Effects of enprofylline on A1 and A2 adenosine receptors

The effects of enprofylline were studied on A₁ adenosine receptors of rat fat cells and on A₂ adenosine receptors of human platelets and of guinea-pig lung. Enprofylline antagonized the 5′-N-ethylcarboxamidoadenosine (NECA)-induced stimulation of platelet adenylate cyclase activity with a K_B of 130 μM. In human platelets, enprofylline did not antagonize but potentiated the NECA-induced inhibition of aggregation. This potentiation was abolished in the presence of the phosphodiesterase inhibitor papaverine. An adenosine antagonistic effect of enprofylline could not be evaluated on A₂ receptors of guinea-pig lung because the xanthine enhanced basal and NECA-stimulated cyclic AMP accumulation. Enprofylline antagonized the N⁶-R-(−)-phenylisopropyladenosine (R-PIA)-induced inhibition of rat fat cell adenylate cyclase with a K_B of 32 μM. The K_i value for inhibition of [³H]PIA binding to fat cell membranes was 45 μM. Enprofylline inhibited cyclic AMP phosphodiesterase activity of human platelets, guinea-pig lung and rat fat cells with K_i values of 15, 130 and 110 μM, respectively. The results show that enprofylline was nearly equipotent as antagonist at A₁ and A₂ adenosine receptors. Mechanisms other than adenosine antagonism or phosphodiesterase inhibition may be involved in the pharmacological effects of enprofylline.     

Rabbit isolated vas deferens possess A1 and A2 adenosine receptors.

1. The effects of adenosine, 5'-N-ethylcarboxamidoadenosine (NECA), 2-chloroadenosine, N6-phenylisopropyladenosine (L-PIA and D-PIA) and N6-cyclohexyladenosine (CHA) were examined on the rabbit isolated vas deferens. 2. All the analogues in a concentration-dependent manner inhibited contractile response to electrical stimulation. 3. 8-Phenyltheophylline caused a rightward shift of all the adenosine and its analogues' concentration-response curves. 4. The order of potency for the adenosine and its analogues on the rabbit isolated vas deferens was: CHA = NECA greater than L-PIA greater than 2-chloroadenosine greater than D-PIA greater than adenosine. 5. It is concluded that CHA, NECA, L-PIA, 2-chloroadenosine, D-PIA and adenosine mediate their inhibitory effects on the rabbit isolated muscle via both A1 and A2 adenosine receptors.     

Potent adenosine A1 and A2A receptors antagonists: recent developments

This review summarizes the current tendencies observed in the past 5 years in the development of A(1) and A(2A) adenosine receptor antagonists performed in various academia and industry. A(1) and A(2A) AR antagonists are as well xanthines as heteroaromatic derivatives and are most commonly 6:5 fused heteroatomic compounds. Among xanthine-based compounds, some common features could be pointed out. The recent A(1) AR ligands which show good biological profile, possess long alkyl chains in position 1 and 3 as well as bulky C(8)-substituent, while A(2A) AR antagonists with a high A(2A) AR affinity are C(8)-styryl substituted with N(1)-alkyl/alkynyl moiety or fused tricyclic xanthines possessing heteroatom(s) in the third cycle. The research in the field of heteroaromatic A(1) and A(2A) ARs antagonists impressively has a wide range. Ligands are as well non-fused monocyclic substituted compounds as fused bi- and tricyclic derivatives with the nitrogen, oxygen and sulfur heteroatoms. Most often, adenosine A(1) receptor non-xanthine antagonists are adenine-based, having substituted amino group and variable nitrogen atoms positions in the molecules. A(2A) AR ligands show good affinity when furanyl function, which is crucial for binding, is present in the fused bicyclic and tricyclic analogs. Moreover, tricyclic nitrogen containing antagonists in order to be active, frequently possess long-alkylphenyl moiety.     

Molecular characterization of A1 and A2a adenosine receptors

Detailed amino acid sequence analyses of A₁ and A_2a adenosine receptors were assembled by analogy to other G-protein-coupled receptors and correlated with pharmacological observations. Sites for phosphorylation, palmitoylation, and sodium binding have been proposed. Striatal A_2a receptors from human and other species were photoaffinity-labeled using the selective, radioiodinated agonist PAPA-APEC. Selective chemical affinity labels for A₁ and A_2a receptors have been introduced. For example, an isothiocyanate, ρ-DITC-APEC (100 nM), irreversibly diminished the B_max for [³H]CGS 21680 (2-[4-[(2-carboxyethyl) phenyl] ethylamino]-5′-N-ethylcarboxamido-adenosine) binding in rabbit striatal membranes by 71% (K_d unaffected), suggesting a direct modification of the ligand binding site. Novel trifunctional affinity labels have been designed. Rabbit and human A_2a receptors were characterized using [³H]XAC binding in the presence of 50 or 25 nM CPX (8-cyclopentyl-1,3-dipropylxanthine), respectively. The inhibition of A₂ radioligand binding by the histidyl-modifying reagent diethylpyrocarbonate suggested the involvement of His residues in interactions with adenosine agonists and antagonists. Properties of transiently expressed mutants of bovine A₁ receptors in which either His²⁵¹ or His²⁷⁸ residues have been substituted with Leu suggest that both histidines are important in binding. © 1993 Wiley-Liss, Inc.     

Pharmacokinetic-haemodynamic relationships of 2-chloroadenosine at adenosine A1 and A2a receptors in vivo.

1. The purpose of the present study was to develop an experimental strategy for the quantification of the cardiovascular effects of non-selective adenosine receptor ligands at the adenosine A1 and A2a receptor in vivo. 2-Chloroadenosine (CADO) was used as a model compound. 2. Three groups of normotensive conscious rats received an short intravenous infusion of 1.4 mg kg-1 CADO during constant infusions of the A1-selective antagonist, 8-cyclopentyltheophylline (CPT; 20 micrograms min-1 kg-1), the A2a-selective antagonist, 8-(3-chlorostyryl) caffeine (CSC; 32 micrograms min-1 kg-1) or the vehicle. The heart rate (HR) and mean arterial blood pressure (MAP) were recorded continuously during the experiment and serial arterial blood samples were taken for analysis of drug concentrations. The ratio MAP/HR was also calculated, which may reflect changes in total peripheral resistance on the assumption that no changes in stroke volume occur. 3. During the infusion of CPT, CADO produced a reduction in both blood pressure and MAP/HR by activation of the A2a receptor. The concentration-effect relationships were described according to the sigmoidal Emax model, yielding potencies based on free drug concentrations (EC50,u) of 61 and 68 ng ml-1 (202 and 225 nM) for the reduction of blood pressure and MAP/HR, respectively. During the infusion of CSC, an EC50,u value of 41 ng ml-1 (136 nM) was observed for the A1 receptor-mediated reduction in heart rate. The in vivo potencies correlated with reported receptor affinities (Ki(A1) = 300 nM and Ki(A2a) = 80 nM). The maximal reductions in MAP/HR and heart rate were comparable to those of full agonists, with the Emax values of -12 +/- 1 x 10(-2) mmHg b.p.m.-1 and -205 b.p.m. respectively. 4. It is concluded that this integrated pharmacokinetic-pharmacodynamic approach can be used to obtain quantitative information on the potency and intrinsic activity of new non-selective adenosine receptor agonists at different receptor subtypes in vivo.     

Effects of selective agonists and antagonists for A1 or A2A adenosine receptors on sleep-waking patterns in rats

There is evidence that adenosine may participate in the regulation of sleep and wakefulness in the mammalian central nervous system. To understand whether the adenosine receptor subtypes, A₁ and A_2A, are involved in the modulation of sleep and waking, we carried out electroencephalographic (EEG) studies in the rat using selective agonists and antagonists for either receptor. EEG activity was recorded for 6 h after intraperitoneal administration of drugs, and the stages of wakefulness, rapid eye movements (REM) sleep and non-REM sleep were classified thereafter. The dose-response effects of the A₁ agonist, 2-chloro-N⁶-cyclopentyladenosine (CCPA), and the A_2A agonist, 2-hexynyl-5′-N-ethylcarboxamido-adenosine (2HE-NECA), were examined. Both drugs, CCPA (0.003–0.03 mg/kg) and 2HE-NECA (0.03–0.3 mg/kg), given at a dose range known to be effective pharmacologically did not significantly modify the sleep patterns in the whole recording period. We have also studied the effects of the selective A₁ antagonist, 8-cyclopentyl-1,3-dipropylxanthyne (DPCPX), and the A_2A antagonist, 7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-c]-1,2,4,-triazolo-[1,5-c]-pyrimidine (5CH 58261). The results obtained with antagonists were compared with those of two non-selective adenosine antagonists, caffeine (10 mg/kg) and 9-chloro-2-(2-furyl)-5,6-dihydro-[1,2,4]-triazolo-[1,5-c]-quinazolin-5-imine (CGS 15943). Like caffeine, both CGS 15943 (0.3–10 mg/kg) and SCH 58261 (0.3–10 mg/kg), at the highest dose, increased wakefulness, while DPCPX (0.3–10 mg/kg) did not affect sleep parameters. The data indicate that potent and selective adenosine agonists have little or no effect on sleep states in the rat, while using adenosine antagonists it seems that A_2A receptors are primarily involved in the modulation of wakefulness. © 1996 Wiley-Liss, Inc.     

Receptor crosstalk: characterization of mice deficient in dopamine D1 and adenosine A2A receptors.

Here we report the development of D1A2A receptor knockout mice to investigate whether interactions between dopamine D1 and adenosine A2A receptors participate in reward-related behavior. The combined deletion of D1 and A2A receptors resulted in mice with decreased weight and appetitive processes, reduced rearing and exploratory behaviors, increased anxiety, and a significantly poorer performance on the rotarod, compared to wild-type littermates. D1A2A receptor knockout mice shared phenotypic similarities with mice deficient in D1 receptors, while also paralleling behavioral deficits seen in A2A receptor knockout mice, indicating individual components of the behavioral phenotype of the D1A2A receptor knockout attributable to the loss of both receptors. In contrast, ethanol and saccharin preference in D1A2A receptor knockout mice were distinctly different from that observed in derivative D1 or A2A receptor-deficient mice. Compared to wild types, preference and consumption of ethanol were decreased in D1A2A receptor knockout mice, the reduction in ethanol consumption greater even than that seen in D1 receptor-deficient mice. Preference and consumption of saccharin were also reduced in D1A2A receptor knockout mice, whereas saccharin preference was similar in wild-type, D1, and A2A receptor knockout mice. These data suggest an interaction of D1 and A2A receptors in the reinforcement processes underlying the intake of rewarding substances, whereby the A2A receptor seems involved in goal-directed behavior and the motor functions underlying the expression of such behaviors, and the D1 receptor is confirmed as essential in mediating motivational processes related to the repeated intake of novel substances and drugs.     

Effects of urea pretreatment on the binding properties of adenosine A1 receptors

The effect of denaturation and/or extraction of nonintegral membrane proteins by 7 M urea on the binding of the antagonist [3H]cyclopentyl-1,3-dipropylxanthine 8 dipropyl-2,3 ([3H]DPCPX), and the agonists adenosine, (-)-N6-(2-phenylisopropyl)-adenosine (R-PIA) and N6-cyclohexyladenosine (CHA), was investigated at human A1 adenosine receptors stably expressed in CHO cells. Pretreatment with urea caused a 56% reduction in membrane proteins. Compared to controls, the use of adenosine deaminase (ADA), 100 microM 5'-guanylylimidodiphosphate (Gpp(NH)p) or urea each caused equivalent increases in specific [3H]DPCPX binding. Neither the binding kinetics nor the affinity of [3H]DPCPX were significantly different in urea-pretreated compared to ADA-pretreated membranes. At 25 degrees C in ADA-pretreated membranes, the competition isotherms for R-PIA and CHA were characterized by two affinity states. Gpp(NH)p (100 microM) reduced, but did not abolish, the value of the high-affinity dissociation constant. Similar results were obtained after treatment with urea for R-PIA, whereas the high-affinity state for CHA was abolished. At 37 degrees C, urea pretreatment, but not 100 microM Gpp(NH)p, abolished high-affinity agonist competition binding. There was no significant effect of any of the treatments on the low-affinity agonist binding state. In urea-pretreated membranes, exogenously added adenosine competed according to a simple mass-action model with a pK(L) of 5.66+/-0.05 (n=3). Compared to the more common approaches of ADA treatment and/or use of guanine nucleotides, our findings suggest that urea pretreatment represents an inexpensive and useful approach for investigating the binding properties of adenosine A1 ligands (including adenosine) to the G protein-uncoupled form of the receptor.     

Xanthine derivatives as antagonists at A1 and A2 adenosine receptors

Summary A variety of alkylxanthines has been comparatively examined as antagonists of A₁ adenosine receptors in rat fat cells, rat and bovine cerebral cortex and of A₂ adenosine receptors in human platelets. With few exceptions all xanthine derivatives with 7-position substituents such as diprophylline, proxyfylline, pentoxifylline and etofylline were less potent antagonists than xanthine itself which hadK _i-values of 170 mol/l (A₁) and 93 mol/l (A₂). Theophylline, caffeine and 3-isobutyl-1-methylxanthine were more potent than xanthine but nearly equipotent antagonists at both receptor subtypes. 8-Phenyl substituents considerably increased the antagonist potency at A₁ and A₂ receptors. 1,3-Diethyl-8-phenylxanthine was the most potent A₂ antagonist (K _i 0.2 mol/l) in human platelets. At A₁ receptors 1,3-dipropyl-8-(2-amino-4-chlorophenyl)xanthine (PACPX) was the most potent antagonist in all three tissues withK _i-values from 0.3 to 8.6 nmol/l. Several 8-phenylxanthine derivatives were remarkably selective antagonists at A₁ receptors. 8-Phenyltheophylline was approximately 700 times more potent as antagonist at A₁ receptors (bovine brain) than at A₂ receptors (human platelets), and PACPX was even 1,600 times more potent as A₁ adenosine receptor antagonist. These compounds offer a possibility for a subtype-selective blockade of adenosine receptors.     

Differential development of adenosine A1 and A2b receptors in the rat duodenum.

1. The development of the adenosine A1 and A2b receptors inducing relaxation of the rat duodenum was studied by use of a combination of functional and radioligand binding assays on rats aged between 5 and 30 days and compared with results previously found in adult rat duodenum. 2. 1,3-[3H]-dipropyl-8-cyclopentylxanthine ([3H]-DPCPX) bound with high affinity to a single site in duodenum preparations from rats aged 20, 25 and 30 days. At 10 and 15 days there was no detectable specific binding of [3H]-DPCPX. 3. The affinity (KD) of the binding site for [3H]-DPCPX was similar in membrane preparations from 20, 25 and 30 day old animals (1.58-2.27 nM), but the density (Bmax) of binding sites was found to increase up to 25 days where peak levels (72.0 +/- 9.5 fmol mg-1 protein) were observed and then decline at 30 days (45.5 +/- 2.9 fmol mg-1 protein) to levels commensurate with those previously determined in the adult rat duodenum. 4. In duodenum from 10 day old rats no responses to N6-cyclopentyladenosine (CPA, 1 nM-10 microM) were observed, at 15 days the duodenum responded to the highest concentration of CPA (3 microM) only, and at 20-30 days concentration-related responses were observed, with the potency of CPA increasing with an increase in age. DPCPX (10 nM) abolished the responses to CPA except at the highest concentration of CPA (3 microM) where the response was markedly attenuated, suggesting the presence of an A1, receptor. 5. In rat duodenum from animals of all ages (5-30 days), concentration-related responses to 5'-N-ethylcarboxamidoadenosine (NECA) were observed. The potency of NECA remained constant with an increase in age, whereas the maximum relaxation response increased from 20% at 5 days to 110% at 25 and 30 days. In the presence of 1 microM DPCPX a right-ward shift in the concentration-response curve to NECA was observed at all ages. In the presence of 10 nM DPCPX, the response to NECA was unaffected in the duodenum from animals aged 10 and 15 days. However, in duodenum from animals aged 20-30 days the concentration-response curve to NECA was shifted to the right suggesting that there is an A1 component to the action of NECA at these ages. Schild analysis of the effects of increasing concentrations of DPCPX versus NECA on the duodenum from 25 day old animals generated a slope of 0.62 suggesting that NECA acts at A1 and A2b receptors as in the adult. 6. The A2b-selective analogue, 2-[p-(carboxyethyl)-phenylethylamino]-5'-N-ethylcarboxamidoadenosi ne (CGS 21680) (10 nM-10 microM) was without effect on the carbachol-contracted duodenum from 15 day old rats and the duodenum from 25 day old rats responded to the highest concentration of CGS 21680 only, suggesting that the A2 receptors here, as in the adult, are not of the A2a subtype. The adenosine antagonist, 8-phenyltheophylline (8-PT) (10 microM), abolished the inhibitory effects of NECA (100 nM-100 microM) on 10, 15 and 25 day old rat duodenum indicating that the responses to NECA were not mediated via an adenosine A3 receptor. 7. These results show that adenosine A1 receptors in rat duodenum are present and functionally viable from day 20 onwards and that the density of A1 receptors varies with age, increasing up to day 25 and then declining at day 30 to a density commensurate with that found in the adult. The responses to CPA, mediated via the A1 receptor, increase with age in a similar fashion. In contrast however, the response to NECA was evident from day 5, the earliest age studied, and from days 5-15 NECA acted via the A2b receptor subtype. However, from day 20 onwards NECA acted at a mixed population of A1 and A2b receptors. These results demonstrate the differential development of the A1 and the A2b receptors in the rat duodenum.     

Modulating effect of adenosine deaminase on function of adenosine A1 receptors

AIM: To study the modulating effect of adenosine deaminase (ADA) on the adenosine A1 receptor (A1R) in HEK293 cells stably expressing the human A1R. METHODS: cDNA was amplified by RT-PCR using total RNA from human embryo brain tissue as the template. The PCR products were subcloned into the plasmid pcDNA3 and cloned into the plasmid pcDNA3.1. The cloned A1R cDNA was sequenced and stably expressed in HEK293 cells. The modulating effect of adenosine deaminase on A1R was studied by using [3H]DPCPX binding assay and an intracellular calcium assay. RESULTS: HEK293 cells stably expressing human A1R were obtained. Saturation studies showed that the K(D) value and B(max) value of [3H]DPCPX were 1.6+/-0.2 nmol/L and 1.819+/-0.215 nmol/g of protein respectively, in the absence of ecto-ADA respectively, and 1.3+/-0.2 nmol/L and 1.992+/-0.130 nmol/g of protein in the presence of ecto-ADA respectively, suggesting that the K(D) value and B(max) value of [3H]DPCPX were unaffected by ecto-ADA. In the case of [3H]DPCPX competition curves obtained from intact cells or membranes, A1R agonist CCPA/[3H]DPCPX competition curve could be fitted well to a one-site model in the absence of ecto-ADA and a two-site model in the presence of ecto-ADA with a K(H) value of 0.74 (0.11+/-4.8) nmol/L (intact cells) or 1.8 (0.25+/-10) nmol/L (membrane) and a K(L) value of 0.94 (0.62+/-1.41) micromol/L (intact cells) or 0.77 (0.29+/-0.99) micromol/L (membrane). The K(L) value is not significantly different from the IC50 value of 0.84(0.57+/-1.23) micromol/L (intact cells) or 0.84 (0.63+/-1.12) micromol/L (membrane) obtained in the absence of ecto-ADA. Similar results were obtained from the CPA/[3H]DPCPX competition curve in the absence or presence of ecto-ADA on intact cells or membranes. Intracellular calcium assay demonstrated that the EC50 value of CPA were 10 (5+/-29) nmol/L and 94 (38+/-229) nmol/L in the presence or absence of ecto-ADA, respectively. CONCLUSION: A1R stably expressed in the HEK293 cells display a low affinity for agonists in the absence of ADA and high and low affinities for agonists in the presence of ADA. The presence of ADA may promote the signaling through the adenosine A1 receptor in HEK293 cells.     

The coexistence of adenosine A1 and A2 receptors in guinea-pig aorta

The effects of adenosine, 5'-(N-ethyl)carboxamidoadenosine (NECA), 2-chloroadenosine (2-CA), No-cyclohexyladenosine (CHA) and N6(R-2-phenylisopropyl)-adenosine (R-PIA) on the tone of phenylephrine-constricted guinea-pig isolated aorta have been examined. For aortic relaxation the analogues exhibited the following rank order of potency: NECA > adenosine > 2-CA > R-PIA > CHA. This is consistent with previous reports that relaxation of this tissue is mediated by the adenosine A₂ receptor. An unexpected finding was that R-PIA, 2-CA and CHA all induced contractions at concentrations lower than were required for relaxation, giving a biphasic dose-response curve. Neither NECA nor adenosine contracted the aorta. This is consistent with activation of vascular A₁ receptors. An A₁-selective concentration of the antagonist l,3-dipropyl-8-cyclopentyl xanthine abolished the contraction elicited by R-PIA in the guinea-pig aorta. This further suggests that the contraction is mediated by a₁ receptors.     

Regulation of dopamine D1 receptors by chronic administration of structurally different D1 receptor antagonists: a quantitative autoradiographic study.

The effects of chronic treatment (18 days) with the novel D1 antagonists, the benzonaphthazepine SCH 39166 (2 mg/kg per day) and the tetrahydroisoquinoline A-69024 (10 mg/kg per day), on D1 and D2 receptor binding in the rat brain were studied by quantitative receptor autoradiography. The benzazepine derivatives, SCH 23390 (0.5 mg/kg per day) and SKF 38393 (20 mg/kg per day), the prototype D1 antagonist and agonist, respectively, were also included in the experiment. Chronic treatment with SCH 23390 increased D1 receptor binding, studied with [3H]SCH 23390, in the nucleus accumbens and in all subregions of the anterior caudatus-putamen. However, chronic treatment with SKF 38393 did not alter D1 receptor binding in the brain areas studied. Interestingly, chronic treatment with SCH 39166 increased D1 receptor binding in the anterior caudatus-putamen but not in the nucleus accumbens. In contrast, chronic treatment with A-69024 did not alter D1 receptor binding in the brain areas studied. Treatment with SCH 23390, SCH 39166, A-69024 or SKF 38393 failed to alter D1 receptor binding in the posterior caudatus-putamen and the tuberculum olfactorium. Neither the D1 antagonists nor the D1 agonist investigated altered D2 receptor binding, studied with [125I]sulpiride, in the caudatus-putamen and nucleus accumbens. In summary, the benzonaphthazepine D1 antagonist, SCH 39166, as well as the benzazepine D1 antagonist, SCH 23390, can increase D1 receptor binding without influencing D2 receptor binding. However, a tetrahydroisoquinoline, A-69024, failed to increase D1 receptor binding, suggesting a differential regulation of D1 receptors after treatment with this putative D1 antagonist.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential effects of early chronic lead exposure on postnatal rat brain NMDA,PCP, and adenosine A1 receptors: An autoradiographic study

The deleterious effects of postnatal lead (Pb) exposure on neural development, synaptic plasticity, and cognitive function have been well documented in laboratory animals. While the exact mechanisms by which Pb produces long-lasting neurotoxicity remain unknown, recent evidence suggests that Pb may interact with and/or disrupt the N-methyl-D-aspartate/phencyclidine receptor complex and the associated ion channel. In addition to perturbations of excitatory amino acid neurotransmission, chronic Pb exposure may also have deleterious effects on inhibitory mechanisms such as that provided by purinergic neuromodulation. In order to further examine the possibility that alterations of both excitatory and inhibitory neurotransmission may contribute to the neurotoxic actions of Pb, the effects of early Pb exposure on ligand binding to postnatal rat brain N-methyl-D-aspartate (NMDA), phencyclidine (PCP), and adenosine A₁ receptors were examined using quantitative autoradiography techniques. Rat pups nursed mothers exposed to 4% PbCO₃ in their diet or a Na₂CO₃ control diet from postnatal day 1 (P1) to P25. At P25, rats were sacrificed and the regional distributions of brain NMDA, PCP, and adenosine A₁ receptors were examined. Chronic lead exposure was found to produce a specific increase in [³H]CGP 39653 binding to NMDA receptors in the hippocampus. [³H]1-(1-[2-thienyl)cyclohexyl]-piperdine ([³H]TCP) binding to PCP receptors was largely unaffected by the chronic Pb treatment. In contrast, [³H]cyclohexyladenosine ([³H]CHA) binding to adenosine A₁ receptors was markedly reduced in many brain regions with the largest decreases observed in the cerebellum. These results indicate that neonatal Pb exposure produces a specific alteration of both excitatory and inhibitory neuromodulatory mechanisms in the postnatal rat forebrain that may underlie the behavioral hyperactivity and increased seizure sensitivity associated with Pb neurotoxicity. © 1993 Wiley-Liss, Inc.     

Stimulation of nucleoside efflux and inhibition of adenosine kinase by A1 adenosine receptor activation

Adenosine is produced intracellularly during conditions of metabolic stress and is an endogenous agonist for four subtypes of G-protein linked receptors. Nucleoside transporters are membrane-bound carrier proteins that transfer adenosine, and other nucleosides, across biological membranes. We investigated whether adenosine receptor activation could modulate transporter-mediated adenosine efflux from metabolically stressed cells. DDT1 MF-2 smooth muscle cells were incubated with 10 microM [3H]adenine to label adenine nucleotide pools. Metabolic stress with the glycolytic inhibitor iodoacetic acid (1AA, 5 mM) increased tritium release by 63% (P < 0.01), relative to cells treated with buffer alone. The IAA-induced increase was blocked by the nucleoside transport inhibitor nitrobenzylthioinosine (1 microM), indicating that the increased tritium release was primarily a purine nucleoside. HPLC verified this to be [3H]adenosine. The adenosine A1 receptor selective agonist N6-cyclohexyladenosine (CHA, 300 nM) increased the release of [3H]purine nucleoside induced by IAA treatment by 39% (P < 0.05). This increase was blocked by the A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (10 microM). Treatment of cells with UTP (100 microM), histamine (100 microM), or phorbol-12-myristate-13-acetate (PMA, 10 microM) also increased [3H]purine nucleoside release. The protein kinase C inhibitor chelerythrine chloride (500 nM) inhibited the increase in [3H]purine nucleoside efflux induced by CHA or PMA treatment. The adenosine kinase activity of cells treated with CHA or PMA was found to be decreased significantly compared with buffer-treated cells. These data indicated that adenosine A1 receptor activation increased nucleoside efflux from metabolically stressed DDT1 MF-2 cells by a PKC-dependent inhibition of adenosine kinase activity.     

Mechanism of A2 adenosine receptor activation. I. Blockade of A2 adenosine receptors by photoaffinity labeling

It has previously been shown that covalent incorporation of the photoreactive adenosine derivative (R)-2-azido-N6-p-hydroxy-phenylisopropyladenosine [(R)-AHPIA] into the A1 adenosine receptor of intact fat cells leads to a persistent activation of this receptor, resulting in a reduction of cellular cAMP levels [Mol. Pharmacol. 30:403-409 (1986)]. In contrast, covalent incorporation of (R)-AHPIA into human platelet membranes, which contain only stimulatory A2 adenosine receptors, reduces adenylate cyclase stimulation via these receptors. This effect of (R)-AHPIA is specific for the A2 receptor and can be prevented by the adenosine receptor antagonist theophylline. Binding studies indicate that up to 90% of A2 receptors can be blocked by photoincorporation of (R)-AHPIA. However, the remaining 10-20% of A2 receptors are sufficient to mediate an adenylate cyclase stimulation of up to 50% of the control value. Similarly, the activation via these 10-20% of receptors occurs with a half-life that is only 2 times longer than that in control membranes. This indicates the presence of a receptor reserve, with respect to both the extent and the rate of adenylate cyclase stimulation. These observations require a modification of the models of receptor-adenylate cyclase coupling, which is described in the accompanying paper [Mol. Pharmacol. 39:524-530 (1991)].     

Glucocorticoid receptor activation leads to up-regulation of adenosine A1 receptors and down-regulation of adenosine A2 responses in DDT1 MF-2 smooth muscle cells.

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.