adenosine-5--(n-ethylcarboxamide) and 1-3-dipropyl-8-cyclopentylxanthine

Adenosine has long been regarded as a crucial anti-inflammatory agent that protects the host from excessive damage. It has been reported to play an important role in suppressing immune activation, particularly that of T cells. However, it is a general observation that induction of T-cell activation is an efficient event despite the high adenosine levels that are often present in the affected host due to injury or stress. We report here that prior to antigenic stimulation via TCR/CD3, exposure of T cells to adenosine desensitizes adenosine receptors, so as to create a window of time where the T cells are insensitive to this ubiquitous suppressor. T cells from mice that were pre-exposed to this manipulation showed stronger responses to antigenic stimulation; therefore, the P1 adenosine receptor desensitization demonstrated an adjuvant-like effect. Our results suggest that adenosine receptor desensitization may be a mechanism for T cells to escape the general suppression during early points of T-cell activation and may emerge as a potential alternative for vaccine adjuvants.

Topics: Adenosine; Adenosine A1 Receptor Agonists; Adenosine A1 Receptor Antagonists; Adenosine A2 Receptor Agonists; Adenosine A2 Receptor Antagonists; Adenosine-5'-(N-ethylcarboxamide); Animals; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cells, Cultured; Cyclic AMP; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Receptor, Adenosine A1; Receptor, Adenosine A2A; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Spleen; T-Lymphocytes; Xanthines

The introduction of fluorescence-based techniques, and in particular the development of fluorescent ligands, has allowed the study of G protein-coupled receptor pharmacology at the single cell and single molecule level. This study evaluated how the physicochemical nature of the linker and the fluorophore affected the pharmacological properties of fluorescent agonists and antagonists.. Chinese hamster ovary cells stably expressing the human adenosine A(1) receptor and a cyclic 3',5' adenosine monophosphate response element-secreted placental alkaline phosphatase (CRE-SPAP) reporter gene, together with whole cell [(3)H]-8-cyclopentyl-1,3-dipropylxanthine (DPCPX) radioligand binding, were used to evaluate the pharmacological properties of a range of fluorescent ligands based on the antagonist xanthine amine congener (XAC) and the agonist 5' (N-ethylcarboxamido) adenosine (NECA).. Derivatives of NECA and XAC with different fluorophores, but equivalent linker length, showed significant differences in their binding properties to the adenosine A(1) receptor. The BODIPY 630/650 derivatives had the highest affinity. Linker length also affected the pharmacological properties, depending on the fluorophore used. Particularly in fluorescent agonists, higher agonist potency could be achieved with large or small linkers for dansyl and BODIPY 630/650 derivatives, respectively.. The pharmacology of a fluorescent ligand was critically influenced by both the fluorophore and the associated linker. Furthermore, our data strongly suggest that the physicochemical properties of the fluorophore/linker pairing determine where in the environment of the target receptor the fluorophore is placed, and this, together with the environmental sensitivity of the resulting fluorescence, may finally decide its utility as a fluorescent probe.

Topics: Adenosine-5'-(N-ethylcarboxamide); Alkaline Phosphatase; Animals; Boron Compounds; CHO Cells; Cricetinae; Cricetulus; Cyclic AMP; Dansyl Compounds; Fluorescent Dyes; Genes, Reporter; GPI-Linked Proteins; Humans; Isoenzymes; Ligands; Microscopy, Confocal; Molecular Imaging; Molecular Probe Techniques; Molecular Structure; Radioligand Assay; Receptor, Adenosine A1; Response Elements; Transfection; Xanthines

Adenosine mediates vascular responses through four receptor subtypes: A(1), A(2A), A(2B), and A(3). The role of A(2A) receptors in aortic vascular tone was investigated using A(2A) adenosine receptor (AR) knockout (A(2A)KO) and corresponding wild-type (A(2A)WT) mice. Isolated aortic rings from A(2A)WT and A(2A)KO mice were precontracted with phenylephrine (10(-7) M), and concentration responses for adenosine analogs and selective agonists/antagonists were obtained. Nonselective adenosine analog (NECA; EC(50) = 6.78 microM) and CGS-21680 (A(2A)AR selective agonist; EC(50) = 0.013 microM) produced concentration-dependent relaxation (maximum of 25% and 28% relaxation at 10(-5) M NECA and CGS-21680, respectively) in A(2A)WT aorta. In A(2A)KO aorta, NECA (EC(50) = 0.075 microM) induced concentration-dependent contraction (maximum contraction of 47% at 10(-6) M; P < 0.05 compared with A(2A)WT), whereas CGS-21680 produced no response. SCH-58261 (10(-6) M; A(2A)AR selective antagonist) abolished both NECA- and CGS-21680-mediated vasorelaxation in A(2A)WT (P < 0.05), whereas no change was observed in A(2A)KO. When DPCPX (10(-5) M; A(1) selective antagonist) was used in NECA concentration response, greater vasorelaxation was observed in A(2A)WT (50% vs. 25% in controls at 10(-5) M; P < 0.05), whereas lower contraction was seen in A(2A)KO tissues (5% vs. 47% in controls at 10(-6) M; P < 0.05). Aortic endothelial function, determined by response to acetylcholine, was significantly higher in WT compared with KO (66% vs. 51%; P < 0.05). BAY 60-6583 (A(2B) selective agonist) produced similar relaxation in both KO and WT tissues. In conclusion, A(2A)AR KO mice had significantly lower aortic relaxation and endothelial function, suggesting that the A(2A)AR plays an important role in vasorelaxation, probably through an endothelium-dependent mechanism.

Topics: Acetylcholine; Adenosine; Adenosine-5'-(N-ethylcarboxamide); Aminopyridines; Animals; Aorta; Dose-Response Relationship, Drug; Endothelium, Vascular; Female; Flavins; Gene Expression Regulation; In Vitro Techniques; Male; Mice; Mice, Knockout; Phenethylamines; Pyrimidines; Receptor, Adenosine A1; Receptor, Adenosine A2A; Receptor, Adenosine A2B; Triazoles; Vasoconstriction; Vasoconstrictor Agents; Vasodilation; Vasodilator Agents; Xanthines

1. The effects of adenosine and ATP receptor agonists on the release of endogenous noradrenaline from electrically stimulated (2 Hz, 0.1 msec) rat prostate were examined in order to clarify the pharmacological properties of prejunctional receptors for adenosine and ATP on the adrenergic nerve varicosities in the prostate. Noradrenaline was quantified by HPLC coupled with electrochemical detection techniques. 2. Both adenosine and ATP receptor agonists (1 micromol/L) inhibited noradrenaline release and the relative order of inhibitory effect was N(6)-cyclopentyl-adenosine (CPA) > 5'-N-ethylcarboxamidoadenosine > 2-chloroadenosine > adenosine > 2-methylthio-ATP (2mSATP) > AMP > ATP. 3. The adenosine receptor agonist CPA (1 nmol/L-1 micromol/L) and the ATP receptor agonist 2mSATP (100 nmol/L-100 micromol/L) inhibited the stimulation-induced release of noradrenaline in a concentration-dependent manner. The concentrations of CPA and 2mSATP that produced 50% inhibition of noradrenaline release were 9.6 nmol/L and 1.4 micromol/L, respectively. 4. 1,3-Dipropyl-8-cyclopentylxanthine, an adenosine A(1) receptor antagonist, significantly reduced the inhibitory effects of not only CPA, but also 2mSATP. 5. Suramin, an ATP receptor antagonist, significantly reduced the inhibitory effects of 2mSATP, but not those of CPA. 6. Pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid, another ATP receptor antagonist, had no effect on the inhibitory action of either agonist. 7. These results suggest that, in the sympathetic nerve terminals of rat prostate, adenosine and ATP induce inhibition of noradrenaline release via the activation of adenosine A(1) and/or xanthine-sensitive ATP receptors, which play an inhibitory regulatory role in adrenergic neurotransmission in the prostate.

Topics: 2-Chloroadenosine; Adenosine; Adenosine Monophosphate; Adenosine Triphosphate; Adenosine-5'-(N-ethylcarboxamide); Animals; Chromatography, High Pressure Liquid; Dose-Response Relationship, Drug; Electric Stimulation; In Vitro Techniques; Male; Norepinephrine; Presynaptic Terminals; Prostate; Rats; Rats, Wistar; Receptor, Adenosine A1; Receptors, Presynaptic; Receptors, Purinergic P2; Suramin; Sympathetic Nervous System; Thionucleotides; Xanthines

To evaluate anti-inflammatory effects of several novel adenosine receptor agonists and to determine their specificity for various adenosine receptor subtypes on neutrophils, cells heterologously expressing equine adenosine receptors, or equine brain membranes.. Neutrophils isolated from 8 healthy horses.. Radioligand binding experiments were performed to compare binding affinities of adenosine receptor agonists to equine adenosine A(1), A(2A), and A(3) receptor subtypes. Effects of these agonists on endotoxin-induced production of reactive oxygen species (ROS) by equine neutrophils and roles of specific adenosine receptor subtypes and cAMP production in mediating these effects were determined.. Radioligand binding experiments yielded a ranked order of affinity for the brain equine A(2A) receptor on the basis of 50% inhibitory concentrations (IC(50)) of the agonists as follows: ATL307 (IC(50) = 1.9nM) and ATL313 > ATL309 and ATL310 > ATL202 > 2-([p-2- carboxyethyl] phenylethylamino)-5'-N-ethylcarboxyamidoadenosine > 5'-N-ethylcarboxamidoadenosine. Furthermore, ATL313 had approximately 100-fold greater selectivity for A(2A) over A(1) and A(3) receptors. In functional assays with equine neutrophils, the compounds inhibited endotoxin-induced ROS production and stimulated production of cAMP with the same ranked order of potency. Results of experiments performed with selective adenosine receptor antagonists indicated that functional effects of ATL313 were via stimulation of A(2A) receptors.. Results indicated that activation of A(2A) receptors exerted anti-inflammatory effects on equine neutrophils and that stable, highly selective adenosine A(2A) receptor agonists may be developed for use in management of horses and other domestic animals with septic and nonseptic inflammatory diseases.

Topics: Adenosine; Adenosine A2 Receptor Agonists; Adenosine-5'-(N-ethylcarboxamide); Animals; Binding, Competitive; Cyclic AMP; Horses; Inhibitory Concentration 50; Kinetics; Lipopolysaccharides; Neutrophils; Phenethylamines; Piperidines; Radioligand Assay; Reactive Oxygen Species; Receptor, Adenosine A2A; Xanthines

Initial mutagenesis studies exploring the ligand recognition model of A1 adenosine receptor (A1R) mainly focused on the residues in the 5th-7th transmembrane domains (TMs5-7). Little is known about the role of residues in TM2. To explore the importance of reserved hydrophobic region in TM2 of A1R, we mutated the hydrophobic residues at positions 65 and 69 to hydrophilic residues (L65T, Leu-65 to Thr-65; I69T, Ile-69 to Thr-69; I69S, Ile-69 to Ser-69) to change the hydrophobicity at the outer end of TM2. Binding assays showed that the affinities of mutant receptors were significantly decreased for ribose group-containing agonists (2-chloro-N6-cyclopentyladenosine (CCPA) and 5'-N-ethyl-carboxamidoadenosine (NECA)) but not for antagonists, N6-cyclopentyl-9-methyladenine (N-0840), an adenine derivative lacking ribose group, and 8-cyclopentyl-1, 3-dipropylxanthine (DPCPX), a xanthine derivative. This observation suggests that the hydrophobic region at the outer end of TM2 may mediate the recognition of the ribose group of CCPA and NECA.

Topics: Adenine; Adenosine; Adenosine A1 Receptor Agonists; Adenosine A1 Receptor Antagonists; Adenosine-5'-(N-ethylcarboxamide); Amino Acid Sequence; Animals; Cell Line; Cell Membrane; Humans; Hydrophobic and Hydrophilic Interactions; Ligands; Mice; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; Protein Structure, Tertiary; Rats; Receptor, Adenosine A1; Xanthines

Ischemic postconditioning protects the reperfused heart from infarction, and this protection is dependent on the occupancy of adenosine receptors. We further explored the role of adenosine receptors in this salvage.. In situ rabbit hearts underwent 30 min of regional ischemia and 3 h of reperfusion, and postconditioning was effected with four cycles of 30-s reperfusion/30-s coronary artery occlusion at the end of ischemia.. Postconditioning reduced infarct size from 40.2+/-3.4% of the risk zone in untreated hearts to 15.5+/-2.5%. Protection by postconditioning was blocked by either the non-selective adenosine receptor blocker 8-p-(sulfophenyl)theophylline or the A2b-selective antagonist MRS 1754, injected intravenously 5 min before reperfusion. The protein kinase C (PKC) antagonist chelerythrine also aborted postconditioning's salvage, indicating a PKC-dependent mechanism. Neither the A1-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine nor the A2a-selective antagonist 8-(13-chlorostyryl)caffeine had an effect on protection. The non-selective but A2b-potent adenosine agonist 5'-(N-ethylcarboxamido)adenosine (NECA) infused from 5 min before to 1h after reperfusion mimicked postconditioning's effect on infarct size (17.2+/-2.7% infarction) and MRS 1754 blocked the NECA-induced cardioprotection, confirming that A2b activation was protective. The PKC activator phorbol 12-myristate 13-acetate delivered just before reperfusion also duplicated the protective effect of postconditioning (16.3+/-4.1% infarction), and co-administration of the PKC antagonist chelerythrine aborted PMA's protection, confirming that the protection was the result of PKC activation. NECA's protective effect was not affected by chelerythrine, but rather MRS 1754 blocked PMA's salutary effect (42.8+/-1.0% infarction), suggesting that the A2b receptor's effect is under control of PKC. Finally, wortmannin, a blocker of phosphatidylinositol 3-kinase, also abrogated protection by PMA.. Salvage of ischemic myocardium by postconditioning is dependent on activation of A2b receptors, which in turn depends on activation of PKC. It is still unclear why PKC activation is required to make the heart's adenosine become protective.

Topics: Acetamides; Adenosine; Adenosine A2 Receptor Antagonists; Adenosine-5'-(N-ethylcarboxamide); Alkaloids; Androstadienes; Animals; Aorta; Benzophenanthridines; Constriction; Enzyme Activators; Female; Male; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Perfusion; Phenanthridines; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase C; Purines; Rabbits; Receptor, Adenosine A2B; Signal Transduction; Tetradecanoylphorbol Acetate; Wortmannin; Xanthines

The molecular and pharmacological properties of adenosine receptors in the T24 human bladder epithelial carcinoma cell line were assessed by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR), Ca2+ Flux, cAMP production and interleukin-8 measurements. RT-PCR experiments detected the presence of transcripts for the adenosine A1, A2A and A2B receptors but not for the adenosine A3 subtype. Application of specific adenosine receptor ligands resulted in concentration-dependent increases in intracellular calcium ([Ca2+]i) with the following order of potency and EC50 values: 5'-N-Ethylcarboxamidoadenosine (NECA) (1153+/-214)>5'-(N-Cyclopropyl)carboxamidoadenosine (CPCA) (1436+/-186)>adenosine (4823+/-932). This rank order of potency is typical of adenosine A2B receptors. In addition, select adenosine receptor antagonists N-(4-acetylphenyl)-2-[4-(2,3,6,7-tetrahydro-2,6 dioxo-1,3-dipropyl-1H-purin-8-yl)phenoxy]acetamide (MRS 1706), 8-[4-[((4-Cyano[2,6-]-phenyl)carbamoylmethyl)oxy]phenyl]-1,3-di(n-propyl)-xanthine (MRS 1754), 1,3-Diethyl-8-phenylxanthine (DPCPX), 1,3-Diethyl-8-phenylxanthine (DPX), Alloxazine, 8-(3-Chlorostyryl)caffeine (CSC), and Theophylline blocked the NECA-induced calcium responses. Additionally, NECA, CPCA, and adenosine stimulated cAMP formation with a rank order of potency characteristic of adenosine A2B receptors. The select adenosine A2A antagonist, 5-amino-7-(phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c] pyrimidine (SCH 58261) failed to antagonize the NECA response, whereas the potent and highly selective adenosine A2B antagonists MRS 1754 and MRS 1706 inhibited NECA-stimulated cAMP production. Lastly, NECA-induced interleukin-8 secretion was inhibited by MRS 1754. Taken together, these data indicate that [Ca2+]i accumulation and cAMP production as well as interleukin-8 secretion is mediated through the adenosine A2B receptor in the T24 cell line.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Calcium; Cell Line, Tumor; Cyclic AMP; Dose-Response Relationship, Drug; Gene Expression Regulation, Neoplastic; Humans; Interleukin-8; Intracellular Fluid; Protein Isoforms; Purines; Receptors, Purinergic P1; Reverse Transcriptase Polymerase Chain Reaction; RNA, Neoplasm; Urinary Bladder Neoplasms; Xanthines

To investigate the effects of age on adenosine A1 receptor (ADORA1) mediated vascular, inotropic and chronotropic functional responses in isolated rat hearts.. NECA (5'-(N-ethylcarboxamido)adenosine) and R-PIA (R-N6-(1-methyl-2-phenylethyl)adenosine) concentration-response curves were produced in Langendorff prepared hearts isolated from immature (6 weeks), young (16 weeks) and mature (52 weeks) male Wistar rats and the effects of DPCPX (ADORA1 antagonist, 8-cyclopentyl-1,3-dipropylxanthine, 30 nM) and pertussis toxin pre-treatment (PTX, 48 h, 10 microg/kg i.p., inhibits G(i/o)-protein) were observed.. NECA mediated coronary vasodilation and induced biphasic concentration-response curves in hearts from immature rats (pEC50 8.5 (8.1-8.9) and 11.3 (10.3-12.3)). At the low sensitivity site, the potency of NECA increased in young but not mature rats and remained unchanged at the high sensitivity site. Both DPCPX and PTX each blocked NECA at the high sensitivity site in immature rats, producing monophasic concentration-response curves (pEC50 8.6 (8.5-9.9) for DPCPX and pEC50 8.7 (8.3-9.0) for PTX), but not in young and mature rats. A vasoconstrictor response was observed at low NECA concentrations in hearts from PTX pre-treated immature rats, but not in hearts from young and mature rats, and the response was inhibited by DPCPX. No age related changes were observed in R-PIA mediated negative inotropic and chronotropic responses (P>0.05).. ADORA1 mediates a vasodilator response as well as a vasoconstrictor response in the coronary resistance vessels; the latter occurs via a PTX-insensitive pathway and declines with age.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Age Factors; Aging; Animals; Coronary Circulation; Coronary Vessels; Dose-Response Relationship, Drug; Down-Regulation; Heart; Heart Rate; In Vitro Techniques; Male; Pertussis Toxin; Rats; Rats, Wistar; Receptor, Adenosine A1; Vasoconstriction; Vasodilation; Vasodilator Agents; Ventricular Function, Left; Xanthines

Adenosine and ATP/UTP are main components of the purinergic system that modulate cellular and tissue functions via specific adenosine and P2 receptors, respectively. Here, we explored the possibility that A(1) adenosine receptor (A(1)R) and P2Y(2) receptor (P2Y(2)R) form heterodimers with novel pharmacological properties. Coimmunoprecipitation showed these receptors directly associate in A(1)R/P2Y(2)R-cotransfected HEK293T cells. Agonist binding by the A(1)R was significantly inhibited by P2Y(2)R agonists only in membranes from cotransfected cells. The functional activity of A(1)R, as indicated by the G(i/o)-mediated inhibition of adenylyl cyclase, in the cotransfected cells was attenuated by the simultaneous addition of A(1)R and P2Y(2)R agonists. The increase in intracellular Ca(2+) levels induced by P2Y(2)R activation of G(q/11) was synergistically enhanced by the simultaneous addition of an A(1)R agonist in the coexpressing cells. These results suggest that oligomerization of A(1)R and P2Y(2)R generates a unique complex in which the simultaneous activation of the two receptors induces a structural alteration that interferes signaling via G(i/o) but enhances signaling via G(q/11).

Topics: Adenosine A1 Receptor Agonists; Adenosine-5'-(N-ethylcarboxamide); Animals; Calcium; Cell Line; Cell Membrane; Cloning, Molecular; Cyclic AMP; Humans; Protein Binding; Purinergic P2 Receptor Agonists; Rats; Receptor, Adenosine A1; Receptors, Purinergic P2; Receptors, Purinergic P2Y2; Uridine Triphosphate; Xanthines

The Cambridge Structural Database (CSD) was searched through two 3D queries based on substructures shared by well-known antagonists at the A(1) and A(3) adenosine receptors (ARs). Among the resulting 557 hits found in the CSD, we selected five compounds to purchase, synthesize, or translate synthetically into analogues better tailored to interact with the biological targets. Binding experiments using human ARs showed that four out of five tested compounds turned out to be antagonists at the A(1)AR or A(3)AR with K(i) values between 50 and 440 nM. Lead optimizations of 2-(benzimidazol-2-yl)quinoxalines (BIQs, 3) gave the best results in terms of potency and selectivity at the A(1) and A(3) ARs. Particularly, 2-(4-ethylthiobenzimidazol-2-yl)quinoxaline (3e) exhibited K(i) values at the A(1)AR, A(2A)AR, and A(3)AR of 0.5, 3440, and 955 nM, respectively, whereas 2-(4-methylbenzimidazol-2-yl)quinoxaline (3b) displayed at the same ARs K(i) values of 8000, 833, and 26 nM, respectively.

Topics: Adenosine; Adenosine A1 Receptor Antagonists; Adenosine A3 Receptor Antagonists; Adenosine-5'-(N-ethylcarboxamide); Animals; Benzimidazoles; Binding, Competitive; CHO Cells; Cricetinae; Databases, Factual; Drug Design; Humans; Quinoxalines; Receptor, Adenosine A1; Receptor, Adenosine A2A; Receptor, Adenosine A3; Xanthines

The vascular response to adenosine and its analogs is mediated by four adenosine receptors (ARs), namely, A(1), A(2A), A(2B), and A(3). A(2A)ARs and/or A(2B)ARs are involved in adenosine-mediated vascular relaxation of coronary and aortic beds. However, the role of A(1)ARs in the regulation of vascular tone is less well substantiated. The aim of this study was to determine the role of A(1)ARs in adenosine-mediated regulation of vascular tone. A(1)AR-knockout [A(1)AR((-/-))] mice and available pharmacological tools were used to elucidate the function of A(1)ARs and the impact of these receptors on the regulation of vascular tone. Isolated aortic rings from A(1)AR((-/-)) and wild-type [A(1)AR((+/+))] mice were precontracted with phenylephrine, and concentration-response curves for adenosine and its analogs, 5'-N-ethyl-carboxamidoadenosine (NECA, nonselective), 2-chloro-N(6)-cyclopentyladenosine (CCPA, A(1)AR selective), 2-(2-carboxyethyl)phenethyl amino-5'-N-ethylcarboxamido-adenosine (CGS-21680, A(2A) selective), and 2-chloro-N(6)-3-iodobenzyladenosine-5'-N-methyluronamide (Cl-IBMECA, A(3) selective) were obtained to determine relaxation. Adenosine and NECA (0.1 microM) caused small contractions of 13.9 +/- 3.0 and 16.4 +/- 6.4%, respectively, and CCPA at 0.1 and 1.0 microM caused contractions of 30.8 +/- 4.3 and 28.1 +/- 3.9%, respectively, in A(1)AR((+/+)) rings. NECA- and CCPA-induced contractions were eliminated by 100 nM of 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, selective A(1)AR antagonist). Adenosine, NECA, and CGS-21680 produced an increase in maximal relaxation in A(1)AR((-/-)) compared with A(1)AR((+/+)) rings, whereas Cl-IBMECA did not produce contraction in either A(1)AR((+/+)) or A(1)AR((-/-)) rings. CCPA-induced contraction at 1.0 microM was eliminated by the PLC inhibitor U-73122. These data suggest that activation of A(1)ARs causes contraction of vascular smooth muscle through PLC pathways and negatively modulates the vascular relaxation mediated by other adenosine receptor subtypes.

Topics: Adenosine; Adenosine A1 Receptor Agonists; Adenosine A1 Receptor Antagonists; Adenosine-5'-(N-ethylcarboxamide); Animals; Aorta; Female; In Vitro Techniques; Male; Mice; Mice, Knockout; Muscle, Smooth, Vascular; Receptor, Adenosine A1; Type C Phospholipases; Vasodilation; Vasodilator Agents; Xanthines

Because adenosine is a vascular tone modulator, we examined the effect of adenosine and congeners in the vascular reactivity of isolated human placental vessels and in perfused cotyledons. We characterized its vasomotor action and tentatively identified the receptor subtypes and their intracellular signaling mechanisms. We recorded isometric tension from the circular layer of chorionic vessel rings maintained under 1.5 g of basal tension or precontracted with KCl. The relative order of potency of adenosine and structural analogs is consistent with the expression of A2B receptors, 5'-(N-ethylcarboxamido)adenosine (NECA) being the most potent. The maximal contraction ranged from 45% to 60% of the KCl standard response, except for an A2A receptor agonist that did not exceed 15%. Consistently, NECA was 100-fold more potent than adenosine to raise the perfusion pressure of ex vivo perfused cotyledons. In contrast, a selective A3 receptor agonist relaxed precontracted rings of chorionic vessels. Whereas a selective A3 receptor antagonist was ineffective to antagonize adenosine-induced contraction, A2 or A1 receptor antagonists reduced adenosine-induced vasoconstriction concentration-dependently. Denudation of the endothelial layer reduced adenosine- and NECA-induced contractions by 50-70%. Furthermore, indomethacin reduced adenosine- or NECA-induced contractions concentration-dependently in intact and endothelium-denuded rings. A thromboxane receptor antagonist blocked adenosine- and NECA-induced contractions in intact and endothelium-denuded rings, suggesting the involvement of an arachidonic acid metabolite as the mediator of the vasoconstriction. We propose that adenosine A2B receptors mediate the adenosine-induced contraction vasomotor effect in human chorionic vessels and that this involves synthesis of a thromboxane receptor activator or a related prostanoid.

Topics: Adenosine; Adenosine A2 Receptor Antagonists; Adenosine-5'-(N-ethylcarboxamide); Arachidonic Acid; Chorion; Endothelium, Vascular; Female; Humans; In Vitro Techniques; Placenta; Pregnancy; Receptor, Adenosine A2B; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Triazines; Triazoles; Vasoconstriction; Vasodilator Agents; Xanthines

To investigate the effect of an adenosine agonist, 2-5'-N-ethylcarboxamidoadenosine (NECA), on the outward active transport of fluorescein across the retinal pigment epithelium (RPE) in rabbits. High (5x10(-4)-2x10(-3) M) and low (1x10(-5)-1x10(-4) M) concentrations of NECA or phosphate buffered saline (PBS) were intravitreously injected into Dutch-belted rabbits. Sodium fluorescein was injected intravenously 180 min after NECA. Differential vitreous fluorophotometry was performed 3 hr after the sodium fluorescein injection and the vitreal fluorescein/fluorescein monoglucuronide (F/FG) ratio then was calculated. The F/FG ratios are inversely proportional to the outward active transport of fluorescein across the RPE. Retinal detachments were induced by injection of PBS into the subretinal space after the intravitreous injection of low- or high-dose NECA or PBS, and the size of the blebs was monitored. In eyes that received a low-dose injection of NECA, the F/FG ratio was higher compared with controls (P<0.05); in eyes that received a high-dose intravitreal injection, the F/FG ratio was significantly lower compared with controls (P<0.05). The effect of low-dose NECA on the F/FG ratio was suppressed by the A2 receptor antagonist, ZM241385, and the effect of high-dose NECA was suppressed by the A1 receptor antagonist, 8-cyclopentyl-1, 3-dipropylxanthine. The A3 receptor antagonist MRS1191 did not influence the effect of low- or high-dose NECA. Intravitreal injection of high-dose NECA enhanced the reabsorption of subretinal fluid compared with PBS; however, low-dose NECA inhibited reabsorption of subretinal fluid (P<0.02 and 0.05, respectively). Intravitreous injection of high-dose NECA accelerates the active outward transport across the RPE via A1 receptors and low-dose NECA decelerates it via A2 receptors.

Topics: Adenosine; Adenosine A1 Receptor Antagonists; Adenosine A2 Receptor Antagonists; Adenosine A3 Receptor Antagonists; Adenosine-5'-(N-ethylcarboxamide); Animals; Biological Transport, Active; Dihydropyridines; Fluorescein; Fluoresceins; Fluorophotometry; Injections, Intravenous; Male; Pigment Epithelium of Eye; Purinergic P1 Receptor Antagonists; Rabbits; Retinal Detachment; Triazines; Triazoles; Xanthines

The effects of paeoniflorin (PF), a compound isolated from Paeony radix, on neurological impairment and histologically measured infarction volume following transient and permanent focal ischemia were examined in Sprague-Dawley rats. In transient ischemia model, rats were subjected to a 1.5-h occlusion of the middle cerebral artery (MCA). The administration of PF (2.5 and 5 mg kg(-1), s.c.) produced a dose-dependent decrease in both neurological impairment and the histologically measured infarction volume. Similar results were also obtained when PF (2.5, 5, and 10 mg kg(-1), s.c.) was given in permanent ischemia model. The neuroprotective effect of PF (10 mg kg(-1), s.c.) was abolished by pretreatment of DPCPX (0.25 mg kg(-1), s.c.), a selective adenosine A1 receptor (A1R) antagonist. PF (10, 40, and 160 mg kg(-1), i.v.) had no effect on mean arterial pressure (MAP) and heart rates (HR) in the conscious rat. Additionally, PF (10(-3) mol l(-1)) had no effect on noradrenaline- (NA-) or high K+ concentration-induced contractions of isolated rabbit primary artery. In competitive binding experiments, PF did not compete with the binding of [3H]DPCPX, but displaced the binding of [3H]NECA to the membrane preparation of rat cerebral cortex. This binding manner was distinguished from the classical A1R agonists. The results demonstrated that activation of A1R might be involved in PF-induced neuroprotection in cerebral ischemia in rat. However, PF had no 'well-known' cardiovascular side effects of classical A1R agonists. The results suggest that PF might have the potential therapeutic value as an anti-stroke drug.

Topics: Adenosine-5'-(N-ethylcarboxamide); Animals; Benzoates; Binding, Competitive; Bridged-Ring Compounds; Cerebral Cortex; Disease Models, Animal; Dose-Response Relationship, Drug; Glucosides; Infarction, Middle Cerebral Artery; Inhibitory Concentration 50; Ischemic Attack, Transient; Male; Monoterpenes; Neuroprotective Agents; Paeonia; Plant Roots; Rats; Rats, Sprague-Dawley; Receptor, Adenosine A1; Time Factors; Xanthines

1. This study investigated the possibility that adenosine receptors modulate the alpha(1)-adrenoceptor-mediated contractility of human cultured prostatic stromal cells (HCPSC). 2. The nonselective adenosine receptor agonist, 5'-N-ethylcarboxamido-adenosine (NECA; 10 nm-10 microm), and the A(1) adenosine receptor selective agonist, cyclopentyladenosine (CPA; 10 nm-10 microm), elicited significant contractions in HCPSC, with maximum contractile responses of 18+/-3% and 17+/-2% reduction in initial cell length, respectively. 3. In the presence of a threshold concentration of phenylephrine (PE) (100 nm), CPA (1 nm-10 microm) caused contractions, with an EC(50) of 124+/-12 nm and maximum contractile response of 37+/-4%. The A(1) adenosine receptor-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX 100 nm) blocked this effect. In the presence of DPCPX (100 nm), NECA (1 nm-10 microm) inhibited contractions elicited by a submaximal concentration of PE (10 microm), with an IC(50) of 48+/-2 nm. The A(2A) adenosine receptor-selective antagonist 4-(2-[7-amino-2-[furyl][1,2,4]triazolo[2,3-alpha][1,3,5,]triazin-5-yl amino]ethyl)phenol (Zm241385 100 nm) blocked this effect. 4. In BCECF-AM (10 microm)-loaded cells, both CPA (100 pM-1 microm) and NECA (100 pm-10 microm) elicited concentration-dependent decreases in intracellular pH (pH(i)), with EC(50) values of 3.1+/-0.3 and 6.0+/-0.3 nm, respectively. The response to NECA was blocked by Zm241385 (100 nm; apparent pK(B) of 9.4+/-0.4), but not by DPCPX (100 nm). The maximum response to CPA was blocked by DPCPX (100 nm), and unaffected by Zm241385 (100 nm). 5. NECA (10 nm-10 microm) alone did not increase [(3)H]-cAMP in HCPSC. In the presence of DPCPX (100 nm), NECA (10 nm-10 microm) caused a concentration dependent increase in [(3)H]-cAMP, with an EC(50) of 1.2+/-0.1 microm. This response was inhibited by Zm241385 (100 nm). CPA (10 nm-10 microm) had no effect on cAMP, in the presence or absence of forskolin (1 microm). 6. These findings are consistent with a role for adenosine receptors in the modulation of adrenoceptor-mediated contractility in human prostate-derived cells.

Topics: Adenosine A1 Receptor Agonists; Adenosine A1 Receptor Antagonists; Adenosine A2 Receptor Agonists; Adenosine A2 Receptor Antagonists; Adenosine-5'-(N-ethylcarboxamide); Aged; Cells, Cultured; Dose-Response Relationship, Drug; Humans; Male; Prostate; Receptor, Adenosine A1; Receptor, Adenosine A2A; Receptors, Adrenergic, alpha-1; Stromal Cells; Xanthines

Adenosine receptors involved in modulation of contractions were characterized in the bisected rat vas deferens by combining pharmacological and immunohistochemical approaches. In both portions, noradrenaline-elicited contractions were enhanced by the adenosine A(1) receptor agonist N(6)-cyclopentyladenosine (CPA), and inhibited by the non-selective adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA) in the presence of the adenosine A(1) receptor antagonist 1,3-dipropyl-8-cyclopentyl-l,3-dipropylxanthine (DPCPX). The adenosine A(2A) receptor agonist 2-p-(2-carboxyethyl)phenethyl-amino-5'-N-ethylcarboxamidoadenosine (CGS 21680) also inhibited noradrenaline-elicited contractions but only in the prostatic portion. Contractions elicited by the stable ATP analogue alpha,beta-methyleneATP (alpha,beta-MeATP) were inhibited only by NECA in the presence of DPCPX and only in the prostatic portion. This study provides functional evidence for the presence, in both portions of the rat vas deferens, of an adenosine A(1) receptor-mediated enhancement and of an adenosine A(2) receptor-mediated inhibition of contractions. The latter effect is mediated by both A(2A) and A(2B) subtypes in the prostatic portion but only by the A(2B) subtype in the epididymal portion. This regional variation is supported by the immunohistochemical results that revealed an adenosine A(2A) receptor immunoreactivity not co-localized with nerve fibres more abundant in the prostatic than in the epididymal portion.

Topics: Adenosine; Adenosine Triphosphate; Adenosine-5'-(N-ethylcarboxamide); Animals; Epididymis; Immunohistochemistry; In Vitro Techniques; Male; Muscle Contraction; Norepinephrine; Phenethylamines; Prostate; Rats; Rats, Wistar; Receptor, Adenosine A2A; Receptors, Purinergic P1; Theobromine; Triazines; Triazoles; Vas Deferens; Xanthines

Under normoxic conditions, macrophages from C57BL mice produce low levels of vascular endothelial growth factor (VEGF). Hypoxia stimulates VEGF expression by approximately 500%; interferon-gamma (IFN-gamma) with endotoxin [lipopolysaccharide (LPS)] also stimulates VEGF expression by approximately 50 to 150% in an inducible nitric oxide synthase (iNOS)-dependent manner. Treatment of normoxic macrophages with 5'-N-ethyl-carboxamido-adenosine (NECA), a nonselective adenosine A(2) receptor agonist, or with 2-[p-(2-carboxyethyl)-phenylethyl amino]-5'-N-ethyl-carboxamido-adenosine (CGS21680), a specific adenosine A(2A) receptor agonist, modestly increases VEGF expression, whereas 2-chloro-N(6)-cyclopentyl adenosine (CCPA), an adenosine A(1) agonist, does not. Treatment with LPS (0 to 1000 ng/ml), or with IFN-gamma (0 to 300 U/ml), does not affect VEGF expression. In the presence of LPS (EC(50) < 10 ng/ml), but not of IFN-gamma, both NECA and CGS21680 synergistically up-regulate VEGF expression by as much as 10-fold. This VEGF is biologically active in vivo in the rat corneal bioassay of angiogenesis. Inhibitors of iNOS do not affect this synergistic induction of VEGF, and macrophages from iNOS-/- mice produce similar levels of VEGF as wild-type mice, indicating that NO does not play a role in this induction. Under hypoxic conditions, VEGF expression is slightly increased by adenosine receptor agonists but adenosine A(2) or A(1) receptor antagonists 3,7-dimethyl-1-propargyl xanthine (DMPX), ZM241385, and 8-cyclopentyl-1,3-dipropylxanthine (DCPCX) do not modulate VEGF expression. VEGF expression is also not reduced in hypoxic macrophages from A(3)-/- and A(2A)-/- mice. Thus, VEGF expression by hypoxic macrophages does not seem to depend on endogenously released or exogenous adenosine. VEGF expression is strongly up-regulated by LPS/NECA in macrophages from A(3)-/- but not A(2A)-/- mice, confirming the role of adenosine A(2A) receptors in this pathway. LPS with NECA strongly up-regulates VEGF expression by macrophages from C(3)H/HeN mice (with intact Tlr4 receptors), but not by macrophages from C(3)H/HeJ mice (with mutated, functionally inactive Tlr4 receptors), implicating signaling through the Tlr4 pathway in this synergistic up-regulation. Finally, Western blot analysis of adenosine A(2A) receptor expression indicated that the synergistic interaction of LPS with A(2A) receptor agonists does not involve up-regulation of A(2A) receptors by LPS. These results indi

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Blotting, Western; Cells, Cultured; Drosophila Proteins; Endothelial Growth Factors; Female; Interferon-gamma; Lipopolysaccharides; Lymphokines; Macrophages, Peritoneal; Male; Membrane Glycoproteins; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Knockout; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Phenethylamines; Protein Kinase Inhibitors; Purinergic P1 Receptor Agonists; Receptor, Adenosine A2A; Receptors, Cell Surface; Receptors, Purinergic P1; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Theobromine; Toll-Like Receptor 4; Toll-Like Receptors; Triazines; Triazoles; Up-Regulation; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors; Xanthines

Electrophysiological mechanisms by which adenosine may activate cardiac afferent neurons are unknown. Slow afterhyperpolarizations (AHPs) follow action potentials in a subset of vagal C afferents, rendering them inexcitable. The purpose of this study was to test the hypothesis that adenosine increases vagal neuronal excitability by blocking slow AHPs and to determine the adenosine receptor subtype mediating these effects.. Using the perforated patch-clamp technique, we identified cultured adult rabbit nodose ganglion cells with slow AHPs in current-clamp mode. Trains of 100 current pulses at 20% above threshold were injected, with an interspike interval of 100 ms, and the number of action potentials triggered were counted and reported as the action potential response rate. During adenosine (10 micromol/L), slow AHPs were suppressed and action potential response rate was augmented from 3.8+/-0.5% at baseline to 28+/-7% after adenosine (P:=0.0009). The selective A(2)-adenosine receptor agonist NECA but not the A(1)-adenosine agonist CCPA replicated the adenosine effect. The selective A(2A)-adenosine antagonist ZM 241385 (10 nmol/L) but not the A(1) adenosine antagonist DPCPX (5 micromol/L) abolished the adenosine effect. We considered two alternative hypotheses: (1) A(2)-receptor-mediated suppression of I(Ca) leading to smaller increases in intracellular Ca during stimulation, resulting in less activation of I(K(Ca)) and consequent suppression of slow AHPs, or (2) A(2)-receptor-mediated elevation of cAMP directly suppressing slow AHPs. Under voltage-clamp conditions, adenosine did not significantly inhibit I(Ca), making the latter hypothesis more likely.. Adenosine inhibits slow AHPs in vagal afferent neurons. This effect is most likely caused by A(2A)-receptor-mediated stimulation of cAMP production.

Topics: Action Potentials; Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Cadmium; Colforsin; Neurons, Afferent; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Rabbits; Receptors, Purinergic P1; Time Factors; Vagus Nerve; Xanthines

The effects of purinoceptor agonists on noradrenaline NA release by electrical stimulation in rat mesenteric arteries were examined to clarify the pharmacological properties of prejunctional purinoceptors on adrenergic nerves. Adenosine and the other P1-receptor agonists, 5'-(N-ethylcarboxamido) adenosine and 2-chloroadenosine, significantly inhibited the release of NA. Also beta,gamma-methylene ATP and 2-methylthio ATP, P2-receptor agonists, significantly inhibited NA releases. The inhibitory effect of adenosine was significantly reduced by adenosine deaminase, but those of beta,gamma-methylene ATP and 2-methylthio ATP were not affected. This suggests that the inhibitory effects of P2-receptor agonists are not due to conversion into adenosine. 1,3-Dipropyl-8-cyclopentylxanthine (DPCPX), a P1 (A1)-receptor antagonist, significantly reduced the inhibitory effects of not only the P1- but also P2-receptor agonists. Therefore, DPCPX appears to act on both prejunctional P1- and P2-receptor as an antagonist. Pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), a P2-receptor antagonist, significantly reduced the inhibitory effects of the P2-receptor agonists, but not those of the P1-receptor agonists. From these findings in the rat mesenteric artery, the P1-receptor agonist-induced inhibition of NA-release appears to be mediated via a well-known prejunctional P1-receptor of the A1-subtype, but the P2-receptor agonist-induced inhibition appears to be mediated via an unidentified purinoceptor that is blocked not only by P2-receptor antagonists but also by P1-receptor antagonists.

Topics: 2-Chloroadenosine; Adenosine; Adenosine Deaminase; Adenosine Triphosphate; Adenosine-5'-(N-ethylcarboxamide); Animals; Clonidine; Dose-Response Relationship, Drug; Electric Stimulation; In Vitro Techniques; Male; Mesenteric Arteries; Norepinephrine; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Purinergic P2 Receptor Agonists; Purinergic P2 Receptor Antagonists; Pyridoxal Phosphate; Rats; Rats, Wistar; Tetrodotoxin; Xanthines

The aim of this study was to investigate whether the A1/A2 receptor agonist, 5'-N-ethylcarboxamidoadenosine (NECA), and the selective A1 agonist, N6-cyclopentyladenosine (CPA), induced physical dependence by quantifying specific antagonist-precipitated withdrawal syndromes in conscious rats. In addition, the presence of bidirectional cross-withdrawal was also investigated. The agonists were administered s.c. to groups of rats at 12 h intervals. Antagonists were administered s.c., 12 hours after the last dose, followed by observation and measurement of faecal output for 20 min. NECA (4 x 0.03 mg kg(-1), s.c) and CPA (4 x 0.03, 0.1 and 0.3 mg kg(-1), s.c.) induced physical dependence, as shown by the expression of a significant withdrawal syndrome when challenged with the adenosine A1/A2 receptor antagonist, 3,7-dimethyl-1-propargylxanthine (DMPX, 0.1 mg kg(-1), s.c.) and the A1 antagonist, 8-cyclopentyl-1,3-dipropylxanthine (CPDPX, 0.1 mg kg(-1), s.c.) respectively. The syndromes consisted of teeth chattering and shaking behaviours shown to occur in morphine-dependent animals withdrawn with naloxone viz, paw, body and 'wet-dog' shakes, but with the additional behaviours of head shaking and yawning. In further contrast to the opiate withdrawal syndrome, no diarrhoea occurred in the groups of animals treated with adenosine agonists and withdrawn with their respective antagonists. Bidirectional cross-withdrawal syndromes were also revealed when naloxone (3 mg kg(-1), s.c.) was administered to adenosine agonist pre-treated rats and adenosine antagonists were given to morphine pre-treated rats. This study provides further information illustrating that close links exist between the adenosine and opiate systems.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Behavior, Animal; Female; Injections, Subcutaneous; Male; Morphine; Morphine Dependence; Muscle Contraction; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Purinergic P2 Receptor Agonists; Purinergic P2 Receptor Antagonists; Rats; Rats, Wistar; Substance Withdrawal Syndrome; Theobromine; Xanthines

We have demonstrated that ethanol-induced motor incoordination is modulated by cerebellar adenosine A(1) receptor. This study represents an extension into another important brain motor area, the striatum that, unlike cerebellum, has high density of both A(1) and A(2A) receptors. Direct intra-striatal micro-infusion of Ro15-4513 (0.05, 0.5, 1 ng), a partial inverse-agonist of benzodiazepine, significantly and nearly dose-dependently attenuated ethanol-induced motor incoordination indicating mediation of ethanol's motor incoordination by striatum. Intra-striatal A(1)-selective agonist N(6)-cyclohexyladenosine (CHA; 1, 2, 4 ng), A(1) = A(2A) non-selective agonist, 5'-N-ethylcarboxamidoadenosine (NECA; 1.5, 3, 6 ng), and A(1)-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 25, 50, 100 ng) dose-dependently accentuated and attenuated, respectively, ethanol-induced motor incoordination, strongly suggesting modulation by striatal adenosine A(1) receptor. Intra-striatal DPCPX significantly antagonized not only ethanol-induced motor incoordination but also its potentiation by intra-striatal CHA, R-(+)-N(6)-(2-phenylisopropyladenosine) (R-PIA), or NECA. No change in motor coordination occurred after the highest dose of CHA, R-PIA, or NECA followed by saline. Similarly, the highest intra-striatal dose of Ro15-4513 or DPCPX neither altered motor coordination or locomotor activity indicating relative selectivity of interaction with ethanol. Nearly 25-fold higher dose of A(2A)-selective agonist, CGS-21680, compared to CHA was necessary to produce a comparable potentiation of ethanol's motor incoordination perhaps suggesting a lack of or less significant striatal A(2A) involvement. Intra-striatal pertussis toxin (0.5 microg) pre-treatment markedly attenuated ethanol-induced motor incoordination as well as its potentiation by intra-striatal CHA. These results support that striatum is one of the brain motor areas mediating the motor impairing effects of acute ethanol and that the latter's modulation occurs via A(1)-selective receptors coupled to pertussis toxin-sensitive G proteins.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Affinity Labels; Animals; Antihypertensive Agents; Ataxia; Azides; Benzodiazepines; Central Nervous System Depressants; Corpus Striatum; Ethanol; Male; Mice; Mice, Inbred ICR; Microinjections; Motor Activity; Pertussis Toxin; Phenethylamines; Receptor, Adenosine A2A; Receptors, Purinergic P1; Vasodilator Agents; Virulence Factors, Bordetella; Xanthines

1. Adenosine produced a biphasic lowering of the mean BP with a drastic bradycardic effect at the highest doses. The first phase hypotensive response was significantly reduced by the nitric oxide (NO) synthase inhibitor L-NAME. 2. The A(2a)/A(2b) agonist NECA produced hypotensive and bradycardic responses similar to those elicited by adenosine, which were not significantly modified by the A(2b) antagonist enprofylline. 3. The A(2a) agonist CGS 21680 did not significantly influence basal HR while induced a hypotensive response antagonized by the A(2a) selective antagonist ZM 241385, and reduced by both L-NAME and the guanylate cyclase inhibitor methylene blue. 4. The A(1) agonist R-PIA showed a dose-dependent decrease in BP with a drastic decrease in HR at the highest doses. The A(1) selective antagonist DPCPX significantly reduced the bradycardic activity and also the hypotensive responses obtained with the lowest doses while it increased those obtained with the highest ones. 5. The A(1)/A(3) agonist APNEA, in the presence of the xanthinic non-selective antagonist 8-pSPT, maintained a significant hypotensive, but not bradycardic, activity, not abolished by the histamine antagonist diphenhydramine. 6. The selective A(3) agonist IB-MECA revealed a weak hypotensive and bradycardic effect, but only at the highest doses. 7. In conclusion, in the systemic cardiovascular response to adenosine two major components may be relevant: an A(2a)- and NO-mediated hypotension, and a bradycardic effect with a consequent hypotension, via atypical A(1) receptors. Finally, an 8-pSPT-resistant hypotensive response not attributable to A(3) receptor-stimulation or to release of histamine by mastocytes or other immune cells was observed.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Blood Pressure; Diphenhydramine; Dose-Response Relationship, Drug; Enzyme Inhibitors; Guinea Pigs; Heart Rate; Hypotension; Male; Methylene Blue; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Phenethylamines; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Receptors, Purinergic P1; Theophylline; Triazines; Triazoles; Vasodilator Agents; Xanthines

In neuronal and atrial tissue, G protein-gated inwardly rectifying K(+) channels (Kir3.x family) are responsible for mediating inhibitory postsynaptic potentials and slowing the heart rate. They are activated by Gbetagamma dimers released in response to the stimulation of receptors coupled to inhibitory G proteins of the G(i/o) family but not receptors coupled to the stimulatory G protein G(s). We have used biochemical, electrophysiological, and molecular biology techniques to examine this specificity of channel activation. In this study we have succeeded in reconstituting such specificity in an heterologous expression system stably expressing a cloned counterpart of the neuronal channel (Kir3.1 and Kir3.2A heteromultimers). The use of pertussis toxin-resistant G protein alpha subunits and chimeras between G(i1) and G(s) indicate a central role for the G protein alpha subunits in determining receptor specificity of coupling to, but not activation of, G protein-gated inwardly rectifying K(+) channels.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Cell Line; G Protein-Coupled Inwardly-Rectifying Potassium Channels; GTP-Binding Protein alpha Subunits, Gi-Go; GTP-Binding Protein alpha Subunits, Gs; Humans; Ion Channel Gating; Kinetics; Membrane Potentials; Pertussis Toxin; Phenethylamines; Point Mutation; Potassium Channels; Potassium Channels, Inwardly Rectifying; Radioligand Assay; Recombinant Fusion Proteins; Recombinant Proteins; Transfection; Virulence Factors, Bordetella; Xanthines

The role of adenosine on regulation of the (Na(+)+K(+))ATPase activity present in the Malpighian tubules isolated from Rhodnius prolixus was investigated. Adenosine decreases the (Na(+)+K(+)) ATPase specific activity by 88%, in a dose-dependent manner, with maximal effect at a concentration of 10(-9) M. This effect was mimicked by N(6)-cyclohexyladenosine (CHA) at 10(-8) M, an agonist for A(1) adenosine receptor, and was reversed by 10(-9) M 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), an antagonist for A(1) adenosine receptor. On the other hand, 5'-N-ethyl-carboxamide adenosine (NECA), an agonist for A(2) adenosine receptor, used in the range of 10(-9)-10(-5) M, did not change the (Na(+)+K(+))ATPase specific activity. In the same way, 10(-8) M 3, 7-dimethyl-1-propargylxanthine (DMPX), an antagonist for A(2) adenosine receptor, did not modify the inhibitory effect of adenosine. These data suggest that the inhibitory effect of adenosine on the (Na(+)+K(+))ATPase specific activity present in Malpighian tubules from Rhodnius prolixus is mediated by A(1) adenosine receptor activation. Arch.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Malpighian Tubules; Receptors, Purinergic P1; Rhodnius; Sodium-Potassium-Exchanging ATPase; Theobromine; Xanthines

HT29 cells display an undifferentiated phenotype in culture. However, numerous treatments are able to induce both epithelial differentiation and cell growth inhibition. We have previously demonstrated that adenosine and its analogues act through specific adenosine receptors to modulate cell proliferation in HT29 and other human colon adenocarcinoma cell lines. Among the treatments tested, the most potent inhibition of HT29 cell growth was induced by deprivation of extracellular adenosine using adenosine deaminase. Here, we investigated the capacity of adenosine deaminase to initiate epithelial differentiation. After 1 month of daily addition of 10 U/ml adenosine deaminase to the culture medium, HT29 cells were cloned by limited dilution. Among the clones obtained, we focused our attention on clone 13. Microscopic visualization and proliferation studies indicated that cells from this clone grew very slowly and in a pseudo-monolayer, in marked contrast with the situation observed in the mother HT29 cell line. In addition, clone 13 cells displayed epithelial features that mimic the enterocytic differentiation of Caco-2 cells. These modifications were accompanied by dramatic changes in the activity of adenosine receptors, as demonstrated by pharmacological studies. In contrast to the original HT29 cells, clone 13 as well as Caco-2 cells displayed (i) a very low number of adenosine A(1) receptors, and (ii) increases in intracellular cAMP levels when challenged with adenosine analogues. It is hypothesized that a loss of adenosine A(1) receptors, with no change or a concomitant increase in adenosine A(2) receptors, results in the emergence of adenosine A(2) receptor-mediated differentiation and inhibition of proliferation, through a cAMP-dependent pathway.

Topics: Adenosine; Adenosine Deaminase; Adenosine-5'-(N-ethylcarboxamide); Caco-2 Cells; Cell Differentiation; Cell Division; Clone Cells; Culture Media; Cyclic AMP; Epithelial Cells; HT29 Cells; Humans; Purinergic P1 Receptor Antagonists; Receptors, Purinergic P1; Xanthines

Adenosine A(2a)-receptor activation enhances shortening of isolated cardiomyocytes. In the present study the effect of A(2a)-receptor activation on the contractile performance of isolated rat hearts was investigated by recording left ventricular pressure (LVP) and the maximal rate of LVP development (+dP/dt(max)). With constant-pressure perfusion, adenosine caused concentration-dependent increases in LVP and +dP/dt(max), with detectable increases of 4.1 and 4.8% at 10(-6) M and maximal increases of 12.0 and 11.1% at 10(-4) M, respectively. The contractile responses were prevented by the A(2a)-receptor antagonists chlorostyryl-caffeine and aminofuryltriazolotriazinyl-aminoethylphenol (ZM-241385) but were not affected by the beta(1)-adrenergic antagonist atenolol. The adenosine A(1)-receptor antagonist dipropylcyclopentylxanthine and pertussis toxin potentiated the positive inotropic effects of adenosine. The A(2a)-receptor agonists ethylcarboxamidoadenosine and dimethoxyphenyl-methylphenylethyl-adenosine also enhanced contractility. With constant-flow perfusion, 10(-5) M adenosine increased LVP and +dP/dt(max) by 5.5 and 6.0%, respectively. In the presence of the coronary vasodilator hydralazine, adenosine increased LVP and +dP/dt(max) by 7.5 and 7.4%, respectively. Dipropylcyclopentylxanthine potentiated the adenosine contractile responses with constant-flow perfusion in the absence and presence of hydralazine. These increases in contractile performance were also antagonized by chlorostyryl-caffeine and ZM-241385. The results indicate that adenosine increases contractile performance via activation of A(2a) receptors in the intact heart independent of beta(1)-adrenergic receptor activation or changes in coronary flow.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Adrenergic beta-Antagonists; Animals; Atenolol; Drug Synergism; Heart; In Vitro Techniques; Male; Myocardial Contraction; Perfusion; Pertussis Toxin; Pressure; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Rats; Rats, Sprague-Dawley; Receptor, Adenosine A2A; Receptors, Purinergic P1; Vasodilation; Vasodilator Agents; Virulence Factors, Bordetella; Xanthines

The development of adenosine A1 and A2B receptors on the longitudinal muscle and muscularis mucosae of the neonatal rat distal colon has been investigated using homogenate binding, quantitative autoradiography and functional studies. In homogenate binding studies 1,3-[3H]-dipropyl-8-cyclopentylxanthine ([3H]DPCPX) bound with high affinity to A1 receptors in the muscularis mucosae and intact colon from rats aged 10, 15, 20, 25 and 30 days. The affinity of [3H]DPCPX was similar to that in the adult at all ages, but the density of binding sites was higher in the neonatal tissues. Quantitative autoradiography showed a higher density of [3H]DPCPX binding sites in the longitudinal muscle than in the muscularis mucosae at all ages studied (day 10 to adult), and this binding was concentration-dependently displaced by N6-cyclopentyladenosine (CPA). In functional studies the longitudinal muscle relaxed in response to 5'-N-ethylcarboxamidoadenosine (NECA) and CPA at all ages studied (15-30 days), NECA being more potent than CPA. The potency of NECA remained constant and it was antagonised by 1 microM DPCPX at all ages with pA2-values consistent with activation of A2 receptors. The inactivity of 2-[p-(carboxyethyl)-phenylethylamino]-5'-N-ethylcarboxamidoadenosi ne (CGS 21680) indicated that the A2 receptors were of the A2B subtype. The muscularis mucosae contracted in response to CPA at all ages studied (day 15 to adult) and the antagonism by DPCPX (10 nM) were consistent with activation of A1 receptors. In conclusion, binding, autoradiographic and functional studies identified A1 receptors on the rat colon muscularis mucosae at all ages studied. However, while binding and autoradiographic localisation showed the presence of A1 receptors in the longitudinal muscle at all ages studied, functional data only revealed the presence of A2B receptors.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Aging; Animals; Autoradiography; Binding Sites; Binding, Competitive; Colon; Male; Muscle Contraction; Muscle Development; Muscle, Smooth; Radioligand Assay; Rats; Rats, Wistar; Receptors, Purinergic P1; Subcellular Fractions; Vasodilator Agents; Xanthines

1. The present study was designed to characterize the adenosine receptors involved in the relaxation of the pig intravesical ureter, and to investigate the action of adenosine on the non adrenergic non cholinergic (NANC) excitatory ureteral neurotransmission. 2. In U46619 (10(-7) M)-contracted strips treated with the adenosine uptake inhibitor, nitrobenzylthioinosine (NBTI, 10(-6) M), adenosine and related analogues induced relaxations with the following potency order: 5'-N-ethylcarboxamidoadenosine (NECA) = 5'-(N-cyclopropyl)-carboxamidoadenosine (CPCA) = 2-chloroadenosine (2-CA) > adenosine > cyclopentyladenosine (CPA) = N6-(3-iodobenzyl)-adenosine-5'-N-methylcarboxamide (IB-MECA) = 2-[p-(carboxyethyl)-phenylethylamino]-5'-N-ethylcarboxamidoaden os ine (CGS21680). 3. Epithelium removal or incubation with indomethacin (3 x 10(-6) M) and L-N(G)-nitroarginine (L-NOARG, 3 x 10(-5) M), inhibitors of prostanoids and nitric oxide (NO) synthase, respectively, failed to modify the relaxations to adenosine. 4. 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 10(-8) M) and 4-(2-[7-amino-2-(2-furyl) [1,2,4]-triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385, 3 x 10(-8) M and 10(-7) M), A1 and A2A receptor selective antagonists, respectively, did not modify the relaxations to adenosine or NECA. 8-phenyltheophylline (8-PT, 10(-5) M) and DPCPX (10(-6) M), which block A1/A2-receptors, reduced such relaxations. 5. In strips treated with guanethidine (10(-5) M), atropine (10(-7) M), L-NOARG (3 x 10(-5) M) and indomethacin (3 x 10(-6) M), both electrical field stimulation (EFS, 5 Hz) and exogenous ATP (10(-4) M) induced contractions of preparations. 8-PT (10(-5) M) increased both contractions. DPCPX (10(-8) M), NECA (10(-4) M), CPCA, (10(-4) M) and 2-CA (10(-4) M) did not alter the contractions to EFS. 6. The present results suggest that adenosine relaxes the pig intravesical ureter, independently of prostanoids or NO, through activation of A2B-receptors located in the smooth muscle. This relaxation may modulate the ureteral NANC excitatory neurotransmission through a postsynaptic mechanism.

Topics: Adenosine; Adenosine Triphosphate; Adenosine-5'-(N-ethylcarboxamide); Animals; Dose-Response Relationship, Drug; Electric Stimulation; Female; Indomethacin; Male; Muscle Relaxation; Receptors, Purinergic P1; Swine; Synaptic Transmission; Theophylline; Ureter; Xanthines

1. The aim of the present investigation was to characterize and determine the tissue location of the adenosine receptors present in the rat ileum using a method that detects drug action on the cholinergic nerves innervating the longitudinal and circular muscles. 2. The non-selective adenosine agonist, NECA (10 and 100 nM) caused significant concentration-related reductions in the circular muscle responses to transmural stimulation over the frequency range of 2.5-40 Hz, but did not affect the responses of the longitudinal muscle, nor did it reduce the muscle responses of the guinea-pig ileum. 3. The affinity order of antagonists at inhibiting the effect of NECA on the circular muscle was: CPDPX>8-PT>DMPX with apparent pA2 values of 9.31, 7.54 and 5.63 respectively. CPDPX (10-100 nM) caused parallel displacements of the concentration-effect curves to CPA with a pKb value of 9.15 and Schild slope of 1.03. 4. The agonists previously tested in the rat jejunum peristaltic reflex preparation were also shown to inhibit responses of the rat ileum in the following decreasing order of potency: CPA>NECA>2-CADO>R-PIA>S-PIA>>PAA. In addition, CHA and CCPA were also potent agonists. NECA (100 nM) and CPA (32 nM) did not inhibit carbachol (1 microM)-induced tone of tissues pre-treated with TTX (1 microM). 5. In conclusion, the rat ileum contains inhibitory A1 adenosine receptors situated on cholinergic nerve endings innervating the circular muscle.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Analysis of Variance; Animals; Female; Ileum; Male; Neurons; Rats; Rats, Wistar; Receptors, Purinergic P1; Xanthines

The adenosine receptor agonist N(6)-cyclohexyl[(3)H]adenosine ([(3)H]CHA) was used to identify and pharmacologically characterize adenosine A1 receptors in brown trout (Salmo trutta) brain. In membranes prepared from trout whole brain, the A1 receptor agonist [(3)H]CHA bound saturably, reversibly and with high affinity (K(d)=0. 69+/-0.04 nM; B(max)=0.624+/-0.012 pmol/mg protein) to a single class of binding sites. In equilibrium competition experiments, the adenosine agonists and antagonists all displaced [(3)H]CHA from high-affinity binding sites with the rank order of potency characteristic for an adenosine A1 receptors. A1 receptor density appeared not age-related (from 3 months until 4 years), and was similar in different brain areas. The specific binding was inhibited by guanosine 5'-triphosphate (IC(50)=0.778+/-0.067 microM). GTP (5 microM) induced a low affinity state of A1 receptors. In superfused trout cerebral synaptosomes, 30 mM K(+) stimulated the release of glutamate in a calcium dependent manner. Glutamate-evoked release was dose-dependently reduced by CHA, and the inhibition was reversed by the A1 antagonist 8-cyclopentyltheophylline (CPT). In the same synaptosomal preparation, 30 mM K(+) as well as 1 mM glutamate stimulated the release of adenosine in a Ca(2+)-independent manner and tetrodotoxin insensitive. These findings show that in trout brain adenosine A1 receptors are present which are involved in the modulation of glutamate transmitter release. Moreover, the stimulation of adenosine release by K(+) depolarisation or glutamate support the hypothesis that, as in mammalian brain, a cross-talk between adenosine and glutamate systems exists also in trout brain.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Binding, Competitive; Brain; Cell Membrane; Glutamic Acid; Guanosine Diphosphate; Kinetics; Neuroprotective Agents; Radioligand Assay; Receptors, Purinergic P1; Synaptosomes; Theophylline; Tritium; Trout; Xanthines

Adenosine is known to produce biphasic effects in the renal tissues via adenosine receptors. However, the presence of more than one subtype of adenosine receptor on a type of kidney cell or tissue has not been conclusively demonstrated. To address this issue, we investigated the presence of A1 and A2 adenosine receptors in baby hamster kidney (BHK) cells by use of radioligand binding and the reverse transcription-polymerase chain reaction. Ligand binding studies with (3H)-DPCPX revealed a single class of binding site with a K(D) of 9.2 +/- 2.0 nM, a Bmax of 1.7 +/- 0.2 pmol/mg protein and a pharmacological profile characteristic of A1 adenosine receptor on the BHK cell membrane. As the presence of A2 adenosine receptors could not be conclusively determined by ligand binding studies, the more sensitive method of RT-PCR was employed. The presence of A1 and A2B adenosine receptors was detected by RT-PCR with specific primers and the subsequent sequencing of the resultant amplification product. The sequences obtained were 75-90% homologous to the respective adenosine receptor mRNA of rat, mouse and human.

Topics: Adenosine-5'-(N-ethylcarboxamide); Animals; Base Sequence; Cell Line; Cell Membrane; Cricetinae; Humans; Kidney; Mice; Molecular Sequence Data; Radioligand Assay; Rats; Receptor, Adenosine A2B; Receptors, Purinergic P1; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sequence Homology; Tritium; Xanthines

1. Bovine adrenal zona fasciculata (AZF) cells express a non-inactivating K+ current (IAC) that sets the resting potential while it is activated by intracellular ATP. In whole-cell patch clamp recordings from bovine AZF cells, we found that adenosine selectively inhibited IAC by a maximum of 78.4 +/- 4.6 % (n = 8) with an IC50 of 71 nM. The non-selective adenosine receptor agonist NECA effectively inhibited IAC by 79.3 +/- 2.9 % (n = 24) at a concentration of 100 nM. 2. Inhibition of IAC was mediated through multiple P1 adenosine receptor subtypes. The A1-selective agonist CCPA (10 nM), the A2A-selective agonist CGS 21680 (100 nM) and the A3-selective agonist IB-MECA (10 nM) inhibited IAC by 64.8 +/- 8.4, 78.4 +/- 4.6 and 69.3 +/- 6.9 %, respectively. 3. Specific adenosine receptor subtype antagonists including DPCPX (A1), ZM 241385 (A2A) and MRS 1191 (A3) effectively blocked inhibition of IAC by adenosine receptor-selective agonists. 4. A mixture of the three adenosine receptor antagonists completely suppressed inhibition of IAC by adenosine, but failed to alter inhibition by external ATP which acts through a separate P2 nucleotide receptor. 5. Inhibition of IAC by adenosine or NECA was eliminated by substituting GDP-beta-S for GTP in the pipette, or by replacing ATP with AMP-PNP or UTP. 6. In addition to inhibiting IAC, adenosine (10 microM) depolarized AZF cells by 46.2 +/- 5.8 mV (n = 6). 7. These results show that bovine AZF cells express at least three adenosine receptor subtypes (A1, A2A, A3), each of which is coupled to the inhibition of IAC K+ channels through a G-protein-dependent mechanism requiring ATP hydrolysis. Adenosine-mediated inhibition of IAC is associated with membrane depolarization. Adenosine and other purines may co-ordinate the stress-induced secretion of corticosteroids and catecholamines from the adrenal gland.

Topics: Adenosine; Adenosine Triphosphate; Adenosine-5'-(N-ethylcarboxamide); Animals; Cattle; GTP-Binding Proteins; In Vitro Techniques; Male; Membrane Potentials; Patch-Clamp Techniques; Potassium; Potassium Channel Blockers; Potassium Channels; Receptors, Purinergic P1; Signal Transduction; Triazines; Triazoles; Xanthines; Zona Fasciculata

Adenosine is known to exert dual actions on the afferent arteriole, eliciting vasoconstriction, by activating A1 receptors, and vasodilation at higher concentrations, by activating lower-affinity A2 receptors. We could demonstrate both of these known adenosine responses in the in vitro perfused hydronephrotic rat kidney. Thus, 1.0 microM adenosine elicited a transient vasoconstriction blocked by 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), and 10-30 microM adenosine reversed KCl-induced vasoconstriction. However, when we examined the effects of adenosine on pressure-induced afferent arteriolar vasoconstriction, we observed a third action. In this setting, a high-affinity adenosine vasodilatory response was observed at concentrations of 10-300 nM. This response was blocked by both 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3, 5]triazin-5-yl-amino]ethyl)phenol (ZM-241385) and glibenclamide and was mimicked by 2-phenylaminoadenosine (CV-1808) (IC50 of 100 nM), implicating adenosine A2a receptors coupled to ATP-sensitive K channels (KATP). Like adenosine, 5'-N-ethylcarboxamidoadenosine (NECA) elicited both glibenclamide-sensitive and glibenclamide-insensitive vasodilatory responses. The order of potency for the glibenclamide-sensitive component was NECA > adenosine = CV-1808. Our findings suggest that, in addition to the previously described adenosine A1 and low-affinity A2b receptors, the renal microvasculature is also capable of expressing high-affinity adenosine A2a receptors. This renal adenosine receptor elicits afferent arteriolar vasodilation at submicromolar adenosine levels by activating KATP.

Topics: 4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone; Adenosine; Adenosine Triphosphate; Adenosine-5'-(N-ethylcarboxamide); Animals; Arterioles; Blood Pressure; Enzyme Inhibitors; Hydronephrosis; Kidney; Perfusion; Potassium Channels; Potassium Chloride; Rats; Receptor, Adenosine A2A; Receptors, Purinergic P1; Renal Artery; Renal Circulation; Triazines; Triazoles; Vasoconstriction; Vasodilation; Vasodilator Agents; Xanthines

The present study had employed in vitro receptor autoradiography with [3H]DPCPX to visualise the presence of adenosine A1 receptors on the rat nodose ganglion, which contains the perikarya of vagal afferent neurons projecting the the nucleus tractus solitarius (NTS). In addition, unilateral vagal ligation resulted in an accumulation of [3H]DPCPX binding adjacent to the ligatures, indication that adenosine A1 receptors are subject to axoplasmic flow along the rat vagus nerve. Radioligand binding assays were utilised to characterise the properties of adenosine A1 receptors in the dorsal vagal complex (NTS, area postrema and dorsal motor nucleus of the vagus) of pup and adult normotensive (Wistar Kyoto, WKY) and hypertensive (spontaneously hypertensive, SHR) rats. Saturation binding indicated that the affinity (KD) of [3H]DPCPX, and the binding site density (Bmax) were not different between the adult WKY and SHR, although the pup SHR had a lower KD value than the pup WKY rat. Competition binding assays revealed complex differences between the two rat strains; however, with respect to hypertension, the affinity of the selective adenosine A1 agonist, cyclohexyladenosine (CHA), was markedly reduced in the membranes from SHR (Ki approximately 93 nM) compared to WKY (approximately 6 nM). Such an observation is consistent with the attenuated responses of SHRs to intra-NTS injections of adenosine.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Autoradiography; Axonal Transport; Binding, Competitive; Hypertension; Male; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Receptors, Purinergic P1; Reference Values; Solitary Nucleus; Vagus Nerve; Xanthines

Adenosine is a potent vasodilator of vascular smooth muscle. Endothelium-derived nitric oxide (NO) elicits vasodilation. We have previously reported that adenosine stimulates the production of NO from porcine carotid arterial endothelial cells (PCAEC) via a receptor-mediated mechanism. This study was to determine whether adenosine also enhances NO production from human arterial endothelium and to define the involvement of adenosine A1 and A2 receptors.. Human iliac arterial endothelial cells (HIAEC) and PCAEC were harvested and cultured in dishes. NO production was evaluated with a NO electrode sensor which measured continuously real-time NO production.. NO content of the medium bathing HIAEC and PCAEC was significantly increased with adenosine (100 micromol/L). Ethylcarboxamidoadenosine (NECA), a nonselective adenosine receptor agonist, and carboxyethyl-phenethylamino-ethylcarboxamidoadenosine (CGS-21680), a selective adenosine A2a receptor agonist, increased NO production by HIAEC and PCAEC with respective EC50 values of 3.32 and 6.96 nmol/L for NECA and 30.97 and 29.47 nmol/L for CGS-21680. Chlorofuryl-triazolo-quinazolinamine (CGS-15943; 1 micromol/L), an adenosine A1 and A2 receptor antagonist, and aminofuryltriazolotriazinyl-aminoethylphenol (ZM-241385; 1 micromol/L), a selective adenosine A2a receptor antagonist, inhibited the effect of CGS-21680. Chlorocyclopentyl-adenosine (CCPA; 1 micromol/L), an adenosine A1 receptor agonist, significantly depressed NO production by both HIAEC and PCAEC: This effect was inhibited by cyclopentyl-dipropylxanthine (DPCPX), a selective adenosine A1 receptor antagonist.. The results demonstrate that adenosine A2a receptors increase, and adenosine A1 receptors decrease, the production of NO by human and porcine arterial endothelial cells.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Carotid Arteries; Cells, Cultured; Endothelium, Vascular; Humans; Iliac Artery; Nitric Oxide; Phenethylamines; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Quinazolines; Receptor, Adenosine A2A; Receptors, Purinergic P1; Swine; Triazines; Triazoles; Xanthines

The vasoactive properties of P1,P4-diadenosine tetraphosphate (Ap4A) were studied by measuring the effects of perfusion pressure of a rat isolated perfused kidney.. The vasoconstrictive response to Ap4A was mediated to a large extent to a P2X receptor which could be shown by inhibition with pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid tetrasodium. The remaining vasoconstriction of Ap4A could be blocked by a 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), a selective A1 receptor antagonist In raised tone preparation Ap4A evoked vasodilation when P2 receptors were blocked by suramin. The dilation was not mediated by a P2Y receptor as the effect could not be blocked by suramin.. Ap4A induces vasoconstriction via A1 and P2X receptors and vasodilatation via an unidentified receptor which is not a P2Y receptor. Ap4A may play an important role in kidney perfusion and, thus, in blood-pressure control.

Topics: Adenosine Triphosphate; Adenosine-5'-(N-ethylcarboxamide); Animals; Dinucleoside Phosphates; In Vitro Techniques; Male; Perfusion; Pyridoxal Phosphate; Rats; Rats, Inbred WKY; Receptors, Purinergic; Receptors, Purinergic P1; Receptors, Purinergic P2; Renal Artery; Thionucleotides; Vasoconstrictor Agents; Vasodilator Agents; Xanthines

The effect of adrenalectomy on the expression of adenosine receptors and their mRNA in rat brain was examined using quantitative autoradiography and in situ hybridization. 1,3-[3H]Dipropyl-8-cyclopentylxanthine ([3H]DPCPX), a selective adenosine A1 receptor antagonist, and [3H]CGS 21680, a selective adenosine A(2A) receptor agonist, were used as radioligands. One week after adrenalectomy, the expression of mRNA for adenosine A1 receptors was significantly decreased, as was the number of binding sites for [H]DPCPX. These effects were significantly counteracted by replacement treatment with dexamethasone (1.5 mg/kg i.p., twice daily). Addition of GTP caused a similar increase of [3H]DPCPX binding in sham-operated rats, adrenalectomized rats and rats adrenalectomized and treated with dexamethasone. Moreover, no differences in displacement of [3H]DPCPX by the adenosine receptor agonist N6-(R-phenylisopropyl)adenosine were found among these groups. Adrenalectomy did not significantly affect the number of [3H]CGS 21680 binding sites in striatum or the mRNA encoding adenosine A(2A) receptors. No changes in the affinity of [3H]CGS 21680 for adenosine A(2A) receptors or in the potency of the adenosine receptor agonist 2-chloroadenosine to displace [3H]CGS 21680 were found. Dexamethasone treatment decreased cAMP formation induced by the nonselective adenosine agonist 5'-N-ethylcarboxamidoadenosine in Jurkat cells, which express adenosine A(2B) receptors, but did not alter it in PC-12 cells, which express mostly A(2A) receptors. The results suggest that endogenous corticosteroids positively regulate the expression of adenosine A1 receptors, at least partly at the transcriptional level. In contrast, corticosteroids do not regulate the expression of adenosine A(2A) receptors.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Adrenalectomy; Analysis of Variance; Animals; Autoradiography; Brain; Cyclic AMP; Dexamethasone; Enkephalins; Gene Expression; Glucocorticoids; Humans; In Situ Hybridization; Jurkat Cells; Male; Organ Specificity; PC12 Cells; Phenethylamines; Protein Precursors; Rats; Rats, Sprague-Dawley; Receptor, Adenosine A2A; Receptors, Purinergic P1; RNA, Messenger; Transcription, Genetic; Tritium; Xanthines

This study investigates the proliferative effect of adenosine (ADO) in cultured mesangial cells, and the possible mediation of A1 and/or A2 receptors in this proliferative effect of ADO. ADO (10(-5) M) induced a significant increase in the [3H]thymidine incorporation into DNA with respect to quiescent cells. This increase was similar to that obtained with the ADO A1 receptor agonist, R-PIA (10(-5) M), and with the ADO A2 receptor agonist, NECA (10(-5) M). Theophylline (10(-4) M), and ADO receptors inhibitor, completely inhibits the ADO-induced proliferation. The combinations NECA + A2 receptor antagonist, PD 116,948 (AT1, 10(-6) M) and PIA + A2 receptor antagonists, PD 115,199 (AT2, 10(-2) M) did not induce any significant difference with respect to cells maintained in control conditions. These findings demonstrate the proliferative effect of ADO in cultured mesangial cells, and that this effect is not specific to either of A1 or A2 receptors activation.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Calcium; Cell Division; Cells, Cultured; Cyclic AMP; Glomerular Mesangium; Phenylisopropyladenosine; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Purines; Rats; Rats, Wistar; Sulfonamides; Theophylline; Thymidine; Xanthines

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Analgesics, Non-Narcotic; Animals; Blood Pressure; Body Temperature Regulation; Dose-Response Relationship, Drug; Energy Metabolism; Female; Fever; Male; Purinergic P1 Receptor Agonists; Rabbits; Receptors, Purinergic P1; Xanthines

The behavioral effect of the adenosine antagonists CPT, PACPX, DPCPX and PD 115,199 on spontaneous locomotor activity was investigated in mice after parenteral administration. CPT, PACPX and PD 115,199 affected locomotor activity in a biphasic way. Doses in the nanomolar/kg range significantly reduced locomotion (PACPX> or =PD 115,199>>CPT). Higher doses were progressively less active until they became ineffective or slightly stimulated locomotion. NECA, a mixed A1/A2 agonist, and CCPA, a highly selective A1 agonist, also induced a biphasic behavior, with low doses stimulating and high doses inhibiting locomotion. The stimulant effect of 1 nmol/kg NECA was antagonized by depressant doses of antagonists, whereas antagonists-induced hypomotility was potentiated by a depressant dose of NECA (20 nmol/kg). It is suggested that the blockade of A1 receptors by antagonists is probably responsible for reducing locomotor activity, whereas the activation of A2 receptors by agonists is likely responsible for reducing locomotion in mice.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Antineoplastic Agents; Male; Mice; Motor Activity; Purinergic P1 Receptor Agonists; Purines; Sulfonamides; Theophylline; Xanthines

1. Fluid secretion was induced in the jejunum of anesthetised rats using vasoactive intestinal peptide. 2. The adenosine antagonist, DPCPX (0.1 mg/kg), suppressed the antisecretory action of morphine (10 mg/kg), but naloxone (80 micrograms/kg) did not inhibit the antisecretory response of the adenosine agonist, NECA (40 micrograms/kg), at a dose previously shown to antagonize the antisecretory response of morphine. 3. NECA (40 (micrograms/kg) reversed secretion in pithed and reserpine-pretreated (5 mg/kg subcutaneously) rats. 4. It is proposed that adenosine acts as a mediator of the morphine antisecretory effect at a site distal to the noradrenergic neurons involved in the action of morphine.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Decerebrate State; Female; Intestinal Mucosa; Jejunum; Male; Morphine; Naloxone; Narcotic Antagonists; Norepinephrine; Purinergic P1 Receptor Agonists; Rats; Rats, Wistar; Reserpine; Sympatholytics; Vasoactive Intestinal Peptide; Xanthines

The effect of adenosine A1 and A2 receptor agonists and antagonists was investigated on haloperidol-induced catalepsy in rats. Pretreatment (i.p.) with the non-selective adenosine receptor antagonist, theophylline, or the selective adenosine A2 receptor antagonist, 3,7-dimethyl-1-propargylxanthine (DMPX), significantly reversed haloperidol-induced catalepsy, whereas the selective adenosine A1 receptor antagonists, 8-phenyltheophylline and 8-cyclopentyl-1,3-dipropylxanthine produced no effect. Similar administration of the adenosine A2 receptor agonists, 5'-(N-cyclopropyl)-carboxamidoadenosine and 5'-N-ethylcarboxamidoadenosine (NECA), and the mixed agonists with predominantly A1 site of action, N6-(2-phenylisopropyl) adenosine or 2-chloroadenosine, potentiated haloperidol-induced catalepsy. Higher doses of the adenosine agonists produced catalepsy when given alone. However, N6-cyclopentyladenosine, a highly selective adenosine A1 receptor agonist, was ineffective in these respects. The per se cataleptic effect of adenosine agonists was blocked by DMPX and the centrally acting anticholinergic agent, scopolamine. Scopolamine also attenuated the potentiation of haloperidol-induced catalepsy by adenosine agonists. Further, i.c.v. administration of NECA and DMPX produced a similar effect as that produced after their systemic administration. These findings demonstrate the differential influence of adenosine A1 and A2 receptors on haloperidol-induced catalepsy and support the hypothesis that the functional interaction between adenosine and dopamine mechanisms might occur through adenosine A2 receptors at the level of cholinergic neurons. The results suggest that adenosine A2, but not A1, receptor antagonists may be of potential use in the treatment of Parkinson's disease.

Topics: 2-Chloroadenosine; Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Catalepsy; Dopamine Antagonists; Haloperidol; Male; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Rats; Rats, Wistar; Receptors, Purinergic P1; Scopolamine; Theobromine; Theophylline; Xanthines

1. The binding of 1,3-[3H]-dipropyl-8-cyclopentylxanthine ([3H]-DPCPX), a specific adenosine A1 receptor antagonist, was examined in rat vas deferens membrane preparations using radioligand binding techniques. 2. 1,3-[3H]-Dipropyl-8-cyclopentylxanthine bound to these preparations with a KD of 1.07 +/- 0.14 nmol/L (n = 6). The density of [3H]-DPCPX binding sites was 133.38 +/- 5.57 fmol/mg protein. 3. Computer analysis indicated that nucleosides competed for [3H]-DPCPX binding at two distinct sites. The rank order of potency at the higher affinity site corresponded to R-phenylisopropyladenosine (R-PIA) > or = 2-chloroadenosine (2-CIADO) > or = cyclopentyladenosine (CPA) > or = N-ethylcarboxamidoadenosine (NECA) > s-phenylisopropyladenosine (s-PIA). Ki values were in the low nmol/L range. The rank order of nucleoside potency at the lower affinity site corresponded to R-PIA > or = CPA > or = NECA > or = 2-CIADO > S-PIA. Ki values were in the low mumol/L range. 4. Nucleotides competed for [3H]-DPCPX binding at a single site only. The rank order of potency at this site corresponded to alpha, beta-methylene ATP > or = beta, gamma-methylene ATP > or = ATP. Ki values were in the high mumol/L range. The site seemed to correspond with one of the two binding sites predicted by nucleoside competition binding. 5. The ATP-regenerating compound myokinase did not significantly change the competition curve for ATP, indicating that the competition for [3H]-DPCPX binding observed in the presence of ATP was due to an effect of ATP per se and not to an action of a degradation product. 6. The results demonstrate that in rat vasa deferentia there exist two distinct binding sites for [3H]-DPCPX. One of these sites binds only nucleosides and may represent an adenosine A1 receptor, as usually defined. The other site binds both nucleosides and nucleotides and may represent an atypical adenosine A1 receptor, an atypical P2 or a P3 purinoceptor.

Topics: 2-Chloroadenosine; Adenine Nucleotides; Adenosine; Adenosine Triphosphate; Adenosine-5'-(N-ethylcarboxamide); Animals; Binding Sites; Binding, Competitive; In Vitro Techniques; Kinetics; Male; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Radioligand Assay; Rats; Rats, Sprague-Dawley; Receptors, Purinergic P1; Vas Deferens; Xanthines

Microglial response to stimuli is characterized by secretion of both neurotoxic and neurotrophic factors. Various adenosine receptor agonists stimulated the production of nerve growth factor (NGF) in microglia. Using reverse transcription-polymerase chain reaction (RT-PCR)- and ELISA-techniques, we show that the mixed A1- and A2-agonist 5'-(N-ethylcarboxamido)-adenosine (NECA) induces an increase in NGF mRNA expression and NGF protein release. Whereas the A1-specific agonist cyclopentyladenosine (CPA) only minimally affected NGF release, the A2a-specific agonist CGS-21680 triggered the greatest increase in microglial NGF synthesis. Analyzing the selective antagonist (E)-8-(3,4-dimethoxystyryl)-1,3-dipropyl-7-methylxanthine (EXIP), as well as modulators of the cyclic AMP (cAMP) pathway, we identified an adenosine A2a-receptor-sensitive, cAMP-mediated mechanism of microglial NGF synthesis. Our results indicate that A2a-adenosine receptors modulate microglial neurotrophin expression and release.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Animals, Newborn; Antihypertensive Agents; Bucladesine; Colforsin; Gene Expression; Microglia; Nerve Growth Factors; Phenethylamines; Polymerase Chain Reaction; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Rats; Rats, Wistar; Receptor, Adenosine A2A; Receptors, Purinergic P1; Vasodilator Agents; Xanthines

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

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Bronchi; Cholinergic Fibers; Dipyridamole; Dose-Response Relationship, Drug; Electric Stimulation; Guinea Pigs; In Vitro Techniques; Male; Muscle Contraction; Muscle, Smooth; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Receptors, Purinergic P1; Substance P; Theobromine; Vasodilator Agents; Xanthines

Topics: Adenosine-5'-(N-ethylcarboxamide); Aminoimidazole Carboxamide; AMP-Activated Protein Kinases; Animals; Bronchi; Cattle; Enzyme Activation; In Vitro Techniques; Multienzyme Complexes; Phenylisopropyladenosine; Protein Kinases; Protein Serine-Threonine Kinases; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Receptors, Purinergic P1; Ribonucleotides; Triazines; Triazoles; Xanthines

Adenosine receptors were studied in isolated rat ileum, ileal longitudinal muscle and muscularis mucosae, using a range of agonists and an antagonist. In the rat ileal longitudinal muscle adenosine receptor agonists relaxed the tissues. N6-cyclopentyladenosine (CPA) was more potent than 5'-N-ethylcarboxamidoadenosine (NECA) or adenosine and 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) (1 nM) gave a 5-fold parallel shift to the right of the concentration-response curves to both CPA and NECA corresponding to an apparent pA2 value of 9.6 suggesting that the agonists relax via adenosine A1 receptors. In the intact ileum adenosine receptor agonists also relaxed the tissue but NECA and CPA were equipotent. DPCPX (3 nM) however inhibited responses to both CPA and NECA with dose-ratios of 8 and 15.6, corresponding to pA2 values of 9.3 and 9.7, respectively. DPCPX (300 nM) gave a much greater shift to the right of the concentration-response curve to NECA with a dose-ratio of 769, corresponding to an apparent pA2 of 9.4. This suggests that the agonists are acting at adenosine A1 receptors to cause relaxation of the whole tissue. Adenosine receptor agonists contracted rat ileal muscularis mucosae with a potency order indicative of an A adenosine receptor. DPCPX (3-100 nM) antagonized responses to CPA giving a linear Schild plot with a slope close to unity and a pA2 of 8.4 suggesting an action on adenosine A1 receptors.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Electric Stimulation; Ileum; Intestinal Mucosa; Male; Muscle, Smooth; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Rats; Rats, Wistar; Receptors, Purinergic P1; Vasodilator Agents; Xanthines

In the rat vas deferens there are prejunctional A1 receptors mediating inhibition of transmitter release and post-junctional A1 and A2 receptors mediating enhancement and inhibition of contractions respectively. In this study the distribution of adenosine receptors in the prostatic and epididymal portions of the bisected rat vas deferens was investigated. The pre- and post-junctional A1 receptors were present on both portions of the bisected tissue. However, post-junctional A2 receptors appear to be present only in the prostatic region, showing that adenosine receptors are differentially distributed along the length of the rat vas deferens.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Dose-Response Relationship, Drug; Drug Interactions; Male; Rats; Rats, Wistar; Receptors, Purinergic P1; Vas Deferens; Xanthines

The neuromodulator adenosine is one of the major endogenous inhibitors of overactive excitatory neurotransmission. Adenosine receptors have been identified on neuronal but also on glial surfaces, indicating a role of glial cells in mediation of adenosine effects. Microglia, the immunocompetent cells of the brain, typically respond with proliferation, migration and production of inflammatory substances to viral or bacterial stimuli or to cell damage and degeneration. Since adenosine is released in large amounts in conditions of, for example, hypoxic or ischemic stress, it might be involved in the activation process of microglia. Proliferation of microglia was determined by incorporation of [3H]thymidine into microglial DNA after stimulation with adenosine A1- and A2-receptor agonists. N6-Cyclopentyl adenosine (CPA) and CGS-21680, a specific adenosine A2-receptor agonist had no effect on microglial proliferation. However, combinations of CPA and CGS-21680 as well as the mixed agonist, N6-ethyl-carboxamido adenosine (NECA) increased incorporation of radiolabel above controls. The effect of NECA was inhibited by the adenosine A1-receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). From these results, it is concluded that proliferation of microglia can be increased only by simultaneous stimulation of both adenosine A1- and A2-receptors. Targeted interference with the activation of A1-adenosine receptors by specific drugs appears to be sufficient to reduce microglial activation. The findings may have implications for the treatment of neurodegenerative diseases in which microglial activation is supposed to play a causative role.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Animals, Newborn; Cell Division; Cells, Cultured; Microglia; Phenethylamines; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Rats; Receptors, Purinergic P1; Thymidine; Xanthines

5'-N-Ethylcarboxamidoadenosine (NECA) a non-selective adenosine A1 and A2a receptor agonist was employed in stimulated (3 Hz, 25 mA, 1 min) superfused nucleus tractus solitarius (NTS) brain slices loaded with [3H]5-hydroxytryptamine ([3H]5-HT), and ligand binding with [3H]NECA on NTS membranes. Superfusion of NTS slices with 1.0 and 3.0 nM NECA for 5 min prior to S2 stimulation produced an augmented release of [3H]5-HT (35.7%) above the control S2/S1 ratio. When the duration of NECA perfusion was increased to 20 min prior to S2, the S2/S1 ratio was depressed 21% at 1.0 nM for [3H]5-HT release. The augmented release of [3H]5-HT by NECA at 5 min was blocked by the adenosine A2a antagonist 8-(3-chlorostyryl)caffeine (CSC; 100 nM), and was reduced but not blocked by the A1 antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 10 nM). Saturation binding assays with [3H]NECA on NTS membranes showed two binding sites, a high affinity site with a KD 2.18 nM and low affinity site with a KD of 44.9 nM. With the selective adenosine A2a antagonist CSC the high affinity site was blocked while 10 nM DPCPX, the A1 antagonist, reduced binding of the low affinity site, but did not abolish it. NECA binds to two different adenosine receptor sites in NTS membranes with the A2a receptor being the high affinity site. The same A2a site is associated with the augmented neurotransmitter release of [3H]5-HT with 5 min tissue exposure since it is blocked by CSC. Longer tissue exposure to NECA resulted in desensitization and finally inhibition of release possibly associated with adenosine A1 receptors.

Topics: 2-Chloroadenosine; Acetylcholine; Adenosine; Adenosine-5'-(N-ethylcarboxamide); Analysis of Variance; Animals; Antihypertensive Agents; Binding, Competitive; Dopamine; Dose-Response Relationship, Drug; Electric Stimulation; gamma-Aminobutyric Acid; Male; Neurotransmitter Agents; Organ Culture Techniques; Phenethylamines; Purinergic P1 Receptor Agonists; Rats; Rats, Sprague-Dawley; Receptor, Serotonin, 5-HT2A; Receptors, Serotonin; Sensitivity and Specificity; Serotonin; Solitary Nucleus; Tritium; Vasodilator Agents; Xanthines

Adenosine A1 receptors as well as other components of the adenylate cyclase system have been studied in cultured cerebellar granule cells. No significant changes in adenosine A1 receptor number, assayed by radioligand binding in intact cells, were detected from 2 days in vitro (DIV) until 7 DIV. Nevertheless, a decline in this parameter was detected at 9 DIV. The steady-state levels of alpha-Gg and alpha-Gi, detected by immunoblotting, showed similar profiles, increasing from 2 to 5 DIV and decreasing afterward. Forskolin-stimulated adenylate cyclase levels also showed an increase until 5 DIV, decreasing at 7 and 9 DIV. The adenosine A1 receptor analogue cyclopentyladenosine (CPA) was able to inhibit cyclic AMP accumulation at 2, 5, and 7 DIV but failed to do so at 9 DIV. This inhibition was prevented by the specific adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine. The presence of adenosine deaminase in the culture increased adenosine A1 receptor number during the period studied and induced recovery of the inhibitory effect of CPA, lost after 7 DIV. These data suggest that functional expression of adenosine A1 receptors and the other components of the adenylate cyclase system is subjected to regulation during the maturation of cultured cerebellar granule cells and demonstrates a key role for endogenous adenosine in the process.

Topics: Adenosine; Adenosine Deaminase; Adenosine-5'-(N-ethylcarboxamide); Adenylyl Cyclases; Animals; Animals, Newborn; Cell Membrane; Cells, Cultured; Cerebellum; Colforsin; Cyclic AMP; GTP-Binding Proteins; Kinetics; Neurons; Phenylisopropyladenosine; Rats; Rats, Sprague-Dawley; Receptors, Purinergic P1; Time Factors; Xanthines

1. The ontogenetic profiles of the prejunctional A1 and postjunctional A1 and A2 receptors on the rat vas deferens were investigated, using a combination of functional and radioligand binding assays to follow the A1 receptors and functional assays alone to follow the development of the A2 receptors. 2. The prejunctional A1 receptor, assessed by the inhibitory action of N6-cyclopentyladenosine (CPA) (3 nM-3 microM) on nerve-mediated contractions, was present from day 15 onwards, day 15 being the earliest age at which nerve-mediated contractions could be detected. The potency of CPA was constant across the ages studied, with pD2 values ranging from 6.4-7.1, not significantly different from that previously observed in adult rat vas deferens. 3. The postjunctional A2 receptors, assessed by the inhibitory action of 5'-N-ethylcarboxamidoadenosine (NECA) (10 nM-30 microM) on KCl-induced contractions were present from day 10 onwards, day 10 being the earliest age at which responses to KCl could be observed. The potency of NECA remained constant with an increase in age, with potency values, expressed as pEC25 values, ranging from 6.5-7.0. 4. The postjunctional A1 receptor displayed a different development profile from that of the prejunctional A1 and postjunctional A2 receptors. Postjunctional A1 receptors were identified by the enhancement of KCl-induced contractions by CPA (10 nM-0.3 microM). At 10 and 15 days, CPA failed to enhance KCl-induced contractions. From day 20 to day 40, this enhancement increased with an increase in age and the level of enhancement achieved statistical significance from day 30. 5. Radioligand binding studies using 1,3-[3H]-dipropyl-8-cyclopentylxanthine ([3H]-DPCPX) revealed binding sites characteristic of A1 receptors on the vas deferens from rats aged 20 days onwards. The density (Bmax) of A1 receptors expressed relative to protein content was greatest at day 20 (153 +/- 33 fmol mg-1 protein) and declined at day 30 (43.9 +/- 3.7 fmol mg-1 protein) to a level commensurate with that previously determined in adult rat vas deferens (43.3 +/- 12 fmol mg-1 protein). However, when expressed relative to tissue wet weight little variation in receptor density was observed between these ages (Bmax 0.13 +/- 0.02 fmol mg-1 wet weight at 20 days; 0.17 +/- 0.01 fmol mg-1 wet weight at 30 days). The binding affinity (KD) remained constant with an increase in age and was similar to the KD value previously generated for adult rat vas deferens (approx

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Age Factors; Animals; Animals, Suckling; Dose-Response Relationship, Drug; In Vitro Techniques; Male; Muscle Development; Muscle, Smooth; Neuroeffector Junction; Rats; Rats, Wistar; Receptors, Purinergic P1; Vas Deferens; Xanthines

The expression and function of stimulatory adenosine A2 receptor on the cardiac myocyte is not well defined. The objective of the present study is to characterize the A2a receptor in adult rat cardiac ventricular myocytes. After selection of an optimal lot of collagenase for myocyte isolation and for consistent measurement of adenosine-mediated responses, the A1-receptor pathway was inactivated by pertussis toxin and by the A1-selective antagonist 1,3-dipropyl-8-cyclopentylxanthine. Effects of the adenosine agonist and antagonist or cardiac myocyte contractile amplitude and on adenosine 3',5'-cyclic monophosphate (cAMP) levels were determined. The A2a-receptor-selective agonist 2-[p-(2-carboxyethyl)phenylethylamino]-5'-N-ethylcarboxamidoade nos ine (CGS-21680) caused a pronounced stimulation of myocyte contractile amplitude and an increase in the cAMP level, as did the nonselective agonists 5'-(N-ethylcarboxamido) adenosine (NECA) and adenosine. The A2a-receptor-selective antagonist 8-(3-chlorostyryl)caffeine blocked the NECA- and adenosine-induced positive inotropic response. Probing of myocyte RNA with a rat A2a-receptor cDNA demonstrated a 2.6-kb mRNA, corresponding to that encoding the A2a receptor. Together, data from contractile, cAMP, and RNA studies indicate that A2a receptors are expressed and are functionally coupled to stimulation of cAMP accumulation and cardiac contractility in adult rat ventricular myocytes.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Cyclic AMP; Heart Ventricles; Myocardial Contraction; Myocardium; Pertussis Toxin; Phenethylamines; Purinergic P1 Receptor Agonists; Rats; Rats, Sprague-Dawley; Receptors, Purinergic P1; RNA, Messenger; Virulence Factors, Bordetella; Xanthines

The effects of repeated administration of the selective adenosine A1 receptor agonist 2-chloro-N6-cyclopentyladenosine (CCPA), the selective adenosine A2 receptor agonist 2-hexynyl-5'-N-ethylcarboxamidoadenosine (2HE-NECA), the non-selective adenosine A1/A2 receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA), the selective adenosine A1 receptor antagonist 8-cyclopentyl-1,3 dipropylxanthine (DPCPX) and the selective adenosine A2 receptor antagonist 5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo-(4,3-e)1,2,4-triazolo(1,5 -c)pyrimidine (SCH 58261) on the anticonvulsant activity of 3-(2-carboxypiperazine-4y)propenyl-1-phosphonic acid (CPPene), a selective N-methyl-D-aspartate receptor antagonist, were evaluated in audiogenic sensible dilute brown agouti mice DBA/2J (DBA/2). Mice were treated intraperitoneally twice daily for 7 days with CCPA 0.11 mg/kg, 2HE-NECA 0.056 mg/kg, NECA 0.11 mg/kg, DPCPX 0.5 mg/kg and SCH 58261 0.5 mg/kg followed by 2 vehicle injections (the wash-out period of 1 day) and subsequently CPPene was administered intracerebroventricularly. Audiogenic seizures were delivered 30 min after CPPene administration. Repeated treatment with CCPA significantly reduced the anticonvulsant properties of CPPene against audiogenic seizures. A weak and not significant reduction of anticonvulsant effects of CPPene was observed following repeated administration of NECA, whilst the repeated administration of 2HE-NECA did not decrease the antiseizure activity of CPPene. Conversely, repeated administration of DPCPX markedly potentiated the anticonvulsant properties of CPPene, whilst the repeated treatment with SCH 58261 did not increase the anticonvulsant activity of CPPene. The present results indicate that repeated treatment with CPPA, a selective adenosine A1 receptor agonist, decreases the anticonvulsant properties of CPPene, whilst the repeated administration of DPCPX, a selective adenosine A1 receptor antagonist, potentiates the anticonvulsant effects of CPPene. The compounds acting as selective agonists or antagonists of adenosine A2 receptors do not affect the antiseizure activity of CPPene. In conclusion, the repeated interaction of agonists or antagonists with adenosine A1 receptors seems to induce changes on anticonvulsant activity of CPPene, whereas drugs acting at adenosine A2 receptors do not.

Topics: Acoustic Stimulation; Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Anticonvulsants; Dose-Response Relationship, Drug; Excitatory Amino Acid Antagonists; Injections, Intraventricular; Male; Mice; Mice, Inbred DBA; Piperazines; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Pyrimidines; Triazoles; Xanthines

The ontogeny of P1 purinoceptors in the separated layers of the rat duodenum was investigated using functional assays. In the longitudinal muscle N6-cyclopentyladenosine (CPA) caused relaxations from day 20 that were inhibited by 1,3-dipropyl-8-cyclopentyl-xanthine (DPCPX) (10 nM) indicating an action via adenosine A1 receptors. 5'-N-ethylcarboxamidoadenosine (NECA) caused relaxations at day 15 that were inhibited by DPCPX (1 microM) while 2-p-(2-carboxyethl)phenylethylamino-5'-N-ethylcarboxamidoade nosine (CGS 21680) was almost inactive, indicating an action at adenosine A2B receptors. From day 20 NECA was inhibited by DPCPX (10 nM) but was not antagonised by DPCPX (1 microM) to the extent expected for an adenosine A1 receptor, suggesting activation of adenosine A1 and adenosine A2B receptors. In the muscularis mucosae, CPA and NECA caused contractions from day 10 inhibited by DPCPX (1 microM) while CGS 21680 was less potent, indicating activation of adenosine A2B receptors. These results show that adenosine A2B receptors are present early in the postnatal period, whereas adenosine A1 receptors develop after day 20.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Duodenum; Intestinal Mucosa; Male; Muscle Development; Muscle Relaxation; Muscle, Smooth; Phenethylamines; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Rats; Rats, Wistar; Receptors, Purinergic P1; Xanthines

In isolated guinea pig pulmonary arteries (precontracted with 1 microM noradrenaline) N6-cyclopentyladenosine (CPA), a selective A1 adenosine receptor agonist, exerted a concentration-dependent contraction, whereas 5'-N-ethylcarboxamidoadenosine (NECA), a non-selective A1/A2 receptor agonist, in the presence of DPCPX (a highly selective A1 receptor antagonist), produced a concentration-related rapid relaxation. Pulmonary arteries obtained from guinea pigs treated with aminophylline (APH) or 8-phenyltheophylline (8-PT) for 10 consecutive days, displayed more pronounced contraction in response to CPA compared to those of solvent-treated animals. Relaxant action of NECA was, however, attenuated in arteries prepared from methylxanthine-treated guinea pigs. Opposite changes were found in vascular tissues excised from chronically dipyridamole(DP)-treated guinea pigs.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Female; Guinea Pigs; Pulmonary Artery; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Receptors, Purinergic; Vasoconstriction; Vasodilation; Xanthines

The thermodynamic parameters delta G degree, delta H degree, and delta S degree of the binding equilibrium of seven adenosine agonists and five xanthine antagonists binding specifically to adenosine A2a receptors were determined by means of affinity measurements at six different temperatures (0, 10, 20, 25, 30 and 35 degrees) and van't Hoff plots. Affinity constants were measured on rat striatum membranes by saturation experiments for the selective A2a agonist 2-[p-(carboxy-ethyl)-phenethylamino-]5'-(N-ethyl)carboxamidoadenos ine ([3H]CGS 21680) and by inhibition assays of [3H]CGS 21680 binding for all other compounds. Scatchard plots were monophasic in the full range of temperatures, indicating a single class of high affinity binding sites whose receptor density, BMAX, is essentially temperature independent. Van't Hoff plots were linear in the temperature range 0-30 degrees for agonists and 0-35 degrees for antagonists; their thermodynamic parameters fall, respectively, in the ranges 7 < or = delta H degree < or = 50 kJ/mol and 177 < or = delta S degree < or = 278 J K-1 mol-1 and -36 < or = delta H degree < or = -7 kJ/mol and -33 < or = delta S degree < or = 94 J K-1 mol-1, showing that agonist binding is entropy-driven while antagonist binding is enthalpy-driven. The results are compared with those already reported for the binding of the same compounds to rat brain minus striatum adenosine A1 receptors obtained by displacing [3H]CHA as A1 selective radioligand (Borea PA et al., Mol Neuropharmacol 2: 273-281, 1992). The comparison suggests that the two receptors are very similar as far as their binding sites are concerned and possibly philogenetically related. The analysis of thermodynamical data makes it possible to propose an analogical model of drug-receptor interaction which may account for both affinity and intrinsic activity properties.

Topics: 1-Methyl-3-isobutylxanthine; Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Binding Sites; Brain Chemistry; In Vitro Techniques; Ligands; Male; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Rats; Rats, Wistar; Receptors, Purinergic P1; Temperature; Thermodynamics; Xanthines

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

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Binding, Competitive; Cecum; Dipyridamole; Dose-Response Relationship, Drug; Drug Interactions; Guinea Pigs; Heart Atria; Isoproterenol; Male; Muscle Contraction; Muscle Relaxation; Muscle, Smooth; Myocardial Contraction; Phosphodiesterase Inhibitors; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Theophylline; Xanthines

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

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Colon; Cyclooxygenase Inhibitors; In Vitro Techniques; Indomethacin; Intestinal Mucosa; Muscle Contraction; Muscle, Smooth; Phosphodiesterase Inhibitors; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Quinazolines; Rats; Theophylline; Triazoles; Vasodilator Agents; Xanthines

Inhibition by zatebradine, a specific bradycardic agent, of the negative inotropic but not chronotropic responses to adenosine has been briefly reported in the isolated, perfused dog heart. We therefore investigated whether subtypes of adenosine receptors or postreceptor transduction mechanisms differentiated the negative chronotropic and inotropic responses to adenosine in the isolated, blood-perfused atrial and ventricular preparations of the dog. Adenosine (1-3000 nmol), adenosine A1 receptor agonists, 2-chloroadenosine (CAD, 0.1-300 nmol) and N6-cyclohexyladenosine (CHA, 1-300 nmol) and a nonselective adenosine receptor agonist, 5'-N-ethyl-carboxamidoadenosine (NECA, 0.1-100 nmol), induced the negative chronotropic and inotropic responses. The potency order was NECA > CAD > adenosine > or = CHA. An adenosine A1 receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 10-300 nmol), dose-dependently inhibited the negative chronotropic and inotropic responses to adenosine, CAD and NECA in the isolated, perfused right atrium. DPCPX also blocked the negative inotropic responses to adenosine, CAD and NECA in the isolated left ventricle. However, an adenosine A2 receptor antagonist, 3,7-dimethyl-1-propargylxanthine (DMPX, 300 nmol), did not affect the negative cardiac responses to adenosine and NECA. Although the negative inotropic but not chronotropic responses to CAD and adenosine were dose-dependently inhibited by zatebradine, K+ channel inhibitors 4-aminopyridine and E-4031 did not modify the cardiac responses to adenosine and CAD. These results suggest that the negative cardiac responses to adenosine are mediated by adenosine A1 receptors and the negative chronotropic and inotropic responses to adenosine are differentiated at the postreceptor transduction level(s) in the dog heart.

Topics: 2-Chloroadenosine; 4-Aminopyridine; Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Benzazepines; Depression, Chemical; Dogs; Heart Rate; In Vitro Techniques; Myocardial Contraction; Piperidines; Pyridines; Receptors, Purinergic P1; Xanthines

1. We have assessed the effects of adenosine receptor agonists and antagonists on collagen-induced 5-hydroxytryptamine (5-HT) release and cyclic AMP generation in human platelets. 2. 5'-N-ethylcarboxamidoadenosine (NECA) and CGS 21680 elicited accumulations of cyclic AMP with mean EC50 values of 2678 and 980 nM, respectively. The maximal response to CGS 21680 was approximately half that of the response to 10 microM NECA. 3. NECA and CGS 21680 inhibited collagen-induced 5-hydroxytryptamine release with mean EC50 values of 960 and 210 nM, respectively. The maximal response to CGS 21680 was approximately 25% of the response to 10 microM NECA. 4. The A1/A2a-selective adenosine receptor antagonist PD 115,199 was more potent as an inhibitor of NECA-elicited responses than the A1-selective antagonist DPCPX with calculated Ki values of 22-32 nM and > 10 microM, respectively. 5. In the presence of a cyclic AMP phosphodiesterase inhibitor, the effects of CGS 21680 on cyclic AMP accumulation and 5-HT release were enhanced to levels similar to those elicited by 10 microM NECA. In the absence of phosphodiesterase inhibition, CGS 21680 did not antagonise the effects of NECA. Furthermore, endogenous adenosine did not contribute to the effects of CGS 21680 when phosphodiesterase was inhibited. 6. We conclude that an A2a adenosine receptor appears to be involved in the NECA-elicited increases in cyclic AMP levels and inhibition of 5-HT release in human platelets.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; Adenosine; Adenosine-5'-(N-ethylcarboxamide); Adolescent; Adult; Antihypertensive Agents; Cyclic AMP; Humans; Middle Aged; Phenethylamines; Phenylisopropyladenosine; Platelet Aggregation Inhibitors; Purinergic P1 Receptor Agonists; Purines; Receptors, Purinergic P1; Serotonin; Sulfonamides; Theophylline; Vasodilator Agents; Xanthines

1. The effect of adenosine A1-receptor and P2-purinoceptor agonists on [3H]-inositol phosphate accumulation has been investigated in CHO-K1 cells transfected with the human adenosine A1-receptor. 2. Adenosine receptor agonists stimulated [3H]-inositol phosphate accumulation in CHO-K1 cells with a rank potency order of N6-cyclopentyladenosine (CPA) > 5'-N-ethylcarboxamidoadenosine (NECA) > 2-chloroadenosine > N6-2-(4-aminophenyl) ethyladenosine (APNEA). The responses to both CPA and APNEA were antagonized by the A1 selective antagonist, 1,3-dipropylcyclopentylxanthine (DPCPX) yielding KD values of 1.2 nM and 4.3 nM respectively. 3. ATP, UTP and ATP gamma S were also able to stimulate [3H]-inositol phosphate accumulation in these cells with EC50 values of 1.9 microM, 1.3 microM and 5.0 microM respectively. 2-Methyl-thio-ATP was a weak agonist of this response (EC50 > 100 microM). 4. The [3H]-inositol phosphate response to CPA was completely attenuated by pertussis toxin treatment (24 h; 100 ng ml-1). In contrast, the responses to ATP, UTP and ATP gamma S were only reduced by circa 30% in pertussis toxin-treated cells. 5. The simultaneous addition of CPA and either ATP, UTP or ATP gamma S produced a large augmentation of [3H]-inositol phospholipid hydrolysis. This was due to an increase in the maximal response and was significantly greater than the predicted additive response for activation of these two receptor systems. The synergy was not observed in pertussis toxin-treated cells. 6. No synergy was observed between the [3H]-inositol phosphate responses to histamine and ATP in CHO-K1 cells transfected with the bovine histamine H1-receptor. In these cells the response to histamine was completely resistant to inhibition by pertussis toxin treatment. 7. This study provides a clear demonstration of a synergy between pertussis toxin-sensitive and insensitive receptor systems in a model cell system which is an ideal host for transfected cDNA sequences. This model system should provide a unique opportunity to unravel the mechanisms underlying this example of receptor cross-talk involving phospholipase C.

Topics: 2-Chloroadenosine; Adenosine; Adenosine Triphosphate; Adenosine-5'-(N-ethylcarboxamide); Animals; Cattle; Cells, Cultured; CHO Cells; Cricetinae; Dose-Response Relationship, Drug; Drug Interactions; Drug Synergism; Humans; Inositol Phosphates; Pertussis Toxin; Purinergic P1 Receptor Agonists; Purinergic P2 Receptor Agonists; Uridine Triphosphate; Virulence Factors, Bordetella; Xanthines

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

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Antihypertensive Agents; Carbachol; Colforsin; Hydrogen-Ion Concentration; In Vitro Techniques; Male; Mesenteric Arteries; Methoxamine; Nitroprusside; Perfusion; Phenethylamines; Purinergic P1 Receptor Agonists; Rats; Rats, Wistar; Receptors, Purinergic P1; Vascular Resistance; Vasoconstrictor Agents; Vasodilator Agents; Xanthines

Adenosine receptor (AR) agonists and antagonists are approximately 100-fold and 100,000-fold, respectively, more potent at the bovine A1AR as compared to the rat A3AR. To determine regions of ARs involved in ligand recognition, chimeric receptors composed of bovine A1AR and rat A3AR sequence were constructed and their ligand binding properties examined following expression in COS-7 cells. Substitutions of the second extracellular loop or a region encompassing transmembrane domains 6 and 7 of the A1AR into the A3AR resulted in enhanced affinities of both agonists and antagonists compared to wild-type A3AR. The region of the second extracellular loop of the A1AR responsible for this effect was identified as the distal eleven amino acids of the loop. Replacement of this segment of the A3AR with that of the A1AR in combination with the regions encompassing transmembrane domains 6 and 7 resulted in a 50,000-fold increase in the Kd for antagonist radioligand, [3H]1,3-dipropyl-8- cyclopentylxanthine. Agonist affinity at this chimeric was over 100-fold greater than that displayed by wild-type A3AR. Thus, multiple regions of ARs including a segment of the second extracellular loop are involved in ligand recognition, and considerable overlap exists in structural features required for agonist and antagonist binding.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Amino Acid Sequence; Animals; Binding Sites; Cattle; Cells, Cultured; Molecular Sequence Data; Rats; Receptors, Purinergic P1; Recombinant Fusion Proteins; Structure-Activity Relationship; Xanthines

1. Some postganglionic sympathetic axons possess P2Y-like P2-purinoceptors which, when activated, decrease the release of noradrenaline. We examined the question of whether such receptors also occur at the noradrenergic axons in the rat brain cortex. Slices of the brain cortex were preincubated with [3H]-noradrenaline, then superfused with medium containing desipramine (1 microM) and stimulated electrically, in most experiments by trains of 4 pulses/100 Hz. 2. The selective adenosine A1-receptor agonist, N6-cyclopentyl-adenosine (CPA; 0.03-3 microM) as well as the non-subtype-selective agonist 5'-N-ethylcarboxamido-adenosine (NECA; 0.3-3 microM) reduced the evoked overflow of tritium, whereas the adenosine A2a-receptor agonist, 2-p-(2-carbonylethyl)-phenethylamino-5'-N-ethylcarboxamido-a denosine (CGS-21680; 0.003-30 microM) and the adenosine A3-receptor agonist N6-2-(4-aminophenyl)ethyl-adenosine (APNEA; 0.03-3 microM) caused no change. Of the nucleotides tested, ATP (30-300 microM), adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S; 30-300 microM), adenosine-5'-O-(2-thiodiphosphate) (ADP beta S; 30-300 microM), P1,P4-di(adenosine-5')-tetraphosphate (Ap4A; 30-300 microM) and the preferential P2Y-purinoceptor agonist, 2-methylthio-ATP (300 microM) decreased the evoked overflow of tritium. The P2X-purinoceptor agonist, alpha,beta-methylene-ATP (3-300 microM) caused no change. 3. The A1-selective antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 10 nM) attenuated the effects of the nucleosides CPA (apparent pKB value 9.8) and NECA as well as of the nucleotides ATP (apparent pKB 9.3), ATP gamma S (apparent pKB 9.2) and ADP beta S (apparent pKB 8.7). CGS-21680 and APNEA were ineffective also in the presence of DPCPX. The A2-selective antagonist 1,3-dipropyl-8-(3,4-dimethoxystyryl)-7-methylxanthine (KF-17837) reduced the effects of CPA, NECA and ATP gamma S only when given at a concentration of 300 nM but not at 1O nM.4. The P2-purinoceptor antagonists, suramin (300 micro M), reactive blue 2 (30 micro M) and cibacron blue 3GA(30 micro M) did not change the effect of CPA. Suramin and cibacron blue 3GA shifted the concentration response curve of ATP gamma S to the right (apparent pKB values 3.7 and 5.0, respectively). Reactive blue 2 also attenuated the effect of ATPyS, and cibacron blue 3GA attenuated the effect of ATP, but in these cases the agonist concentration-response curves were not shifted to the right. There was no antagonistic effect of suramin aga

Topics: Adenosine; Adenosine Triphosphate; Adenosine-5'-(N-ethylcarboxamide); Animals; Cerebral Cortex; Male; Norepinephrine; Rats; Rats, Wistar; Receptors, Purinergic P1; Receptors, Purinergic P2; Tetrodotoxin; Xanthines; Yohimbine

1. A1 adenosine receptors were investigated by radioligand binding and functional studies in slices and particulate preparations from guinea-pig cerebral cortex. 2. Binding of the adenosine receptor antagonist radioligand, 8-cyclopentyl-[3H]-1,3-dipropylxanthine (DPCPX) to guinea-pig cerebral cortical membranes exhibited high density (1410 +/- 241 fmol mg-1 protein) and high affinity (Kd 3.8 +/- 0.3 nM). 3. [3H]-DPCPX binding to guinea-pig cerebral cortical membranes was displaced in a monophasic manner by adenosine receptor antagonists with the rank order of affinity (Ki values, nM): DPCPX (6) < xanthine amine congener (XAC, 153) < PD 115,199 (308). 4. Agonist displacement of [3H]-DPCPX binding was biphasic and exhibited the following rank order at the low affinity site (Ki values): 2-chloro-N6-cyclopentyl-adenosine (CCPA, 513 nM) = N6-R-phenylisopropyladenosine (R-PIA, 526 nM) = N6-cyclopentyladenosine (CPA, 532 nM) < 2-chloroadenosine (2CA, 3.2 microM) = 5'-N-ethylcarboxamidoadenosine (NECA, 4.6 microM) < N6-S-phenylisopropyladenosine (S-PIA, 19.9 microM). 5. In cerebral cortical slices, [3H]-DPCPX binding was displaced by antagonists and agonists in an apparently monophasic manner with the rank order of affinity (Ki values, nM): DPCPX (14) < XAC (45) < R-PIA (266) < PD 115,199 (666) < S-PIA (21000). 6. Cyclic AMP accumulation stimulated by 30 microM forskolin in guinea-pig cerebral cortical slices was inhibited by R-PIA, CCPA and CPA up to 1 microM in a concentration-dependent fashion with IC50 values of 14, 18, and 22 nM, respectively. All three analogues inhibited the forskolin response to a similar extent (82-93% inhibition). NECA, S-PTA and 2CA failed to inhibit the forskolin response, but rather enhanced the accumulation of cyclic AMP at concentrations of 100 nM or greater, presumably through activation of A2b adenosine receptors coupled to stimulation of cyclic AMP accumulation in guinea-pig cerebral cortical slices.7. The inhibition of forskolin-stimulated cyclic AMP accumulation by CPA was antagonized with the rank order of affinity (Ki values, nM): DPCPX (6)

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Binding, Competitive; Cerebral Cortex; Colforsin; Cyclic AMP; Guinea Pigs; Histamine; In Vitro Techniques; Phenylisopropyladenosine; Phosphatidylinositols; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Purines; Radioligand Assay; Receptors, Purinergic P1; Sulfonamides; Vasodilator Agents; Xanthines

Adenosine receptor agonists inhibited electrically-evoked contractions of the rat isolated anococcygeus muscle. The compounds tested were: N6-cyclopentyladenosine (CPA), N((S, trans)-2-hydroxycyclopentyl)adenosine (GR79236), the R- and S-isomers of phenylisopropyladenosine (PIA), 5'-N-ethylcarboxamidoadenosine (NECA), ((2-(4-(2-carboxyethyl)phenyl)ethyl)amino)-N-ethylcarbox-amidoa denosine (CGS 21680) and N-((2-methylphenyl)methyl)adenosine (metrifudil). The rank order of agonist potency was: CPA = R-PIA = GR79236 = NECA >> S-PIA > metrifudil > CGS 21680, which is consistent with an effect mediated by adenosine A1 receptors. A similar rank order of potency was obtained for inhibition of electrically-evoked contractions of the guinea-pig ileum. However, there may be a lower receptor reserve in rat anococcygeus compared with the guinea-pig ileum, since higher concentrations of agonists were necessary to produce effects in the anococcygeus than in the guinea-pig ileum and S-PIA behaved as a partial agonist. The effect of NECA was antagonized in rat anococcygeus and guinea-pig ileum by the mixed A1/A2 receptor antagonist, 8-phenyltheophylline (pA2 values of 6.8 and 6.9, respectively). The selective A1-receptor antagonist, 8-cyclopentyl-1,3- dipropylxanthine (DPCPX), also blocked the inhibitory response to NECA in both tissues. Here, however, the pA2 values (9.6 and 8.6, respectively) were slightly but significantly different. These values confirm that the prejunctional adenosine receptors of the rat anococcygeus are of the A1 type, and suggest that they are similar but not necessarily identical to those of the guinea-pig ileum. The differing potencies of DPCPX as an antagonist of NECA between the preparations may reflect a tissue-dependent variation in sensitivity to this antagonist.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Electric Stimulation; Guinea Pigs; Ileum; In Vitro Techniques; Male; Muscle, Smooth; Phenylephrine; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Rats; Rats, Inbred Strains; Receptors, Purinergic P1; Theophylline; Vasodilator Agents; Xanthines

The activation of A1 and A2 adenosine receptors was shown to modulate DNA synthesis. In A431 cells, low concentrations of adenosine (approximately 10 microM) inhibited while higher concentrations (approximately 100 microM) stimulated [14C]thymidine incorporation. Epidermal growth factor (EGF) (10 ng/ml) could partially alleviate the inhibitory effects and further enhance the stimulatory effects of adenosine. Treatment of the cells with adenosine analogues (R-PIA and NECA) and antagonists (theophylline and DPCPX), which are selective for either the A1 (inhibitory) or A2 (stimulatory) receptors, indicated the involvement of these two receptors in the biphasic response to adenosine.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Carcinoma, Squamous Cell; DNA, Neoplasm; Epidermal Growth Factor; Humans; Phenylisopropyladenosine; Purinergic P1 Receptor Antagonists; Receptors, Purinergic P1; Theophylline; Tumor Cells, Cultured; Xanthines

The aim was to determine a role of ATP sensitive potassium (KATP) channels in adenosine A2 receptor mediated coronary vasodilatation in anaesthetised dogs in vivo.. Coronary blood flow in the left circumflex coronary artery, aortic pressure, and left ventricular pressure were measured during intracoronary infusions of the drugs into the left circumflex artery.. A non-selective A2 receptor agonist NECA (5'-N-ethylcarboxamidoadenosine) at 10(-10)-10(-8) mol.min-1 before and after an A1 receptor antagonist DPCPX (8-cyclopentyl-1,3-dipropylxanthine) increased coronary blood flow in a dose dependent manner, without affecting other haemodynamic variables. Glibenclamide at 10 micrograms.kg-1.min-1, which did not alter baseline haemodynamic variables, markedly inhibited the increases in coronary blood flow caused by NECA alone and after DPCPX (p < 0.01). A non-selective adenosine receptor antagonist 8-phenyltheophylline abolished the NECA induced increases in coronary blood flow after DPCPX. These results suggest that A2 receptor mediated coronary vasodilatation was mediated largely by opening of KATP channels. Glibenclamide did not alter the increase in coronary blood flow evoked by forskolin or acetylcholine, suggesting that KATP channels may not be involved in coronary vasodilatation induced by activation of adenylate cyclase or guanylate cyclase. Furthermore, DPCPX increased basal coronary blood flow, which was blocked by 8-phenyltheophylline and by glibenclamide, suggesting that it may have unmasked A2 receptor mediated coronary vasodilatation by inhibiting the A1 receptor mediated vasoconstricting action of endogenous adenosine.. Opening of KATP channels may be involved importantly in adenosine A2 receptor mediated coronary vasodilatation in canine hearts.

Topics: Acetylcholine; Adenosine; Adenosine Triphosphate; Adenosine-5'-(N-ethylcarboxamide); Animals; Colforsin; Coronary Circulation; Dogs; Dose-Response Relationship, Drug; Glyburide; Guanidines; Ion Channel Gating; Pinacidil; Potassium Channels; Purinergic P1 Receptor Antagonists; Receptors, Purinergic P2; Theophylline; Vasodilator Agents; Xanthines

Studies were undertaken in the rat isolated renal artery in order to determine if adenosine receptor agonists were capable of inducing the release of nitric oxide from the renovascular endothelium. N6-cyclopentyladenosine (CPA) and 5'-N-ethylcarboxamidoadenosine (NECA) produced concentration-dependent relaxations in endothelium intact renal artery rings. The NECA curve was biphasic with a first phase pA50 of 6.05. The CPA curve was monophasic with a pA50 of 4.35. In the absence of endothelium the curves to both NECA and CPA were monophasic with pA50 values of 3.37 and 3.50, respectively. The A2a adenosine receptor-selective agonist CGS21680 (2-[p-(2-carboxyethyl)-phenethylamino]-5'-N-ethylcarboxamidoadenos ine) was inactive in endothelium intact tissues. Relaxant responses to CPA and NECA in the presence of endothelium were antagonized by 8-p-sulfophenyltheophylline and by 1,3-dipropyl-8-cyclopentylxanthine only at a nonselective concentration (3 x 10(-6) M) suggesting activation of A2 adenosine receptors. The responses to CPA and NECA in the absence of endothelium are not due to activation of A1 or A2 adenosine receptor subtypes because they are resistant to blockade by these xanthines. CPA and NECA responses in the presence of endothelium were inhibited by NG-nitro-L-arginine methylester (L-NAME), a nitric oxide synthase inhibitor, but not by the cyclooxygenase inhibitor indomethacin or the K+ATP channel antagonist glibenclamide. These results suggest that the rat renal artery contains A2b adenosine receptors that are located exclusively on the endothelium and cause the release of nitric oxide.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Arginine; Endothelium, Vascular; Glyburide; In Vitro Techniques; Indomethacin; Isoproterenol; Male; Muscle Relaxation; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitroprusside; Purinergic P1 Receptor Agonists; Rats; Rats, Sprague-Dawley; Receptors, Purinergic P1; Renal Artery; Renal Circulation; Theophylline; Vasodilator Agents; Xanthines

This study was undertaken to characterize the adenosine receptors in the rat vas deferens. Because adenosine receptors have been well characterized in the cardiovascular system of the guinea pig, antagonist dissociation constants (pKB values) in the rat vas deferens were compared with those from the left atrium (A1) and the aorta (A2) of the guinea pig. The A1-selective agonists (+/-)-N6-endonorbornan-2-yl-5'-N-hydroxy ethylcarboxamidoadenosine (N-0723) and N6-cyclohexyladenosine (CHA) and the nonselective agonist 5'-N-ethylcarboxamidoadenosine (NECA) inhibited the electrically evoked contractions of both the vas deferens and left atrium with a potency order of N-0723 > NECA = CHA. The A2a-selective agonist 2-[p-(2-carboxyethyl)-phenethylamino]5'-N-ethylcarboxamidoadenosin e (CGS21680) was equipotent to NECA in the vas deferens but was 500-fold less potent than NECA in the left atrium. In the aorta only NECA was a potent agonist. The nonselective adenosine receptor antagonist 8-phenyltheophylline antagonized the responses in all three tissues with approximately equal potency (pKB approximately 6.6). In the rat vas deferens, the A1-selective antagonists (+/-)-N6-endonorboman-2-yl-9-methyladenine (N-0861) and 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) were more potent at antagonizing the responses to A1-selective agonists (pKB approximately 8.8 and 6.4, respectively) than they were at antagonizing the responses to NECA and CGS21680 (pKB = 6.3 and < 5, respectively). However, in the left atrium, N-0861 (pKB = 6.2) and DPCPX (pKB = 8.9) were no more potent in antagonizing responses to the A1-selective agonists than they were in antagonizing responses to NECA.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenine; Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Aorta; Guinea Pigs; Heart; In Vitro Techniques; Male; Norbornanes; Phenethylamines; Rats; Receptors, Purinergic P1; Theophylline; Vas Deferens; Xanthines

1. The effects of the adenosine receptor agonists, 5'-N-ethyl-carboxamidoadenosine (NECA) and 2-[4-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamidoadenosin e (CGS-21680) on ventricular automaticity induced by a local injury in the isolated right ventricle of the rat were studied. 2. In concentrations ranging from 0.1 to 100 nM, NECA significantly increased ventricular automaticity. This effect was more reproducible when the adenosine receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) was present at 5 nM, a concentration that blocks A1 adenosine receptors. 3. The excitatory effect of NECA was not observed when DPCPX was present at a concentration of 10 microM, which antagonizes both A1 and A2 adenosine receptors, as well as when rats were reserpinized. 4. In reserpinized rats, NECA increased ventricular automaticity in the presence of isoprenaline and the beta 2-adrenoceptor antagonist, ICI-118,551, but not in the presence of the beta 1-adrenoceptor antagonists, bisoprolol or atenolol. 5. The A 2s-selective adenosine receptor agonist, CGS-21680 (0.1 nM-10 microM) was devoid of excitatory effect on ventricular automaticity. Binding studies of this compound to the rat ventricular membranes revealed that in the preparation there was no specific binding. 6. These results suggest that the excitatory effect of NECA on ectopic ventricular automaticity is dependent on endogenous catecholamines and is mediated by an A2 adenosine receptor which is in some way 'linked' to the beta 1-adrenoceptor. These A2 receptors do not appear to be of the A2a adenosine receptor subtype.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Female; Isoproterenol; Male; Phenethylamines; Rats; Rats, Sprague-Dawley; Receptors, Adrenergic, beta-1; Receptors, Purinergic P1; Ventricular Function; Xanthines

This report describes the synthesis of 29 xanthines containing a chemoreactive chloroaryl, beta-chloroethylamino, alpha,beta-unsaturated carbonyl, bromoacetyl, 3-(fluorosulfonyl)benzoyl, or 4-(fluorosulfonyl)benzoyl group as part of an exocyclic 1-, 3-, or 8-substituent. The xanthines inhibited the binding of [3H]-8-cyclopentyl-1,3-dipropylxanthine ([3H]CPX) to the A1 adenosine receptor (A1AR) of DDT1 MF2 cells at IC50s in the low-nanomolar to low-micromolar range. Seven of the 29 analogues irreversibly inhibited the binding of [3H]CPX without changing the KD of that ligand; five were 1,3-dipropylxanthines having the following reactive groups as 8-substituents: (bromoacetamido)methyl (24), (bromoacetamido)ethyl (25), (bromoacetamido)propyl (26), [4-(fluorosulfonyl)benzamido]methyl (33) or 3-[[4-(fluorosulfonyl)benzoyl]oxy]cyclopentyl (42). Both 8-cyclopentyl-3-[3-[[4- (fluorosulfonyl)benzoyl]oxy]propyl]-1-propylxanthine (53) and 8-cyclopentyl-1,3-bis[3-[[4- (fluorosulfonyl)benzoyl]oxy]propyl]xanthine (55) inhibited [3H]CPX binding irreversibly. Five of the ligands, including 26, 33 (IC50 = 49 microM), and 53 (IC50 = 9 microM), antagonized the binding of [3H]NECA to the A2aAR of PC12 cells, but unlike binding to the A1AR, binding to the A2aAR was completely reversible. The potency of 33 (IC50 = 2 microM, 72% loss of CPX binding at 1 microM) and 53 (IC50 = 0.01 microM, 74% loss of CPX binding at 0.05 microM) and their selectivity for the A1AR suggest that those two ligands may be useful in studies of the structure and function of that receptor.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Cell Membrane; Drug Design; Indicators and Reagents; Magnetic Resonance Spectroscopy; Molecular Structure; PC12 Cells; Purinergic P1 Receptor Antagonists; Radioligand Assay; Structure-Activity Relationship; Xanthines

The effects of adenosine on glomerular filtration rate and renal blood flow are well documented, but its effects on water and sodium excretion are less well established. Previous studies in the rat have shown that i.v. and intra-aortic administration of adenosine decrease water and sodium excretion. The validity of these findings was challenged recently when it was found that intrarenal administration of adenosine in the rat induced marked diuresis and natriuresis. The aim of the current study was to investigate further the effects of intrarenal administration of adenosine on renal excretory function in the rat. Intrarenal infusion of 2 to 15 micrograms/min of adenosine, although having no effect on systemic arterial pressure, induced a 4-fold increase in water and sodium excretion. Intravenous infusion of adenosine at equivalent doses in the same species and under similar experimental conditions resulted in a 1-fold increase in water excretion, and only a transient increase in sodium excretion, whereas intraaortic adenosine had no effect on either variable. During infusion of adenosine by all three routes, there was a significant decline in glomerular filtration rate, but no change in renal plasma flow. The diuretic and natriuretic effects of adenosine during intrarenal infusion were of a similar order of magnitude in animals maintained for 3 weeks on no sodium, normal sodium or high sodium diet, and did not correlate with plasma renin activity. Simultaneous infusion of 10(-7) M 9-cyclopentyl-1,3-dipropylxanthine, a selective adenosine A1 receptor antagonist, markedly inhibited the diuretic and natriuretic effects of intrarenal adenosine. Intrarenal infusion of N6-cyclohexyladenosine, an adenosine A1 receptor agonist, but not of N' ethylcarboxamidoadenosine, a potent A2 receptor agonist, significantly increased water and sodium excretion. These findings suggest that, in the rat, the diuretic and natriuretic effects of adenosine are 1) fully expressed only during intrarenal administration, 2) absent during intra-aortic administration, 3) not related to prior sodium intake or sodium balance, 4) mediated by the adenosine A1 receptor and 5) dissociated from its effects on glomerular filtration and renal plasma flow.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Body Water; Diuresis; Infusions, Intra-Arterial; Infusions, Intravenous; Kidney; Male; Natriuresis; Rats; Rats, Wistar; Sodium; Xanthines

In vitro contractility studies were conducted in canine gastric muscularis mucosae muscle strips with the adenosine analogues 2-chloroadenosine (CIAD), 5'-N-ethylcarboxamidoadenosine (NECA), 5'-(N-cyclopropyl)-carboxamidoadenosine (CPCA), R-N6-(2-phenylisopropyl)adenosine (R-PIA), S-PIA, N6-cyclohexyladenosine (CHA) and (2-p-carboxyethyl)phenylamino-5'-N-carboxamidoadenosine (CGS21680) as well as the A1-selective antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX). Adenosine analogues contracted the muscle strips with the following rank order of potency: CPCA > NECA > CIAD > R-PIA > CHA > S-PIA > CGS21680. CPCA, R-PIA, and CHA were partial agonists. At a concentration selective for adenosine A1 receptors (50 nM), DPCPX did not alter the concentration-response curves to CIAD or CHA. However, at higher concentrations (1-10 microM), DPCPX antagonized CIAD-mediated contractions in a competitive manner (pA2 = 6.96; slope = 0.93). CIAD-mediated contraction was not altered by treatment of the muscle strips with tetrodotoxin (1 microgram/ml) or mepyramine (1 microM). Our results indicate that adenosine A1 receptors, nerves or mast cells are not involved in contraction of canine gastric muscularis mucosae by adenosine analogues.

Topics: 2-Chloroadenosine; Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Dogs; Drug Interactions; Female; Gastric Mucosa; In Vitro Techniques; Male; Muscle Contraction; Muscle, Smooth; Purinergic P1 Receptor Antagonists; Pyrilamine; Receptors, Purinergic P1; Stimulation, Chemical; Tetrodotoxin; Xanthines

We examined the effects of adenosine and adenosine analogues on arginine vasopressin (AVP)-induced increases in osmotic water permeability (Pf; micron/s) and adenosine 3',5'-cyclic monophosphate (cAMP) accumulation in rat inner medullary collecting ducts (IMCDs). When added to the bath, the A1 receptor agonist N6-cyclohexyladenosine (CHA) produced a rapid and reversible inhibition of AVP-stimulated (10 pM) Pf (1,781 +/- 195 to 314 +/- 85 microns/s at 0.3 microM CHA; n = 9). The inhibitory effect of CHA was concentration dependent, with a 50% inhibitory concentration of 10 nM. The effect of CHA was inhibited by prior exposure of IMCDs to the A1 receptor antagonist 1,3-dipropylxanthine-8-cyclopentylxanthine (DP-CPX; 1 microM) or by preincubation with pertussis toxin. CHA had no effect on cAMP-induced increases in Pf. In addition to CHA, adenosine and the nonselective agonist 5'-(N-ethylcarboxamido)-adenosine (NECA) inhibited AVP-dependent Pf by > or = 70%, whereas the A2 receptor agonist CGS-21680 had no effect. Luminal adenosine (0.1 mM) had no effect on basal or AVP-stimulated Pf. CHA, NECA, and adenosine but not CGS-21680 inhibited AVP-stimulated cAMP accumulation in a concentration-dependent manner (50% inhibitory concentrations 0.1-300 nM). The inhibitory effect of CHA on AVP-stimulated cAMP accumulation was attenuated by DPCPX. We conclude that adenosine, acting at the basolateral membrane, inhibits AVP action in the IMCD via interaction with A1 receptors. The inhibition occurs proximal to cAMP generation and likely involves an inhibitory G protein.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Arginine Vasopressin; Cyclic AMP; In Vitro Techniques; Kidney Medulla; Kidney Tubules, Collecting; Kinetics; Male; Pertussis Toxin; Phenethylamines; Rats; Rats, Sprague-Dawley; Receptors, Purinergic P1; Virulence Factors, Bordetella; Xanthines

In rat cerebral cortical slices, the 1-aminocyclopentyl-1S,3R-dicarboxylate (1S,3R-ACPD) isomer of the selective metabotropic excitatory amino acid agonist ACPD inhibited forskolin-stimulated cyclic AMP (cAMP) accumulation in a concentration-dependent manner with a maximal inhibition of 51 +/- 3% and a half-maximally effective concentration of 8.8 +/- 3.4 microM. Similarly, 1R,3S-ACPD inhibited the forskolin response in a concentration-dependent manner, but with an inhibition of 80 +/- 5% at 3 mM. In addition to inhibiting forskolin-stimulated cAMP levels, 1S,3R-ACPD, but not 1R,3S-ACPD, enhanced the cAMP response to A2b adenosine receptor activation. In the presence of 1.2 U/ml of adenosine deaminase (included to reduce the contribution of endogenous adenosine), the efficacy of 1S,3R-ACPD was increased (88 +/- 3% inhibition), but the potency was unchanged. The adenosine receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine also increased the inhibitory effect of 100 microM 1S,3R-ACPD, from 57 +/- 1 to 78 +/- 5%. These results indicate that endogenous adenosine plays an important role in regulating the apparent efficacy of 1S,3R-ACPD inhibition of forskolin-stimulated cAMP accumulation in rat cerebral cortical slices and that previous studies in rat hippocampus and hypothalamus in the absence of added adenosine deaminase may have underestimated the efficacy of this compound.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Cerebral Cortex; Colforsin; Cyclic AMP; Cycloleucine; Female; In Vitro Techniques; Kinetics; Male; Neurotoxins; Rats; Rats, Inbred Strains; Xanthines

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.

Topics: Acetylcholine; Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Carrier Proteins; Colforsin; Depression, Chemical; Enzyme Activation; Enzyme Inhibitors; Female; Guinea Pigs; Heart; Heart Ventricles; In Vitro Techniques; Intracellular Signaling Peptides and Proteins; Isoproterenol; Male; Myocardial Contraction; Myocardium; Phenylisopropyladenosine; Phosphoprotein Phosphatases; Protein Kinases; Proteins; Receptors, Muscarinic; Receptors, Purinergic; Stimulation, Chemical; Ventricular Function; Xanthines

Adenosine causes airway obstruction in asthmatics and smokers. Theophylline and cromolyn, drugs used to treat these patients, bind to human lung adenosine receptors (ARs). This study investigated whether A1ARs and/or A2ARs are functionally present in human lung and airways, and whether theophylline and/or cromolyn antagonize their function. Peripheral lung or airway fragments from 21 people were incubated for 15 min with (1) an A1AR agonist, N6-cyclopentyladenosine (CPA, 5 to 1,000 nM), or (2) an A2AR agonist, either 5'-N-ethylcarboxamido adenosine (NECA, 0.5 to 20 microM) or 2-[p-(2-carboxyethyl)-phenethyl amino]-5'-N-ethylcarboxamido adenosine (CGS 21680, 0.5 to 28 microM), in the presence of the A1AR antagonist 8-cyclopentyl-1,3-dipropylxanthine (50 nM) and/or (3) theophylline (1 mM) and/or (4) cromolyn (500 microM). Adenosine deaminase (2 U/ml) and the phosphodiesterase inhibitor Ro 20-1724 (2 mM) were present in all incubations. Cyclic adenosine monophosphate (cAMP) was measured by radioimmunoassay. In peripheral lung, CPA did not change baseline or isoproterenol-stimulated cAMP content. However, both NECA (20 microM) and CGS 21680 (28 microM) significantly (P < 0.05) increased cAMP content 220 +/- 4% and 201 +/- 32%, respectively (mean +/- SEM). In airways, 20 microM NECA increased cAMP content 129 +/- 34%, and 28 microM CGS 21680 increased it 52 +/- 20% (both P < 0.05). In both peripheral lung and airway tissue, NECA-induced increase in cAMP was antagonized by theophylline (P < 0.05) but not cromolyn. The lungs of younger, nonsmokers had lower baseline cAMP content but did not respond differentially to A2AR stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Adolescent; Adult; Aged; Child; Cromolyn Sodium; Cyclic AMP; Female; Humans; Ligands; Lung; Male; Middle Aged; Phenethylamines; Receptors, Purinergic; Respiratory System; Theophylline; Xanthines

The present study in rats was designed to identify the respective roles of A1 and A2 adenosine receptor activation in the anticonvulsant and behavioral actions of adenosine. Intracaudate injections of the highly selective A2 agonist, CGS 21680, did not affect caudate seizures. However, seizure threshold was increased in the presence of CGS 21680 after blockade of the A1 receptor with CPX, or following activation of the A1 receptor with R-PIA or NECA. Additionally, CGS 21680 led to a dose-related inhibition of locomotor activity when injected into the caudate. These results implicate the involvement of the A2 adenosine receptor in the locomotor depressant actions of adenosine and also suggest possible A2 anticonvulsant effects may depend upon the activation of the A1 receptor.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Anticonvulsants; Caudate Nucleus; Electric Stimulation; Electrodes, Implanted; Electroencephalography; Injections; Male; Motor Activity; Phenethylamines; Phenylisopropyladenosine; Rats; Receptors, Purinergic P1; Stereotaxic Techniques; Xanthines

1. The P1-purinoceptors which mediate the inhibition by adenosine of nerve-mediated contraction of the rat vas deferens have been investigated by use of the agonists N6-cyclopentyladenosine (CPA) and 5'-N-ethylcarboxamidoadenosine (NECA) and the A1-selective antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX). The ontogeny of the responses to adenosine and to the two co-transmitters which induce the contractions in this tissue, adenosine 5'-triphosphate (ATP) and noradrenaline (NA), have also been studied. 2. The order of potency for the adenosine agonists in inhibiting the nerve-mediated contractions was CPA = NECA > adenosine. Micromolar concentrations of DPCPX were required to antagonize the inhibition by adenosine and NECA of nerve-mediated responses, whereas the inhibitory effect of CPA was antagonized by nanomolar concentrations of the antagonist. 3. NECA and adenosine inhibited contractions induced by ATP (10 microM) or by NA (10 microM), NECA being at least ten fold more potent than adenosine, whereas CPA was inactive. Micromolar concentrations of DPCPX were required to antagonize the effect of adenosine on the contractions induced by ATP (10 microM). 4. Nerve-stimulated contractions could be observed in neonatal tissues from day 15 and increased with age, and could be inhibited by adenosine from this time, the potency of adenosine decreasing with age. Responses to ATP also appeared at day 15 and increased with age up to day 25, while responses to NA were present from day 10 (the earliest day tested) and decreased with age. 5. These results show that the rat vas deferens contains both prejunctional Al-receptors and postjunctional A2-receptors, and that adenosine acts on the latter populations to inhibit nerve-mediated contractions.The high potency of adenosine in the neonate and the parallel development of responses to ATP and to nerve-mediated contractions support suggestions that purinergic responses may be particularly important in neonatal tissues.

Topics: Adenosine; Adenosine Triphosphate; Adenosine-5'-(N-ethylcarboxamide); Aging; Animals; Animals, Newborn; Electric Stimulation; In Vitro Techniques; Male; Muscle Contraction; Muscle Development; Muscle, Smooth; Norepinephrine; Rats; Rats, Wistar; Receptors, Purinergic; Tetrodotoxin; Vas Deferens; Vasodilator Agents; Xanthines

To determine the effect of chronic adenosine receptor blockade on atrial responsiveness, we administered theophylline to rats in their drinking water (0.6 mg/ml) for 2 wk. Inotropic and chronotropic responses to the adenosine receptor agonists N6-cyclopentyladenosine (CPA) and 5'-(N-ethylcarboxamido)-adenosine (NECA) were then measured in isolated atria from treated and control animals. The indirect (antiadrenergic) actions of CPA and NECA on force and rate, measured during beta-adrenergic receptor stimulation by isoproterenol, were markedly sensitized (2- to 10-fold reductions in the agonist concentration needed to obtain a half-maximal response) after theophylline. The sensitization was homologous because inotropic and chronotropic responses to carbachol were not affected by theophylline. The direct negative inotropic and chronotropic actions of CPA and NECA, measured without concomitant beta-adrenergic stimulation, were not sensitized after theophylline. The number of atrial A1-receptors, measured by antagonist radioligand binding (maximum specific binding at saturation), was increased by 22% in theophylline-treated rats [66.2 +/- 3.4 vs. 54.3 +/- 1.9 (control) fmol/mg protein, P < 0.05]. Competition binding indicated that the fraction of coupled (high-affinity) receptors was unchanged. The number of ventricular A1-receptors was increased to a similar extent without any change in coupling. Thus chronic dietary theophylline upregulated cardiac A1-adenosine receptors without changing coupling state or affinity and sensitized rat atria to the indirect, antiadrenergic, inhibitory inotropic and chronotropic actions of adenosine receptor agonists.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Heart; Heart Atria; Male; Myocardium; Rats; Rats, Wistar; Receptors, Purinergic; Theophylline; Time Factors; Up-Regulation; Xanthines

Rat glomeruli have been shown to exhibit A1 and A2 adenosine (ADO) receptors. Adenosine also contracts isolated glomeruli and cultured mesangial cells (MC). We studied the effect of the relatively selective ADO agonists R-N6-(1-methyl-1-phenylethyl)adenosine (R-PIA; A1) and 5'-(N-ethylcarboxamido)-adenosine (NECA; A2) on adenosine 3',5'-cyclic monophosphate (cAMP) levels and 45Ca influx in MC. R-PIA (10(-6) M) induced a 55% decrease in cAMP content in 5 microM forskolin-pretreated MC. NECA (10(-6) M) significantly increases by 68% the cAMP levels of forskolin-stimulated MC. NECA-included increase on cAMP was absolutely blocked by the potent A2 antagonist PD115,199 and potentiated by the A1 antagonist PD116,948. Treatment with R-PIA plus the potent A2 antagonist PD115,199 increased 45Ca uptake approximately 40%, and NECA plus A1 antagonist PD116,948 induced a 25% decrease on 45Ca uptake with respect to basal 45Ca uptake. In summary, our studies show the coexistence of functional A1- and A2-like ADO receptors in glomerular MC. The A1 receptor inhibits and the A2 stimulates cAMP accumulation. In addition, the A1 ADO receptor stimulates and the A2 inhibits calcium uptake.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Calcium; Cells, Cultured; Colforsin; Cyclic AMP; Glomerular Mesangium; Purines; Rats; Sulfonamides; Xanthines

1. The effect of adenosine or its stable analogues (2-chloroadenosine, CADO; 5'-N-ethylcarboxamidoadenosine, NECA; and N6-cyclopentyladenosine, CPA) on the release of [3H]-acetylcholine ([3H]-ACh), and on the development of force of contraction evoked by electrical stimulation of the nerve, were studied in the mouse phrenic nerve-hemidiaphragm preparation. Evidence was obtained that the release of ACh is subject to presynaptic modulation through presynaptic A1(P1)-purinoceptors. 2. Adenosine or its stable analogues (CADO, NECA, CPA) inhibited, in a concentration-dependent manner, both the release of ACh and the force of the indirectly elicited contraction of hemidiaphragm preparation, provided in the latter case that the margin of safety was reduced by (+)-tubocurarine or magnesium. The order of potency in reducing ACh release was CPA greater than NECA greater than CADO greater than adenosine with IC50 values of 0.08 +/- 0.01, 0.74 +/- 0.05, 9.05 +/- 0.20, and 410.2 +/- 42.5 mumol/l, respectively. The order of potency in reducing twitch tension was CPA greater than NECA greater than CADO greater than adenosine with IC50 values of 0.11 +/- 0.02, 0.48 +/- 0.03, 2.07 +/- 0.49, and 240.4 +/- 20.0 mumol/l, respectively. 3. 8-Phenyltheophylline (8-PT) antagonized the inhibitory effects of the adenosine receptor agonists on ACh release and twitch tension.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 2-Chloroadenosine; Acetylcholine; Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Diaphragm; Dipyridamole; Electric Stimulation; In Vitro Techniques; Magnesium; Male; Mice; Muscle Contraction; Neuromuscular Junction; Phrenic Nerve; Purinergic Antagonists; Receptors, Purinergic; Synapses; Synaptic Transmission; Theophylline; Tritium; Tubocurarine; Xanthines

The effects of selective adenosine receptor agonists [N6-cyclopentyladenosine (CPA) and N-ethylcarboxamidoadenosine (NECA)] and antagonists [8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and 9-chloro-2-(2-furanyl)-5,6-dihydro-1,2,4-triazolo[1,5-c]quinazoline-5-im ine (CGS-15943A)] on aspartate and glutamate release from the ischemic rat cerebral cortex were studied with the cortical cup technique. Cerebral ischemia (for 20 min) was elicited by four-vessel occlusion. Excitatory amino acid releases were compared from control ischemic rats and drug-treated rats. Basal levels of aspartate and glutamate release were not greatly affected by pretreatment with the adenosine receptor agonists or antagonists. However, CPA (10(-10) M) and NECA (10(-9) M) significantly inhibited the ischemia-evoked release of aspartate and glutamate into cortical superfusates. The ability to block ischemia-evoked release of excitatory amino acids was not evident at higher concentrations of CPA (10(-6) M) or NECA (10(-5) M). The selective A1 receptor antagonist DPCPX also had no effect on release when administered at a low dosage (0.01 mg/kg, i.p.) but blocked the ischemia-evoked release of aspartate and glutamate at a higher dosage (0.1 mg/kg). Evoked release was inhibited by the selective A2 receptor antagonist CGS-15943A (0.1 mg/kg, i.p.). Thus, adenosine and its analogs may suppress ischemia-evoked release of excitatory neurotransmitter amino acids via high-affinity A1 receptors, whereas coactivation of lower-affinity A2 receptors may block (or reverse) the A1-mediated response.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Aspartic Acid; Brain Ischemia; Cerebral Cortex; Glutamates; Glutamic Acid; Male; Neurotransmitter Agents; Purinergic Antagonists; Quinazolines; Rats; Rats, Inbred Strains; Receptors, Purinergic; Triazoles; Xanthines

The adenosine receptors from the isolated dog left atrium were characterized using the non-selective agonists 5'-N-ethyl-carboxamidoadenosine (NECA) and adenosine, the A1-selective agonist N6-R-phenylisopropyladenosine (R-PIA), and the A2 adenosine receptor agonist C2-naphthylethoxyadenosine (NEA). The potency order of the agonists in the dog left atrium was NECA greater than adenosine greater than R-PIA = NEA. This potency order was the same as that found in the guinea pig aorta (A2) but different from that in the guinea pig left atrium (A1). In the guinea pig left atrium the potency order was NECA greater than R-PIA greater than adenosine much greater than NEA. The negative inotropic responses to NECA in the dog left atrium were antagonised by the non-selective antagonist 8-phenyltheophylline (8-PT) and the A1-selective antagonists 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) and N6-endonorbornan-2-yl-9-methyladenine (N-0861), giving pKB values of 6.3, 7.3 and 5.1, respectively. These values are significantly different from those estimates determined in either the guinea pig left atrium or guinea pig aorta. The potency order of the agonists and the relatively low potencies of the A1-selective antagonists suggests that the adenosine receptors in the dog left atrium are not of the classical A1 adenosine receptor subclass and may instead be more closely related to the A2 adenosine receptor.

Topics: Adenine; Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Dogs; Female; Heart Atria; In Vitro Techniques; Male; Myocardium; Norbornanes; Receptors, Purinergic; Theophylline; Vasodilation; Xanthines

1. The P1-purinoceptors mediating relaxation of the rat duodenum and inhibition of contraction of the rat urinary bladder were characterized by use of adenosine and its analogues 5'-N-ethylcarboxamidoadenosine (NECA), N6-cyclopentyladenosine (CPA) and 2-p-((carboxyethyl)phenethylamino)-5'- carboxamidoadenosine (CGS 21680), as well as the A1-selective antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX). The stable analogue of adenosine 5'-triphosphate (ATP), adenylyl 5'-(beta,gamma-methylene)diphosphonate (AMPPCP), was also used as previous work had indicated that it has a direct action on some P1 receptors in addition to its P2-purinoceptor activity. 2. In the rat duodenum, the order of potency of the adenosine agonists was NECA greater than or equal to CPA greater than AMPPCP = adenosine greater than CGS 21680, and DPCPX antagonized CPA and AMPPCP at a concentration of 1 nM whereas equivalent antagonism of NECA and adenosine required a concentration of 1 microM. This suggests the presence of a mixture of A1 and A2 receptors in this tissue, with CPA and AMPPCP acting on the A1 and NECA and adenosine acting on the A2 receptors. 3. In the rat bladder, the order of potency of the adenosine agonists for inhibition of carbachol-induced contractions was NECA much greater than adenosine greater than CPA = CGS 21680, and a concentration of DPCPX of 1 microM was required to antagonize responses to NECA and adenosine. This suggests the presence of A2 receptors in this tissue. ATP and AMPPCP each caused contractions which were not enhanced by DPCPX (1 microM) which suggests that in this tissue AMPPCP was acting only via P2 receptors and had no P1 agonist activity. That AMPPCP was active on the A1 receptors in the duodenum but inactive on the A2 receptors in the bladder implies that it has selectivity for the A1 subtype.4. That CGS 21680, which has been reported to bind selectively to the high affinity A2a subclass of A2 receptors, had a very low potency on the A2 receptors in the duodenum and in the bladder suggests that these receptors are of the low affinity A2b subclass.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Duodenum; In Vitro Techniques; Male; Muscle Relaxation; Muscle, Smooth; Phenethylamines; Rats; Rats, Inbred Strains; Receptors, Purinergic; Urinary Bladder; Vasodilator Agents; Xanthines

The adenosine receptors in the plasma membrane of chromaffin cells from bovine adrenal medulla were characterized. The presence of A1 receptors was discounted owing to the absence of R-[3H]phenylisopropyladenosine (R-PIA) and [3H]8-cyclopentyl-1,3-dipropylxanthine ([3H]-DPCPX) binding. The binding of the specific A2a ligand CGS-21680 was low. In contrast, the binding of 5'-(N-[3H]-ethylcarboxamidoadenosine ([3H]NECA) was relatively high (1.7 pmol/mg of protein at a ligand concentration up to 90 nM). This binding did not correspond to non-adenosine receptor NECA binding sites because the specific [3H]-NECA binding was similar when unlabeled adenosine, NECA, or R-PIA was used to measure the nonspecific binding. The rank order of potency of different ligands for the displacement of specific [3H]NECA binding was DPCPX greater than NECA greater than chloroadenosine greater than R-PIA greater than theophylline = CGS-21680. These results indicate that the receptors present on the plasma membrane of chromaffin cells are exclusively of the A2b subtype.

Topics: 2-Chloroadenosine; Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Cattle; Cell Fractionation; Cell Membrane; Chromaffin System; Phenylisopropyladenosine; Radioligand Assay; Receptors, Purinergic; Tritium; Vasodilator Agents; Xanthines

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.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Diaphragm; In Vitro Techniques; Male; Motor Endplate; Nerve Endings; Neurotransmitter Agents; Phenylisopropyladenosine; Phrenic Nerve; Purines; Rats; Rats, Inbred Strains; Receptors, Purinergic; Sulfonamides; Vasodilator Agents; Xanthines

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Binding Sites; Coronary Disease; In Vitro Techniques; Kinetics; Myocardium; Rats; Rats, Inbred Strains; Receptors, Purinergic; Reference Values; Xanthines

Adenosine and its agonists exert a depressant effect on neuronal activity by interacting with the adenosine A1 receptor. There is now also evidence for electrophysiological effects mediated by adenosine receptors other than the A1 type, possibly A2 receptors. A1 and A2 receptor-induced changes in the electrical properties of neuronal membranes were investigated by intracellularly recording from rat hippocampal CA1 neurons and using the adenosine agonists, 5'-N-ethylcarboxamidoadenosine (NECA) and R-phenylisopropyladenosine (PIA), and the unselective A1 and A2 receptor antagonist, theophylline and the selective A1 receptor antagonist, 8-cyclopenthyl-1,3-dipropylxanthine (DPCPX). PIA and NECA produced an inhibitory effect which was blocked by DPCPX and thus was mediated by A1 receptors. PIA produced inhibition at lower concentrations (0.1-1 mumol/l) than NECA (0.5-10 mumol/l), whereas at high concentrations (2.5 mumol/l) it exerted a dual effect, i.e. either an inhibitory or an excitatory one. During simultaneous perfusion with the A1 receptor antagonist DPCPX, PIA produced concentration-dependent excitatory effects at concentrations above 1 mumol/l. These excitatory effects were blocked by theophylline. DPCPX produced excitation that was enhanced by NECA. Forskolin caused no change in the membrane properties. It is concluded that (1) NECA and PIA affect the membrane properties not only by an action on the A1 but also on non-A1 receptors, because the excitatory effects of PIA and NECA were insensitive to DPCPX and abolished by theophylline; (2) PIA and NECA are more potent at A1 than at A2 receptors; (3) PIA is more potent than NECA at A1 and A2 receptors; (4) effects mediated by A2 receptors prevail over those mediated by A1 receptors when A2 receptors are activated; and (5) the non-A1 receptor-mediated effects are independent of an increased formation of cAMP.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Colforsin; Electric Stimulation; Electrodes; Hippocampus; Male; Membrane Potentials; Neurons; Phenylisopropyladenosine; Purinergic Antagonists; Rats; Rats, Inbred Strains; Receptors, Purinergic; Synapses; Theophylline; Xanthines

The present study compares the characteristics of radioligand binding to adenosine receptors and adenosine uptake sites in 100- and 50-day-old normal and narcoleptic dogs. Binding to A1 receptors was quantified using a selective A1 agonist ([3H]N6-[(R)-1-methyl-2-phenylethyl] adenosine, [3H]R-PIA) and an antagonist ([3H]dipropyl-8-cyclopentyl-xanthine, [3H]CPX). Differences in the binding of [3H]R-PIA and that of [3H]5'-ethylcarboxamide adenosine ([3H]NECA), which binds to both A1 and A2 receptors with similar affinities, were used to quantify A2 receptors. Nucleoside transport sites were labeled with [3H]nitrobenzylthioinosine ([3H]NBTI), a potent inhibitor of nucleoside transport systems. The present study offered no evidence that either adenosine A1 receptors and adenosine uptake sites in the frontal cortex or adenosine A2 receptors in the putamen were altered in narcoleptic dogs. However, we found that adenosine A1 receptors in the dog exist in different affinity states and that the affinity state in which the receptor is found depends on the brain region examined. A characterization of these low- and high-affinity sites was performed and results indicated that these sites cannot be explained by a single interaction of the A1 receptor with a single G-protein population.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Aging; Animals; Brain; Cataplexy; Dogs; GTP-Binding Proteins; Guanylyl Imidodiphosphate; Kinetics; Membranes; Narcolepsy; Phenylisopropyladenosine; Radioligand Assay; Receptors, Purinergic; Thioinosine; Xanthines

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.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Binding, Competitive; Brain Chemistry; Cattle; Cholic Acids; Chromatography, DEAE-Cellulose; Chromatography, Ion Exchange; GTP-Binding Proteins; Guanosine Triphosphate; Hydroxyapatites; In Vitro Techniques; Membranes; Nerve Tissue Proteins; Phenylisopropyladenosine; Phosphatidylcholines; Receptors, Purinergic; Xanthines

On the basis of a model we recently developed for the antagonist binding site of the adenosine A1 receptor (J. Med. Chem. 1990, 33, 1708-1713), it was predicted that 1H-imidazo[4,5-c]quinolin-4-amines would be antagonists of the A1 receptor. Furthermore, it was expected that certain hydrophobic substitutions at the 2- and 4-positions would enhance affinity. Here, we report on the synthesis and the adenosine A1 and A2 receptor affinity of substituted 1H-imidazo[4,5-c]quinolin-4-amines. Some of these compounds have nanomolar affinity for the A1 receptor. The structure-activity relationships (SAR) of these compounds are discussed in relation to SAR for other adenosine receptor ligands. The 1H-imidazo[4,5-c]quinolin-4-amines constitute a novel class of non-xanthine adenosine antagonists.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Cattle; Cell Membrane; Cerebral Cortex; Chemical Phenomena; Chemistry; Imidazoles; Molecular Structure; Quinolines; Rats; Receptors, Purinergic; Structure-Activity Relationship; Xanthines

Carbamazepine (CBZ), an anticonvulsant with psychotropic and anti-pain properties, has been reported to displace ligands at adenosine binding sites. This paper describes biochemical and behavioural studies in rodents comparing CBZ to the adenosine agonists L-phenylisopropyl-adenosine (L-PIA) and N-ethylcarboxamido-adenosine (NECA), the new antagonists PD116,948 and PD115,199 which are also relatively A1 and A2 specific respectively, and the mixed antagonists theophylline and caffeine, attempting to determine functional correlates of the binding studies. Changes in cAMP synthesis and behavioural syndromes produced by the drugs, alone and in combination, were monitored. Classification of the observed effects in terms of A1 and A2 activity was complex, probably due to functional interactions between A1 and A2 subtypes. Nevertheless, it was found that chronic CBZ administration (0.25% in food for 3 days, followed by 0.5% for 11 days) produced a pattern of interaction identical to that of PD115,199 (10-100 mg/kg IP). Thus, both treatments attenuated the behavioural syndrome produced by L-PIA (0.1 or 0.5 mg/kg SC), but did not affect that produced by NECA (0.03 mg/kg SC). CBZ mildly increased hypoactivity after clonidine (0.2 mg/kg IP) which was used as a control. By contrast, the A1 antagonist PD116,948 (0.1-10 mg/kg IP) antagonised both behavioural syndromes. Similarly in the biochemical experiments both chronic CBZ and PD115,199 (10-100 microM) reduced stimulation of cAMP synthesis by L-PIA (confirming that this is mediated by A2 receptors), while only basal cAMP synthesis was affected by PD116,948 (10 microM) and theophylline (60 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Behavior, Animal; Brain Chemistry; Caffeine; Carbamazepine; Clonidine; Cyclic AMP; Down-Regulation; Male; Mice; Mice, Inbred C57BL; Motor Activity; Phenylisopropyladenosine; Purines; Receptors, Adrenergic; Sulfonamides; Theophylline; Xanthines

The antagonism by the A1-adenosine receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) and the A2-adenosine receptor antagonist [9-chloro-2-(2-furanyl)-5,6-dihydro-1,2,4-triazolo (1,5-c)quinazolin-5-imine] (CGS 15943A) of the effects of the A1-adenosine receptor agonist (-)-N6-phenylisopropyladenosine (R-PIA) and the A2-adenosine receptor agonist 5'-N-ethyl-carboxamideadenosine (NECA) in the presence of isoprenaline on contractile response and cyclic AMP (cAMP) content in cardiomyocytes from guinea pig cardiac ventricles were studied. In electrically driven (1 Hz) guinea pig ventricular cardiomyocytes R-PIA concentration-dependently (0.0001-100 microM) reduced the stimulatory effects of isoprenaline (0.01 microM) on contractile response and on cAMP content. The A1-adenosine receptor antagonist DPCPX (0.3 microM) antagonized the effects of R-PIA on contractile reponse and on cAMP content, whereas the A2-adenosine receptor antagonist CGS 15943A (0.01 microM) was ineffective. NECA (0.0001-100 microM) reduced the effects of isoprenaline (0.01 microM) on contractile response to about the same extent as R-PIA. However, NECA did not change cAMP content. DPCPX (0.3 microM) antagonized the effects of NECA on contractile response and evoked a cAMP-increasing effect of NECA, which was 38% of the isoprenaline value at most. In contrast, CGS 15943A did not affect the reduction of contractile response induced by NECA, whereas CGS 15943A revealed a cAMP-decreasing effect of NECA (0.1-10 microM). This study provides functional evidence that both, cAMP-decreasing A1- and cAMP-increasing A2-adenosine receptors are present on ventricular cardiomyocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Cyclic AMP; Drug Interactions; Electric Stimulation; Female; Guinea Pigs; Heart; Isoproterenol; Male; Myocardial Contraction; Quinazolines; Receptors, Purinergic; Triazoles; Vasodilator Agents; Xanthines

Several 2-amino-3-benzoylthiophenes were found to increase the binding of [3H]N6-cyclohexyladenosine to A1 adenosine receptors in rat brain membranes. Concentration-response curves were bell-shaped, with up to 45% stimulation of binding at 10 microM followed by inhibition at higher concentrations. Because these compounds originated from a series of nonxanthine adenosine antagonists, the inhibition of binding was attributed to the presence of interfering adenosine antagonist activity. The compounds stimulated binding of several A1 agonist ligands but only inhibited binding of the A1 antagonist ligand [3H]8-cyclopentyl-1,3-dipropylxanthine, indicating that enhancement was specific for the agonist conformation of the receptor. The enhancement was also specific for the A1 receptor, because agonist binding to A2 adenosine, M2 muscarinic, alpha 2 adrenergic, and delta opiate receptors showed little or no enhancement. Uncoupling of the A1 receptor from the inhibitory guanine nucleotide-binding protein did not prevent enhancement. The enhancers slowed the dissociation of [3H]N6-cyclohexyladenosine from the A1 receptor, implying an allosteric mechanism of action. The inhibition of forskolin-stimulated cyclic AMP accumulation in FRTL-5 cells was employed as a functional index of A1 receptor activation. The enhancers caused up to 19-fold leftward shifts in the concentration-response curve for N6-cyclopentyladenosine and also caused up to 55% inhibition of cyclic AMP accumulation in the absence of agonist. The binding and functional results are consistent with a model in which the enhancers bind preferentially to the agonist conformation of the A1 receptor, thereby shifting the receptor equilibrium in favor of agonist binding. Adenosine enhancers may be useful for ischemia and other conditions involving local energy deficits. More generally, allosteric enhancers may provide a means for strengthening physiological control circuits in a variety of receptor systems.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Allosteric Regulation; Animals; Cyclic AMP; Dogs; Dose-Response Relationship, Drug; Humans; Models, Biological; Rats; Receptors, Purinergic; Species Specificity; Structure-Activity Relationship; Xanthines

Intracellular recordings were performed in a pontine slice preparation of the rat brain containing the locus coeruleus (LC). Adenosine (100, 300 mumol/l) and its structural analogues, namely (-)-N6-(R-phenylisopropyl)-adenosine (R-PIA; 3-30 mumol/l) and S-PIA (10, 30 mumol/l), as well as 5'-N-ethylcarboxamido-adenosine (NECA; 3-30 mumol/l) inhibited the firing rate of spontaneous action potentials and produced hyperpolarization; their rank order of potency was R-PIA congruent to NECA greater than S-PIA greater than adenosine. When applied by superfusion, all agonists strongly desensitized the LC cells; the hyperpolarization never surmounted 6 mV. Upon pressure ejection of adenosine 10 mmol/l from a micropipette positioned close to an LC neurone, the membrane potential was raised by 14 mV and the apparent input resistance decreased by 20%. When the membrane potential was hyperpolarized by current injection to a similar extent as adenosine did, the fall in input resistance was only 7%. The adenosine uptake inhibitor S-(p-nitrobenzyl)-6-thioguanosine (NBTG) 30 mumol/l decreased the frequency of action potentials alone; on simultaneous bath-application with adenosine 300 mumol/l it potentiated the hyperpolarization caused by the purine derivative. 8-Cyclopentyl-1,3-dipropylxanthine (CPDPX) 0.1 mumol/l had no effect on its own, but it antagonized both R-PIA 30 mumol/l and NBTG 30 mumol/l. A higher concentration of CPDPX (1 mumol/l) facilitated the spontaneous firing. In conclusion, both exogenous and endogenous adenosine activates somatic and/or dendritic A1-receptors of LC neurones leading to an enhancement of potassium conductance and thereby to a decreased firing rate and a hyperpolarization.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Electrophysiology; Guanosine; In Vitro Techniques; Locus Coeruleus; Male; Membrane Potentials; Neurons; Phenylisopropyladenosine; Rats; Rats, Inbred Strains; Receptors, Purinergic; Thionucleosides; Xanthines

Adenosine (1 microM to 1 mM) depressed spontaneous transmitter release from frog motor nerve terminals without producing any observable postsynaptic effects. Since this action of adenosine was blocked by 20 microM theophylline and 1 microM 8-phenyltheophylline, adenosine probably acts at a specific receptor on motor nerve terminals to reduce spontaneous transmitter output. The effects of the adenosine analogs, L-N6-phenylisopropyladenosine (L-PIA, 100 pM to 1 microM), D-PIA (100 nM to 100 microM), and 5'-N-ethylcarboxamidoadenosine (NECA, 10nM to 100 microM), were tested on spontaneous transmitter release at the frog neuromuscular junction. L-PIA depressed mepp frequency at a threshold concentration of about 1 nM, was thirteen times more potent than NECA, and was 294 times more effective than D-PIA. The rank-order potency of these analogs indicates that adenosine acts at an A1-like receptor to depress spontaneous transmitter release. Inhibitory actions of maximally effective concentrations of adenosine and L-PIA were also blocked by the A1-specific antagonist, 1-3-dipropyl-8-cyclopentylxanthine (DPCPX) at a concentration of 100 nM. Micromolar concentrations of NECA, an agonist with approximately equal affinity for the A1 and A2 receptors, produced biphasic effects on mepp frequency. Thus, a second adenosine receptor, perhaps of the A2 subtype, may be present on motor nerve terminals and may mediate an increase in spontaneous transmitter release.

Topics: Acetylcholine; Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Membrane Potentials; Motor Neurons; Phenylisopropyladenosine; Purinergic Antagonists; Rana pipiens; Receptors, Purinergic; Theophylline; Xanthines

1. White adipocytes were found to be more responsive than brown adipocytes to inhibition of lipolysis by the A1 adenosine receptor agonist phenylisopropyladenosine. 2. Radioligand binding studies with plasma membranes isolated from the two adipocyte types indicated differences in the properties of the A1 receptors. Kd values (high and low affinity) for phenylisopropyladenosine were higher in membranes from brown adipocytes. The Kd values for the antagonist dipropylcyclopentylxanthine were also higher in brown adipocyte membranes. 3. The effects of guanine nucleotides in converting adipocyte A1 receptors into a low-affinity state were enhanced by dithiothreitol.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Adipose Tissue; Adipose Tissue, Brown; Animals; Binding, Competitive; Cell Membrane; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Lipolysis; Male; Norepinephrine; Phenylisopropyladenosine; Purinergic Antagonists; Rats; Rats, Inbred Strains; Receptors, Purinergic; Xanthines

The effects of the A1 adenosine receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) on force of contraction were examined in isolated electrically driven auricles and papillary muscles from guinea-pigs in the absence and presence of (-)-N6-phenylisopropyladenosine (PIA) and 5'-N-ethylcarboxamidadenosine (NECA). In auricles DPCPX (30-1000 mmol/l) alone increased force of contraction. DPCPX produced only a minor inhibition of phosphodiesterase I-III activity. PIA and NECA alone exerted concentration-dependent negative inotropic effects and the concentration-response curves for PIA and NECA were shifted competitively to the right by the adenosine receptor antagonist DPCPX with similar potency and efficacy. The pA2-value for the inhibition of the effects of PIA and NECA were 9.1 and 8.8, respectively. In papillary muscles DPCPX alone had no inotropic effect. In the presence of isoprenaline PIA and NECA alone exerted concentration-dependent negative inotropic effects and again DPCPX shifted the concentration-response curves for PIA and NECA competitively to the right with similar potency and efficacy. The pA2-value for the inhibition of the effects of PIA and NECA were 9.3 and 9.0, respectively. It is concluded that DPCPX is a potent competitive A1 adenosine receptor antagonist in guinea-pig atrial and ventricular cardiac preparations. Since PIA and NECA were equally potent the cardiac adenosine receptor may constitute a subtype of A1 adenosine receptors differing from the receptor in other tissues such as fat cells. Furthermore, DPCPX has a positive inotropic effect in atrial tissue which cannot be attributed to the A1 receptor antagonism.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Female; Guinea Pigs; Heart; In Vitro Techniques; Isoenzymes; Male; Myocardial Contraction; Papillary Muscles; Phenylisopropyladenosine; Phosphoric Diester Hydrolases; Receptors, Purinergic; Theophylline; Xanthines

Two cell cultures, NEP2 and NEM2, isolated from human foetal brain have been maintained through several passages and found to express some properties of astrocytes. Both cell cultures contain adenylate cyclase stimulated by catecholamines with a potency order of isoprenaline greater than adrenaline greater than salbutamol much greater than noradrenaline, which is consistent with the presence of beta 2-adrenergic receptors. This study reports that the beta 2-adrenergic-selective antagonist ICI 118,551 is approximately 1,000 times more potent at inhibiting isoprenaline stimulation of cyclic AMP (cAMP) formation in both NEP2 and NEM2 than the beta 1-adrenergic-selective antagonist practolol. This observation confirms the presence of beta 2-adrenergic receptors in these cell cultures. The formation of cAMP in NEP2 is also stimulated by 5'-(N-ethylcarboxamido)adenosine (NECA) more potently than by either adenosine or N6-(L-phenylisopropyl)adenosine (L-PIA), which suggests that this foetal astrocyte expresses adenosine A2 receptors. Furthermore, L-PIA and NECA inhibit isoprenaline stimulation of cAMP formation, a result suggesting the presence of adenosine A1 receptors on NEP2. The presence of A1 receptors is confirmed by the observation that the A1-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine reverses the inhibition of isoprenaline stimulation of cAMP formation by L-PIA and NECA. Additional evidence that NEP2 expresses adenosine receptors linked to the adenylate cyclase-inhibitory GTP-binding protein is provided by the finding that pretreatment of these cells with pertussis toxin reverses the adenosine inhibition of cAMP formation stimulated by either isoprenaline or forskolin.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Adenylate Cyclase Toxin; Adrenergic beta-Antagonists; Astrocytes; Brain; Cells, Cultured; Cyclic AMP; Humans; Isoproterenol; Pertussis Toxin; Phenylisopropyladenosine; Propanolamines; Receptors, Adrenergic, beta; Receptors, Purinergic; Virulence Factors, Bordetella; Xanthines

The binding of the adenosine receptor agonists, [3H]N-ethylcarboxamidoadenosine (NECA) and [3H]cyclohexyladenosine (CHA) to membrane preparations and to cryostat sections of the rat brain was examined. The xanthine derivative, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) was ca. 500-fold more effective at A1 than at A2 sites. [3H]CHA binding to A1 adenosine receptors was virtually eliminated by the inclusion of DPCPX (50 nM), while [3H]NECA binding was only partially inhibited. The pattern of DPCPX-insensitive [3H]NECA binding sites was strikingly different from that of A1 receptors and is believed to represent an association with A2 type adenosine receptors and perhaps another or several, previously undescribed non-A1 sites.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Autoradiography; Binding, Competitive; Brain Chemistry; Membranes; Rats; Receptors, Cell Surface; Receptors, Purinergic; Subcellular Fractions; Xanthines