affinity binding were S-PIA > or =R-PIA > or = CGS 21680 (circular myometrium), R-PIA > CGS 21680 > or = S-PIA (longitudinal myometrium) and CGS 21680 > > S-PIA > or = R-PIA (endometrium). 5. In preparations of circular myometrium, longitudinal myometrium and endometrium the selective A1-purinoceptor antagonist, 1,3-dipropyl-8-(2-amino-4-chloro)-phenylxanthine (PACPX), competed for two [3H]-NECA binding sites, the non-selective antagonist 3,7-dimethyl-1-propargylxanthine (DMPX), competed for one site only.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding of [3H]cyclohexyladenosine (CHA) to the cellular fractions and P2 subfractions of the goldfish brain was studied. The A1 receptor density was predominantly in synaptosomal membranes. In goldfish brain synaptosomes (P2), 30 mM K+ stimulated glutamate, taurine and GABA release in a Ca(2+)-dependent fashion, whereas the aspartate release was Ca(2+)-independent. Adenosine, R-phenylisopropyladenosine (R-PIA) and CHA (100 microM) inhibited K(+)-stimulated glutamate release (31%, 34% and 45%, respectively). All of these effects were reversed by the selective adenosine A1 receptor antagonist, 8-cyclopentyltheophylline (CPT). In the same synaptosomal preparation, K+ (30 mM) stimulated Ca2+ influx (46.8 +/- 6.8%) and this increase was completely abolished by pretreatment with 100 nM omega-conotoxin. Pretreatment with 100 microM R-PIA or 100 microM CHA, reduced the evoked increase of intra-synaptosomal Ca2+ concentration, respectively by 37.7 +/- 4.3% and 39.7 +/- 9.0%. A possible correlation between presynaptic A1 receptor inhibition of glutamate release and inhibition of calcium influx is discussed.

Langendorff-perfused guinea pig ventricles were used to examine the effects of the adenosine agonists, (-)-N-6-phenylisopropyl-adenosine (PIA) and 5'-N-ethylcarboxamidoadenosine and the muscarinic cholinergic agonist, acetylcholine, on the rate of tension development, protein phosphatase inhibitor-1 (PPI-1) activity, and cyclic AMP-dependent protein kinase (PKA) activity ratio. Isoproterenol (10 nM) and forskolin (1 microM) stimulated rate of tension development, PKA activity ratio and PPI-1 activity each approximately 2-fold. Acetylcholine (1 microM) by itself was not effective, but when administered with isoproterenol for forskolin reduced the rate of tension development and PPI-1 activity without decreasing PKA activity ratio. Similarly, both PIA and 5'-N-ethylcarboxamidoadenosine alone were ineffective, but when simultaneously applied with isoproterenol attenuated the isoproterenol-stimulated rate of tension development and PPI-1 activity. PIA reduced PKA activity ratio, whereas 5'-N-ethylcarboxyamidoadenosine failed to do so. However, the effect of PIA on PKA activity ratio was smaller than those seen on rate of tension development and PPI-1 activity. Hence, the present data do not support a cyclic AMP-dependent regulation of PPI-1 activity by adenosine and muscarinic agonists. It is tempting to speculate that adenosine and muscarinic agonists reduce PPI-1 activity by a cyclic AMP-independent mechanism.

Signal transduction by A1 adenosine receptors was investigated by measuring the modulation by adenosine agonists of guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTP[S]) binding to guanine nucleotide-binding proteins (G proteins). The extent of stimulation of [35S]GTP[S] binding was dependent on the presence of high concentrations of Mg2+ (1-10 mM), GDP (10 microM), and NaCl (100 mM). Under optimal conditions, the agonist (R)-N6-phenylisopropyladenosine [(R)-PIA] stimulated binding of [35S]GTP[S] to G proteins approximately 2.3-fold. All adenosine receptor agonists tested stimulated the binding of [35S]GTP[S] with a rank order of potency typical of A1 adenosine receptors in bovine tissues, (R)-PIA > 2-chloro-N6-cyclopentyladenosine > (S)-PIA > 5'-N-ethylcarboxamidoadenosine > 2-[4-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamidoadenosin e. The EC50 values for G protein activation correlated with the Ki value of agonists for inhibition of radioligand binding to the high affinity state of the A1 adenosine receptor. The inclusion of 100 mM NaCl as well as increasing GDP concentrations led to a parallel increase of Ki values and EC50 values. In addition, both compounds induced a shift of A1 adenosine receptors from the high affinity state for agonists to the low affinity state.

Multiple physiological functions have been described to be affected by adenosine in numerous cell types. A comparative study of the expression of adenosine receptors has been performed in preadipocytes and adipocytes from rat epididymal fat pad. The results show that, in agreement with its well known antilipolytic effect, adenosine induces a negative modulation of adenylate cyclase via the A1 receptor present in adipocytes. By contrast, the A2 receptor subtype, which is positively coupled to adenylate cyclase, is herein demonstrated to be only expressed in adipose precursor cells. This expression allows, in chemically defined medium, the adenosine analogue NECA, by means of its ability to elevate cAMP concentration, to potentiate differentiation. These findings emphasize the role that adenosine might play as a bimodal regulatory extracellular signal in adipose tissue development.

1. Radioligand binding properties of the adenosine receptor ligands, [3H]-1,3-dipropyl-8-cyclopentylxanthine ([3H]-DPCPX), and [3H]-R-phenylisopropyladenosine ([3H]-R-PIA) were investigated in frog brain membranes. 2. The specific binding of the adenosine antagonist, [3H]-DPCPX to frog brain membranes showed one binding site with Kd and Bmax values of 43.8 nM and 0.238 +/- 0.016 pmol mg-1 protein, respectively. Guanosine 5'-triphosphate (GTP, 100 microM) decreased to 72 +/- 7% and Mg2+ (8 mM) increased to 121 +/- 3% [3H]-DPCPX (40 nM) binding to frog brain membranes. 3. [3H]-DPCPX saturation binding experiments performed in the presence of Mg2+ (8 mM), or in the presence of GTP showed that Mg2+ ions decreased the Kd value of [3H]-DPCPX to 14 nM, and GTP increased this value to 65.6 nM. Bmax values were not significantly (P > 0.05) modified (0.261 +/- 0.018 pmol mg-1 protein, with Mg2+, and 0.266 +/- 0.026 pmol mg-1 protein, in presence of GTP) by the presence of Mg2+ or GTP. 4. The specific binding of [3H]-R-PIA (15 nM) was decreased to 37 +/- 6% by GTP (100 microM) and increased to 123 +/- 4% by Mg2+ (8 mM). [3H]-R-PIA saturation binding experiments performed in the presence of Mg2+ (8 mM) showed one binding site with Kd and Bmax values of 0.9 nM and 0.229 +/- 0.008 pmol mg-1 of protein, respectively. 5. The concentration-inhibition curves of adenosine agonists and antagonists versus [3H]-DPCPX binding showed the following order of potencies: CPA> R-PIA~ NECA> S-PIA> > CGS 21680, for the agonists, and XAC ~-DPCPX> > XCC> PACPX, for the antagonists.6. The present results suggest that the adenosine binding site in the frog brain membranes is G-protein coupled, but that the antagonist affinities and the pharmacological profile is different from the Al or A2 adenosine receptors.

The accumulation of cyclic adenosine 3',5'-phosphate (cAMP) elicited by antidiuretic hormone (arginine vasopressin, AVP) in the medullary collecting tubule (OMCD) microdissected from the rat kidney is inhibited by different factors: the A1 agonist of adenosine (-)-N6-(R-phenylisopropyl) adenosine (PIA), an alpha 2-adrenergic agonist clonidine (CLO), and prostaglandin E2 (PGE2). The negative regulation elicited by PGE2 was further characterized by measuring summation of inhibition with other inhibitors, by testing the effect of pertussis toxin and by studying the part played by extracellular calcium. Inhibitors were used at concentrations inducing maximum effects. The simultaneous addition of 0.3 microM PGE2 with either 0.1 microM PIA or 1 microM CLO led to an inhibition of the response to AVP (80.0 +/- 3.5%, SEM, N = 7 and 92.6 +/- 0.8%, N = 5, respectively) greater than those elicited by each agent alone. In contrast, PIA and CLO added together induced an inhibition similar to that due to CLO alone. The action of PGE2 in combination with either PIA or CLO corresponded to a partial summation fitting with the values calculated by assuming a cumulative inhibition. Preincubation of OMCD samples with pertussis toxin (100 ng/ml or 1 micrograms/ml) relieved the inhibitory effects of CLO and PIA but did not affect the action of PGE2. PGE2-induced inhibition was prevented in a calcium-free medium [0 Ca2+ + 0.1 mM [ethylene-bis (oxyethylene-nitrilo)] tetraacetate (EGTA)]: values were 67.0 +/- 2.1% and 5.8 +/- 8.7% (+/- SEM) in 2 mM Ca2+ and 0 Ca2+ medium, respectively, N = 7.(ABSTRACT TRUNCATED AT 250 WORDS)

5'-(N-ethylcarboxamido)-adenosine (NECA) and N-[(R)-(phenylisopropyl)]-adenosine (PIA) were incubated in an adenylate cyclase assay of a particulate fraction of caudate-putamen tissue of the rat in order to examine the effect of somatostatin on adenosine receptors coupled adenylate cyclase subunits in vitro. Somatostatin was able to inhibit the enhancement of cyclic AMP formation induced by NECA in the presence of the hydrolysable guanine nucleotide guanosine-triphosphate. The adenosine receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine as well as the somatostatin receptor antagonist cyclo (7-aminoheptanoyl-Phe-D-Trp-Lys-O-benzyl-Thr) did not influence somatostatin induced inhibition of NECA-activated adenylate cyclase. Somatostatin did not modulate the effect mediated by the A-1 adenosine receptor agonist PIA. Both pertussis toxin and cholera toxin activated striatal adenylate cyclase acting on the guanine nucleotide regulatory subunit of the enzyme. The stimulation induced by pertussis toxin was antagonized by somatostatin, while in presence of cholera toxin somatostatin enhanced cyclic AMP formation. These results suggest that somatostatin acts through a stimulatory as well as an inhibitory guanine nucleotide regulatory protein subtype to affect probably postsynaptic A-2 adenosine receptor coupled adenylate cyclase activity.

The cardiovascular effects of N6-2-(4-aminophenyl)ethyladenosine (APNEA), which when radiolabelled with 125I shows high affinity for the newly described adenosine A3 receptor, have been investigated in the angiotensin II-supported circulation of the pithed rat. APNEA induces hypotensive responses which are unaffected by high doses (20-40 mg kg-1) of the broad spectrum, adenosine receptor antagonist, 8-(p-sulphophenyl)theophylline (8-SPT). 8-SPT-resistant falls in blood pressure are also seen, in the absence of bradycardia, with 5'-N-ethylcarboxamidoadenosine (NECA) and the R- and S-enantiomers of N6-phenylisopropyladenosine (PIA). Xanthine insensitivity, high potencies of APNEA, NECA and R-PIA, and an enantiomeric selectivity favouring R- over S-PIA are distinguishing features of the adenosine A3 receptor. We suggest that hypotension in the pithed rat may be a functional correlate of this site.

The influence of pH on the equilibrium dissociation constant and on kinetic association and dissociation constants was studied for adenosine receptor agonist L-N6-[adenine-2,8-3H, ethyl-2-3H]phenylisopropyladenosine ([3H]R-PIA) and antagonist 8-cyclopentyl-1,3-[3H]-dipropylxanthine ([3H]DPCPX). Two ionizable groups, of pK 7.0 and pK 7.4, are involved in the [3H]R-PIA associations with high- and low-affinity states of the receptor, and another group, of pK 6.0, is involved in the association with the low-affinity state. No ionizable group is involved in the dissociation process for the high-affinity state, whereas two ionizable groups, of pK 6.0 and 6.5, are involved in the low-affinity state. For [3H]DPCPX, three ionizable groups (pK 6.0, 7.4, and 8.0) are involved in the association process and only one group, (pK 6.0), is involved in the dissociation step. The apparent pK values obtained agree with histidine residues. We thus studied the effect of diethylpyrocarbonate (DEP), which reacts irreversibly with histidine residues, on agonist and antagonist binding to A1 adenosine receptors from pig brain cortical membranes. DEP treatment of membrane reduced the affinity (KD) and the total binding (R) of the agonist and the antagonist. Membrane preincubation with unlabeled ligand (R-PIA or DPCPX) prevented the effect of DEP modification observed when the same ligand, but with label, is added to the same membranes, but did not prevent the DEP modification on different, labeled ligand.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of forskolin (FSK) on [3H]-acetylcholine release ([3H]-ACh) from the phrenic motor nerve terminals, and its modification by adenosine deaminase (ADA), by the A2-adenosine receptor agonist 2-[p-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamide adenosine (CGS 21680C), by the A1-adenosine receptor agonist R-N6-phenylisopropyl adenosine (R-PIA), by the A2-antagonist N-(2-(dimethylamino)-ethyl)-N-methyl-4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3 -dipropyl-1H-purine-8-yl)-benzene sulphonamide (PD 115,199), and by the A1-antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) were studied on the rat phrenic-hemidiaphragm preparation. It is concluded that the excitatory effect of FSK on evoked [3H]-ACh release depends on tonic A2-adenosine receptor activation.

The negative inotropic responses of guinea pig isolated left atria to the P1-receptor agonists adenosine and L-N6-phenylisopropyladenosine (L-PIA), the muscarinic receptor agonist carbachol, and the potassium channel activator cromakalim were examined. The potassium channel blocker 4-aminopyridine (4-AP, 10 mM) decreased the concentration-response curve for L-N-6-phenylisopropyladenosine (L-PIA) so that the maximum reduction in atrial tension was significantly reduced from 77.8 to 61.9%. The concentration-response curve for the negative inotropic response to carbachol was displaced to the right by a substantially greater amount (521-fold), partly due to the muscarinic receptor blocking activity of 4-AP. The response to a single submaximum dose of L-PIA (520 nM) was slow in onset and monophasic, whereas adenosine induced a rapid reduction in tension followed by a gradual return toward the resting level. In the presence of 4-AP (10 mM), the peak response to L-PIA was significantly reduced from a 59.7 to 31.0% inhibition of tension. In addition, the rate of development of the response was significantly slowed. The peak inhibition of tension by adenosine (112 microM) (60.0%) was also significantly reduced to 37.4% in the presence of 4-AP (10 mM). Furthermore, there was no rapid decrease in tension but a gradual decrease to a peak effect which was no different from that in the absence of 4-AP. These results suggest that the P1-receptor agonists exert negative inotropy through a K+ channel-dependent component which is blocked by 4-AP and a slower-onset K+ channel-independent component. The potassium channel blocker, bromobenzoylmethyladamantylamine (BMA 100 microM) also shifted (11-fold) the concentration-response curve for the negative inotropic response to adenosine, and to a lesser extent that to cromakalim (2.1-fold) and carbachol. Glibenclamide (0.3, 3, and 30 microM), a selective antagonist of ATP-regulated potassium channels, was a potent antagonist of the negative inotropic responses to cromakalim. However, 30 microM failed to antagonize the responses to L-PIA, adenosine, or carbachol, indicating that they do not mediate their responses through ATP-regulated potassium channels. 4-AP (10 mM) caused a slight shift (2.3-fold) of the concentration-response curve for cromakalim, indicating weak activity against ATP-regulated potassium channels in the left atrium. This study showed that P1-receptor agonists exert negative inotropic responses in guinea pig left atria only in part through activation of potassium channels. These channels differ from the ATP-regulated potassium channels activated by cromakalim.

cAMP content and protein phosphorylation were determined in unlabeled and 32P-labeled guinea pig ventricular myocytes. Isoproterenol (10 nM, 37 degrees C, 10 seconds) increased cAMP content (236%) and phospholamban (265%) and troponin I (135%) phosphorylation in ventricular myocytes. When isoproterenol (0-300 nM) and the A1-adenosine receptor agonist (-)-N6-phenylisopropyladenosine (PIA, 1 microM) or the A1- and A2-adenosine receptor agonist 5'-(N-ethylcarboxamido)-adenosine (NECA, 1 microM) were administered simultaneously, both adenosine receptor agonists attenuated phospholamban phosphorylation to approximately the same extent (40%). The EC50 value for isoproterenol to phosphorylate phospholamban was 8 +/- 1 nM (n = 3), which increased to 31 +/- 4 nM (n = 3) in the presence of PIA or NECA. IC50 values for PIA or NECA to decrease the phosphorylation of phospholamban were 30 or 32 nM in 10 nM isoproterenol-stimulated cells and 80 or 85 nM in 30 nM isoproterenol-stimulated cells. Both adenosine receptor agonists failed to inhibit the phosphorylation of troponin I. However, acetylcholine (2 microM) in the presence of 10 nM isoproterenol inhibited phosphorylation of phospholamban as well as troponin I in ventricular cells. These effects were antagonized by 10 microM atropine. The effects of PIA and NECA on phosphorylation were antagonized by the A1-selective adenosine receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (1 microM) but not by the A2-selective adenosine receptor antagonist 9-chloro-2-(2-furanyl)-5,6-dihydro-1,2,4,triazolo(1,5-c)quinazolin -5-imine (1 microM). PIA and NECA did not reduce cAMP levels in isoproterenol-stimulated cells. We conclude that phospholamban phosphorylation was inhibited by A1-adenosine receptor activation and that these effects on phospholamban phosphorylation cannot be explained by a reduction in cAMP levels.

Objective: The aim was to characterise the process and the mechanisms of sensitisation of the atrial myocyte to adenosine receptor agonist., Methods: The ability of adenosine A1 receptor to mediate inhibition of adenylyl cyclase activity and myocyte contractility was determined in atrial myocytes cultured from 14 d chick embryos. Under conditions in which the myocytes were sensitised to the effects of A1 agonist, changes in the levels of adenosine A1 receptor and pertussis toxin sensitive G proteins were determined and correlated with alterations in the adenylyl cyclase activity and contractile responses of the myocyte to the A1 agonist., Results: Removal of adenosine from the culture medium with adenosine deaminase resulted in an enhanced ability of the adenosine A1 receptor agonist R-N6-(2-phenylisopropyl)-adenosine to exert a direct, negative inotropic effect and to inhibit isoprenaline stimulated adenylyl cyclase activity. The increase in the extent of maximum inhibition of adenylyl cyclase activity and of myocyte contractility was 215(30), n = 5, and 90(10)%, n = 14, respectively. Binding of the antagonist radioligand [3H]-8-cyclopentyl-1,3-dipropylxanthine in membranes from myocytes pre-exposed to adenosine deaminase showed a 70% increase in the adenosine A1 receptor density and a 54% increase in the proportion of the high affinity adenosine A1 receptor: control 33(5)%, n = 5, versus sensitised 49(3)%, n = 5, p < 0.01. The increase in the total number of adenosine receptors and the proportion of the high affinity form was associated with a similar increase in the level of pertussis toxin sensitive G protein(s), as determined by pertussis toxin mediated 32P-ADP ribosylation and by immunoblotting. Prior exposure of the culture to the adenosine receptor antagonist 8-(p-sulphophenyl)theophylline also led to similar results., Conclusions: The data indicate that the increased level of pertussis toxin sensitive G protein(s) results in an enhanced coupling to form the high affinity adenosine A1 receptor, that newly formed high affinity receptors are linked to an enhanced sensitivity of atrial myocytes to A1 adenosine agonist stimulation, and that upregulation of the G protein is a mechanism mediating the homologous sensitisation of cardiac adenosine A1 receptor pathway.

Objectives: Adenosine has previously been shown to protect against ischaemia induced ventricular arrhythmias. The aim of this study was to determine whether stimulation of A1 adenosine receptors with a selective agonist also protects against arrhythmias, and to attempt to elucidate the underlying mechanisms., Methods: Large White/Welsh Landrace cross breed domestic pigs (25-40 kg) subjected to left anterior descending coronary artery occlusion were used. R-PIA [R(-)N6-(2-phenylisopropyl) adenosine; 5 micrograms.kg-1] was given prior to coronary artery occlusion and the effects on haemodynamic variables and early ischaemic arrhythmias in the absence and presence of right atrial pacing were studied. The three experimental groups were: solvent controls, n = 10; R-PIA without pacing, n = 10; R-PIA with atrial pacing, n = 10. Ex vivo assessment of platelet aggregation was also performed to determine any inhibitory effects of R-PIA on platelets., Results: Administration of R-PIA without atrial pacing reduced the total number of ventricular ectopic beats induced by coronary occlusion, from 326(SEM 71) in controls to 121(30) (p < 0.05) and the incidence of ventricular fibrillation from 70% to 20% (p < 0.05). At the dose used, R-PIA reduced heart rate from 102(7) to 74(3) beats.min-1, with a consequent reduction in mean arterial blood pressure from 95(4) to 81(3) mm Hg. Atrial pacing following drug administration, at a rate of 109(5) beats.min-1, restored blood pressure and abolished the antiarrhythmic effects of R-PIA. Drug intervention had no effect on either ex-vivo platelet aggregation or coronary sinus oxygen content, suggesting a lack of activity at A2 adenosine receptors., Conclusions: An A1 adenosine receptor agonist exerts a marked protection against ischaemia induced ventricular arrhythmias and fibrillation in pigs. The reversal of this effect by restoring heart rate suggests that the drug induced bradycardia may be important in the antiarrhythmic action of R-PIA, possibly via an anti-ischaemic effect.

Adenosine receptor agonists and agents enhancing pericellular concentrations of adenosine possess antiinflammatory properties. In the present study, we found that R-phenylisopropyladenosine (R-PIA), 5'-N-ethylcarboxamido adenosine (NECA), other agonists of adenosine receptors and dipyridamole, an adenosine uptake inhibitor, inhibited tumor necrosis factor (TNF) production by endotoxin-stimulated human monocytes in a concentration-dependent manner with no inhibition of interleukin-6. The rank order of agonist potency is characteristic of neither A1 nor A2 receptors and suggests the involvement of another receptor subtype. The effect of R-PIA on TNF was in part abolished by the antagonist 8-sulfophenyltheophylline. In endotoxin-treated rats, R-PIA pretreatment (2.5 mg/kg) reduced serum TNF levels by 98%, with no modification of serum IL6 levels. TNF inhibition could be an important mechanism by which adenosine analogs exert their antiinflammatory action.

Adenosine has been shown to protect the ischemic and reperfused myocardium. To examine whether the protective effect of the nucleoside is mediated by modulation of oxidative stress, isolated rat hearts were perfused for 30 minutes with 100 microM H2O2 or an exogenous free radical-generating system consisting of purine (3.06 mM) and xanthine oxidase (10 units/l) in the presence or absence of drugs acting on adenosine A1 or A2 receptors. H2O2 alone produced a greater than 90% loss in contractility concomitant with a threefold elevation in resting tension, although these effects occurred in the absence of ultrastructural damage. Two A1 receptor agonists N6-cyclopentyladenosine (CPA, 1 microM) and R(-)-N6-(2-phenylisopropyl)adenosine (R-PIA, 1 microM) significantly attenuated the cardiodepressant effects of H2O2 and depressed the elevation in resting tension; however, only the effect of CPA was found to be significant with regard to the latter parameter. A similar concentration of S(+)-N6-(2-phenylisopropyl)adenosine (S-PIA), a markedly less potent A1 receptor agonist, was found to be without beneficial effect. However, a significant protective effect against both the reduction in contractility and the elevation in resting tension was seen with a 10-fold elevation in the concentration of S-PIA (10 microM). The protective effects on functional parameters were associated with preservation of high-energy phosphate and adenine nucleotide contents after 30 minutes of H2O2 treatment. The salutary effects of all drugs were reversed in the presence of the A1 receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (0.5 microM). An A2 receptor agonist 2-[p-(carboxyethyl)phenethylamino]-5'-N-ethylcarboxamidoadenosine, termed CGS 21680 (1 microM), failed to alter the cardiac response to H2O2 with regard to all parameters studied. Neither a 50% reduction in external CaCl2 concentration nor treatment with 10 microM DL-propranolol exerted salutary effects against H2O2-induced dysfunction. None of the A1 receptor agonists modulated the response to purine plus xanthine oxidase. Our results demonstrate a selective protective effect of adenosine A1 receptor activation against the cardiac toxicity of H2O2 and provide, at least in part, a basis for the cardioprotective actions of adenosine and its analogues.

Background: A brief period of myocardial ischemia can result in an increased resistance to subsequent, more severe episodes of ischemia. Recent studies have indicated that activation of adenosine A1-receptors may mediate this preconditioning effect. It is also known that A1-activation can lead to ATP-sensitive potassium channel (KATP) opening via a G(i) protein-mediated effect. Thus, we determined whether the KATP blocker glyburide could abolish preconditioning or the protective effects of A1-receptor activation., Methods and Results: Anesthetized dogs were subjected to 5 minutes of left circumflex coronary artery (LCx) occlusion (or sham) followed by 10 minutes of reperfusion. The hearts were then subjected to 60 minutes of LCx occlusion and 5 hours of reperfusion. Glyburide (5 micrograms/kg/min) or vehicle was given directly into the LCx 20 minutes before preconditioning or sham preconditioning. Preconditioning resulted in a significantly reduced infarct size compared with nonpreconditioned animals. Glyburide abolished the protective effect of preconditioning. To establish a link between KATP and A1-receptor activation, the effect of the A1-agonist R-PIA with or without glyburide on infarct size was determined. R-PIA (0.4 microgram/kg/min, directly into the LCx) significantly reduced infarct size, and this protective effect was abolished by glyburide. None of the treatments described above had a significant effect on peripheral hemodynamic status or myocardial blood flow., Conclusions: Preconditioning may be mediated by KATP activation, and this may be linked to A1-receptor stimulation.

The effects of adenosine (A) receptor agonists on ischemia-induced impairment of 2-deoxyglucose (2-DG) uptake by rat hippocampal slices was evaluated. Hippocampal slices were exposed to 20-min hypoxia + hypoglycemia (ischemia) and then returned to oxygenated and glucose-containing Krebs-Ringer solution for 6 h. Ischemia reduced 2-DG uptake in the hippocampal slices. The ischemia-induced reduction in 2-DG uptake was attenuated by pretreatment with A1 receptor agonists but not with A2 receptor agonists. 8-Phenyltheophylline, an A1 receptor antagonist, exacerbated the ischemia-induced decrease. The A1 receptor agonist-induced neuroprotective effect was blocked by co-treatment with 8-phenyltheophylline. The present study suggests that the A1 receptor-mediated function has a protective role in ischemia-induced decreases in glucose metabolism in hippocampal slices.

1. The nerve-evoked contractions elicited by transmural electrical stimulation of mouse urinary bladders superfused in modified Krebs Ringer buffer containing 1 microM atropine plus 3.4 microM guanethidine were inhibited by adenosine (ADO) and related nucleoside analogues with the following rank order of potency: R-phenylisopropyladenosine (R-PIA) greater than cyclohexyladenosine (CHA) greater than 5'N-ethylcarboxamido adenosine (NECA) greater than ADO greater than S-phenylisopropyladenosine (S-PIA). Tissue preincubation with 8-phenyltheophylline (8-PT) displaced to the right, in a parallel fashion, the NECA concentration-response curve. 2. The contractions elicited by application of exogenous adenosine 5'-triphosphate (ATP) were also inhibited by ADO and related structural analogues. The rank order of potency to reduce the motor response to ATP was: NECA greater than 2-chloroadenosine (CADO) greater than R-PIA greater than ADO greater than CHA greater than S-PIA. 3. The ADO-induced ATP antagonism was of a non-competitive nature and was not specific. Tissue incubation with 10 microM NECA not only reduced the motor responses elicited by ATP, but also 5-hydroxytryptamine, acetylcholine and prostaglandin F2 alpha. The action of NECA was antagonized following tissue preincubation with 8-PT. The inhibitory action of NECA was not mimicked by 10 microM CHA. 4. The maximal bladder ATP contractile response was significantly increased by tissue preincubation with 5-30 microM 8-PT. 5. The 0.15 Hz evoked muscular twitch was significantly increased by 8-PT while dipyridamole consistently reduced the magnitude of the twitch response. These results are consonant with the hypothesis that an endogenous ADO tone modulates the bladder neurotransmission. 6. A working model is proposed suggesting the presence of ADO-Al and A2 receptors in the mouse urinary bladder. The A1 receptor subpopulation is probably of presynaptic origin whereas the smooth muscle membranes contain a population of the A2 receptor subtype.

1. The indirect negative inotropic effects of P1- and muscarinic-receptor agonists were compared by examining how the responses of isolated guinea-pig left atria and papillary muscles to positive inotropes were affected by the presence of the P1-receptor agonist, L-PIA or the muscarinic-receptor agonist, carbachol. 2. The indirect negative inotropic effects of L-PIA and carbachol were similar: both attenuated the responses of left atria more effectively than those of papillary muscles; and both more effectively attenuated responses to the cAMP-dependent positive inotropes, isoprenaline and forskolin. 3. However, there were differences: L-PIA, but not carbachol, was able at high concentrations to inhibit the responses of left atria to the calcium channel opener Bay K 8644; and at concentrations that produced similar direct negative inotropic effects, L-PIA consistently attenuated the positive inotropes more effectively than carbachol. 4. These findings are consistent with L-PIA being able to activate an additional negative inotropic mechanism that carbachol cannot.

The effect of a systematically administered adenosine analogue upon neuronal damage induced by kanic acid has been examined using the binding of a peripheral benzodiazepine receptor ligand as a marker for the gliosis following neuronal death. Intraperitoneal (i.p.) injections of R-phenylisopropyladenosine (R-PIA), at a dose of 100 micrograms kg-1 or 25 micrograms kg-1 reduced substantially the hippocampal neurotoxicity induced by intraperitoneal kainate 100 mg kg-1. The effect of R-PIA was prevented by 8-phenyl-theophylline, confirming the involvement of a purine P1 receptor.

The effects of two adenosine analogs on cyclic AMP (cAMP) accumulation and DNA synthesis were studied in cultured smooth muscle cells (SMCs) isolated from adult and neonatal rat arteries. N-ethylcarboxamido adenosine (NECA) dose-dependently increased intracellular cAMP levels and appeared to be more potent in adult than in neonatal SMCs. R-phenylisopropyl adenosine (R-PIA), in nanomolar concentrations, counteracted the increase in cAMP evoked by 10 microM forskolin in adult but not in neonatal SMCs, indicating that the enhanced "A2" response seen in adult SMCs was not due to a lack of "A1" receptors in these cultures. Binding experiments performed using the adenosine antagonist XAC did not reveal any differences in the number or affinity of the adenosine receptors between neonatal and adult SMCs. This indicates effects presumably on the G-protein level. A high capacity to spontaneously synthesize DNA and a weak response to platelet-derived growth factor (PDGF) were seen in the neonatal SMCs. Furthermore, NECA had no effect on PDGF-induced DNA synthesis in these cells. In contrast, adult SMCs presented a low rate of spontaneous DNA synthesis and a marked proliferative response to PDGF, which was inhibited by NECA. This inhibition paralleled the increase in cAMP elicited by NECA. Our findings suggest that neonatal and adult SMCs differ both in their response to growth factors and growth inhibitors.

1. It has been suggested that adenosine A1 receptors may be sub-divided into A1 and A3 types, based on the relative potencies of 5'-N-ethylcarboxamidoadenosine (NECA) and selected N6-substituted adenosine analogues. At A1 receptors (rat adipocytes) N6-phenylisopropyladenosine (PIA) was reported to be approx. 100-fold more potent than NECA, whereas the compounds were equipotent at A3 receptors (those in cardiac and neuronal preparations). 2. The study reported here has systematically evaluated this proposal, the rank orders of potency of NECA, R- and S-PIA, N6-cyclopentyladenosine (CPA) and N6-cyclohexyladenosine (CHA) being determined in rat adipocytes, guinea-pig ileum and rat and guinea-pig atria. 3. R-PIA, CHA and CPA were found to have consistent potencies relative to NECA across all 6 tissues, including rat adipocytes. The rank order was CPA greater than or equal to CHA, R-PIA greater than or equal to NECA greater than S-PIA. 4. We conclude that the relative potencies of these agonists do not support the concept that adenosine A1 receptors in rat adipocytes differ from those in neuronal and cardiac tissues.

We have recently demonstrated that human neutrophils (PMN) possess two different classes of adenosine receptors (A1 and A2) that, when occupied, promote chemotaxis and inhibit the generation of reactive oxygen species (e.g., O2- and H2O2), respectively. We have previously demonstrated that adenosine protects endothelial cells (EC) from injury by stimulated neutrophils (PMN) both by diminishing generation of H2O2 and inhibiting adherence of PMN to EC. We therefore determined whether occupancy of A1 or A2 adenosine receptors regulated adherence of PMN to EC. At concentrations similar to those required to inhibit release of O2- by ligation of A2 receptors, both adenosine (IC50 = 56 nM) and 5'N-ethylcarboxamidoadenosine (NECA, IC50 = 8 nM), the most potent A2 agonist, inhibited adherence to EC by stimulated PMN (FMLP, 0.1 microM). In direct contrast, the specific A1 agonists N6-phenylisopropyladenosine and N6-cyclopentyladenosine (CPA) promoted PMN adherence to EC at concentrations of 1-100 nM. To further investigate the mechanisms by which adenosine receptor agonists affected the adherence of stimulated PMN we examined the effect of NECA (A2) and CPA (A1) on the adherence of PMN to fibrinogen (a ligand for the beta 2 integrin CD11b/CD18) and to gelatin. In a dose-dependent manner (IC50 = 2 nM), NECA inhibited the adherence of FMLP-treated PMN to fibrinogen- but not gelatin-coated plates. In contrast, CPA (A1) promoted adherence of stimulated PMN to gelatin-(EC50 = 13 pM) but not fibrinogen-coated plates. Theophylline (10 microM), an adenosine receptor antagonist, reversed the inhibition by NECA (0.3 microM) of stimulated neutrophil adherence to fibrinogen. These observations not only confirm the presence of A1 and A2 receptors on PMN but also suggest two opposing roles for adenosine in inflammation. Occupancy of A1 receptors promotes neutrophil adherence to endothelium and chemotaxis (a proinflammatory role) whereas occupancy of A2 receptors inhibits adherence and generation of toxic oxygen metabolites (an antiinflammatory role).

The effect of selective adenosine receptor agonists on nociceptive responses of mice and rats and on morphine analgesia was investigated. All compounds used: phenylisopropyladenosine (R-PIA), adenosine ethylcarboxamide (NECA), cyclohexyladenosine (CHA) and 2-chloroadenosine (2-CADO) exhibited antinociceptive action in mice and rats in the hot-plate (56 degrees C) and tail-immersion (52 degrees C) tests. R-PIA, CHA and NECA potentiated the antinociceptive action of morphine in mice, and R-PIA and NECA--in rats. 2-CADO did not affect the morphine action in the tests.

Amiloride, a potassium sparing diuretic, inhibits adenosine A1 receptor-radioligand binding in calf and rat brain membranes in the low micromolar range. The drug interacted with the A1 receptor in a manner different from classical A1 ligands, but structure-activity relationship studies indicated that this inhibitory effect is not related to the ion transport inhibiting properties of amiloride (Garritsen et al., 1990a,b) In the present study, the question is addressed how amiloride interacts with the adenosine A1 receptor. Amiloride and two of its analogues, in concentrations equivalent to their Ki values in displacement studies, decrease the affinity of the A1 antagonist [3H]8-cyclopentyl-1,3-dipropylxanthine, but not the maximal binding capacity of the radioligand. Furthermore, the dissociation rate of the receptor-ligand complex is unaltered in the presence of amiloride or its analogues in a concentration exceeding the Ki value 10-fold. These characteristics argue for a purely competitive mode of interaction. The functional consequences of the interaction between amiloride analogues and the A1 receptor were investigated at the level of cyclic adenosine 3',5'-monophosphate (cAMP) formation. The amiloride analogue 5-(N-butyl-N-methyl) amiloride (MBA) reversed A1-receptor mediated inhibition of forskolin-stimulated cAMP formation in rat fat cell membranes. In this model, the antagonist potency of MBA is ca 5 microM. This value is in fair agreement with a Ki value of 3.5 microM in binding assays under similar conditions. In conclusion, amiloride inhibits A1 receptor binding in an apparently competitive manner. This suggests that the binding sites of amiloride and the classic A1 receptor ligands may at least partially overlap.(ABSTRACT TRUNCATED AT 250 WORDS)

In human D384 astrocytoma cells, cyclic AMP accumulation can be conveniently studied after labelling of the adenosine triphosphate pool (15 fmol cell-1) with [3H]adenine. In this study, adenosine had a biphasic effect on cyclic AMP accumulation, which was scarcely altered by blocking adenosine uptake and metabolism. Low concentrations of adenosine led to an inhibition of cyclic AMP accumulation, and higher concentrations led to stimulation. No effect of adenosine on cyclic AMP was observed unless phosphodiesterase was inhibited by rolipram. The A1 receptor antagonist DPCPX attenuated the inhibitory phase of adenosine response, and enhanced the cyclic AMP accumulation induced by adenosine analogues. The cyclic AMP accumulation was stimulated by NECA greater than ADO greater than CGS 21680 greater than CV 1808 greater than CPA greater than or equal to CHA, indicating mediation by A2 receptors. The stimulatory effect of NECA was much more effectively blocked by the combined A1 and A2 receptor antagonist CGS 15943 (KB 4 nmol l-1) than by the A1 antagonist DPCPX (KB 110 nmol l-1). Treatment of the cells with pertussis toxin (0.2 microgram ml-1 for 2.5 h) potentiated the cyclic AMP response to adenosine analogues significantly. The cyclic AMP response to NECA was enhanced by the protein kinase C activator phorbol dibutyrate even after pertussis toxin treatment. By contrast, nanomolar concentrations of bradykinin, which increases Ca(2+)-levels and protein kinase C activity in D384 cells, reduced NECA-induced cyclic AMP accumulation in control and pertussis toxin-treated cells. Thus, D384 cells possess both A1 and A2 adenosine receptors influencing cyclic AMP in opposite directions.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolated, endothelium-free rings of vascular smooth muscle (VSM) from the ventral aorta of the dogfish shark, Squalus acanthias, were used to examine the vasoactive effects of various adenosine agonists. Cumulative addition of 2-chloroadenosine (2 Cl-ADO) over the concentration range 10 nM-1 mM resulted in a biphasic response, with a significant increase in tension at 1 microM and a more significant decline in tension at 100 microM and 1 mM, suggesting that this tissue may possess both A1 and A2 adenosine receptors. N6-Cyclopentyladenosine (N-6 CPA) and N6-(2-phenylisopropyl)adenosine, R(-)isomer (R-PIA), generally considered to be more A1 specific, also produced slight, but significant increases in tension, but only at relatively high concentrations. The more specific A1 agonist, N6-(25)-[2-endo-norbonyl] adenosine [(S)-ENBA] produced a significant increase in tension at 1 pM, reaching 28% above control at 10 nM. The response to (S)-ENBA was also biphasic, with a fall in tension at 10 microM. The relatively non-specific agonist 5'-N-ethylcarboxamidoadenosine (NECA) produced a small, but significant, increase in tension at 1 microM, with no subsequent decline in tension at higher concentrations. These results allow us to assign a tentative structure-activity relationship (SAR) for an increase in tension of (S)-ENBA much much greater than R-PIA greater than or equal to 2-Cl ADO = N-6 CPA = NECA; for the decrease, the SAR is (S)-ENBA greater than 2-Cl ADO greater than R-PIA greater than N-6 CPA = NECA.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine (10(-9)-10(-6) mol/l) and R-phenylisopropyladenosine (10(-9)-10(-7) mol/l) partially inhibited the intracellular accumulation of cyclic AMP induced by isoproterenol, prostaglandin E1, histamine and 5'-N-ethylcarboxamidoadenosine in lymphocytes. In contrast, S-phenylisopropyladenosine, which is a poor agonist of the adenosine A1/Ri receptor, had essentially no inhibitory effect. 8-Phenyltheophylline, in low concentrations that do not inhibit cyclic AMP phosphodiesterase, completely blocked the inhibitory effect of R-phenylisopropyladenosine on the increase in cyclic AMP induced by prostaglandin E1. R-Phenylisopropyladenosine (10(-8)-10(-6) mol/l) also inhibited the cyclic AMP accumulation in lymphocytes induced by forskolin (10(-5) mol/l), which activates adenylate cyclase through direct interaction with the enzyme. We also investigated the presence of the adenosine A1/Ri receptor on human polymorphonuclear leukocytes. R-Phenylisopropyladenosine (3 x 10(-9)-10(-7) mol/l) abolished the stimulating effects of prostaglandin and forskolin on cyclic AMP accumulation in polymorphonuclear leukocytes. This effect was blocked by 8-phenyltheophylline and was not observed with the stereoisomer S-phenylisopropyladenosine. The results support the existence of an A1/Ri receptor that regulates cyclic AMP metabolism of human lymphocytes and polymorphonuclear leukocytes.

Studies were done to determine whether the apparent changes in behavioral sensitivity to adenosine receptor ligands that occur with age and with neonatal caffeine exposure were due to a change in sensitivity of the receptor for the ligand or to a more fundamental change in the receptor. Using an animal model that mimics the brain developmental period and level of caffeine exposure in human premature neonates treated with caffeine for apnea of prematurity, behavioral and neurochemical investigations were undertaken. The locomotor responses to acute challenge with caffeine (15, 30 and 60 mg/kg) and with D-phenylisopropyladenosine (D-PIA) (0.038 and 0.38 mg/kg), an adenosine receptor agonist, were measured in control and neonatally caffeine-exposed rats at 12, 15, 18, and 28 days of age. The dissociation constants (Kd) and maximal binding densities (Bmax) for agonist binding at the adenosine A1 receptor site were determined over a similar time period. Caffeine displacement of an adenosine A1 agonist was also measured to examine in vitro sensitivity to caffeine as a function of age and neonatal caffeine exposure. Our studies demonstrated that the differential responses to adenosine receptor ligands seen as a function of both age and neonatal caffeine exposure could not be overcome by merely increasing the doses of ligand administered. In addition, the results of the binding studies indicated that changes in the adenosine receptor are occurring as a function of age in different regions of the brain of control animals and that this development is influenced by neonatal caffeine exposure.

A1 adenosine receptors and associated guanine nucleotide-binding proteins (G proteins) were purified from bovine cerebral cortex by affinity chromatography (Munshi, R., and Linden, J. (1989) J. Biol. Chem. 264, 14853-14859). In this study we have identified the pertussis toxin-sensitive G protein subunits that co-purify with A1 adenosine receptors by immunoblotting with specific antipeptide antisera. Gi alpha 1, Gi alpha 2, Go alpha, G beta 35, and G beta 36 were detected. Of the total [35S]guanosine 5'-O-(3-thio)triphosphate [( 35S]GTP gamma S) binding sites, Gi alpha 1 and Go alpha each accounted for greater than 37% whereas Gi alpha 2 comprised less than 13%. G beta 35 was found in excess over G beta 36. Low molecular mass (21-25 kDa) GTP-binding proteins were not detected. We also examined the characteristics of purified receptors and various purified bovine brain G proteins reconstituted into phospholipid vesicles. All three alpha-subunits restored GTP gamma S-sensitive high affinity binding of the agonist 125I-aminobenzyladenosine to a fraction (25%) of reconstituted receptors with a selectivity order of Gi2 greater than Go greater than or equal to Gi1 (ED50 values of G proteins measured as fold excess over the receptor concentration were 4.7 +/- 1.2, 24 +/- 5, and 34 +/- 7, respectively). Furthermore, receptors occupied with the agonist R-phenylisopropyladenosine catalytically increased the rate of binding of [35S]GTP gamma S to reconstituted G proteins by 6.5-8.5-fold. These results suggest that A1 adenosine receptors couple indiscriminately to pertussis toxin-sensitive G proteins.

Desensitization of adenosine receptors (ARs) was studied in DDT1 MF-2 cells, which possess both A1- and A2AR, differentially coupled to adenylate cyclase. (-)-N6-(R)-Phenylisopropyladenosine (R-PIA), an A1AR-selective agonist at the appropriate concentrations, desensitized A1AR-mediated inhibition of adenylate cyclase activity in a time- (t1/2, 8 hr) and dose-dependent and reversible fashion. This was associated with significant decreases in total A1AR number and in the number of receptors possessing a high affinity for agonist in membrane preparations. The decrease in total A1AR in the membranes from the desensitized cells (approximately 40%) was associated with a 37% increase in A1AR measured in light vesicle preparations, compared with control cells. To test a possible role of phosphorylation in A1AR desensitization, cells were incubated with [32P]orthophosphate, followed by exposure to R-PIA for 18 hr. Subsequent purification of the A1AR indicated a 3-4-fold increase in phosphorylation of A1AR in cells treated with R-PIA, compared with control cells. Desensitization of the A1AR did not alter the levels of alpha s and alpha 12 proteins or affect the ability of stimulatory effectors, such as isoproterenol, sodium fluoride, and forskolin, to activate adenylate cyclase. These results suggest that uncoupling, down-regulation, and phosphorylation of the A1AR contribute, at least in part, to desensitization of this inhibitory receptor. Desensitization of the A2AR was characterized using an A2-selective agonist, 2-[4-(2-(4-aminophenyl]methylcarbonyl)ethyl)phenyl]ethylamino- 5'-N-ethylcarboxamidoadenosine (PAPA-APEC). Pretreatment of cells with PAPA-APEC (100 nM) resulted in a rapid loss of agonist stimulation of adenylate cyclase activity (t1/2 of this effect, 45 min). This effect was dose dependent (EC50, approximately 10 nM) and rapidly reversible. Interestingly, desensitization of the A2AR resulted in no change in receptor number, affinity, or mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Taken together, these data suggest distinct mechanisms of desensitization of A1- and A2ARs in a single cell type.

The effect of phospholipases and proteases on the membrane-bound and solubilized A1 adenosine receptor has been studied. Phospholipids modulate the [3H]N6-(R)-phenylisopropyladenosine binding to A1 adenosine receptors in crude membranes and in soluble preparations, because changes in the phospholipid environment decrease both the binding capacity and the affinity for the ligand. It has become clear that 1) there is co-solubilization of receptor and phospholipids; 2) the phospholipid requirements are different for the coupled and the uncoupled receptor; 3) a net charge in the polar head produced by phospholipase D prevents the agonist binding to the receptor-G protein complex; alternatively, when the whole polar head is removed by phospholipase C the uncoupled receptor is altered; and 4) the protease action upon the receptor suggests that receptor coupled to G protein is more protected by the membrane than the uncoupled receptor. In kinetic experiments performed on membranes it was demonstrated that phospholipase C and trypsin increased the Kd value of the high-affinity state by modifying both k1 and k-1. In contrast they only modified the dissociation constant of the low-affinity state. In conclusion it should be noted that phospholipids play a key role for the binding of R-PIA to A1 adenosine receptor. Also, a different disposition within the membrane of the coupled and uncoupled receptor is encountered.

In this article the existence of A1 adenosine receptors and the absence of A2 adenosine receptors in myelin membranes purified from pig brain white matter are demonstrated. The characterization of (R)-[3H]phenylisopropyladenosine ([3H]R-PIA) binding to purified myelin fractions was performed. The distribution of high- and low-affinity species of the A1 adenosine receptor was different in heavy, medium, and light myelin. The fluidity of myelin subfractions and of pig brain cortical membranes was estimated; the microviscosity of heavy myelin (5.4 poises) and of cortical membranes (5.1 poises) was similar and less than that of medium (7.8 poises) and light (8.2 poises) myelin. It was also demonstrated that the agonist R-PIA modifies the microviscosity of myelin membranes and that the degree of modification depends on the fluidity of the membrane assayed. These results suggest that adenosine receptors may have an important role in the functionality of myelin membranes.

Adenosine contracts pregnant and nonpregnant guinea pig myometrial smooth muscle (MSM). We have 1) described dissociation of A1-adenosine receptors from adenylate cyclase inhibition in nonpregnant MSM (M. A. Smith, J. L. Silverstein, D. P. Westfall, and I. L. O. Buxton, Cell. Signal. 1: 357-365, 1989); 2) described appearance of such inhibitory coupling in pregnant MSM [W. P. Schiemann, D. P. Westfall, and I. L. O. Buxton, Am. J. Physiol. 261 (Endocrinol. Metab. 24): E141-E150, 1991]; and 3) demonstrated a role for myometrial A1 receptors in the rapid formation of D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] in nonpregnant MSM and the cyclooxygenase dependence of this effect (W. P. Schiemann, K. O. Doggwiller, and I. L. O. Buxton. J. Pharmacol. Exp. Ther. 258: 429-437, 1991). To further characterize adenosine action in pregnant tissue, we explored A1 coupling to increased phosphoinositide hydrolysis in near-term pregnant MSM. The A1-receptor agonist (+)-N6-(2-phenylisopropyl)adenosine stimulates the rapid dose-dependent formation of Ins(1,4,5)P3 and stimulates rapid degradation of uterine inositol monophosphates (InsP) in a manner paralleling increases in inositol polyphosphates. Both A1-mediated responses were blocked by the A1 antagonist 8-(p-sulfophenyl)theophylline, and, unlike the effect observed in nonpregnant MSM, treatment of pregnant MSM with either meclofenamate or indomethacin failed to block A1-mediated increases in Ins(1,4,5)P3. Pretreatment of MSM with either Li+ or pertussis toxin failed to alter either Ins(1,4,5)P3 formation or InsP degradation. Furthermore, assay of inositol phosphomonoesterase (InsPase) activity in the presence or absence of Li+ confirmed the existence of an MSM Li(+)-insensitive InsPase enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

We have previously shown that endogenous adenosine inhibits non-adrenergic, non-cholinergic (NANC) neurotransmission in isolated guinea-pig atria. In the present study the effect of adenosine analogues, such as N6 cyclopentyladenosine (CPA), 5' N-ethylcarboxamide adenosine (NECA), 2 chloroadenosine (2-CADO), R- and S-phenylisopropyladenosine (R- and S-PIA) on the cardiac response to transmural nerve stimulation has been tested in order to characterize the subtype of adenosine receptor involved in the inhibitory control of NANC neurotransmission. The effect of the adenosine antagonist 8-phenyltheophylline (8-PT) was then tested against CPA and NECA. The prototypical A-1 selective agonist CPA was the most active agonist, reducing the response to the stimulation of NANC nerves with an IC50 value of 2.8 nM; R-PIA, NECA and 2-CADO showed IC50 values of 9.5, 13.7 and 35 nM respectively. S-PIA was the least active agonist, showing an IC50 value (306 nM) about 30-fold greater than that of R-PIA (9.5 nM). None of the agonists tested was able to modify cardiac response to exogenous CGRP. Furthermore, 8-PT competitively antagonized the effect of CPA and NECA with very close pA2 values (6.77 +/- 0.01 and 6.63 +/- 0.08 respectively). From these findings we concluded that prejunctional inhibitory adenosine receptors on capsaicin sensitive sensory nerves of cardiac tissue belong to the A-1 subtype.

Fat-cells were isolated from patients of body-mass indices (BMIs) ranging from 17.9 to 83.9 kg/m2. Isoprenaline-stimulated cyclic AMP accumulation in cells prepared from obese subjects as compared with normal-weight subjects, was less sensitive to inhibition by the adenosine agonist N6-(phenylisopropyl)adenosine (PIA) (P = 0.047). The inhibition of 7 beta-desacetyl-7 beta-[gamma-(N-methylpiperazino) butyryl]-forskolin-stimulated adenylate cyclase by PIA in the presence of adenosine deaminase was also much attenuated in crude plasma membranes of adipocytes prepared from massively obese patients as compared with lean controls (P = 0.0143). This difference was probably not due to different cell size, because adenylate cyclase of crude plasma membranes of large adipocytes was actually more sensitive to PIA than was adenylate cyclase of membranes of smaller fat-cells co-isolated from the same individual. The stimulatory effect of PIA on glucose uptake in the presence of adenosine deaminase was depressed in adipocytes prepared from obese subjects and correlated with BMI at r = -0.626 (P = 0.007) at 100 nM-PIA. The adenosine receptors were studied by using the adenosine antagonist 1,3-[3H]dipropyl-8-cyclopentylxanthine. The binding was rapid and proportional to protein concentration. There was no difference in the affinities of receptors in membranes of obese and normal-weight subjects; Kd values of all patients averaged 3.3 nM. Bmax values were 54 and 130 fmol/mg of protein in membranes prepared from seven obese and five control patients respectively. The Bmax values calculated per mg of protein correlated with BMI at r = -0.539 (P = 0.047). The adenosine content of adipose tissue was higher in obese than in control subjects. These results demonstrate an attenuated response of cyclic AMP accumulation, adenylate cyclase and glucose uptake to adenosine in fat-cells prepared from obese subjects, and suggest that this change is at least partly due to changes in the amount of adenosine receptors, but not their affinity. The decreased receptor number could be due to higher adenosine content. A higher adenosine concentration in adipose tissue could explain why lipolysis is inhibited in situ in obesity, and the desensitization could explain the diminished response to adenosine analogues in isolated fat-cells.

Since high-affinity adenosine A2 receptors (A2a) are localized exclusively in dopamine-rich regions in the central nervous system and mediate inhibition of locomotor activity, we have examined the effect of A2a receptor activation on D1 and D2 receptor binding in membrane preparations of the rat striatum. The A2a agonist 2-[p-(2-carboxyethyl)phenethylamino]-5'- N-ethylcarboxamidoadenosine (CGS 21680) increased the Kd of the dopamine D2 agonist L-(-)-N-[3H]propylnorapomorphine without affecting the Bmax. The increase in Kd was maximal (40%) at 30 nM CGS 21680. CGS 21680 (30 nM) decreased the dopamine-induced inhibition of [3H]raclopride (a D2 antagonist) binding due to an increase (about 3-fold) in KH and KL, the dissociation constants of high- and low-affinity binding sites. The effects of CGS 21680 were antagonized by the adenosine antagonist 8-phenyltheophylline (10 microM). (-)-N6-(2-Phenylisopropyl)adenosine produced an effect similar to that of CGS 21680, provided the concentration used was high enough to stimulate A2a receptors (300 nM). GTP (50 microM) also decreased the dopamine-induced inhibition of [3H]raclopride binding but, in contrast to CGS 21680, GTP decreased the proportion of D2 receptors in the high-affinity state. CGS 21680 (30 nM) did not affect the Kd or Bmax of [3H]raclopride and failed to affect ligand binding to D1 receptors. Thus, stimulation of A2a receptors potently reduces the affinity of D2 agonist binding sites within the plasma membrane of striatal neurons. This A2a-D2 interaction may underlie the neuroleptic-like actions of adenosine agonists and the enhancing effects of adenosine antagonists, such as caffeine, on locomotor activity.

The aim of the present study was to obtain a more detailed understanding of the interaction between presynaptic alpha 2-adrenoceptors and A1 adenosine receptors mediating inhibition of noradrenaline release in the central nervous system. Slices of rabbit hippocampus, prelabelled with [3H]noradrenaline, were superfused in the presence of the re-uptake inhibitor (+)-oxaprotiline and electrically stimulated during superfusion. During stimulation with 36 pulses at 3 Hz the alpha 2-adrenoceptor antagonist yohimbine induced a five-fold increase of noradrenaline release indicating a pronounced autoinhibition of approximately 80%. In these experiments the inhibition of release caused by R-PIA, a preferential A1 agonist, as well as its facilitation caused by DPCPX, a selective A1 antagonist, were smaller in comparison to the effects of these compounds on release virtually free of autoinhibition (i.e. by stimulating the tissue with 4 pulses at 100 Hz (POP-stimulation) or with 36 pulses at 3 Hz in presence of yohimbine). Clonidine, an alpha 2-adrenoceptor agonist, was used to impose a distinct alpha 2-adrenoceptor-mediated inhibition of release elicited by POP-stimulation. Only, however, in the presence of 30 nmol/l clonidine, causing maximum inhibition of approximately 80% of 3H-overflow, but not in the presence of 6 nmol/l clonidine, causing approximately 50% inhibition, a significant diminution of the inhibitory effect of R-PIA was seen. Similarly, the alpha 2-adrenoceptor mechanism was affected only by 10 mumols/l R-PIA causing maximum inhibition of approximately 80%, but remained unchanged in the presence of 30 nmol/l R-PIA diminishing release by 50%.(ABSTRACT TRUNCATED AT 250 WORDS)

The smooth muscle of guinea pig uterus is contracted by adenosine in a manner consistent with the presence of a purine nucleoside receptor of the P1-A1 subtype that is uncoupled from adenylate cyclase. Here we investigate the signal transduction mechanism responsible for adenosine's ability to contract uterine smooth muscle. The A1 adenosine receptor antagonist [3H]-8-cyclopentyl-1.3- dipropyl xanthine ([3H]CPX) bound reversibly to a large number (172 +/- 25 fmol/mg of protein) of receptors in myometrial smooth muscle membranes from estrogen-primed virgin guinea pigs with an affinity (KD = 1.77 +/- 0.21 nM) similar to that expected of [3H]CPX binding to both central and peripheral A1 receptors. In the absence of the stable GTP analog, guanosine-5'-O-[3-thiotriphosphate], agonist competition of [3H]CPX binding resulted in a biphasic curve that was best fit assuming the presence of equal populations of two affinity states of the receptor. Addition of guanosine-5'-O-[3-thiotriphosphate] (10 microM) resulted in a monophasic competition curve of low affinity suggesting coupling of this A1 receptor to effector via a GTP binding protein. In [3H]myo-inositol labeled strips of myometrial smooth muscle, the adenosine agonist R-phenylisopropyl adenosine (R-PIA) stimulated the rapid formation of inositol-1,4,5-trisphosphate (InsP3) that was antagonized by addition of the nucleoside receptor antagonist 8-sulfophenyl theophylline. Prostaglandin stimulation of myometrial strips also increased InsP3 formation. Furthermore, R-PIA stimulated the disappearance of inositol phosphate (InsP) in a fashion consistent with agonist stimulation of an inositol phosphatase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of glucocorticoid treatment of DDT1 MF-2 smooth muscle cells on the signaling via two adenosine receptors with opposing actions on cAMP generation was examined. Treatment with dexamethasone caused a dose- and time-dependent increase in the number of adenosine A1 receptors but did not affect the KD or the proportions of receptors in high and low affinity states. The EC50 was 1 nM dexamethasone, and maximal response was achieved after 24 hr. The number of receptors was increased by approximately 50%. Other steroid hormones, including aldosterone, progesterone, testosterone, and estrogen, were much less effective, and addition of the glucocorticoid receptor antagonist RU 486 or the protein synthesis inhibitor cycloheximide prevented the up-regulation, showing that the effect was mediated via a glucocorticoid receptor-specific mechanism that involves protein synthesis. In dexamethasone-treated cells the A1 receptor agonist (-)-N6-phenylisopropyladenosine [(R)-PIA] was 3 times more potent as an inhibitor of cAMP formation induced by isoprenaline than in untreated cells. ADP ribosylation of inhibitory GTP-binding proteins by pertussis toxin completely prevented (R)-PIA from inhibiting cAMP accumulation. A further analysis of the different GTP-binding proteins, including the three Gi subtypes (Gi1, Gi2, and Gi3), revealed no quantitative or qualitative change after dexamethasone treatment. In addition, the adenosine A2 receptors were down-regulated, as indicated by the fact that the ability of the A2 receptor agonist 5'-N-ethylcarboxamidoadenosine to increase cAMP formation was decreased by 20-30% in dexamethasone-treated cells. In summary, we have shown that A1 and A2 receptors on the same cell are differentially regulated by glucocorticoids and that this has functional importance in the regulation of cAMP accumulation.

5'-(N-Ethylcarboxamido)adenosine (NECA) and N6-[(R)-(phenylisopropyl)]adenosine (PIA) were incubated in an adenylate cyclase assay with a particulate fraction of caudate-putamen tissue of the rat, in order to examine the effect of kainic acid and a 6-hydroxydopamine-induced lesion on adenosine receptor coupled adenylate cyclase in vitro. There was an enhancement of formation of cyclic AMP induced by NECA, that was mediated by A2 adenosine receptors. Phenylisopropyl adenosine also stimulated adenylate cyclase in the striatum, with a maximum increase at 0.1 mM. At smaller concentrations, PIA inhibited the basal activity, which was previously described to be an effect mediated by A1 adenosine receptors. In caudate-putamen tissue from rats receiving a unilateral lesion, induced with kainic acid, basal and maximally NECA- and PIA-stimulated activity of adenylate cyclase was decreased. The maximum stimulatory effects of both substances were also significantly decreased, whereas no change of the inhibitory effect of PIA was observed. After unilateral lesion induced with 6-OHDA, basal and maximally NECA- and PIA-activated adenylate cyclase was increased; however, no inhibitory effect of PIA was seen. These results suggest that A2 adenosine receptor-coupled adenylate cyclase was located on neurones intrinsic to the neostriatum and probably postsynaptic to the dopaminergic input. The A1 adenosine receptors seem to be associated with the nigrostriatal pathway implying a presynaptic localization on dopaminergic afferents. In addition, since after both kainic acid- and 6-OHDA-induced lesions, respectively, in caudate-putamen tissue of the contralateral side, PIA no longer inhibited the activity of adenylate cyclase, contralateral structures also appeared to be involved in the regulation of A1 adenosine receptors.

A simple method for solubilization and reconstitution of the A1 adenosine receptor from bovine brain is presented. Solubilization with CHAPS-phosphatidylcholine (CHAPS/PC) mixture did not alter the binding properties of the A1 adenosine receptor antagonist [3H]-DPCPX. The solubilized receptors were chromatographed on hydroxyapatite or DEAE-cellulose to remove native membrane lipids and part of non-receptor proteins. Elution of the receptor fractions was obtained from DEAE-cellulose column with a linear gradient of KCl (0-0.4 M). The fractions corresponding to the peak of [3H]-DPCPX binding activity were then reconstituted in phosphatidylcholine by dialysis. The reconstituted receptor retained all the binding characteristics and the same rank order of competition potency (R-PIA greater than S-PIA greater than NECA) as the native receptor, although its thermal stability was remarkably reduced. The binding of [3H]-DPCPX to A1 adenosine receptors was increased by GTP, probably as result of interactions with coeluted G-proteins.

Adenosine and its derivatives 1 nmol.L-1 -0.1 mmol.L-1 elicited negative chronotropic effect on cultured rat myocardial cells. The respective IC50 were: L-PIA 85 +/- 10 greater than NECA 160 +/- 64 greater than adenosine 361 +/- 41 nmol.L-1. The negative chronotropic effect was antagonized by IBMX. [3H]Adenosine bound to myocardial cells of rat in a saturable way with a Kd of 238 nmol.L-1 and a Bmax of 7.6 pmol/10(8) cells. The binding of [3H]adenosine was competitively antagonized by adenosine, L-PIA, NECA, and IBMX. These results indicated the existence of adenosine receptor in rat myocardial cells.

1. The effects of the adenosine analogues, 5'-N-ethyl-carboxamide adenosine (NECA), R-N6-phenylisopropyladenosine (R-PIA), 2-chloroadenosine (CADO), and CGS 21680C on electrically evoked tritium outflow from preparations loaded with [3H]-choline and on evoked endplate potentials (e.p.ps), as well as the ability of the xanthines, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) and PD 115,199 to antagonize the effects of the adenosine analogues, were investigated in phrenic nerve-diaphragm preparations. 2. NECA, R-PIA and CADO decreased, in a concentration-dependent manner, the evoked tritium outflow from preparations loaded with [3H]-choline. NECA and R-PIA were about equipotent and more potent than CADO. 3. DPCPX shifted to the right in a near parallel fashion the concentration-response curve for the inhibitory effect of R-PIA on evoked tritium outflow. 4. In the presence of DPCPX, NECA increased, rather than decreased, evoked tritium outflow. PD 115,119 antagonized, in a concentration-dependent manner, this excitatory effect of NECA. 5. CGS 21680C, in low nanomolar concentrations, increased evoked tritium outflow, an effect also antagonized by PD 115,119. 6. CGS 21680C increased, and R-PIA decreased, the amplitude of e.p.ps recorded from preparations paralysed with tubocurarine. Both effects could be observed in the same endplate. 7. It is concluded that both inhibitory (probably A1) and excitatory (probably A2) adenosine receptors coexist at the rat neuromuscular junction, modulating the evoked release of acetylcholine.

Caffeine and other methylxanthines elevate reinforcement threshold for electrical brain stimulation with an order of potency suggesting that the effect is mediated by antagonism of adenosine A2 receptors. The purpose of this study was to evaluate further the possible mechanism by which caffeine and other methylxanthines elevate reinforcement thresholds for ICSS. Drugs known to affect adenosinergic transmission in predictable ways, adenosine receptor agonists and antagonists and benzodiazepine agonists and inverse agonists, were tested to determine their effect upon reinforcement threshold. Both the selective A1 adenosine agonist, R(-)-PIA, and the non-selective A1/A2 adenosine agonist NECA failed to alter reinforcement thresholds, as did CGS 15943, a potent non-xanthine non-selective adenosine receptor antagonist. Chlordiazepoxide, a benzodiazepine agonist, lowered reinforcement thresholds and FG 7142, a benzodiazepine inverse agonist, elevated reinforcement thresholds, perhaps corresponding to their anxiolytic and anxiogenic subjective effects in humans. However, another benzodiazepine agonist, midazolam and another inverse agonist, beta-CCE, did not alter reinforcement thresholds. These results fail to support a general role for adenosinergic systems in the threshold-elevating effect of methylxanthines.

1. This study was carried out in order to identify the receptor responsible for adenosine-induced dilatation of the hepatic arterial vascular bed. 2. Livers of 10 New Zealand White rabbits were perfused in vitro with Krebs-Bülbring buffer via the hepatic artery and the portal vein at constant flows of 26 and 77 ml min-1 100 g-1 liver respectively. The tone of the preparation was raised by the presence of noradrenaline in the perfusate (concentration: 10(-5) M). 3. Dose-response curves for adenosine and its analogues 5'-N-ethyl-carboxamido-adenosine (NECA), the 2-substituted NECA analogue CGS 21680C, and R- and S-N6-phenyl-isopropyl-adenosine (R- and S-PIA) were obtained after their injection into the hepatic arterial supply. 4. The order of vasodilator potency of these agents was: NECA greater than CGS 21680C greater than adenosine greater than R-PIA greater than S-PIA. Their potency, expressed relative to that of adenosine, was in the approximate ratio 10:3:1:0.3:0.1, consistent with that resulting from activation of P1-purinoceptors of the A2 sub-type (which mediate vasodilatation due to adenosine). 5. The P1-purinoceptor antagonist 8-phenyltheophylline (10(-5) M) caused significant attenuation of the vasodilatation to adenosine and analogues. 6. It is concluded that adenosine-induced dilatation of the hepatic arterial vascular bed is mediated by P1-purinoceptors of the A2 sub-type.