n(6)-cyclopentyladenosine and 1-3-dipropyl-8-cyclopentylxanthine

We have recently demonstrated that orexin acts centrally through the brain orexin 1 receptors to induce an antinociceptive action against colonic distension in conscious rats. Adenosine signaling is capable of inducing an antinociceptive action against somatic pain; however, the association between changes in the adenosinergic system and visceral pain perception has not been investigated. In the present study, we hypothesized that the adenosinergic system may be involved in visceral nociception, and thus, adenosine signaling may mediate orexin-induced visceral antinociception. Visceral sensation was evaluated based on the colonic distension-induced abdominal withdrawal reflex (AWR) in conscious rats. Subcutaneous (0.04-0.2mg/rat) or intracisternal (0.8-4μg/rat) injection of N(6)-cyclopentyladenosine (CPA), an adenosine A1 receptor (A1R) agonist, increased the threshold volume of colonic distension-induced AWR in a dose-dependent manner, thereby suggesting that CPA acts centrally in the brain to induce an antinociceptive action against colonic distension. Pretreatment with theophylline, an adenosine antagonist, or 1,3-dipropyl-8-cyclopentylxanthine, an A1R antagonist, subcutaneously injected potently blocked the centrally injected CPA- or orexin-A-induced antinociceptive action against colonic distension. These results suggest that adenosinergic signaling via A1Rs in the brain induces visceral antinociception and that adenosinergic signaling is involved in the central orexin-induced antinociceptive action against colonic distension.

Topics: Adenosine; Adenosine A1 Receptor Antagonists; Analgesics; Animals; Colon; Consciousness; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Administration Routes; Male; Orexins; Physical Stimulation; Purinergic P1 Receptor Agonists; Rats; Rats, Sprague-Dawley; Receptor, Adenosine A1; Reflex; Visceral Pain; Xanthines

Breast cancer cells over-express the adenosine receptor A1 and in most of these cells, P53 gene is a wild type. Because of this finding and relationship between A1 receptor and cell apoptosis and proliferation, this study aimed to determine the effect of agonist and antagonist of A1 receptor on cell apoptosis and proliferation and recognize the relationship between this receptor and P53 expression.. We used a Real-Time PCR test for measuring expression of p53 gene also flow cytometry assay for apoptotic and survival cell rate after treatment of MCF-7 cells with A1 receptor agonist CPA (N6-Cyclopentyladenosine) and A1 receptor antagonist DPCPX (1,3-dipropyl-8-cyclopentylxanthine) in 24,48 and 72 hours.. Our flow cytometry findings indicate that DPCPX significantly induces apoptosis in MCF-7. Also the expression of P53 becomes upregulated with time of DPCPX treatment. CPA treatment increased the survival cell rate and down-regulated this apoptosis-relevant gene P53 (p > 0.05).. DPCPX can induce P53 expression which consequently promotes the cell apoptosis in MCF-7. Therefore, DPCPX could be used as an anti-cancer agent (Tab. 1, Fig. 3, Ref. 5).

Topics: Adenosine; Adenosine A1 Receptor Agonists; Adenosine A1 Receptor Antagonists; Apoptosis; Breast Neoplasms; Cell Proliferation; Cell Survival; Gene Expression Profiling; Genes, p53; Humans; MCF-7 Cells; Receptor, Adenosine A1; Xanthines

Amyotrophic lateral sclerosis (ALS) is a disease leading to neuromuscular transmission impairment. A2A adenosine receptor (A2AR) function changes with disease stage, but the role of the A(1) receptors (A1Rs) is unknown and may have a functional cross-talk with A2AR. The role of A1R in the SOD1(G93A) mouse model of ALS in presymptomatic (4-6 weeks old) and symptomatic (12-14 weeks old) phases was investigated by recording endplate potentials (EPPs), miniature endplate potentials (MEPPs), and quantal content (q.c.) of EPPs, from Mg(2+) paralyzed hemidiaphragm preparations. In presymptomatic mice, the A1R agonist, N (6)-cyclopentyladenosine (CPA) (50 nM), decreased mean EPP amplitude, MEPP frequency, and q.c. of EPPs, an effect quantitatively similar to that in age-matched wild-type (WT) mice. However, coactivation of A2AR with CGS 21680 (5 nM) prevented the effects of CPA in WT mice but not in presymptomatic SOD1(G93A) mice, suggestive of A1R/A2AR cross-talk disruption in this phase of ALS. DPCPX (50 nM) impaired CGS 21680 facilitatory action on neuromuscular transmission in WT but not in presymptomatic mice. In symptomatic animals, CPA only inhibited transmission if added in the presence of adenosine deaminase (ADA, 1 U/mL). ADA and DPCPX enhanced more transmission in symptomatic mice than in age-matched WT mice, suggestive of increase in extracellular adenosine during the symptomatic phase of ALS. The data documents that at the neuromuscular junction of presymptomatic SOD1(G93A) mice, there is a loss of A1R-A2AR functional cross-talk, while in symptomatic mice there is increased A1R tonic activation, and that with disease progression, changes in A1R-mediated adenosine modulation may act as aggravating factors during the symptomatic phase of ALS.

Topics: Adenosine; Adenosine A1 Receptor Agonists; Adenosine A2 Receptor Agonists; Amyotrophic Lateral Sclerosis; Animals; Evoked Potentials; Mice; Motor Endplate; Neuromuscular Junction; Phenethylamines; Receptor Cross-Talk; Receptor, Adenosine A1; Receptor, Adenosine A2A; Superoxide Dismutase; Superoxide Dismutase-1; Synaptic Transmission; Xanthines

Adenosine A1 receptor is highly expressed in hippocampus where it inhibits neurotransmitter release and has neuroprotective activity. Similar actions are obtained by increasing cGMP concentration, but a clear link between adenosine A1 receptor and cGMP levels remains to be established. The present work aims to investigate if cGMP formation is modulated by adenosine A1 receptors at the hippocampus and if this effect is gender dependent. cGMP accumulation, induced by phosphodiesterases inhibitors Zaprinast (100 μM) and Bay 60-7550 (10 μM), and cAMP accumulation, induced by Forskolin (20 μM) and Rolipram (50 μM), were quantified in rat hippocampal slices using specific enzymatic immunoassays. N6-cyclopentyladenosine (CPA, 100 nM) alone failed to modify basal cGMP accumulation. However, the presence of adenosine deaminase (ADA, 2 U/ml) unmasked a CPA (0.03-300 nM) stimulatory effect on basal cGMP accumulation (EC50: 4.2±1.4 nM; Emax: 17±0.9%). ADA influence on CPA activity was specific for cGMP, since inhibition of cAMP accumulation by CPA was not affected by the presence of ADA, though ADA inhibited cAMP accumulation in the absence of CPA. Increasing cGMP accumulation, by about four-fold, with sodium nitroprusside (SNP, 100 μM) abolished the CPA (100 nM) effect on cGMP accumulation in males but did not modify the effect of CPA in female rats. This effect was reversed by 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX, 100 nM), indicating an adenosine A1 receptor mediated effect on cGMP accumulation. In conclusion, adenosine A1 receptors increase intracellular cGMP formation at hippocampus both in males and females under basal conditions, but only in females when cGMP levels are increased by SNP.

Topics: Adenosine; Adenosine Deaminase; Animals; Colforsin; Cyclic AMP; Cyclic GMP; Female; Hippocampus; Imidazoles; Male; Nitroprusside; Rats; Rats, Wistar; Receptor, Adenosine A1; Rolipram; Triazines; Xanthines

Ischemic brain injury is a leading cause of sever neurological and neurobehavioral deficits and death. The hippocampus plays vital roles in learning and memory processes and it is impaired by ischemic insults. Cerebral ischemia/reperfusion leads to Oxidative stress damage impairing the hippocampus. Here we tested whether ascorbic acid and adenosine receptor played a neuroprotective role in a mouse brain ischemia model induced by common carotid arteries occlusion. Adult male mice were randomly assigned into nine experimental groups. The animals were subjected to ischemia by the ligation of common carotid arteries for 15 min. Drugs were injected intrapritoneally once daily for 7 days. Behavioral tests performed at day 14 and then mice were killed at day 21 and their brains were fixed for microscopic studies and some samples were prepared for western blot analysis. Western blot analysis utilized to evaluate the expression of apoptosis-related proteinsin the hippocampus. Short-term memory was assessed by shuttle-box test. Our findings revealed that administration of vitamin C and N6-cyclopentyladenosine (CPA) significantly attenuated ischemia-induced brain injury. Vitamin C and CPA administration increased the expression of anti-apoptotic protein Bcl-2 and decreased the expression of pro-apoptotic protein Bax in the ischemic mice. Ischemia caused short-term memory loss that was improved by vitamin c and CPA treatment. Our results demonstrate that treatment with vitamin C and adenosine receptor agonist attenuated cerebral ischemia/reperfusion-induced brain injury as a potential neuroprotective agent.

Topics: Adenosine; Adenosine A1 Receptor Agonists; Adenosine A1 Receptor Antagonists; Animals; Antioxidants; Ascorbic Acid; Avoidance Learning; bcl-2-Associated X Protein; Behavior, Animal; Blotting, Western; CA1 Region, Hippocampal; In Situ Nick-End Labeling; Male; Maze Learning; Memory, Short-Term; Mice; Neuroprotective Agents; Proto-Oncogene Proteins c-bcl-2; Receptors, Purinergic P1; Reperfusion Injury; Xanthines

Adenosine has a key endogenous neuroprotective role in the brain, predominantly mediated by the adenosine A(1) receptor (A(1)R). This has been mainly explored using pharmacological tools and/or receptor knockout mice strains. It has long been suggested that the neuroprotective effects of A(1)R are increased following receptor upregulation, thus attenuating neuronal damage in pathological conditions. We have previously shown that the neuroprotective and neuromodulatory actions of the cytokines IL-6 and oncostatin M are mediated by induction of neuronal A(1)R expression. In order to investigate the direct effects of A(1)R upregulation in neurons, we have generated a tetracycline-regulated expression system with a bidirectional promoter, directing the simultaneous expression of the mouse A(1)R and GFP/mCherry reporter genes. In a first step, we tested the efficacy of the system in transiently transfected human embryonic kidney 293 cells. In addition, we confirmed the functional integrity of the expressed A(1)R by whole-cell patch clamp recordings. We demonstrated that A(1)R-transfected primary neurons show enhanced survival against N-methyl-D-aspartate-induced excitotoxicity. Pretreatment with an A(1)R-selective agonist additionally strongly decreased neuronal cell death, while an A(1)R antagonist completely abolished the neuroprotective effects of A(1)R upregulation. The presented data provide for the first time direct evidence that the upregulation of A(1)R enhances neuronal survival.

Topics: Adenosine; Adenosine Deaminase; Animals; Calcium Channels; Cell Survival; Cerebral Cortex; HEK293 Cells; Humans; Mice; N-Methylaspartate; Neurons; Neuroprotective Agents; Neurotoxins; Receptor, Adenosine A1; Tetracycline; Transfection; Up-Regulation; Xanthines

Activation of adenosine A(1) receptors inhibits excitatory synaptic transmission. Equilibrative nucleoside transporters (ENTs) regulate extracellular adenosine levels; however, the role of neuronal ENTs in adenosine influx and efflux during cerebral ischemia has not been determined. We used mice with neuronal expression of human ENT type 1 and wild type (Wt) littermates to compare responses to in vitro hypoxic or ischemic conditions. Extracellular recordings in the CA1 region of hippocampal slices from transgenic (Tg) mice revealed increased basal synaptic transmission, relative to Wt slices, and an absence of 8-cyclopentyl-1,3-dipropyl-xanthine mediated augmentation of excitatory neurotransmission. Adenosine (10-100 μM) had a reduced potency for inhibiting synaptic transmission in slices from Tg mice; inhibitory concentration 50% values were approximately 25 and 50 μM in Wt and Tg slices, respectively. Potency of the A(1) receptor agonist N(6) -cyclopentyladenosine (1 nM-1 μM) was unchanged. Transient hypoxia or oxygen-glucose deprivation produced greater inhibition of excitatory neurotransmission in slices from Wt than Tg, mice. The ENT1 inhibitor S-(4-nitrobenzyl)-6-thioinosine abolished these differences. Taken together, our data provide evidence that neuronal ENTs reduce hypoxia- and ischemia-induced increases in extracellular adenosine levels and suggest that inhibition of neuronal adenosine transporters may be a target for the treatment of cerebral ischemia.

Topics: Action Potentials; Adenosine; Adenosine A1 Receptor Antagonists; Animals; Dose-Response Relationship, Drug; Equilibrative Nucleoside Transporter 1; Female; Glucose; Glutathione; Hippocampus; Humans; Hypoxia; In Vitro Techniques; Mice; Mice, Transgenic; Neurons; Patch-Clamp Techniques; Protein Binding; Purinergic P1 Receptor Agonists; Statistics, Nonparametric; Synaptic Transmission; Thioinosine; Tritium; Xanthines

In vitro experiments demonstrate that P2X(1) receptor activation is important for normal afferent arteriolar autoregulatory behavior, but direct in vivo evidence for this relationship occurring in the whole kidney is unavailable. Experiments were performed to test the hypothesis that P2X(1) receptors are important for autoregulation of whole kidney blood flow. Renal blood flow (RBF) was measured in anesthetized male Sprague-Dawley rats before and during P2 receptor blockade with PPADS, P2X(1) receptor blockade with IP5I, or A(1) receptor blockade with DPCPX. Both P2X(1) and A(1) receptor stimulation with alpha,beta-methylene ATP and CPA, respectively, caused dose-dependent decreases in RBF. Administration of either PPADS or IP5I significantly blocked P2X(1) receptor stimulation. Likewise, administration of DPCPX significantly blocked A(1) receptor activation to CPA. Autoregulatory behavior was assessed by measuring RBF responses to reductions in renal perfusion pressure. In vehicle-infused rats, as pressure was decreased from 120 to 100 mmHg, there was no decrease in RBF. However, in either PPADS- or IP5I-infused rats, each decrease in pressure resulted in a significant decrease in RBF, demonstrating loss of autoregulatory ability. In DPCPX-infused rats, reductions in pressure did not cause significant reductions in RBF over the pressure range of 100-120 mmHg, but the autoregulatory curve tended to be steeper than vehicle-infused rats over the range of 80-100 mmHg, suggesting that A(1) receptors may influence RBF at lower pressures. These findings are consistent with in vitro data from afferent arterioles and support the hypothesis that P2X(1) receptor activation is important for whole kidney autoregulation in vivo.

Topics: Adenosine; Adenosine A1 Receptor Agonists; Adenosine A1 Receptor Antagonists; Adenosine Triphosphate; Animals; Arterioles; Blood Pressure; Dinucleoside Phosphates; Dose-Response Relationship, Drug; Homeostasis; Kidney; Male; Purinergic P2 Receptor Agonists; Purinergic P2 Receptor Antagonists; Pyridoxal Phosphate; Rats; Rats, Sprague-Dawley; Receptor, Adenosine A1; Receptors, Purinergic P2; Receptors, Purinergic P2X; Renal Circulation; Xanthines

This study investigated the effects of N6-cyclopentyladenosine (CPA), a potent and selective adenosine A1 receptor (A1R) agonist in normal and nerve-injured rats and mechanisms of its action by behavioral tests and electrophysiological technique. The results showed: (1) In normal rats, intraperitoneal administration of CPA (1mg/kg) increased paw withdrawal latencies, in a way blocked by a selective A1R antagonist 8-cyclopentyl-1, 3-dipropylxanthine (DPCPX, 3mg/kg, i.p.), but had no influence on the threshold of mechanical stimulation. (2) In rats with neuropathic pain induced by spinal nerve ligation (SNL), CPA reduced thermal hyperalgesia and mechanical allodynia, which could last 6h and 10h, respectively (n=6/group, P<0.05). Both of the effects could be blocked by pretreatment of DPCPX intraperitoneally. (3) The baseline of C-fiber but not A-fiber evoked field potentials was depressed by spinal application of CPA (0.01 mM), and this effect was prevented by application of DPCPX (0.02 mM) 30 min before CPA. (4) Spinal application of CPA depressed long-term potentiation (LTP) of A- and C-fiber evoked field potentials, and both the depression could be blocked by pretreatment of DPCPX 30 min before CPA. These results suggested that the activation of A1R has different influences on normal and neuropathic rats probably due to the absence and presence of central sensitization in spinal dorsal horn.

Topics: Adenosine; Adenosine A1 Receptor Agonists; Adenosine A1 Receptor Antagonists; Animals; Drug Administration Schedule; Electric Stimulation; Electrophysiology; Escape Reaction; Injections, Intraperitoneal; Long-Term Potentiation; Male; Nerve Fibers, Myelinated; Nerve Fibers, Unmyelinated; Neuralgia; Pain Threshold; Rats; Rats, Sprague-Dawley; Spinal Nerves; Xanthines

Recent reports have shown that adenosine A1 receptor-mediated cardioprotection requires concomitant A2 receptor activation, but no study thus far has shown that this phenomenon occurs using A1 agonists at reperfusion. Thus, we compared adenosine A2A receptor knockout (A2AKO) and wild-type mouse hearts (n = 9-11) subjected to global ischemia (30 minutes) and reperfusion (60 minutes) in the presence and absence of the A1 agonist N-cyclopentlyadenosine (CPA). We also determined the effects of selective antagonists at A2A and A2B receptors on CPA-induced protection. In wild-type hearts, CPA (100 nM) significantly (P < 0.05) improved contractility (52.7 ± 6.2% versus 23.9 ± 4.9% of preischemia), left ventricular developed pressure, end diastolic pressure; reduced infarct size (7.9 ± 1.7% versus 23.9 ± 6.6% area at risk); decreased lactate dehydrogenase efflux; and increased ERK1/2 phosphorylation at 60 minutes of reperfusion. Adenosine A2A (ZM241385, 50 nM) and A2B (MRS1754, 100 nM) receptor antagonists abolished CPA-mediated cardioprotection in wild-type groups as did the A1 receptor antagonist DPCPX (P < 0.05). In A2AKO hearts, CPA did not improve functional parameters and protective signaling with the exception of end diastolic pressure. In this model, using a clinically relevant mode of pharmacologic intervention, pERK 1/2-dependent A1-mediated cardioprotection requires a cooperative activation of A2 receptors, presumably through endogenous adenosine.

Topics: Acetamides; Adenosine; Adenosine A1 Receptor Agonists; Adenosine A1 Receptor Antagonists; Adenosine A2 Receptor Agonists; Adenosine A2 Receptor Antagonists; Animals; Blood Pressure; Cardiotonic Agents; Drug Synergism; In Vitro Techniques; Male; Mice; Mice, Knockout; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Myocardial Contraction; Myocardial Reperfusion Injury; Phosphorylation; Purines; Receptor, Adenosine A2A; Triazines; Triazoles; Xanthines

Adenosine A(1) receptors are inhibitory G-protein coupled receptors that presynaptically regulate neurotransmitter release, but their role in self-regulating adenosine release is not known. In this study, we examined the modulation of evoked adenosine and dopamine efflux by A(1) receptors and studied whether D(1) receptors mediate these effects. Fast-scan cyclic voltammetry at carbon-fiber microelectrodes was used for the simultaneous detection of adenosine and dopamine efflux on a subsecond time scale. Short electrical stimulation trains delivered to the substantia nigra (60 pulses, 60 Hz) were used to evoke dopamine and adenosine release in the striatum. The adenosine A(1) receptor agonist N(6)-cyclopentyladenosine (CPA, 1 mg/kg, i.p.) decreased both adenosine and dopamine efflux, although the effect for adenosine occurred more quickly than for dopamine. The A(1) antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 6 mg/kg, i.p.) increased stimulated adenosine release. The effects of CPA were partially attenuated by the dopamine D(1) receptor antagonist SCH-23390. Thus, A(1) and D(1) receptors have a synergistic interaction that modulates both stimulated adenosine and dopamine. The decrease in adenosine is not a downstream effect of lowered dopamine release, as decreasing dopamine synthesis and release with α-methyl-p-tyrosine or increasing release with haloperidol had no effect on adenosine release. This study shows that A(1) receptors have some characteristics of an autoreceptor, including self-regulation of adenosine release.

Topics: Adenosine; Adenosine A1 Receptor Agonists; Adenosine A1 Receptor Antagonists; Analysis of Variance; Animals; Benzazepines; Corpus Striatum; Dopamine; Dopamine Antagonists; Drug Interactions; Electric Stimulation; Electrochemistry; Electrodes, Implanted; Male; Models, Biological; Neural Pathways; Rats; Rats, Sprague-Dawley; Receptor, Adenosine A1; Substantia Nigra; Time Factors; Ventral Tegmental Area; Xanthines

Extracellular adenosine concentrations increase within the heart during ischemia, and any exogenous adenosine receptor agonists therefore work in the context of significant local agonist concentrations. We evaluated the interactions between A1, A2A, A2B, and A3 receptors in the presence and absence of adenosine deaminase (ADA, which is used to remove endogenous adenosine) in a cardiac cell ischemia model. Simulated ischemia (SI) was induced by incubating H9c2(2-1) cells in SI medium for 12 hours in 100% N2 gas before assessment of necrosis using propidium iodide (5 microM) or apoptosis using AnnexinV-PE flow cytometry. N6-Cyclopentyladenosine (CPA; 10(-7)M) and N6-(3-iodobenzyl) adenosine-5'-N-methyluronamide (IB-MECA; 10(-7)M) reduced the proportion of nonviable cells to 30.87 +/- 2.49% and 35.18 +/- 10.30%, respectively (% of SI group). In the presence of ADA, the protective effect of CPA was reduced (62.82 +/- 3.52% nonviable), whereas the efficacy of IB-MECA was unchanged (35.81 +/- 3.84% nonviable; P < 0.05, n = 3-5, SI vs. SI + ADA). The protective effects of CPA and IB-MECA were abrogated in the presence of their respective antagonists DPCPX (8-cyclopentyl-1,3-dipropylxanthine) and MRS1191 [3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1,4-(+/-)-dihydropyridine-3,5-dicarboxylate], whereas A2A and A2B agonists had no significant effect. CPA-mediated protection was abrogated in the presence of both A2A (ZM241385, 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-lamino]ethyl)phenol; 50 nM) and A2B (MRS1754, 8-[4-[((4-cyanophenyl)carbamoylmethyl)oxy]phenyl]-1,3-di(n-propyl)xanthine; 200 nM) antagonists (n = 3-5, P < 0.05). In the absence of endogenous adenosine, significant protection was observed with CPA in presence of CGS21680 (4-[2-[[6-amino-9-(N-ethyl-b-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid) or LUF5834 [2-amino-4-(4-hydroxyphenyl)-6-(1H-imidazol-2-ylmethylsulfanyl)pyridine-3,5-dicarbonitrile] (P < 0.05 vs. SI + ADA + CPA). Apoptosis (14.35 +/- 0.15% of cells in SI + ADA group; P < 0.05 vs. control) was not significantly reduced by CPA or IB-MECA. In conclusion, endogenous adenosine makes a significant contribution to A1 agonist-mediated prevention of necrosis in this SI model by cooperative interactions with both A2A and A2B receptors but does not play a role in A3 agonist-mediated protection.

Topics: Acetamides; Adenosine; Adenosine A1 Receptor Agonists; Adenosine A2 Receptor Agonists; Adenosine A2 Receptor Antagonists; Adenosine A3 Receptor Agonists; Adenosine A3 Receptor Antagonists; Aminopyridines; Animals; Apoptosis; Cardiotonic Agents; Cell Line; Cell Survival; Dihydropyridines; Imidazoles; Myocardial Ischemia; Phenethylamines; Purines; Rats; Triazines; Triazoles; Xanthines

Adenosine is an endogenous neuromodulator previously shown to suppress synaptic transmission and membrane excitability in the CNS. In this study we have determined the actions of adenosine on excitatory synaptic transmission in the subiculum, the main output area for the hippocampus. Adenosine (10 microM) reversibly inhibited excitatory post synaptic currents (EPSCs) recorded from subiculum neurons. These actions were mimicked by the A(1) receptor-specific agonist, N(6)-cyclopentyl-adenosine (CPA, 10 nM) and blocked by the A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 500 nM), but were unaffected by the A(2A) antagonist ZM 241385 (50 nM). In membrane excitability experiments, bath application of adenosine and CPA reversibly inhibited action potentials (AP) in subiculum neurons that were evoked by stimulation of the pyramidal cell layer of the CA1, but not by depolarizing current injection steps in subiculum neurons, suggesting a presynaptic mechanism of action. In support, adenosine and CPA application reduced mEPSC frequency without modulating mEPSC amplitude. These studies suggest that modulation of subiculum neuron excitability by adenosine is mediated via presynaptic A(1) receptors.

Topics: Action Potentials; Adenosine; Adenosine A1 Receptor Agonists; Adenosine A1 Receptor Antagonists; Adenosine A2 Receptor Agonists; Adenosine A2 Receptor Antagonists; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Central Nervous System Agents; Excitatory Postsynaptic Potentials; Hippocampus; In Vitro Techniques; Membrane Potentials; Neurons; Presynaptic Terminals; Pyramidal Cells; Rats; Rats, Sprague-Dawley; Receptor, Adenosine A1; Receptors, Adenosine A2; Synapses; Synaptic Transmission; Triazines; Triazoles; Xanthines

Although adenosine is an important neuromodulator, its role in modulating motor functions at the level of the spinal cord is poorly understood. In the present study, we investigated the effects of adenosine on excitatory synaptic transmission and neuronal death induced by experimental ischaemia by using whole-cell patch-clamp recordings from lamina IX neurones in spinal cord slices. Adenosine significantly decreased the frequency of miniature excitatory postsynaptic currents (mEPSCs) in almost all neurones examined that could be mimicked by an A(1) receptor agonist, N (6)-cyclopentyladenosine (CPA), and inhibited by an A(1) receptor antagonist, 8-cyclopentyl-1, 3-dipropylxanthine (DPCPX). Interestingly, adenosine increased mEPSC frequency in the presence of DPCPX in a subpopulation of neurones. In these neurones, an A(2A) receptor agonist, 2-[4-(2-carbonylethyl)-phenethylamino]-5'-N-ethylcarboxamidoadenosine (CGS21680), increased mEPSC frequency. Adenosine also induced an outward current that was blocked by the addition of Cs(+) and tetraethylammonium into the patch-pipette solution and inhibited in the presence of Ba(2+). The adenosine-induced outward current was mimicked by CPA, but not CGS21680, and inhibited by DPCPX. Moreover, superfusing with ischaemia simulating medium (ISM) generated an agonal inward current in all of the neurones tested. The latencies of the inward currents induced by ISM were significantly prolonged by adenosine or CPA, but not by CGS21680. These results suggest that adenosine receptors are functionally expressed in both the pre- and postsynaptic sites of lamina IX neurones and that their activation may exert multiple effects on motor function. Moreover, this study has provided a cellular basis for an involvement of A(1) receptors in the neuroprotective actions of adenosine.

Topics: Adenosine; Animals; Cell Death; Excitatory Postsynaptic Potentials; In Vitro Techniques; Lumbar Vertebrae; Miniature Postsynaptic Potentials; Motor Neurons; Patch-Clamp Techniques; Phenethylamines; Rats; Rats, Sprague-Dawley; Receptor, Adenosine A1; Receptor, Adenosine A2A; Receptors, Presynaptic; Receptors, Purinergic P1; Sacrum; Spinal Cord; Spinal Cord Ischemia; Synaptic Transmission; Xanthines

The pre-Bötzinger complex (preBötC) is a central pattern generator within the ventrolateral medulla oblongata's ventral respiratory group that is important for the generation of respiratory rhythm. Activation of adenosine A(1) receptors (A(1)R) depresses preBötC rhythmogenesis. Although it remains unclear whether A(1)R activation is important for organisms in a normal metabolic state, A(1)R activation is important to the response of the preBötC to metabolic stress, such as hypoxia. This study examined mechanisms linking A(1)R activation to depression of preBötC rhythmogenesis in medullary slice and island preparations from neonatal mice.. Converting medullary slices to islands by cutting away much of the medullary tissue adjacent to the preBötC decreased the amplitude of action potential bursts generated by a population of neurons within the preBötC (recorded with an extracellular electrode, and integrated using a hardware integrator), without noticeably affecting burst frequency. The A(1)R agonist N6-Cyclopentyladenosine (NCPA) reduced population burst frequency in slices by ca. 33% and in islands by ca. 30%. As in normal (drug-free) artificial cerebrospinal fluid (aCSF), NCPA decreased burst frequency in slices when GABA(A)ergic or GABA(A)ergic and glycinergic transmission were blocked, and in islands when GABA(A)ergic transmission was antagonized. Converting slices to island preparations decreased synaptic input to inspiratory neurons. NCPA further decreased the frequency of synaptic inputs to neurons in island preparations and lowered the input resistance of inspiratory neurons, even when chemical communication between neurons and other cells was impeded.. Together these data support the suggestion that depression of preBötC activity by A(1)R activation involves both decreased neuronal excitability and diminished inter-neuronal communication.

Topics: Adenosine; Adenosine A1 Receptor Agonists; Adenosine A1 Receptor Antagonists; Animals; Animals, Newborn; Bicuculline; Electrophysiology; Evoked Potentials; Female; In Vitro Techniques; Male; Medulla Oblongata; Mice; Neural Inhibition; Neurons; Patch-Clamp Techniques; Pyridazines; Receptor, Adenosine A1; Respiratory Center; Respiratory Mechanics; Strychnine; Synaptic Transmission; Xanthines

Adenosine is known to modulate neuronal activity within the nucleus tractus solitarius (NTS). The modulatory effect of adenosine A1 receptors (A1R) on alpha2-adrenoceptors (Adr2R) was evaluated using quantitative radioautography within NTS subnuclei and using neuronal culture of normotensive (WKY) and spontaneously hypertensive rats (SHR). Radioautography was used in a saturation experiment to measure Adr2R binding parameters (Bmax, Kd) in the presence of 3 different concentrations of N6-cyclopentyladenosine (CPA), an A1R agonist. Neuronal culture confirmed our radioautographic results. [3H]RX821002, an Adr2R antagonist, was used as a ligand for both approaches. The dorsomedial/dorsolateral subnucleus of WKY showed an increase in Bmax values (21%) induced by 10 nmol/L of CPA. However, the subpostremal subnucleus showed a decrease in Kd values (24%) induced by 10 nmol/L of CPA. SHR showed the same pattern of changes as WKY within the same subnuclei; however, the modulatory effect of CPA was induced by 1 nmol/L (increased Bmax, 17%; decreased Kd, 26%). Cell culture confirmed these results, because 10(-5) and 10(-7) mol/L of CPA promoted an increase in [3H]RX821002 binding of WKY (53%) and SHR cells (48%), respectively. DPCPX, an A1R antagonist, was used to block the modulatory effect promoted by CPA with respect to Adr2R binding. In conclusion, our study shows for the first time an interaction between A1R that increases the binding of Adr2R within specific subnuclei of the NTS. This may be important in understanding the complex autonomic response induced by adenosine within the NTS. In addition, changes in interactions between receptors might be relevant to understanding the development of hypertension.

Topics: Adenosine; Adenosine A1 Receptor Antagonists; Animals; Disease Models, Animal; Dose-Response Relationship, Drug; Hypertension; Intranuclear Space; Male; Protein Binding; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Receptor, Adenosine A1; Receptors, Adrenergic, alpha-2; Solitary Nucleus; Xanthines

The respiratory effects of stimulation of adenosine A(1) receptors were studied in spontaneously breathing rats that were either (1) neurally intact and subsequently bilaterally vagotomized in the neck, or (2) neurally intact and subjected to supranodosal vagotomy or (3) midcervically vagotomized before and after pharmacological blockade of A(1) receptors. Before neural interventions an intravenous bolus of the A(1) receptor agonist N6-cyclopentyladenosine (CPA, 5 microg kg(-1)) decreased breathing rate, tidal volume, mean arterial blood pressure (MAP) and heart rate. After section of the midcervical vagi, CPA still decreased respiratory rate and tidal volume. Supranodose vagotomy abolished the fall in respiratory rate but did not affect the depression of tidal volume. Blockade of A(1) receptors with intravenous doses of DPCPX (100 microg kg(-1)) eliminated all respiratory effects of CPA challenge. In all the neural states, CPA caused significant falls in mean arterial blood pressure and heart rate. DPCPX pre-treatment prevented these cardiovascular effects. The present data suggest that: (1) CPA-evoked activation of A(1) receptors decreases breathing rate and tidal volume and this occurs central to the cervical vagi; (2) supranodosal vagotomy prevents the decrease in breathing rate, which is presumably due to stimulation of nodosal A(1) receptor; and (3) depression of tidal volume and the hypotensive response result from the excitation of central nervous A(1) expressing neurones.

Topics: Adenosine; Animals; Blood Pressure; Heart Rate; Male; Nodose Ganglion; Rats; Rats, Wistar; Receptor, Adenosine A1; Respiratory Mechanics; Tidal Volume; Vagotomy; Xanthines

Vasoactive intestinal peptide (VIP) modulates GABA release from hippocampal nerve terminals and enhances hippocampal synaptic transmission through a pathway dependent on GABAergic transmission. Since VIP modulation of hippocampal synaptic transmission is dependent on the tonic actions of adenosine we investigated if endogenous adenosine could influence VIP enhancement of GABA release from isolated hippocampal nerve endings, and which adenosine receptors could be mediating this influence. When extracellular endogenous adenosine was removed using adenosine deaminase (ADA, 1U/ml), the enhancement (57.2+/-3.7%) caused by VIP on GABA release was prevented. Blockade of adenosine A(1) receptors with 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 10nM) or of A(2A) receptors with ZM241385 (50nM) abolished the effect of VIP. In the presence of ADA, selective A(2A) receptor-activation with CGS21680 (10nM) readmitted most of the enhancement caused by VIP on GABA release (50.7+/-5.3%). Also in the presence of ADA, A(1) receptor activation with N(6)-cyclopentyladenosine (CPA, 50nM) partially readmitted that effect of VIP (32.6+/-3.8%). In conclusion, the enhancement of GABA release caused by VIP in hippocampal nerve terminals is dependent on the tonic actions of adenosine on both A(1) and A(2A) receptors, and this action of adenosine is essential to VIP modulation of GABA release.

Topics: Adenosine; Adenosine Deaminase; Animals; gamma-Aminobutyric Acid; Hippocampus; Male; Phenethylamines; Potassium; Presynaptic Terminals; Rats; Rats, Wistar; Receptor, Adenosine A1; Receptor, Adenosine A2A; Receptors, Purinergic P1; Synaptic Transmission; Synaptosomes; Tritium; Vasoactive Intestinal Peptide; Xanthines

Herein we report the synthesis and biological evaluation of some potent and selective A(1) adenosine receptor agonists, which incorporate a functionalised linker attached to an antioxidant moiety. N(6)-(2,2,5,5-Tetramethylpyrrolidin-1-yloxyl-3-ylmethyl)adenosine (VCP28, 2e) proved to be an agonist with high affinity (K(i)=50nM) and good selectivity (A(3)/A(1) > or = 400) for the A(1) adenosine receptor. N(6)-[4-[2-[1,1,3,3-Tetramethylisoindolin-2-yloxyl-5-amido]ethyl]phenyl]adenosine (VCP102, 5a) has higher binding affinity (K(i)=7 nM), but lower selectivity (A(3)/A(1)= approximately 3). All compounds bind weakly (K(i)>1 microM) to A(2A) and A(2B) receptors. The combination of A(1) agonist activity and antioxidant activity has the potential to produce cardioprotective effects.

Topics: Adenosine; Adenosine A1 Receptor Agonists; Animals; Antioxidants; Binding Sites; Cardiotonic Agents; Cell Line; Indicators and Reagents; Isoindoles; Myocardial Ischemia; Myocardial Reperfusion Injury; Myocytes, Cardiac; Pyrrolidines; Rats; Structure-Activity Relationship; Xanthines

The present study was designed to examine the effects of adenosine A(1) receptor on dopamine D(1) receptor desensitization in a human embryonic kidney 293 cell line stably cotransfected with human adenosine A(1) receptor and dopamine D(1) receptor cDNAs (A(1)D(1) cells) by means of cAMP accumulation assay. Long-term exposure of A(1)D(1) cells to dopamine D(1) receptor agonist (+/-)-1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol hydrochloride (SKF38393) caused a rapid desensitization of dopamine D(1) receptor. Coadministration of adenosine A(1) receptor agonist N(6)-cyclopentyladenosine (CPA) potentiated the effect of SKF38393. This enhancement effect of CPA was blocked by adenosine A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) but not by pertussis toxin, indicating that this effect of CPA was mediated by adenosine A(1) receptor and was G(i) protein independent. Furthermore, the blockade of endogenous adenosine by adenosine deaminase or DPCPX attenuated dopamine D(1) receptor desensitization. Collectively, these results suggest that adenosine A(1) receptor plays an important role in the regulation of dopamine D(1) receptor by potentiating ligand-induced desensitization.

Topics: 2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine; Adenosine; Binding, Competitive; Cell Line; Cyclic AMP; Dopamine Agonists; Drug Synergism; Humans; Receptor, Adenosine A1; Receptors, Dopamine D1; Time Factors; Transfection; 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

The A1 adenosine receptor (A1AR) has been suggested to participate in insulin- and contraction-stimulated glucose transport in skeletal muscle, but the qualitative and quantitative nature of the effect are controversial. We sought to determine if A1AR is expressed in rat soleus muscle and then characterize its role in glucose transport in this muscle. A1AR mRNA and protein expression were determined by RT-PCR and Western blotting, respectively. To examine the role of adenosine in 3-O-methylglucose transport, isolated muscles were exposed to adenosine deaminase and alpha,beta-methylene adenosine diphosphate to remove endogenous adenosine and were left unstimulated (basal) or stimulated with insulin. To assess the functional participation of A1AR in 3-O-methylglucose transport, muscles were incubated with A1-selective agonist and (or) antagonist in the absence of endogenous adenosine and with or without insulin. A1AR mRNA was expressed in soleus muscle and A1AR was present at the plasma membrane. Removal of endogenous adenosine reduced glucose transport in response to 100 microU/mL insulin (approximately 50%). The A1-selective agonist, N6-cyclopentyladenosine, increased submaximal (100 microU/mL) insulin-stimulated glucose transport in a dose-dependent manner (0.001-1.0 micromol/L). This stimulatory effect was inhibited by the A1-selective receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine in a concentration-dependent manner (0.001-1.0 micromol/L). However, neither activation nor inhibition of A1AR altered basal or maximal (10 mU/mL) insulin-stimulated glucose transport. Our results suggest that adenosine contributes approximately 50% to insulin-stimulated muscle glucose transport by activating the A1AR. This effect is limited to increasing insulin sensitivity, but not to either basal or maximal insulin-stimulated glucose uptake in rat soleus muscle.

Topics: 3-O-Methylglucose; Adenosine; Adenosine A1 Receptor Antagonists; Animals; Biological Transport; Cell Membrane; Dose-Response Relationship, Drug; Glucose; Glucose Transporter Type 4; In Vitro Techniques; Insulin; Male; Muscle, Skeletal; Rats; Rats, Wistar; Receptor, Adenosine A1; RNA, Messenger; Xanthines

Failure to autoresuscitate from apnea has been suggested to play a role in sudden infant death. Little is known, however, about factors that influence the gasping and heart rate response to severe hypoxia that are fundamental to successful autoresuscitation in the newborn. The present experiments were carried out on 184 rat pups to investigate the influence of the parasympathetic nervous system, as well as adenosine, in mediating the profound bradycardia that occurs with the onset of hypoxic-induced primary apnea and in modulating hypoxic gasping. On days 1 to 2, days 5 to 6, and days 10 to 11 postpartum and following bilateral cervical vagotomy (VAG) or administration of a selective adenosine A(1) receptor antagonist (8-cyclopentyl-1,3-dipropylxanthine; DPCPX), each pup was exposed to a single period of severe hypoxia produced by breathing an anoxic gas mixture (97% N(2)-3% CO(2)). Exposure to severe hypoxia resulted in an age-dependent decrease in heart rate (P < 0.001), accentuated with increasing postnatal age, that was attenuated in all age groups by DPCPX but not by VAG. Furthermore, DPCPX but not VAG decreased the time to last gasp but increased the total number of gasps in the 1- to 2-day-old and 5- to 6-day-old pups but not in the 10- to 11-day-old pups during exposure to severe hypoxia. Thus our data provide evidence that adenosine acting via adenosine A(1) receptors plays a role in modulating hypoxic gasping and in mediating the profound bradycardia that occurs coincident with hypoxic-induced primary apnea in rats during early postnatal life.

Topics: Adenosine; Adenosine A1 Receptor Agonists; Adenosine A1 Receptor Antagonists; Animals; Animals, Newborn; Dose-Response Relationship, Drug; Forelimb; Heart Rate; Hypoxia; Parasympathetic Nervous System; Rats; Rats, Sprague-Dawley; Receptor, Adenosine A1; Reflex; Remission, Spontaneous; Vagotomy; Xanthines

The interaction of a new nonribose ligand (LUF5831) with the human adenosine A1 receptor was investigated in the present study. Radioligand binding experiments were performed in the absence and presence of diverse allosteric modulators on both wild-type (wt) and mutant (T277A) adenosine A1 receptors. Thermodynamic data were obtained by performing these assays at different temperatures. In addition, cyclic adenosine monophosphate (cAMP) assays were performed. The presence of allosteric modulators had diverse effects on the affinity of LUF5831, N6-cyclopentyladenosine (CPA), a full agonist, and 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), an inverse agonist/antagonist, for the adenosine A1 receptor. PD81,723, for example, increased the affinity of CPA, while the affinity of LUF5831 was decreased. However, the affinity of DPCPX was decreased even more. In addition, LUF5831 was shown to have an affinity for the mutant (T277A) adenosine A1 receptor (Ki=122+/-22 nM), whereas CPA's affinity was negligible. The results of temperature-dependent binding assays showed that the binding of LUF5831 was entropy driven, in between the behaviour of CPA binding to the high- and low-affinity states of the receptor, respectively. The inhibition of the forskolin-induced production of cAMP through activation of the wt adenosine A1 receptor showed that LUF5831 had a submaximal effect (37+/-1%) in comparison to CPA (66+/-5%). On the mutant receptor, however, neither CPA nor LUF5831 inhibited cAMP production. This study indicates that the nonribose ligand, LUF5831, is a partial agonist for the adenosine A1 receptor.

Topics: Adenosine; Adenosine A1 Receptor Agonists; Allosteric Regulation; Animals; CHO Cells; Cricetinae; Cyclic AMP; Humans; Nitriles; Pyridines; Radioligand Assay; Receptor, Adenosine A1; Thermodynamics; Xanthines

The antagonistic interactions between adenosine A1 receptors and dopamine D1 receptors were studied in a human embryonic kidney 293 cell line stably cotransfected with human adenosine A1 receptor and dopamine D1 receptor cDNAs. In the cotransfected cells, but not in control cells only transfected with dopamine D1 receptors, adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA, 10 microM) increased the Kd of dopamine D1 receptor antagonist [N-methyl-3H]R(+)-7-Chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine ([3H]SCH23390) without affecting the Bmax. Moreover, CPA induced a concentration-dependent decrease in the affinity of dopamine D1 receptors for the agonist (+/-)-1-Phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol hydrochloride (SKF38393) and inhibited dopamine D1 receptor-mediated cyclic AMP response element recruitment. Furthermore, pertussis toxin treatment completely counteracted the effects of low concentrations of CPA but only partially counteracted the effects of high concentrations of CPA. These results suggest that adenosine A1 receptors antagonistically modulate dopamine D1 receptors at the level of receptor binding and the second messenger generation. Furthermore, the antagonistic interactions between these two receptors induced by low concentrations of CPA might have a different manner with those induced by high concentrations of CPA.

Topics: 2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine; Adenosine; Adenosine A1 Receptor Agonists; Adenosine A1 Receptor Antagonists; Alkaline Phosphatase; Benzazepines; Cell Line; Cyclic AMP Response Element-Binding Protein; DNA, Complementary; Dopamine Agonists; Dopamine Antagonists; Humans; Receptor, Adenosine A1; Receptors, Dopamine D1; Transfection; Xanthines

We studied the wild-type human adenosine A1 receptor and three mutant receptors, in which the glycine at position 14 had been changed into an alanine, a leucine, or a threonine residue. All receptors were characterized in radioligand binding experiments, the wild-type and the Gly14Thr mutant receptor in greater detail. Both receptors were allosterically modulated by sodium ions and PD81,723 (2-amino-4,5-dimethyl-3-thienyl-[3(trifluoromethyl)-phenyl]methanone), although in a different way. All mutant receptors appeared to be spontaneously or "constitutively" active in a [35S]GTPgammaS binding assay, the first demonstration of the existence of such CAM (constitutively active mutant) receptors for the adenosine A1 receptor. The Gly14Thr mutant receptor was also constitutively active in another functional assay, i.e., the inhibition of forskolin-induced cAMP production in intact cells. Importantly, this mutant displayed a peculiar "locked-on" phenotype, i.e., neither agonist nor inverse agonist was capable of modulating the basal activity in both the GTPgammaS and the cAMP assay, unlike the wild-type and the two other mutant receptors.

Topics: Adenosine; Animals; Binding, Competitive; Cell Line; Cell Membrane; Chlorocebus aethiops; Colforsin; COS Cells; Cyclic AMP; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Humans; Kinetics; Mutation; Plasmids; Radioligand Assay; Receptor, Adenosine A1; Sodium; Sulfur Radioisotopes; Theophylline; Thiophenes; Transfection; Tritium; Xanthines

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

Topics: Adenosine; Adenosine A1 Receptor Agonists; Adenosine A1 Receptor Antagonists; Adenosine Deaminase; Calcium; Cell Line; Cell Membrane; Embryo, Mammalian; Humans; Kidney; Radioligand Assay; Receptor, Adenosine A1; Signal Transduction; Xanthines

Intravenous adenosine in-vivo was shown to potentiate the effects of non-depolarizing neuromuscular blocking agents. This study aimed to determine whether adenosine A1-receptors mediated this potentiation. The authors investigated the effects of intravenous adenosine, N6-cyclopentyladenosine, specific A1-receptor agonist, and 8-cyclopentyl-1,3-dipropylxanthine, specific A1-receptor antagonist, on neuromuscular block by vecuronium, in in-vivo rat sciatic nerve-tibialis anterior preparations. In the presence of 50% steady state block by vecuronium, adenosine, and N6-cyclopentyladenosine caused similar degree of depressions of twitch tension. Twitch tension returned to its pre-injection value more rapidly when 8-cyclopentyl-1,3-dipropylxanthine was given at the maximal block than when it was allowed to recover spontaneously. It was concluded that in in-vivo adenosine potentiated the neuromuscular effects of vecuronium through adenosine A1-receptors in rats.

Topics: Adenosine; Adenosine A1 Receptor Agonists; Adenosine A1 Receptor Antagonists; Animals; Drug Interactions; Drug Synergism; Male; Neuromuscular Junction; Neuromuscular Nondepolarizing Agents; Rats; Rats, Sprague-Dawley; Receptor, Adenosine A1; Sciatic Nerve; Synaptic Transmission; Vecuronium Bromide; Xanthines

Adenosine A1 and A2A receptor agonists and antagonists have been reported to alter learning and memory. The aim of our study was to investigate the involvement of adenosinergic system in memory retrieval into posterior cingulate cortex (PCC) of Wistar rats. To clarify this question, we tested specifics agonist and antagonists of adenosine A1 and A2A receptors in rats submitted to a one-trial inhibitory avoidance task. The stimulation of adenosine A1 and A2A receptors by CPA and CGS21680, respectively, impaired memory retrieval for inhibitory avoidance task, into PCC. These findings provide behavioral evidence for the role of adenosinergic system in the memory retrieval into PCC.

Topics: Adenosine; Animals; Avoidance Learning; Cerebral Cortex; Exploratory Behavior; Gyrus Cinguli; Male; Memory; Microinjections; Phenethylamines; Purinergic Agonists; Purinergic Antagonists; Rats; Rats, Wistar; Receptor, Adenosine A1; Receptor, Adenosine A2A; Triazines; Triazoles; Xanthines

The present study was performed to clarify the mechanism of change in intraocular pressure by 2-(1-hexyn-1-yl)adenosine (2-H-Ado), a selective adenosine A2 receptor agonist, in rabbits. 2-H-Ado (0.1%, 50 microl)-induced ocular hypertension (E(max): 7.7 mm Hg) was inhibited by an adenosine A2A receptor antagonist 1,3,7-trimethyl-8-(3-chlorostyryl)xanthine, ATP-sensitive K+ channel blocker glibenclamide or 5-hydroxydecanoic acid, but not by an adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine, an adenosine A2B receptor antagonist alloxazine or a cyclooxygenase inhibitor indomethacin. The outflow facility induced by 2-H-Ado seems to be independent of increase in intraocular pressure or ATP-sensitive K+ channel. In contrast, the recovery rate in intraocular pressure decreased by hypertonic saline was accelerated by 2-H-Ado, and this response was dependent on ATP-sensitive K+ channel. These results suggest that 2-H-Ado-induced ocular hypertension is mediated via K+ channel opening through adenosine A2A receptor, and this is probably due to aqueous formation, but independent of change in outflow facility or prostaglandin production.

Topics: Adenosine; Adenosine A2 Receptor Agonists; Adenosine A2 Receptor Antagonists; Alkynes; Animals; Antihypertensive Agents; Caffeine; Decanoic Acids; Glyburide; Hydroxy Acids; Hypotonic Solutions; Intraocular Pressure; Male; Ocular Hypertension; Phenethylamines; Pinacidil; Potassium Channel Blockers; Potassium Channels; Rabbits; Receptor, Adenosine A2A; Sodium Chloride; Time Factors; Xanthines

To study the effect of allosteric modulators on the internalization of human adenosine A(1) receptors, the receptor was equipped with a C-terminal yellow fluorescent protein tag. The introduction of this tag did not affect the radioligand binding properties of the receptor. CHO cells stably expressing this receptor were subjected during 16 h to varying concentrations of the agonist N(6)-cyclopentyladenosine (CPA) in the absence or presence of 10 microM of the allosteric enhancer PD 81,723 ((2-amino-4,5-dimethyl-3-thienyl)-[3-(trifluoromethyl)phenyl]methanone) or the allosteric inhibitor SCH-202676 (N-(2,3-diphenyl-1,2,4-thiadiazol-5(2H)-ylidene)methanamine). CPA itself was able to internalize 25% and 40% of the receptors at a concentration of 400 nM or 4 muM, respectively. Addition of either PD 81,723 or SCH-202676 alone had no effect on internalization. However, with PD 81,723 a slight amount of internalization was obtained already at 40 nM of CPA and at 400 nM CPA 59% of the receptors internalized. SCH-202676 on the other hand effectively prevented CPA-induced internalization of the receptor.

Topics: Adenosine; Allosteric Regulation; Animals; Binding, Competitive; CHO Cells; Cricetinae; Cricetulus; Dose-Response Relationship, Drug; Endocytosis; Humans; Luminescent Proteins; Microscopy, Confocal; Radioligand Assay; Receptor, Adenosine A1; Recombinant Fusion Proteins; Thiadiazoles; Thiazoles; Thiophenes; Transfection; Tritium; Xanthines

Adenosine acts as an important protector of ischemic myocardium through coronary vasodilation and the depression of cardiac contractility. The protective effect of adenosine may partly relate to the cardiac hormone atrial natriuretic peptide (ANP). The aim of the present study was to investigate the effects of adenosine and the adenosine receptor subtype on atrial hemodynamics and ANP release using isolated perfused beating rat atria. Adenosine, a nonselective adenosine receptor agonist, increased the ANP release with negative inotropism in a dose-dependent manner. Adenosine-stimulated ANP release was attenuated by a selective A1 antagonist but not A(2A) antagonist or A3 antagonist. The order of potency of the various agonists for the ANP release was A1 agonists>>A3 agonist=adenosine>A(2A) agonist. The order of potency for the negative inotropy was A1 agonists>adenosine=A(2A) agonist>A3 agonist. The negative inotropism and ANP release by a specific A1 agonist (N6-cyclopentyl-adenosine) were also attenuated by A1 antagonist but not A(2A) antagonist or A3 antagonist. Treatment with A1 agonist resulted in a decrease of cAMP contents in atria and perfusates. The agonist-stimulated ANP release was significantly attenuated in the presence of forskolin, isoproterenol 8-Br-cAMP, or an adenylyl cyclase inhibitor. These results suggest that the A1 receptor subtype is responsible for the adenosine-induced ANP release and negative inotropism through adenylyl cyclase-cAMP pathway.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Adenosine; Adenosine A1 Receptor Agonists; Adenosine A1 Receptor Antagonists; Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Animals; Atrial Function; Atrial Natriuretic Factor; Colforsin; Cyclic AMP; Enzyme Inhibitors; In Vitro Techniques; Isoproterenol; Male; Myocardial Contraction; Myocardium; Rats; Rats, Sprague-Dawley; Receptor, Adenosine A1; Xanthines

This study investigated the effect of adenosine in the forced swimming test (FST) and the tail suspension test (TST) in mice, and the contribution of adenosine A1 and A2A receptors to adenosine's antidepressant-like effect. The immobility time in the FST was reduced by adenosine given either by i.p. (5-10 mg/kg) or i.c.v. (0.01-10 microg/site) route. Adenosine (1-10 mg/kg, i.p.) also produced an antidepressant-like effect in the TST. No treatment affected locomotion in an open-field. The anti-immobility effect of adenosine (10 mg/kg, i.p.) in the FST was prevented by i.p. pretreatment of mice with caffeine (3 mg/kg), DPCPX (2 mg/kg) and ZM241385 (1 mg/kg). CHA (0.05 mg/kg, i.p.) and DPMA (1-5 mg/kg, i.p.) also produced an antidepressant-like effect in the FST. This is the first report of an antidepressant-like effect of adenosine in mice, apparently mediated through an interaction with A1 and A2A receptors.

Topics: Adenosine; Animals; Antidepressive Agents; Caffeine; Depressive Disorder; Disease Models, Animal; Female; Male; Mice; Motor Activity; Receptor, Adenosine A1; Receptor, Adenosine A2A; Stress, Psychological; Triazines; Triazoles; Xanthines

Ischemic preconditioning models have been characterized in brain, heart, and other tissues, and previous pharmacologic studies have suggested an involvement of adenosine and ATP dependent potassium (KATP) channels in such tolerance phenomena. This question was reexamined in a reproducible gerbil model in which the duration of ischemic depolarization defined the severity of preconditioning and test insults. Agents studied were glibenclamide, a blocker of KATP channels; 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), an adenosine A1 receptor antagonist; and N6-cyclopentyladenosine (CPA), an A1 agonist. Intraventricular glibenclamide injections aggravated neuron damage after brief priming insults, in parallel with a dose-dependent prolongation of ischemic depolarization. However, the depolarization thresholds for ischemic neuronal injury were identical in vehicle- and glibenclamide-treated animals, and glibenclamide did not affect preconditioning when equivalent insult severity was maintained during priming insults. Neither DPCPX nor CPA had any effect on the onset or duration of depolarization after intraperitoneal injection in this model, and neither drug affected neuron damage. In the case of CPA, it was necessary to maintain temperature for 4 to 6 hours of recirculation to avoid significant confounding hypothermia. These results fail to support a direct involvement of A1 receptors or KATP channels during early stages in the development of ischemic tolerance in vivo, and emphasize the need for robust, well-controlled, and quantitative models in such studies.

Topics: Adenosine; Adenosine A1 Receptor Agonists; Adenosine A1 Receptor Antagonists; Adenosine Triphosphate; Animals; Anti-Arrhythmia Agents; Female; Gerbillinae; Glyburide; Hippocampus; Ischemic Preconditioning; Membrane Potentials; Potassium Channels; Receptor, Adenosine A1; Xanthines

Adenosine has powerful inhibitory effects in the central nervous system. In this study, we aim to understand how adenosine controls the progression of seizure-like events (SLEs) in a seizure-prone region of the brain, the entorhinal cortex. We chose to use a low Mg(2+) model of epilepsy in an in vitro slice preparation where, in the entorhinal cortex, SLEs progress into a type of epileptiform activity called late recurrent discharges (LRDs) that bear resemblance to status epilepticus. Adenosine, acting via its A1 receptor, exerted powerful inhibitory effects to prevent the spontaneous progression to LRDs while the potent A1 receptor antagonist, DPCPX, accelerated the progression in a concentration dependent manner. The spontaneous progression from SLEs to LRDs was associated with a decline in total cellular ATP levels and studies with metabolic inhibitors indicated a key role for the production of endogenous adenosine from ATP. We therefore hypothesise that when ATP becomes rate limiting, extracellular adenosine levels fall, the normal inhibitory brake is removed and the progression from SLEs to LRDs or status epilepticus-like activity can ensue. Moreover, under these conditions, inhibition of the adenine nucleotide salvage pathways reversed the status epilepticus-like activity. Our findings suggest a powerful role for adenosine for the control of the progression to status epilepticus-like activity in an epilepsy model that is refractory to most anti-epileptic drugs. On this basis, manipulation of adenine nucleotide metabolism may represent a potential therapeutic approach for the treatment of status epilepticus.

Topics: Adenosine; Adenosine A1 Receptor Antagonists; Adenosine Triphosphate; Analysis of Variance; Animals; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Inhibitors; Female; Hippocampus; In Vitro Techniques; Rats; Rats, Wistar; Receptor, Adenosine A1; Status Epilepticus; Xanthines

A current response induced by superfusing adenosine was examined in substantia gelatinosa (SG) neurons of adult rat spinal cord slices by using the whole-cell patch-clamp technique. In 78% of the neurons examined, adenosine induced an outward current at -70 mV [18.8 +/- 1.1 pA (n = 98) at 1mM] in a dose-dependent manner (EC(50) = 177 microM). A similar current was induced by A(1) agonist N(6)-cyclopentyladenosine (1 microM), whereas A(1) antagonist 8-cyclopentyl-1,3-dipropylxanthine (1 microM) reversed the adenosine action. The adenosine current reversed its polarity at a potential being close to the equilibrium potential for K(+), and was attenuated by Ba(2+) (100 microM) and 4-aminopyridine (5mM) but not tetraethylammonium (5mM). The adenosine current was enhanced in duration by equilibrative nucleoside-transport (rENT1) inhibitor S-(4-nitrobenzyl)-6-thioinosine (1 microM) and adenosine deaminase (ADA) inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine (1 microM), and slowed in falling phase by adenosine kinase (AK) inhibitor iodotubercidine (1 microM). We conclude that a Ba(2+)- and 4-aminopyridine-sensitive K(+) channel in SG neurons is opened via the activation of A(1) receptors by adenosine whose level is possibly regulated by rENT1, adenosine deaminase and adenosine kinase. Considering that intrathecally-administered adenosine analogues produce antinociception, the regulatory systems of adenosine may serve as targets for antinociceptive drugs.

Topics: 4-Aminopyridine; Adenosine; Animals; Barium; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Inhibitors; Equilibrative Nucleoside Transport Proteins; Excitatory Postsynaptic Potentials; In Vitro Techniques; Male; Membrane Potentials; Patch-Clamp Techniques; Posterior Horn Cells; Potassium; Potassium Channel Blockers; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Rats; Rats, Sprague-Dawley; Substantia Gelatinosa; Xanthines

We studied fusion proteins between the human adenosine A1 receptor and different 351Cys-mutated G(i1) alpha-subunits (A1-Gialpha) with respect to two important concepts in receptor pharmacology, i.e. allosteric modulation and constitutive activity/inverse agonism. The aim of our study was twofold. We first analysed whether such fusion products are still subject to allosteric modulation, and, secondly, we investigated the potential utility of the fusion proteins to study constitutive receptor activity. We determined the pharmacological profile of nine different A1-Gialpha fusion proteins in radioligand binding studies. In addition, we performed [35S]GTPgammaS binding experiments to study receptor and G protein activation of selected A1-Gialpha fusion proteins. Compared to unfused adenosine A1 receptors, the affinity of N6-cyclopentyladenosine (CPA) at wild-type A1-Gialpha fusion proteins (351Cys) increased more than eightfold, while the affinity of 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) did not change significantly. Furthermore, we showed that the allosteric enhancer of agonist binding, PD81,723 (2-amino-4,5-dimethyl-3-thienyl-[3-(trifluoromethyl)-phenyl]methanone), elicited similar effects on ligand binding; i.e. CPA binding to the A1-Gialpha fusion proteins was enhanced, whereas the affinity of DPCPX was hardly affected. Moreover, sodium ions were unable to decrease agonist binding to the majority of the A1-Gialpha fusion proteins, presumably because they exhibit their effect through uncoupling of the R-G complex. From [35S]GTPgammaS binding experiments, we learned that all the A1-Gialpha fusion proteins tested had a higher basal receptor activity than the unfused adenosine A1 receptor, thereby providing improved conditions to observe inverse agonism. Moreover, the maximal CPA-induced stimulation of basal [35S]GTPgammaS binding was increased for the five A1-Gialpha fusion proteins tested, whereas the inhibition induced by 8-cyclopentyltheophylline (CPT) was more pronounced at 351Cys, 351Ile, and 351Val A1-Gialpha fusion proteins. Thus, the maximal receptor (de)activation depended on the amino acid at position 351 of the Gi alpha-subunit. In conclusion, A1-Gialpha fusion proteins, especially with 351Cys and 351Ile, can be used as research tools to investigate inverse agonism, due to their increased readout window in [35S]GTPgammaS binding experiments.

Topics: Adenosine; Allosteric Site; Animals; Binding, Competitive; Cell Membrane; COS Cells; Cysteine; Dose-Response Relationship, Drug; GTP-Binding Protein alpha Subunits, Gi-Go; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Mutation; Radioligand Assay; Receptor, Adenosine A1; Recombinant Fusion Proteins; Sodium Chloride; Sulfur Radioisotopes; Thiophenes; Transfection; Tritium; Xanthines

This study was performed to determine whether intravitreal or intravenous adenosine can alter the microcirculation in the optic nerve head (ONH) of rabbits. Capillary blood flow in the ONH was measured serially with a laser speckle tissue analyser for 2 hr after the intravitreal (0.1, 1.0 and 10 nmol) or intravenous (0.2 and 0.6 mg kg(-1)min) injections of adenosine. In addition, the effect of specific adenosine A(1) and A(2a) antagonists and an adenosine triphosphate (ATP)-sensitive potassium (K(ATP)) channel blockers on the adenosine-induced changes on the ONH blood flow was analysed. Intravitreal adenosine increased the capillary blood flow in the ONH in a dose-dependent manner, while intravenous adenosine had no effect. Co-administration of the specific adenosine A(1) receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 10 nmol) significantly suppressed (P=0.006, ANOVA) the increase in the ONH blood flow induced by adenosine (10 nmol). The specific A(2a) receptor antagonist, 8-(3-chlorostyryl) caffeine (CSC, 10 nmol), had a weak effect in inhibiting the increase but the change was not significant (P=0.08, ANOVA). Both specific A(1) and A(2a) receptor agonists, N(6)-cyclopentyladenosine (CPA, 10 nmol) and 2-p-(2-carboxyethyl) phenethyl-amino-5'-N-ethylcarboxamidoadenosine (CGS-21680, 10 nmol), increased the ONH tissue blood flow (P<0.01, ANOVA). Glibenclamide (10 nmol), a selective K(ATP) channels antagonist, suppressed the increase of ONH blood flow induced by 10 nmol adenosine significantly (P<0.001, ANOVA). On the other hand, 10 nmol of 8-Br-cAMP, a cAMP analog, failed to enhance the capillary blood flow in the ONH. These results indicate that adenosine increases the capillary blood flow in the ONH of rabbits, and it acts through A(1) and A(2a) receptors from the ablumenal side where pericytes are located. Activation of K(ATP) channels is strongly related to the mechanism of adenosine-induced increase in ONH blood flow, while the participation of adenylate cyclase is less likely.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Adenosine; Adenosine A1 Receptor Antagonists; Adenylyl Cyclase Inhibitors; Animals; Caffeine; Capillaries; Dose-Response Relationship, Drug; Glyburide; Injections; Injections, Intravenous; Optic Disk; Phenethylamines; Potassium Channels; Purinergic P1 Receptor Agonists; Purinergic P2 Receptor Antagonists; Rabbits; Regional Blood Flow; Stimulation, Chemical; Vasodilation; Vitreous Body; Xanthines

The involvement of adenosine A(1) and A(2A) receptors in the motor effects of caffeine is still a matter of debate. In the present study, counteraction of the motor-depressant effects of the selective A(1) receptor agonist CPA and the A(2A) receptor agonist CGS 21680 by caffeine, the selective A(1) receptor antagonist CPT, and the A(2A) receptor antagonist MSX-3 was compared. CPT and MSX-3 produced motor activation at the same doses that selectively counteracted motor depression induced by CPA and CGS 21680, respectively. Caffeine also counteracted motor depression induced by CPA and CGS 21680 at doses that produced motor activation. However, caffeine was less effective than CPT at counteracting CPA and even less effective than MSX-3 at counteracting CGS 21680. On the other hand, when administered alone in habituated animals, caffeine produced stronger motor activation than CPT or MSX-3. An additive effect on motor activation was obtained when CPT and MSX-3 were coadministered. Altogether, these results suggest that the motor-activating effects of acutely administered caffeine in rats involve the central blockade of both A(1) and A(2A) receptors. Chronic exposure to caffeine in the drinking water (1.0 mg/ml) resulted in tolerance to the motor effects of an acute administration of caffeine, lack of tolerance to amphetamine, apparent tolerance to MSX-3 (shift to the left of its 'bell-shaped' dose-response curve), and true cross-tolerance to CPT. The present results suggest that development of tolerance to the effects of A(1) receptor blockade might be mostly responsible for the tolerance to the motor-activating effects of caffeine and that the residual motor-activating effects of caffeine in tolerant individuals might be mostly because of A(2A) receptor blockade.

Topics: Adenosine; Amphetamine; Animals; Behavior, Animal; Caffeine; Central Nervous System Stimulants; Dose-Response Relationship, Drug; Drug Administration Schedule; Drug Interactions; Male; Motor Activity; Phenethylamines; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Radioligand Assay; Rats; Rats, Sprague-Dawley; Receptor, Adenosine A2A; Receptors, Purinergic P1; Theophylline; Time Factors; Triazines; Triazoles; Tritium; Xanthines

Compelling clinical evidence implicates the potential role of adenosine in development of airway hyperresponsiveness and suggests involvement of pulmonary sensory receptors. This study was carried out to determine the effect of a low dose of adenosine infusion on sensitivity of pulmonary C-fiber afferents in anesthetized open-chest rats. Infusion of adenosine (40 microg x kg-1x min-1 i.v. for 90 s) mildly elevated baseline activity of pulmonary C fibers. However, during adenosine infusion, pulmonary C-fiber responses to chemical stimulants and lung inflation (30 cmH2O tracheal pressure) were markedly potentiated; e.g., the response to right atrial injection of capsaicin (0.25 or 0.5 microg/kg) was increased by more than fivefold (change in fiber activity = 2.64 +/- 0.67 and 16.27 +/- 3.11 impulses/s at control and during adenosine infusion, n = 13, P < 0.05), and this enhanced response returned to control in approximately 10 min. The potentiating effect of adenosine infusion was completely blocked by pretreatment with 8-cyclopentyl-1,3-dipropylxanthine (100 microg/kg), a selective antagonist of the adenosine A1 receptor, but was not affected by 3,7-dimethyl-1-propargylxanthine (1 mg/kg), an A2-receptor antagonist, or 3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1,4-(+/-)-dihydropyridine-3,5-dicarboxylate (2 mg/kg), an A3-receptor antagonist. This potentiating effect was also mimicked by N6-cyclopentyladenosine (0.25 microg x kg-1 x min-1 for 90 s), a selective agonist of the adenosine A1 receptor. In conclusion, our results showed that infusion of adenosine significantly elevated the sensitivity of pulmonary C-fiber afferents in rat lungs and that this potentiating effect is likely mediated through activation of the adenosine A1 receptor.

Topics: Adenosine; Anesthesia; Animals; Body Weight; Bradycardia; Bronchial Hyperreactivity; Capsaicin; Dose-Response Relationship, Drug; Infusions, Intravenous; Lung; Male; Nerve Fibers, Unmyelinated; Rats; Rats, Sprague-Dawley; Receptor, Adenosine A1; Receptors, Purinergic P1; Xanthines

Adenosine is a neuromodulator that has been proposed to be a major endogenous anticonvulsant acting via A1 receptors. We tested if implementation of kindling through stimulation of the amygdala affected A1 receptor-mediated neuromodulation in hippocampal slices taken from rats 4 weeks after the last stage 5 seizure. The A1 receptor agonist, N6-cyclopentyladenosine (CPA) (6-100 nm), inhibited field excitatory postsynaptic potential (fEPSP) slope with an EC50 of 19.1-19.5 nm in control and sham-operated rats, but was less potent in kindled rats (EC50 = 42.7 nm). This might result from a decreased number of A1 receptors in hippocampal nerve terminal membranes, because A1 receptor immunoreactivity decreased by 28 +/- 3% and the binding density of the A1 receptor agonist [3H]R-PIA decreased from 1702 +/- 64 to 962 +/- 78 fmol/mg protein in kindled compared with control rats. The tonic inhibition of hippocampal synaptic transmission by endogenous adenosine was also lower in kindled rats, because A1 receptor blockade with 50 nm 1,3-dipropyl-8-cyclopentyladenosine (DPCPX) enhanced fEPSP slope by 23 +/- 3% and theta-burst-induced long-term potentiation by 94 +/- 4% in control rats but was virtually devoid of effects in kindled rats. The evoked release of adenosine from hippocampal slices or nerve terminals was 56-71% lower in kindled rats probably due to the combined decrease in the capacity of adenosine transporters and decreased release of adenosine 5'-triphosphate (ATP), which was partially compensated by a higher extracellular catabolism of ATP into adenosine in kindled rats. These results indicate that, although adenosine might inhibit the onset of epileptogenesis, once kindling is installed, the efficiency of the adenosine inhibitory system is impaired.

Topics: Adenosine; Adenosine Triphosphate; Animals; Blotting, Western; Electrophysiology; Excitatory Postsynaptic Potentials; Hippocampus; Immunohistochemistry; Kindling, Neurologic; Long-Term Potentiation; Male; Neuroprotective Agents; Organ Culture Techniques; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Rats; Rats, Wistar; Receptors, Purinergic P1; Synaptic Transmission; Synaptosomes; Xanthines

Chronic treatment with opioids induces adaptations in neurons leading to tolerance and dependence. Studies have implicated the midbrain periaqueductal gray (PAG) in the expression of many signs of withdrawal. Patch-clamp recording techniques were used to examine whether augmentation of adenylyl cyclase signalling produces hyperexcitation in GABAergic nerve terminals within the mouse PAG. Both the rate of mIPSCs and the amplitude of evoked IPSCs during naloxone-precipitated withdrawal was profoundly enhanced in chronically morphine treated mice, compared to vehicle treated controls, in the presence but not the absence an adenosine A(1) receptor antagonist DPCPX. Enhanced GABAergic transmission in the presence of DPCPX was abolished by blocking protein kinase A. Inhibitors of cAMP transport, phosphodiesterase and nucleotide transport mimicked the effect of DPCPX. Coupling efficacy of micro-receptors to presynaptic inhibition of GABA release was increased in dependent mice in the presence of DPCPX. The increased coupling efficacy was abolished by blocking protein kinase A, which unmasked an underlying micro-receptor tolerance. These findings indicate that enhanced adenylyl cyclase signalling following chronic morphine treatment produces (1) GABAergic terminal hyperexcitability during withdrawal that is retarded by a concomitant increase in endogenous adenosine, and (2) enhanced micro-receptor coupling to presynaptic inhibition that overcomes an underlying tolerance.

Topics: Action Potentials; Adenosine; Affinity Labels; Animals; Colforsin; Cyclic AMP; Dipyridamole; Dose-Response Relationship, Drug; Drug Interactions; Enkephalins; Enzyme Inhibitors; gamma-Aminobutyric Acid; In Vitro Techniques; Isoquinolines; Male; Mesencephalon; Mice; Mice, Inbred C57BL; Morphine; Morphine Dependence; Naloxone; Narcotic Antagonists; Narcotics; Neural Inhibition; Neurons; Patch-Clamp Techniques; Periaqueductal Gray; Probenecid; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Substance Withdrawal Syndrome; Sulfonamides; Synaptic Transmission; Thioinosine; Time Factors; Uricosuric Agents; Vasodilator Agents; Xanthines

Hyperalgesia and altered activities of enzymes involved in nucleotide hydrolysis are observed after exposure to repeated restraint in rats. Here, we investigated the effect of an adenosine A(1) receptor agonist, N(6)-cyclopentyladenosine (CPA, 3.35 mg/kg, i.p.), adenosine A(1) receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 0.8 mg/kg, i.p.) as well the effect of an adenosine reuptake blocker, dipyridamole (5 mg/kg, i.p.), on nociception in chronically stressed and control rats. We repeatedly submitted rats to restraint for 40 days. Nociception was assessed with a tail-flick apparatus. The control group presented increased tail-flick latencies after administration of CPA and dipyridamole, but this effect was not observed in the stressed group. DPCPX by itself had no effect on nociception. The analgesic effect of CPA and dipyridamole observed in the control group was reverted by DPCPX. These results indicate the involvement of adenosine A(1) receptor in the antinociception observed in control animals and suggest that the pain signaling induced by chronic stress presents a different modulation involving the adenosinergic system.

Topics: Adenosine; Animals; Chronic Disease; Hyperalgesia; Male; Pain Measurement; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Rats; Rats, Wistar; Receptors, Purinergic P1; Stress, Physiological; Xanthines

Adenosine is known to act as a neuromodulator by suppressing synaptic transmission in the central and peripheral nervous system. Both the release of adenosine within the small intestine and the presence of adenosine receptors on enteric neurons have been demonstrated. The aim of the present study was to characterize a possible involvement of adenosine receptors in the modulation of the myenteric reflex. The experiments were carried out on ileum segments 10 cm in length incubated in an single chambered organ bath, and the reflex response was initiated by electrical stimulation (ES).. ES caused an ascending contraction and a descending relaxation followed by a contraction. All motility responses to ES were completely blocked by tetrodotoxin, indicating that they are mediated by neural mechanisms. Atropine blocked the contractile effects, whereas the descending relaxation was significantly increased. The A1 receptor agonist N6-cyclopentyladenosine increased the ascending contraction, whereas the ascending contraction was reduced by the A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine. Activation of the A1 receptor further reduced the descending relaxation and the latency of the peristaltic reflex. The A2B receptor antagonist alloxazine increased ascending contraction, whereas descending relaxation remained unchanged. For A2A and A3 receptors, we found contradictory effects of the agonists and antagonists, thus there is no clear physiological role for these receptors at this time.. This study suggests that the myenteric ascending and descending reflex response of the rat small intestine is modulated by release of endogenous adenosine via A1 receptors.

Topics: Adenosine; Animals; Dose-Response Relationship, Drug; Electric Stimulation; Flavins; Gastrointestinal Motility; Ileum; In Vitro Techniques; Male; Muscarinic Antagonists; Muscle Contraction; Neural Inhibition; Neurons; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Rats; Rats, Wistar; Receptors, Purinergic P1; Reflex; Tetrodotoxin; Xanthines

Male Wistar rats were bilaterally implanted with indwelling cannulae in the caudal region of the posterior cingulate cortex. After recovery, animals were trained in a step-down inhibitory avoidance task (3.0-s, 0.4-mA foot shock) and received, immediately after training, a 0.5-microl infusion of the adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA; 1, 50 or 100 nM) or of the adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 1, 25 or 50 nM). Animals were tested twice, 1.5 h and, again, 24 h after training, in order to examine the effects of these agents on short- and long-term memory, respectively. Only 50-nM DPCPX was effective in altering memory, promoting a facilitation. These results suggest that adenosine A1 receptors in the posterior cingulate cortex inhibit memory consolidation in a way that their blockade facilitates memory for inhibitory avoidance in rats.

Topics: Adenosine; Animals; Avoidance Learning; Dose-Response Relationship, Drug; Gyrus Cinguli; Male; Memory; Purinergic P1 Receptor Antagonists; Rats; Receptors, Purinergic P1; Retention, Psychology; Xanthines

Intracellular recordings were made in rat brain slice preparations containing pyramidal cells of the associative frontal cortex in order to characterize the action of selective adenosine A(1) and A(3) receptor ligands on synaptic neurotransmission. The selective A(1) receptor agonist N(6)-cyclopentyladenosine (CPA) inhibited concentration-dependently the excitatory postsynaptic potentials (PSPs) which were evoked by focal electrical stimulation. The CPA-mediated inhibition was blocked by 1, 3-dipropyl-8-cyclopentylxanthine (DPCPX), a highly A(1) receptor-selective antagonist. The A(3) receptor agonist N(6)-(3-iodobenzyl)-adenosine-5'-N-methylcarboxamide (IB-MECA) inhibited concentration-dependently the evoked PSPs while the A(1) receptors were blocked continuously by DPCPX. Under these conditions, the A(3) receptor antagonist 9-chloro-2-(2-furanyl)-5-[(phenylacetyl)amino]-1,2,4-triazolo[1, 5-c]quinazoline (MRS 1220) did not influence the PSPs but inhibited completely the effect of IB-MECA. The inhibitory effect of IB-MECA was unaffected by DPCPX. CPA additionally inhibited the PSPs when applied after IB-MECA. Pharmacological dissociation of the N-methyl-D-aspartate (NMDA) and non-NMDA receptor components of the PSPs showed that CPA as well as IB-MECA reduced both. We conclude that adenosine A(1) and A(3) receptors are present on cortical pyramidal cells and involved in the inhibition of excitatory neurotransmission. Our results indicate no interplay between the two receptor subtypes. The separate inhibition may become particularly evident in situations where there are high levels of endogenously released adenosine, as seen in hypoxia.

Topics: Adenosine; Animals; Cerebral Cortex; Drug Interactions; Electric Stimulation; Female; In Vitro Techniques; Male; Neurons; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Quinazolines; Rats; Rats, Wistar; Receptor, Adenosine A3; Receptors, Purinergic P1; Synaptic Transmission; Triazoles; Xanthines

Nonselective adenosine (ADO) receptor antagonists block hypoxia-induced bradycardia and hypertension in fetal sheep. This study was designed to determine the ADO receptor subtype that is involved in these cardiovascular responses. In chronically catheterized fetal sheep (>0.8 term), fetal hypoxemia was induced by having the ewe breathe a hypoxic gas mixture (9% O(2)-3% CO(2)-88% N(2)) for 1 h. Intra-arterial infusion of ZM-241385, an antagonist highly selective for ADO A(2A) receptors, to eight fetuses during normoxia significantly increased mean arterial pressure (MAP) from 42.5 +/- 2.0 to 46.1 +/- 2.0 mmHg without altering heart rate (HR). Infusion of a selective antagonist of ADO A(1) receptors [1, 3-dipropyl-8-cyclopentylxanthine (DPCPX)] elevated MAP and HR only after the infusion was terminated, although administration of the vehicle for ZM-241385 or DPCPX had no effect on MAP or HR. Isocapnic hypoxia with infusion of DPCPX or the vehicle for DPCPX or ZM-241385 produced a transient fall in HR, a rise in MAP, and a decrease in plasma volume. In contrast, ADO A(2A) receptor blockade abolished the hypoxia-induced bradycardia and hypertension and blunted the decline in plasma volume. We conclude that fetal ADO A(2A) receptors: 1) modulate AP during normoxia, and 2) mediate cardiovascular responses during acute O(2) deficiency.

Topics: Adenosine; Animals; Blood Gas Analysis; Blood Pressure; Female; Fetal Hypoxia; Heart Rate; Hydrogen-Ion Concentration; Phenethylamines; Pregnancy; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Receptor, Adenosine A2A; Receptors, Purinergic P1; Sheep; Triazines; Triazoles; Xanthines

Cl(-) currents induced by cell swelling were characterized in an immortalized cell line (DC1) derived from rabbit distal bright convoluted tubule by the whole cell patch-clamp techniques and by (125)I(-) efflux experiments. Exposure of cells to a hypotonic shock induced outwardly rectifying Cl(-) currents that could be blocked by 0.1 mM 5-nitro-2-(3-phenylpropyl-amino)benzoic acid, 1 mM DIDS, and by 1 mM diphenylamine-2-carboxylate. (125)I(-) efflux experiments showed that exposure of the monolayer to a hypotonic medium increased (125)I(-) loss. Preincubation of cells with LaCl(3) or GdCl(3) prevented the development of the response. The addition of 10 microM adenosine to the bath medium activated outwardly rectifying whole cell currents similar to those recorded after hypotonic shock. This conductance was inhibited by the A(1)-receptor antagonist 8-cyclopentyl-1,3-diproxylxanthine (DPCPX), LaCl(3), or GdCl(3) and was activated by GTPgammaS. The selective A(1)-receptor agonist N(6)-cyclopentyladenosine (CPA) mimicked the effect of hypotonicity on (125)I(-) efflux. The CPA-induced increase of (125)I(-) efflux was inhibited by DPCPX and external application of LaCl(3) or GdCl(3). Adenosine also enhanced Mn(2+) influx across the apical membrane. Overall, the data show that DC1 cells possess swelling- and adenosine-activated Cl(-) conductances that share identical characteristics. The activation of both conductances involved Ca(2+) entry into the cell, probably via mechanosensitive Ca(2+) channels. The effects of adenosine are mediated via A(1) receptors that could mediate the purinergic regulation of the volume-sensitive Cl(-) conductance.

Topics: Adenosine; Animals; Cell Line; Cell Membrane; Cell Size; Chloride Channels; Gadolinium; Guanosine 5'-O-(3-Thiotriphosphate); Hypotonic Solutions; Iodine Radioisotopes; Kidney Tubules; Lanthanum; Manganese; Membrane Potentials; Patch-Clamp Techniques; Rabbits; Receptors, Purinergic P1; Recombinant Proteins; Transfection; Xanthines

Activation of adenosine A(1) receptors by endogenous adenosine plays a neuroprotective role under various pathophysiological conditions including hypoxia. Intracellular recordings were made in rat pyramidal cells of the somatosensory cortex. Hypoxia (5 min) induced a membrane depolarization and a decrease of input resistance. The A(1) receptor agonist N(6)-cyclopentyladenosine (CPA, 100 microM) reversibly inhibited the hypoxic depolarization. The inhibition was also present after blockade of the A(2A), A(2B) and A(3) receptor subtypes by selective antagonists. CPA had no effect on the hypoxic decrease of input resistance. 1,3-Dipropyl-8-cyclopentylxanthine (DPCPX), a selective A(1) receptor antagonist, which did not alter hypoxic depolarization when given alone abolished the inhibitory effect of CPA. Neither CPA nor DPCPX influenced membrane potential or apparent input resistance under normoxic conditions. The novel pyrimidoindole (R)-9-(1-methylbenzyl)-2-(4'-pyridyl)-9H-pyrimido[4,5-b]indole-4-amine (APPPI, 1 and 10 microM) reversibly diminished hypoxic depolarization but had no significant effect on input resistance. The effect of APPPI at a concentration of 1 microM, but not at 10 microM, was blocked by DPCPX (0.1 microM). CPA (100 microM) inhibited [(3)H]-noradrenaline ([(3)H]-NA) release from rat hippocampal brain slices significantly only in the presence of rauwolscine (0.1 microM), an alpha(2)-adrenoceptor antagonist. APPPI (1 and 10 microM) exhibited an inhibitory effect similar to that observed with CPA. The effects of both CPA and APPPI were antagonized by DPCPX (0.1 microM). The present data suggest that mainly presynaptic mechanisms prevent neurons from hypoxic changes by an inhibition of transmitter release. However, in contrast to CPA, APPPI exhibited additional effects, which require further investigation.

Topics: Adenosine; Animals; Hippocampus; In Vitro Techniques; Indoles; Male; Membrane Potentials; Neurons; Neurotransmitter Agents; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Pyrimidines; Rats; Rats, Wistar; Somatosensory Cortex; Xanthines

The mitogen-activated protein kinase (MAPK) family consists of the p42/p44 MAPKs and the stress-activated protein kinases, c-Jun N-terminal kinase (JNK) and p38 MAPK. We have previously reported that the human adenosine A(1) receptor stimulates p42/p44 MAPK in transfected Chinese hamster ovary cells. In this study, we have investigated whether the endogenous adenosine A(1) receptor in the smooth muscle cell line, DDT(1)MF-2 activates p42/p44 MAPK, JNK and p38 MAPK. The adenosine A(1) receptor agonist N(6)-cyclopentyladenosine stimulated time and concentration-dependent increases in p42/p44 MAPK and p38 MAPK phosphorylation in DDT(1)MF-2 cells. No increases in JNK phosphorylation were observed following adenosine A(1) receptor activation. N(6)-cyclopentyladenosine-mediated increases in p42/p44 MAPK and p38 MAPK phosphorylation were blocked by the selective adenosine A(1) receptor antagonist 1,3-dipropylcyclopentylxanthine and following pretreatment of cells with pertussis toxin. Furthermore, adenosine A(1) receptor-mediated increases in p42/p44 MAPK were sensitive to the MAPK kinase 1 inhibitor PD 98059 (2'-amino-3'-methoxyflavone), whereas p38 MAPK responses were blocked by the p38 MAPK inhibitor SB 203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole). The broad range protein tyrosine kinase inhibitors genistein and tyrphostin A47 (alpha-cyano-(3,4-dihydroxy)thiocinnamide) did not block adenosine A(1) receptor stimulation of p42/p44 MAPK. For comparison, insulin-mediated increases in p42/p44 MAPK were blocked by genistein and tyrphostin A47. The Src tyrosine kinase inhibitor PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) and the epidermal growth factor receptor tyrosine kinase inhibitor AG1478 (4-(3-chloroanilino)-6,7-dimethoxyquinazoline) also had no effect on adenosine A(1) receptor stimulation of p42/p44 MAPK. Furthermore, the protein kinase C inhibitors Ro 31-8220 (3-[1-[3-(2-isothioureido) propyl]indol-3-yl]-4-(1-methylindol-3-yl)-3-pyrrolin-2,5-dione), chelerythrine and GF 109203X (2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide) were without effect on adenosine A(1) receptor-induced p42/p44 MAPK phosphorylation. In contrast, wortmannin and LY 294002 (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one), inhibitors of phosphatidylinositol 3-kinase, attenuated adenosine A(1) receptor stimulation of p42/p44 MAPK phosphorylation. In conclusion, the adenosine A(1) receptor stimulate

Topics: Adenosine; Animals; Cell Line; Cricetinae; Enzyme Inhibitors; Hypoglycemic Agents; Insulin; JNK Mitogen-Activated Protein Kinases; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Muscle, Smooth; p38 Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinases; Protein Kinase C; Protein-Tyrosine Kinases; Receptors, Purinergic P1; Signal Transduction; Vasodilator Agents; Xanthines

The Na(+)-Ca(2+) exchanger is a protein present in the cell membrane of many cell types. In heart it plays important roles in Ca homeostasis and ionic current generation. Recently, it has been reported that the beta-adrenergic agonist isoprenaline (ISO) can increase directly Na(+)-Ca(2+) exchanger activity in guinea-pig ventricular myocytes. Adenosine (ADO) exerts anti-adrenergic properties that make it effective against some arrhythmias and the aim of the present study was to determine whether or not ADO can antagonize the direct modulatory effect of ISO on the exchanger.Whole-cell patch clamp measurements of Na(+)-Ca(2+) exchanger current (I(NaCa)) were made from guinea-pig ventricular myocytes, with major interfering currents inhibited. I(NaCa) was measured at 378 degrees C as current sensitive to external nickel (Ni(2+), 10 mM) during an applied descending voltage ramp. ISO (1 microM) significantly increased both inward and outward I(NaCa). This effect was abolished in the presence of ADO (200 microM). ADO alone did not significantly alter the amplitude of I(NaCa). The effect of ADO on the response of I(NaCa) to ISO was mimicked by the A(1)ADO receptor agonist N(6)-cyclopentyladenosine (CPA, 10 microM), whereas the effect of ADO on the response of I(NaCa) to ISO was inhibited by the A(1)ADO receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 2 microM). These data suggest that the A(1)ADO receptor mediated the response. The anti-adrenergic effects on I(NaCa) of ADO were not affected by the protein kinase C (PKC) inhibitor, chelerythrine (CLT, 1 microM), nor by the nitric oxide (NO) synthase inhibitor, N (G)-nitro-L-arginine methyl ester((L)-NAME, 0.5 mM). Moreover, in the presence of PKC activator phorbol 12-myristate 13-acetate (PMA, 1 microM) or exogenous NO donor sodium nitroprusside (SNP, 100 microM), ISO preserved its stimulatory effect on I(NaCa). However, prior incubation of myocytes with pertussis toxin (PTX, 5 microg ml(-1) did prevent the effect of ADO. The anti-adrenergic effect of ADO on I(NaCa) was mimicked by externally applied carbachol (CCh, 10 microM), a muscarinic receptor agonist. We conclude that ADO antagonized the effect of beta-adrenergic stimulation of I(NaCa) by directly activating inhibitory G-protein (G(i))-linked A(1) receptors in guinea-pig ventricular myocytes. These findings may suggest a novel mechanism by which adenosine exerts some of its antiarrhythmic effects.

Topics: Adenosine; Adrenergic beta-Agonists; Adrenergic beta-Antagonists; Animals; Carbachol; Cells, Cultured; Cholinergic Agonists; GTP-Binding Protein alpha Subunits, Gi-Go; Guinea Pigs; Heart Ventricles; Isoproterenol; Myocardium; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Protein Kinase C; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Receptors, Adrenergic, beta; Receptors, Purinergic P1; Signal Transduction; Sodium-Calcium Exchanger; Ventricular Function; Xanthines

We examined the effects of paeoniflorin on adenosine A1 receptor-mediated memory disturbance in the mouse passive avoidance test and inhibition of long-term potentiation (LTP) in the rat hippocampal CA1 region. The pretraining administration of the selective adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA) significantly impaired the retention performance determined 24 h after the training test. The intraperitoneal injections of paeoniflorin and the adenosine A1 receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) significantly attenuated the deficit in retention performance caused by CPA. The in vitro studies revealed that adenosine (1 and 10 microM) dose dependently reduced both the population spike (PS) amplitudes and the tetanic stimulation-induced LTP in the hippocampus. DPCPX, at the concentration (0.1 microM) that had no effect on PS amplitudes or LTP induction, significantly reversed the suppressive effects of adenosine on both indices. Paeoniflorin also dose dependently reversed 10 microM adenosine-induced suppression of LTP but had no effect on PS reduced by adenosine. These results suggest that paeoniflorin ameliorates memory disruption mediated by adenosine A1 receptor and that modulation of adenosine-mediated inhibition of LTP in the hippocampus is implicated in its beneficial effect on learning and memory impairment in rodents.

Topics: Adenosine; Animals; Avoidance Learning; Benzoates; Bridged-Ring Compounds; Glucosides; Hippocampus; Long-Term Potentiation; Male; Mice; Monoterpenes; Rats; Rats, Wistar; Receptors, Purinergic P1; Xanthines

A model of electrically evoked release of glutamate from rat hippocampus was developed and used to detect possible changes induced by lesions of hippocampal afferences. Neuronal glutamate in hippocampal slices was labelled by preincubation with [3H]glutamine. The slices were then superfused with physiological medium in the presence of the glutamate uptake inhibitor L-transpyrrolidine-2,4-dicarboxylic acid (100 microM or 3 microM) and stimulated twice electrically (S1, S2: 240 pulses, 3 Hz, 2 ms, 26-30 mA); various drugs were added before S2. In order to determine the basal and evoked outflow of [3H]glutamate only, the mixture of 3H-labelled compounds (glutamine, glutamate and GABA) was separated by ion exchange chromatography in superfusate fractions and slices. The electrically evoked overflow of [3H]glutamate was largely Ca2+-dependent and tetrodotoxin-sensitive and hence represented action potential-induced exocytotic release of [3H]glutamate. Evoked [3H]glutamate release was significantly increased by the adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 0.1 microM), suggesting the presence of endogenous inhibitory adenosine, and reduced by the A1 receptor agonist N6-cyclopentyladenosine (1 microM, antagonized by DPCPX, 0.1 microM). There was no evidence for a cholinergic, serotonergic, or adrenergic modulation of the evoked release of [3H]glutamate: the corresponding selective agonists (or antagonists) were ineffective. After aspirative lesions of the septohippocampal pathways the hippocampal noradrenaline content was markedly increased, whereas cholinergic and serotonergic markers were reduced. The evoked release of [3H]glutamate in hippocampal slices of lesioned rats was significantly increased by a mechanism which still has to be determined, but which is not related to alterations in A1 receptor function. It is concluded that the present model was able to detect lesion-induced differences in electrically evoked release of [3H]glutamate, but the relationship of these differences to changes of noradrenergic, cholinergic or serotonergic hippocampal innervations remains to be established.

Topics: Action Potentials; Adenosine; Animals; Electric Stimulation; Fornix, Brain; Glutamic Acid; Hippocampus; Male; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Rats; Rats, Wistar; Receptors, Purinergic P1; Xanthines

Adenosine and its endogenous precursor ATP are main components of the purinergic system that modulates cellular and tissue functions via specific adenosine and ATP receptors (P1 and P2 receptors), respectively. Although adenosine inhibits excitability and ATP functions as an excitatory transmitter in the central nervous system, little is known about the ability of P1 and P2 receptors to form new functional structures such as a heteromer to control the complex purinergic cascade. Here we have shown that G(i/o) protein-coupled A1 adenosine receptor (A1R) and Gq protein-coupled P2Y1 receptor (P2Y1R) coimmunoprecipitate in cotransfected HEK293T cells, suggesting the oligomeric association between distinct G protein-coupled P1 and P2 receptors. A1R and P2Y2 receptor, but not A1R and dopamine D2 receptor, also were found to coimmunoprecipitate in cotransfected cells. A1R agonist and antagonist binding to cell membranes were reduced by coexpression of A1R and P2Y1R, whereas a potent P2Y1R agonist adenosine 5'-O-(2-thiotriphosphate) (ADPbetaS) revealed a significant potency to A1R binding only in the cotransfected cell membranes. Moreover, the A1R/P2Y1R coexpressed cells showed an ADPbetaS-dependent reduction of forskolin-evoked cAMP accumulation that was sensitive to pertussis toxin and A1R antagonist, indicating that ADPbetaS binds A1R and inhibits adenylyl cyclase activity via G(i/o) proteins. Also, a high degree of A1R and P2Y1R colocalization was demonstrated in cotransfected cells by double immunofluorescence experiments with confocal laser microscopy. These results suggest that oligomeric association of A1R with P2Y1R generates A1R with P2Y1R-like agonistic pharmacology and provides a molecular mechanism for an increased diversity of purine signaling.

Topics: Adenosine; Adenylyl Cyclases; Animals; Cell Line; Cell Membrane; Cyclic AMP; GTP-Binding Protein alpha Subunits, Gi-Go; Heterotrimeric GTP-Binding Proteins; Humans; Inositol 1,4,5-Trisphosphate; Kinetics; Macromolecular Substances; Purinergic P1 Receptor Agonists; Radioligand Assay; Rats; Receptors, Dopamine D2; Receptors, Purinergic P1; Receptors, Purinergic P2; Recombinant Proteins; Transfection; Tritium; Xanthines

This study was designed to determine the adenosine (Ado) receptor subtype that mediates the depressant effects of Ado on fetal breathing and rapid eye movements (REM). In chronically catheterized fetal sheep (>0.8 term), intra-arterial infusion of N(6)-cyclopentyladenosine (CPA), an Ado A(1)-receptor agonist, increased the incidence of high-voltage electrocortical (ECoG) activity while virtually abolishing low-voltage activity, REM, and breathing. These effects were blocked by 9-cyclopentyl-1,3-dipropylxanthine (DPCPX), an Ado A(1)-receptor antagonist. Infusion of DPCPX alone increased breath amplitude but had no significant effect on inspiratory duration, breath interval, incidence of REM, or incidence of low-voltage activity. Ado A(2A)-receptor blockade with ZM-241385 increased the incidence of low-voltage ECoG activity, REM, and breathing but had no effect on breath amplitude or respiratory cycle. Both DPCPX and ZM-241385 eliminated the inhibitory effects of Ado on REM and breathing. We conclude that 1) Ado A(1) receptors tonically inhibit fetal respiratory drive, 2) Ado A(2A) receptors tonically inhibit REM-like behavioral state, and 3) both Ado A(1) and A(2A) receptors mediate the depressant effects of Ado on REM and breathing.

Topics: Adenosine; Animals; Fetus; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Receptors, Purinergic P1; Respiratory Physiological Phenomena; Sheep; Sleep Stages; Triazines; Triazoles; 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

Although adenosine is an important neuromodulator in the CNS, its role in modulating sympathetic outflow at the level of the spinal cord has not been studied. Because very little is known about adenosine A1 receptors (A1Rs) in the spinal cord, we determined their location and role with particular reference to the control of sympathetic preganglionic activity and interneuronal activity in the rat. High levels of immunoreactivity for A1Rs were observed throughout the spinal cord. Immunostaining was dense in the intermediolateral cell column (IML) and intercalated nucleus, regions containing retrogradely labeled sympathetic preganglionic neurons (SPNs). Electron microscopy revealed A1R immunoreactivity (A1R-IR) within presynaptic terminals and (to a lesser extent) postsynaptic structures in the IML, as well as the luminal membrane of endothelial cells lining capillaries. Using double-labeling techniques, some presynaptic terminals were observed to synapse onto SPNs. To investigate the effects of activating these A1Rs, visualized whole-cell patch-clamp recordings were made from electrophysiologically and morphologically identified SPNs and interneurons. Applications of the A1R agonist cyclopentyladenosine (CPA) reduced the amplitude of EPSPs elicited by stimulation of the lateral funiculus, an effect blocked by the A1R antagonist 8-cyclopentyl-1,3-dipropylxanthine. These effects were attributable to adenosine acting at a presynaptic site because CPA application increased the paired-pulse ratio. CPA did not affect evoked IPSPs. These data show that activating A1Rs reduces fast excitatory, but not inhibitory, transmission onto SPNs and interneurons in the IML and that A1Rs may play a protective role on neurons involved in the control of sympathetic outflow.

Topics: Adenosine; Animals; Antibody Specificity; Electric Stimulation; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Immunohistochemistry; In Vitro Techniques; Interneurons; Male; Microscopy, Electron; Neural Inhibition; Patch-Clamp Techniques; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Rats; Reaction Time; Receptors, Purinergic P1; Spinal Cord; Sympathetic Nervous System; Synapses; Synaptic Transmission; Thorax; Xanthines

A study concerning the feasibility of microsphere use as sustained delivery systems for N(6)-cyclopentyladenosine (CPA) administration has been performed. The release of this drug and the related stability effects in human whole blood have been tested. Moreover, the impact of the delivery system on CPA interaction toward human adenosine A1 receptor and the related cellular responses has been analyzed. The microspheres were prepared by an emulsion-solvent evaporation method using poly(lactic acid). Free and encapsulated CPA was incubated in fresh blood and the drug stability was analyzed with HPLC. The affinity of CPA to human A1 receptor expressed by CHO cells was obtained by binding experiments. Activity was evaluated by measurements of the inhibition of forskolin-stimulated 3',5'-cyclic adenosine monophosphate (c-AMP) performing competitive binding assays. Encapsulated CPA was released within 72 h and its degradation in blood was negligible. Affinity and activity values of CPA obtained in the absence and in the presence of unloaded microspheres were the same. CPA encapsulation in microspheres allows its sustained release and its stabilization in human whole blood to be obtained. The presence of this release system does not interfere with the CPA activity at its action site.

Topics: Adenosine; Cyclic AMP; Delayed-Action Preparations; Drug Stability; Humans; Lactic Acid; Microspheres; Polyesters; Polymers; Purinergic P1 Receptor Agonists; 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. Recent studies have shown beneficial effects of an adenosine A(2A) receptor agonist in dt(sz) mutant hamsters, an animal model of paroxysmal dystonia, in which stress and consumption of coffee can precipitate dystonic attacks. This prompted us to examine the effects of adenosine receptor agonists and antagonists on severity of dystonia in dt(sz) hamsters in more detail. 2. The non-selective adenosine A(1)/A(2A) receptor antagonists, caffeine (10 - 20 mg kg(-1) i.p.) and theophylline (10 - 30 mg kg(-1) s.c.), worsened the dystonia in dt(sz) hamsters. 3. Aggravation of dystonia was also caused by the selective adenosine A(1)/A(2A) antagonist CGS 15943 (9-chloro2-2-furyl)[1,2,4]triazolo[1,5-c]quinazolin-5-amine) at a dose of 30 mg kg(-1) i.p. and by the adenosine A(1) antagonist DPCPX (8-cyclopentyl-1,3-dipropylxanthine; 20 - 30 mg kg(-1) i.p.), while the A(2) antagonist DMPX (3,7-dimethyl-1-propargylxanthine; 2 - 4 mg kg(-1) i.p.) and the highly selective A(2A) antagonist ZM 241385 (4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol; 2 - 5 mg kg(-1) i.p.) failed to exert any effects on dystonia. 4. In contrast to the antagonists, both the adenosine A(1) receptor agonist CPA (N(6)-cyclopentyladenosine; 0.1 - 1.0 mg kg(-1) i.p.) and the A(2A) agonist CGS 21680 (2p-(2carboxyethylphen-ethylamino-5'-N-ethylcarboxamindoadenosine; 0.1 - 2.0 mg kg(-1) i.p.) exerted a striking improvement of dystonia. 5. These data suggest that the precipitating effects of methylxanthines are, at least in part, related to their adenosine receptor antagonistic action. 6. Although adenosine receptor agonists can be regarded as interesting candidates for the therapy of paroxysmal dystonia, adverse effects may limit the therapeutic potential of adenosine A(1) agonists, while beneficial effects of the adenosine A(2A) agonist CGS 21680 were already found at well tolerated doses.

Topics: Adenosine; Animals; Caffeine; Cricetinae; Disease Models, Animal; Dose-Response Relationship, Drug; Dystonia; Female; Male; Mesocricetus; Muscle Contraction; Mutation; Phenethylamines; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Quinazolines; Theobromine; Theophylline; Triazines; Triazoles; Xanthines

1. A spindle of fast network oscillations precedes the ischaemia-induced rapid depolarisation in the rat hippocampus in vivo. However, this oscillatory pattern could not be reproduced in slices and the underlying mechanisms remain poorly understood. We have found that anoxia-induced network oscillations (ANOs, 20-40 Hz, lasting for 1-2 min) can be reproduced in the intact hippocampi of postnatal day P7-10 rats in vitro, and we have examined the underlying mechanisms using whole-cell and extracellular field potential recordings in a CA3 pyramidal layer. 2. ANOs were generated at the beginning of the anoxic depolarisation, when pyramidal cells depolarised to subthreshold values. Maximal power of the ANOs was attained when pyramidal cells depolarised to -56 mV; depolarisation above -47 mV resulted in a depolarisation block of pyramidal cells and a waning of ANOs. 3. A multiple unit activity in extracellular field recordings was phase locked to the negative and ascending phases of ANOs. Pyramidal cells recorded in current-clamp mode generated action potentials with an average probability of about 0.05 per cycle. The AMPA receptor-mediated EPSCs and the GABA receptor-mediated IPSCs in CA3 pyramidal cells were also phase locked with ANOs. 4. ANOs were prevented by tetrodotoxin and glutamate receptor antagonists CNQX and APV, and were slowed down by the allosteric GABA(A) receptor modulator diazepam. In the presence of the GABA(A) receptor antagonist bicuculline, ANOs were transformed to epileptiform discharges. 5. In the presence of the A1 adenosine receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), the anoxia induced an epileptiform activity and no ANOs were observed. 6. In normoxic conditions, a rise of extracellular potassium to 10 mM induced an epileptiform activity. Increasing extracellular potassium in conjunction with a bath application of the adenosine A1 receptor agonist cyclopentyladenosine induced oscillations similar to ANOs. 7. Multisite recordings along the septo-temporal hippocampal axis revealed that ANOs and anoxic depolarisation originate in the temporal part, and propagate towards the septal pole at a speed of 1.9 mm x min(-1). 8. ANOs were observed starting from P7, i.e. at a developmental stage when the effects of GABA change from depolarisation to hyperpolarisation. 9. These results suggest that the synchronisation of anoxia-induced oscillations relies on synaptic mechanisms; that the inhibition by GABA and adenosine sets the

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Adenosine; Anesthetics, Local; Animals; Bicuculline; Diazepam; Excitatory Amino Acid Antagonists; GABA Antagonists; GABA Modulators; gamma-Aminobutyric Acid; Hippocampus; Hypoxia, Brain; In Vitro Techniques; Male; Membrane Potentials; Neurons; Patch-Clamp Techniques; Periodicity; Potassium; Rats; Rats, Wistar; Tetrodotoxin; Xanthines

To explore the neuronal signaling mechanisms underlying sleep regulation in the rat, the present study examined continuous intra-third ventricle infusion of N-ethylmaleimide (NEM), a sulphydryl reagent that inhibits G(i/o) protein-coupled receptor-mediated signaling pathways. The diurnal infusion of NEM (0.01-10 micromol/10 h) dose-dependently inhibited both non-rapid eye movement sleep and rapid eye movement sleep. A maximal dose of NEM (10 micromol/10 h) dramatically inhibited day-time sleep (-57% for non-rapid eye movement sleep and -89% for rapid eye movement sleep) with a compensatory increase of sleep during the subsequent night-time (+33% for non-rapid eye movement sleep and +259% for rapid eye movement sleep). The day-time brain temperature was also increased by NEM, demonstrating effects of NEM on both sleep and body temperature levels. Immunostaining of the rat hypothalamus with a monoclonal antibody against the A1 adenosine receptor (A1R) was used to explore the distribution of a sleep-related G(i/o) protein-coupled receptor. Robust A1R-like immunoreactivity was found in the ventromedial preoptic nucleus and the supraoptic nucleus. Fura-2-based Ca(2+) imaging analysis of acute hypothalamic slices further demonstrated that the A1R agonist N(6)-cyclopentyladenosine (CPA; 200 nM) inhibited spontaneous Ca(2+) oscillations and high potassium (80 mM)-induced Ca(2+) flux in the ventromedial preoptic nucleus, while NEM (100-300 microM) and an A1R antagonist 8-cyclopentyl-dipropylxanthine (300 nM) blocked the CPA actions and increased the high potassium-induced Ca(2+) flux. From these results we suggest that NEM-sensitive G protein-coupled receptor(s) may play an important role in the regulation of sleep and body temperature in the rat and one possible mechanism is an A1R-mediated regulation of intracellular Ca(2+) concentrations in the ventromedial preoptic nucleus.

Topics: Adenosine; Animals; Body Temperature; Calcium; Calcium Channels; Calcium Signaling; Circadian Rhythm; Ethylmaleimide; Fura-2; Glutamic Acid; GTP-Binding Proteins; Hypothalamus; Immunohistochemistry; Male; Neurons; Potassium; Preoptic Area; Rats; Rats, Sprague-Dawley; Receptors, Purinergic P1; Second Messenger Systems; Sleep; Sulfhydryl Reagents; Tetrodotoxin; Xanthines

tk;1Adenosine plays a role in the control of water and electrolyte reabsorption in the distal tubule. As the distal convoluted tubule is important in the regulation of renal Mg(2+) balance, we determined the effects of adenosine on cellular Mg(2+) uptake in this segment. The effect of adenosine was studied on immortalized mouse distal convoluted tubule (MDCT) cells, a model of the intact distal convoluted tubule. The rate of Mg(2+) uptake was measured with fluorescence techniques using mag-fura 2. To assess Mg(2+) uptake, MDCT cells were first Mg(2+) depleted to 0.22 +/- 0.01 mM by being cultured in Mg(2+)-free media for 16 h and then placed in 1.5 mM MgCl(2); next, changes in intracellular Mg(2+) concentration ([Mg(2+)](i)) were determined. [Mg(2+)](i) returned to basal levels, 0.53 +/- 0.02 mM, with a mean refill rate, d([Mg(2+)](i))/dt, of 137 +/- 16 nM/s. Adenosine stimulates basal Mg(2+) uptake by 41 +/- 10%. The selective A(1) purinoceptor agonist N(6)-cyclopentyladenosine (CPA) increased intracellular Ca(2+) and decreased parathyroid hormone (PTH)-stimulated cAMP formation and PTH-mediated Mg(2+) uptake. On the other hand, the selective A(2) receptor agonist 2-[p-(2-carbonyl-ethyl)-phenylethylamino]-5'-N-ethylcarboxamidoadenosine (CGS) stimulated Mg(2+) entry in a concentration-dependent fashion. CGS increased cAMP formation and the protein kinase A inhibitor RpcAMPS inhibited CGS-stimulated Mg(2+) uptake. Selective inhibition of phospholipase C, protein kinase C, or mitogen-activated protein kinase enzyme cascades with U-73122, Ro-31-8220, and PD-98059, respectively, diminished A(2) agonist-mediated Mg(2+) entry. Aldosterone potentiated CGS-mediated Mg(2+) entry, and elevation of extracellular Ca(2+) diminished CGS-responsive cAMP formation and Mg(2+) uptake. Accordingly, MDCT cells possess both A(1) and A(2) purinoceptor subtypes with intracellular signaling typical of these respective receptors. We conclude that adenosine has dual effects on Mg(2+) uptake in MDCT cells through separate A(1) and A(2) purinoceptor pathways.

Topics: Adenosine; Adenosine Triphosphate; Aldosterone; Animals; Calcium; Cell Line, Transformed; Cyclic AMP; Enzyme Inhibitors; Ion Transport; Kidney Tubules, Distal; Magnesium; Mice; Parathyroid Hormone; Phenethylamines; Protein Kinase Inhibitors; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Receptors, Purinergic P1; Theobromine; Xanthines

Although intrathecal administration of adenosine analogues or A(1) adenosine receptor agonists is known to result in antinociception, this has not been examined yet at the cellular level. In the present study, we examined in pharmacology an action of adenosine on glutamatergic miniature excitatory postsynaptic currents (mEPSCs) in substantia gelatinosa (SG) neurons of an adult rat spinal cord slice; this was done under the condition where a postsynaptic action of adenosine was blocked. In 65% of the neurons examined (n=72), adenosine at a concentration of 100 microM depressed the frequency of mEPSC in a reversible manner; the remaining neurons exhibited an inhibition followed by potentiation of the frequency. When examined quantitatively in extent in some cells (n=25), the inhibition was 40+/-3% (n=25) while the potentiation was 42+/-8% (n=6). These actions were not accompanied by a change in mEPSC amplitude. The inhibitory action on mEPSC frequency was dose-dependent in a range of 10-500 microM with an EC(50) value of 277 microM. The inhibitory action of adenosine was mimicked by a selective A(1) adenosine receptor agonist, CPA (1 microM; depression: 54+/-9%, n=4); this action of adenosine (100 microM) was not observed in the presence of a specific A(1) adenosine receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) (1 microM; 94+/-4% of control, n=3). The facilitatory action of adenosine (100 microM) was unaffected by an A(2a) antagonist, ZM 241385 (0.1 microM, n=3); an A(2a) agonist, CGS 21680 (0.1-10 microM; n=6), was without actions on mEPSC frequency. It is concluded that adenosine inhibits excitatory transmission to SG neurons through the activation of presynaptic A(1) adenosine receptor and that some of the inhibition is followed by a potentiation of the transmission. It remains to be examined which subtypes of adenosine receptors except for the A(1)- and A(2a)-subtypes are involved in the potentiating action. Considering that adenosine-like immunoreactivity and adenosine receptors are expressed at a high density in the SG, which is thought to play an important role in modulating nociceptive transmission from the periphery to the central nervous system, this inhibitory action of adenosine could contribute to a negative modulation of pain transmission.

Topics: Adenosine; Age Factors; Analgesics; Animals; Antihypertensive Agents; Excitatory Postsynaptic Potentials; In Vitro Techniques; Male; Neural Inhibition; Neurons; Patch-Clamp Techniques; Phenethylamines; Rats; Rats, Sprague-Dawley; Receptors, Purinergic P1; Substantia Gelatinosa; Triazines; Triazoles; Xanthines

A number of cycloalkyl substituents (from C-3 to C-8) have been introduced on the 6-amino group of adenosine, 1-deazaadenosine, and 2'-deoxyadenosine, bearing or not a chlorine atom at the 2-position, to evaluate the influence on the A(1) and A(2A) affinity of steric hindrance and lipophilicity. Furthermore, the guanosine 5'-triphosphate (GTP) shift and the maximal induction of guanosine 5'-(gamma-thio)triphosphate ([(35)S]GTPgammaS) binding to G proteins in rat brain membranes were used to determine the intrinsic activity of these nucleosides at the A(1) adenosine receptor. All compounds of the ribose-bearing series proved to be full agonists, the 1-deaza derivatives showing affinities for the A(1) receptor about 10-fold lower than the corresponding adenosines. On the other hand, all the 2'-deoxyribose derivatives bind to the A(1) receptor with affinities in the high nanomolar range, with the 2-chloro substituted compounds showing slightly higher affinities than the 2-unsubstituted counterparts. In terms of the potencies, the most potent compounds proved to be those bearing four- and five-membered rings. Both GTP shifts and [(35)S]-GTPgammaS experiments showed that most of the 2'-deoxyadenosine derivatives are partial agonists. The 2'-deoxyadenosine derivatives which were identified as partial agonists consistently detected fewer A(1) receptors in the high-affinity state than full agonists. However, it is worthwhile noting that there was not a simple linear relationship between receptor occupancy and activation. These results indicate that a critical density of A(1) adenosine receptors in the high-affinity state is required for G protein activation.

Topics: Adenosine; Animals; Brain; Guanosine 5'-O-(3-Thiotriphosphate); Magnetic Resonance Spectroscopy; Phenethylamines; Purinergic P1 Receptor Agonists; Radioligand Assay; Rats; Tubercidin; Xanthines

The elevated plus-maze and the light/dark box are two established anxiety tests in rodents, which are useful to screen putative anxiogenic effects of drugs.. Caffeine is well known to promote anxious behaviour in humans and animal models, but the precise site of action of the drug is still a matter of debate. The present study investigated whether the anxiogenic effects of caffeine observed in mice depend on the blockade of A(2A) receptor. First, the effects induced by the non-selective drug caffeine were compared with those elicited by two selective A(2A) receptor antagonists over a wide range of doses in the same experimental conditions. The effects of A(2A) or A(1 )adenosine receptor agonists and of a selective A(1) adenosine receptor antagonist were also investigated. Second, wild-type and A(2A) receptor knockout mice offered another approach to delineate the role played by A(2A) receptor in caffeine's anxiogenic effects.. Mice were exposed to the elevated plus-maze or to the light/dark box for 5 min after acute or chronic administration of tested drugs.. Caffeine acutely administered (50 or 100 mg/kg IP) induced anxiety-like effects in both procedures. Its chronic administration (50 mg/kg IP twice daily) for 1 week or consumption in the drinking water (0.3 g/l) for 8 days or 2 months were also anxiogenic in the plus-maze test. The A(2A) receptor antagonists ZM241385 (up to 60 mg/kg IP) and SCH58261 (up to 10 mg/kg IP) were devoid of acute effects in both tests. One week administration of ZM241385 (30 mg/kg IP) or SCH58261 (3 mg/kg IP) had no effects in the plus-maze test. An antagonist (DPCPX) and an agonist (CPA) at A(1) receptors had no acute effects on anxiety-related indices, whereas an A(2A) receptor agonist (CGS 21680) displayed non-specific motor effects in the plus-maze test. Acute administration of caffeine (50 mg/kg IP) induced no clear-cut anxiety-like effects in the plus-maze test in A(2A) receptor knockout mice that exhibited higher basal anxiety levels than wild-type mice. Chronic administration (50 mg/kg IP twice daily) for 1 week elicited less anxiety-like behaviour in A(2A) receptor knockout than in wild-type mice.. Adaptative mechanisms following mutation in A(2A) receptors or their long-term blockade after chronic ingestion of caffeine may be responsible for increase proneness to anxiety. However, the short-term anxiety-like effect of caffeine in mice might not be related solely to the blockade of adenosine A(2A) receptors, since it is not shared by A(2A) selective antagonists.

Topics: Adenosine; Administration, Oral; Animals; Anxiety; Behavior, Animal; Caffeine; Darkness; Drug Administration Schedule; Injections, Intraperitoneal; Light; Male; Maze Learning; Mice; Mice, Knockout; Neuroprotective Agents; Phenethylamines; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Pyrimidines; Reaction Time; Receptor, Adenosine A2A; Triazines; Triazoles; Xanthines

In this study we evaluated the role of adenosine receptor activation on the K+-evoked D-[3H]aspartate release in cultured chick retina cells exposed to oxidant conditions. Oxidative stress, induced by ascorbate (3.5 mM)/Fe2+ (100 microM), increased by about fourfold the release of D-[3H]aspartate, evoked by KCl 35 mM in the presence and in the absence of Ca2+. The agonist of A1 adenosine receptors, N6-cyclopentyladenosine (CPA; 10 nM), inhibited the K+-evoked D-[3H]aspartate release in control in oxidized cells. The antagonist of A1 adenosine receptor, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 50 nM), potentiated the release of D-[3H]aspartate in oxidized cells, and reverted the effect observed in the presence of CPA 10 nM. However, in oxidized cells, when DPCPX was tested together with CPA 100 nM the total release of D-[3H]aspartate increased from 5.1 +/- 0.4% to 11.4 +/- 1.0%, this increase being reverted by 3,7-dimethyl-1-propargylxanthine (DMPX; 100 nM), an antagonist of A2A adenosine receptors. In cells of both experimental conditions, the K+-evoked release of D-[3H]aspartate was potentiated by the selective agonist of A2A adenosine receptors, 2-[4-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamidoadenosin e (CGS 21680; 10 nM), whereas the antagonist of these receptors, DMPX (100 nM), inhibited the release of D-[3H]aspartate in oxidized cells, but not in control cells. Adenosine deaminase (ADA; 1 U/ml), which is able to remove adenosine from the synaptic space, reduced the K+-evoked D-[3H]aspartate release, from 5.1 +/- 0.4% to 3.1 +/- 0.3% in oxidized cells, and had no significant effect in control cells. The extracellular accumulation of endogenous adenosine, upon K+-depolarization, was higher in oxidized cells than in control cells, and was reduced by the inhibitors of adenosine transporter (NBTI) and of ecto-5'-nucleotidase (AOPCP). This suggests that adenosine accumulation resulted from the outflow of adenosine mediated by the transporter, and from extracellular degradation of adenine nucleotide. Our data show that both inhibitory A1 and excitatory A2A adenosine receptors are present in cultured retina cells, and that the K+-evoked D-[3H]aspartate release is modulated by the balance between inhibitory and excitatory responses. Under oxidative stress conditions, the extracellular accumulation of endogenous adenosine seems to reach levels enough to potentiate the release of D-[3H]aspartate by the tonic activation of A2A adenosine receptors.

Topics: Adenosine; Adenosine Deaminase; Animals; Ascorbic Acid; Aspartic Acid; Calcium; Cells, Cultured; Chick Embryo; Drug Synergism; Kinetics; Oxidative Stress; Phenethylamines; Potassium; Potassium Chloride; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Receptor, Adenosine A2A; Receptors, Purinergic P1; Retina; Theobromine; Tritium; Xanthines

A1 adenosine receptors (A1ARs) are heavily expressed in adipocytes and influence fat cell metabolism. Because increasing evidence suggests a role for leptin in mediating appetite and fat cell metabolism, we tested whether ALARs regulate leptin production. Rats were treated with the A1AR agonist N6-cyclopentyladenosine (CPA), and changes in circulating levels of leptin and leptin gene expression were examined. Serum leptin levels rose 2- to 10-fold, with peak increases seen 8-16 h after injection of CPA (P < 0.05). In contrast, CPA did not alter steady state levels of adipose tissue leptin mRNA. To assess the influence of endogenous adenosine on circulating leptin levels, rats were also injected with dipyridamole (DPY), an adenosine reuptake blocker. DPY induced 80% increases in serum levels at 8 h after injections (P < 0.05). Supporting the idea that stimulation of leptin production is A1AR mediated, pretreatment with the A1AR antagonist 8-cyclopentyl-1,3-dipropylxanthine completely blocked increases in leptin levels after DPY treatment. To complement in vivo studies, the effect of A1AR activation on leptin secretion was also studied in epididymal fat pad cultures. In cultures, CPA treatment increased leptin secretion by 37% (P < 0.05). Collectively, these data show that the adenosinergic system can increase leptin secretion by directly activating A1ARs in fat tissue.

Topics: Adenosine; Adipocytes; Animals; Culture Techniques; Dipyridamole; Gene Expression; Leptin; Male; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Rats; Rats, Sprague-Dawley; Receptors, Purinergic P1; RNA, Messenger; Xanthines

To characterize age-related changes in beta-adrenergic responsiveness and to test the hypothesis that an increase in the effects of adenosine contribute to impaired beta-adrenergic responsiveness, Fischer 344 rat right atria (RA), left atria (LA), and left ventricular trabeculae carnae were exposed to the beta-receptor agonist isoproterenol (ISO), followed by four doses of the selective adenosine A(1) receptor agonist cyclopentyladenosine (CPA). Spontaneous contractile rates of adult RA were inhibited more than senescent RA by CPA. Contractility (+dF/dt) of adult LA was reduced more than senescent LA by CPA. Left trabeculae carnae tissue responded weakly to CPA, but senescent tissue was less responsive than adult tissue. Senescent atrial A(1) receptor density was 56% greater than in adult tissue, whereas the density in senescent ventricles was 39% lower than in adult tissue. No significant difference in antagonist affinities (K(d)) of A(1) receptor was observed between adult and senescent atria. In addition, agonist competition curves indicated a significant increase in senescent atrial and a decrease in senescent ventricular tissue in the affinity of agonist for high-affinity A(1) receptors with no difference in dissociation constant (K(i)). No significant age-related differences in atrial or ventricular tissues occurred in either the antagonist affinity (K(d)) or density (B(max)) of the beta-adrenergic receptors. CPA was found to inhibit ISO-stimulated adenylate cyclase activity more in senescent than in adult atrial and ventricular membrane preparations. We conclude that age-related differences in functional response to ISO and CPA, A(1) receptor density, and ISO-stimulated adenylate cyclase activity differ in atrial and ventricular myocardium.

Topics: Adenosine; Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Adrenergic beta-Agonists; Aging; Animals; Cardiotonic Agents; Electric Stimulation; Heart; Heart Atria; Heart Ventricles; In Vitro Techniques; Isoproterenol; Myocardial Contraction; Myocardium; Purinergic P1 Receptor Agonists; Radioligand Assay; Rats; Rats, Inbred F344; Receptors, Adrenergic, beta; Xanthines

The release of cortical acetylcholine from the intracortical axonal terminals of cholinergic basal forebrain neurons is closely associated with electroencephalographic activity. One factor which may act to reduce cortical acetylcholine release and promote sleep is adenosine. Using in vivo microdialysis, we examined the effect of adenosine and selective adenosine receptor agonists and antagonists on cortical acetylcholine release evoked by electrical stimulation of the pedunculopontine tegmental nucleus in urethane anesthetized rats. All drugs were administered locally within the cortex by reverse dialysis. None of the drugs tested altered basal release of acetylcholine in the cortex. Adenosine significantly reduced evoked cortical acetylcholine efflux in a concentration-dependent manner. This was mimicked by the adenosine A(1) receptor selective agonist N(6)-cyclopentyladenosine and blocked by the selective A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). The A(2A) receptor agonist 2-[p-(2-carboxyethyl)-phenethylamino]-5'-N-ethylcarboxamidoadenosi ne hydrochloride (CGS 21680) did not alter evoked cortical acetylcholine release even in the presence of DPCPX. Administered alone, neither DPCPX nor the non-selective adenosine receptor antagonist caffeine affected evoked cortical acetylcholine efflux. Simultaneous delivery of the adenosine uptake inhibitors dipyridamole and S-(4-nitrobenzyl)-6-thioinosine significantly reduced evoked cortical acetylcholine release, and this effect was blocked by the simultaneous administration of caffeine. These data indicate that activation of the A(1) adenosine receptor inhibits acetylcholine release in the cortex in vivo while the A(2A) receptor does not influence acetylcholine efflux. Such inhibition of cortical acetylcholine release by adenosine may contribute to an increased propensity to sleep during prolonged wakefulness.

Topics: Acetylcholine; Adenosine; Analysis of Variance; Animals; Caffeine; Cerebral Cortex; Chromatography, High Pressure Liquid; Dipyridamole; Male; Microdialysis; Phenethylamines; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Rats; Rats, Wistar; Synapses; Xanthines

In this study the effect of insulin and A(1)-adenosine receptor stimulation on protein kinase B (PKB) activation has been investigated in the hamster vas deferens smooth muscle cell line DDT(1)MF-2. Increases in PKB phosphorylation were determined by Western blotting using an antibody that detects PKB phosphorylation at Ser(473). Insulin, a recognized activator of PKB, stimulated a concentration-dependent increase in PKB phosphorylation in DDT(1)MF-2 cells (EC(50) 5+/-1 pM). The selective A(1)-adenosine receptor agonist N(6)-cyclopentyladenosine (CPA) stimulated time and concentration-dependent increases in PKB phosphorylation in DDT(1)MF-2 cells (EC(50) 1.3+/-0.5 nM). CPA-mediated increases in PKB phosphorylation were antagonized by the A(1)-adenosine receptor selective antagonist 1,3-dipropylcyclopentylxanthine (DPCPX) yielding an apparent K(D) value of 2.3 nM. Pre-treatment of DDT(1)MF-2 cells with pertussis toxin (PTX, 100 ng ml(-1) for 16 h), to block G(i)/G(o)-dependent pathways, abolished CPA (1 microM) induced phosphorylation of PKB. In contrast, responses to insulin (100 nM) were resistant to PTX pre-treatment. The phosphatidylinositol 3-kinase (PI-3K) inhibitors wortmannin (IC(50) 10.3+/-0.6 nM) and LY 294002 (IC(50) 10.3+/-1.2 microM) attenuated the phosphorylation of PKB elicited by CPA (1 microM) in a concentration-dependent manner. Wortmannin (30 nM) and LY 294002 (30 microM) also blocked responses to insulin (100 nM). Removal of extracellular Ca(2+) and chelation of intracellular Ca(2+) with BAPTA had no significant effect on CPA-induced PKB phosphorylation. Similarly, pretreatment (30 min) with inhibitors of protein kinase C (Ro 31-8220; 10 microM), tyrosine kinase (genistein; 100 microM), mitogen-activated protein (MAP) kinase kinase (PD 98059; 50 microM) and p38 MAPK (SB 203580; 20 microM) had no significant effect on CPA-induced PKB phosphorylation. In conclusion, these data demonstrate that A(1)-adenosine receptor stimulation in DDT(1)MF-2 cells increases PKB phosphorylation through a PTX and PI-3K-sensitive pathway.

Topics: Adenosine; Androstadienes; Animals; Calcium; Cell Line; Chromones; Cricetinae; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Flavonoids; Genistein; Imidazoles; Indoles; Insulin; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Morpholines; Pertussis Toxin; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Protein Kinase C; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Pyridines; Receptors, Purinergic P1; Virulence Factors, Bordetella; Wortmannin; Xanthines

We compared the modulation of synaptic transmission by adenosine A(1) receptors in the hippocampus of aged (24 months) and young adult rats (6 weeks). The adenosine A(1) receptor agonist, N(6)-cyclopentyladenosine, was less potent (P:<0.05) to inhibit synaptic transmission in aged (EC(50)=53 nM) than young adult (EC(50)=14 nM) hippocampal slices, these effects being prevented by the A(1) receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX). In contrast with the lower effect of the A(1) receptor agonist, it was observed that blockade of A(1) receptors with DPCPX (50 nM), or removal of endogenous extracellular adenosine with adenosine deaminase (2 u ml(-1)), caused a more pronounced disinhibition of synaptic transmission in aged rats. Also consistent with a more intense A(1) receptor-mediated inhibitory tonus by endogenous adenosine in aged rats was the finding that to fully prevent the depression of synaptic transmission induced by 3 min hypoxia, a higher concentration of DPCPX was required in slices from aged (100 nM) than from young (50 nM) rats. It is concluded that in hippocampal slices of aged rats the efficiency of A(1) receptors to modulate synaptic transmission is reduced, but this may be compensated by an enhanced inhibitory tonus by endogenous adenosine.

Topics: Adenosine; Adenosine Deaminase; Aging; Animals; Dose-Response Relationship, Drug; Excitatory Postsynaptic Potentials; Hippocampus; Hypoxia; In Vitro Techniques; Male; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Rats; Rats, Wistar; Receptors, Purinergic P1; Synaptic Transmission; Xanthines

To examine the role of the adenosine A1 receptor in glucose regulation in the absence of insulin, the present study investigated the changes of plasma glucose in male streptozotocin-induced diabetic rats (STZ-diabetic rats) using dipyridamole to increase endogenous adenosine and N6-cyclopentyladenosine (CPA) to activate the adenosine A1 receptor. Intravenous injections of dipyridamole or CPA induced a dose-dependent decrease of plasma glucose in fasting STZ-diabetic rats. Plasma glucose lowering action of dipyridamole, like that of CPA, was inhibited in a dose-dependent manner by pre-treatment with 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) or 8-(p-sulfophenyl)theophylline (8-SPT) at which block the adenosine A1 receptors. Action of the adenosine A1 receptors can thus be considered. In isolated skeletal muscle, CPA enhanced the glucose uptake in a concentration-dependent manner. Blockade of this action by DPCPX and 8-SPT again supported the mediation of the adenosine A1 receptor. Also, CPA produced an increase of glycogen synthesis in isolated soleus muscle. Moreover, CPA decreased plasma triglyceride and cholesterol levels significantly in STZ-diabetic rats. These results suggest that activation of adenosine A1 receptors can increase glucose utilization in peripheral tissues by increasing tissue uptake and glycogen synthesis to lower plasma glucose in rats lacking insulin.

Topics: Adenosine; Animals; Blood Glucose; Cholesterol; Diabetes Mellitus, Experimental; Dipyridamole; Disease Models, Animal; Dose-Response Relationship, Drug; Glucose; Glycogen; Hypoglycemic Agents; Insulin; Male; Muscle, Skeletal; Purinergic P1 Receptor Antagonists; Rats; Rats, Wistar; Receptors, Purinergic P1; Streptozocin; Theophylline; Triglycerides; 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 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

Adenosine has been demonstrated to have an anticonvulsant action which is mediated predominantly by the adenosine A1 receptor subtype. The present study was conducted to determine if the adenosinergic system and adenosine A1 receptors are involved in the anticonvulsant action of the antiepileptic drugs phenobarbitone and carbamazepine, in pentylenetetrazole (PTZ)-induced seizures in rats. The specific adenosine A1 receptor antagonist, DPCPX (1 mg/kg i.p.), had no effect on the anticonvulsant action of the two antiepileptic drugs. However, the nonspecific adenosine receptor antagonist, theophylline (50 and 100 mg/kg i.p.), reversed the anticonvulsant action of carbamazepine completely and that of phenobarbitone partially. This suggests that adenosine A1 receptors do not mediate the anticonvulsant effects of these agents. When phenobarbitone/carbamazepine were coadministered with adenosine/N6-cyclopentyladenosine (CPA), a specific adenosine A1 receptor agonist, an enhancement in protection against PTZ-induced seizures was observed. The diversity of anticonvulsant mechanism of carbamazepine/phenobarbitone and that of adenosinergic agents could be responsible for this effect.

Topics: Adenosine; Animals; Anticonvulsants; Carbamazepine; Convulsants; Drug Combinations; Male; Pentylenetetrazole; Phenobarbital; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Rats; Rats, Wistar; Seizures; Theophylline; Xanthines

It is well-known that nucleotides, nucleosides and purine/pyrimidine bases enhance cell proliferation in vitro. Nevertheless, the molecular mechanisms involved in this mitogenic activity is still controversial, since these compounds are reported both to synergize with growth factor, and to act directly on purinergic receptor inducing per se a proliferative response. It was suggested that cell growth enhancement could be mediated by the A2 purinergic receptor activation. Here we report that a polydeoxyribonucleotide (PDRN) and adenosine are able to increase, the growth rate of human skin fibroblasts in primary cultures. The proliferative activity exerted by PDRN was significantly counteracted by the A2 antagonist 3, 7-Dimethyl-1-propargylxanthine (DMPX), but not by the A1 antagonist 8-cyclopentyl-1,3-dipropylxanthine (PD 116,948, DPCPX). Accordingly, the trophic action of PDRN was mimicked by the A2 agonist N6-[2-(3,5-Dimethoxyphenyl)-2-(methylphenyl)-ethyl]adenosine (DPMA), while the A1 agonist N6-Cyclopenthyladenosine (CPA) did not show any effect. In microfluorimetric studies, we observed that PDRN and adenosine increased the concentration of cytosolic calcium ions. The PDRN-evoked calcium rise was dose-dependent and DMPX sensitive. Taken together, our results suggest that PDRN may operate as a pro-drug providing the cultured cells with an effective amount of mitogenic deoxyribonucleotides, deoxyribonucleosides and bases; moreover, cell proliferation enhancement that has been induced by PDRN seems to be mediated, at least in part, by the activation of purinergic receptors of the A2 subtype.

Topics: Adenosine; Calcium; Cell Division; Cytophotometry; Deoxyribonucleosides; Deoxyribonucleotides; Fibroblasts; Fura-2; Humans; Oligodeoxyribonucleotides; Placenta; Receptors, Purinergic P1; Second Messenger Systems; Theobromine; Xanthines

Novel allosteric enhancers of agonist binding to the rat adenosine A(1) receptor are described. The lead compound for the new series was PD 81,723 ((2-amino-4, 5-dimethyl-3-thienyl)[3-(trifluoromethyl)phenyl]methanone), a compound previously reported by Bruns and co-workers (Mol. Pharmacol. 1990, 38, 950-958). The 4,5-dimethyl group and the benzoyl moiety were targets for further modifications, leading to series of 4, 5-dialkyl (12a-g), of tetrahydrobenzo (12h-u), and of tetrahydropyridine (13a-g) derivatives. A number of compounds, in particular 12b, 12e, 12j, 12n, and 12u, proved superior to PD 81,723. Their EC(50) values for enhancing the binding of the adenosine A(1) receptor agonist N(6)-cyclopentyladenosine to the receptor were lower, and/or their antagonistic activity on the adenosine A(1) receptor was shown to be diminished.

Topics: Adenosine; Allosteric Regulation; Animals; Binding, Competitive; Brain; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Rats; Thiophenes; Xanthines

Ischemic preconditioning (IPC) prevents the deleterious effects of prolonged ischemia and reperfusion (I/R). Because leukocyte infiltration is required to produce the microvascular dysfunction induced by I/R in the small intestine, and P-selectin-dependent leukocyte rolling is a requisite step in this process, we hypothesized that IPC would attenuate postischemic P-selectin expression. To address this postulate, P-selectin expression was evaluated in nonischemic (control) rat jejunum and in rat jejunum subjected to I/R alone (20 min ischemia/60 min reperfusion), or IPC (5 min ischemia/10 min reperfusion) + I/R using a dual radiolabeled monoclonal antibody approach. I/R was associated with a sevenfold increase in jejunal P-selectin expression, an effect that was completely abolished by IPC. Exposing the bowel to adenosine deaminase or an adenosine A1, but not an A2, receptor antagonist during the period of preconditioning ischemia or to selective PKC antagonists during prolonged ischemia prevented the beneficial effect of IPC to limit I/R-induced P-selectin expression. Our data indicate that P-selectin expression is a novel downstream effector target of the adenosine-initiated, PKC-dependent, anti-inflammatory signaling pathway in IPC.

Topics: Adenosine; Adenosine Deaminase; Animals; Antibodies, Monoclonal; Ischemia; Ischemic Preconditioning; Jejunum; Male; Organ Specificity; P-Selectin; Protein Kinase C; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Rats; Rats, Sprague-Dawley; Reperfusion; Theobromine; Xanthines

Adenosine modulates synaptic transmission by acting on inhibitory A(1) and facilitatory A(2A) receptors, the densities of which are modified in aged animals. We investigated how A(2A) receptor activation influences A(1) receptor function and whether this interaction is modified in aged rats. In hippocampal and cortical nerve terminals from young adult (6 wk), but not old rats (24 mo), the A(2A) receptor agonist, 2-[4-(2-carboxyethyl) phenethylamino]-5'-N-ethylcarboxamidoadenosine (CGS 21680; 30 nM) decreased the binding affinity of a selective A(1) receptor agonist, cyclopentyladenosine (CPA), an effect prevented by the A(2A) antagonist, (4-(2-[7-amino-2-(2-furyl (1,2,4)-triazolo(2,3-a (1,3,5)triazin-5-yl-aminoethyl)phenol (ZM 241385, 20 nM). This effect of CGS 21680 required intact nerve terminals and was also observed in the absence of Ca(2+). This A(2A)-induced "desensitization" of A(1) receptors was prevented by the protein kinase C inhibitor, chelerythrine (6 microM), and was not detected in the presence of the protein kinase C activator, phorbol-12,13-didecanoate (250 nM), which itself caused a reduction in binding affinity for CPA. The protein kinase A inhibitor, N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (10 microM), and the protein kinase A activator, 8-Br-cAMP (1 mM), had no effects on the A(2A)-induced A(1) receptor desensitization. This A(2A)-induced A(1) receptor desensitization had a functional correlation because CGS 21680 (10 nM) attenuated by 40% the inhibition caused by CPA (10 nM) on CA1 area population spike amplitude in hippocampal slices. This A(2A)/A(1) interaction may explain the attenuation by adenosine deaminase (2 U/ml), which removes tonic A(1) inhibition, of the facilitatory effect of CGS 21680 on synaptic transmission. The requirement of tonic A(1) receptor activation for CGS 21680 to induce facilitation of synaptic transmission was reinforced by the observation that the A(1) receptor antagonist, 1, 3-dipropyl-8-cyclopentylxanthine (20 nM) prevented CGS 21680 (10 nM) facilitation of population spike amplitude. The present results show the ability of A(2A) receptors to control A(1) receptor function in a manner mediated by protein kinase C, but not protein kinase A, in young adult but not in aged rats.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Adenosine; Aging; Animals; Binding, Competitive; Cerebral Cortex; Electrophysiology; Hippocampus; In Vitro Techniques; Male; Phenethylamines; Protein Kinase C; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Rats; Rats, Wistar; Receptor Cross-Talk; Receptor, Adenosine A2A; Receptors, Purinergic P1; Synaptic Membranes; Triazines; Triazoles; Xanthines

A thermodynamic analysis of the binding of a full agonist (N6-cyclopentyladenosine), a partial agonist (8-butylamino-N6-cyclopentyladenosine) and an antagonist (8-cyclopentyltheophylline) to human wild-type and mutant (mutation of a threonine (Thr) to an alanine (Ala) residue at position 277) adenosine A1 receptors expressed on Chinese hamster ovary (CHO) cells, and to rat brain adenosine A1 receptors was undertaken. The thermodynamic parameters deltaGo (standard free energy), deltaHo (standard enthalpy) and deltaSo (standard entropy) of the binding equilibrium to rat brain receptors were determined by means of affinity measurements carried out at four different temperatures (0, 10, 20 and 25 degrees) and van't Hoff plots. Two temperatures (0 and 25 degrees) were considered for human receptors. Affinity constants were obtained from inhibition assays on membrane preparations of rat brain and CHO cells by use of the antagonist [3H]1,3-dipropyl-8-cyclopentylxanthine ([3H]DPCPX) as selective adenosine A1 receptor radioligand. As for rat brain receptors, full agonist binding was totally entropy driven, whereas antagonist binding was essentially enthalpy driven. Partial agonist binding appeared both enthalpy and entropy driven. As for human receptors, full agonist affinity was highly dependent on the presence of Thr277. Moreover, affinity to both wild-type and mutant receptors was enhanced by temperature increase, suggesting a totally entropy-driven binding. Antagonist binding did not depend on the presence of Thr277. Antagonist affinity decreased with an increase in temperature, suggesting a mainly enthalpy-driven binding. Partial agonist binding was significantly dependent on the presence of Thr277 at 25 degrees, whereas such a dependence was not evident at 0 degrees. It is concluded that Thr277 contributes only to the binding of adenosine derivatives and that its role changes drastically with the receptor conformation and with the type of agonist (full or partial) interacting with the adenosine A1 receptors.

Topics: Adenosine; Alanine; Amino Acid Substitution; Animals; Binding, Competitive; CHO Cells; Cricetinae; Entropy; Humans; Kinetics; Point Mutation; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Radioligand Assay; Rats; Receptors, Purinergic P1; Theophylline; Thermodynamics; Threonine; Transfection; Xanthines

The effects of the selective adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA; 1 and 0.1 mg/kg, i.p.) and the A1 selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (CPX) have been examined on the electroencephalogram (EEG) of intact rats. From four EEG leads the EEG signal was subjected to Fast Fourier Transform and analysed both in narrow (0.01629638 Hz) and wide frequency bands. CPA tended to increase EEG power at low frequencies, and in several of the narrow frequency bands significantly shifted peak frequencies to lower values. The agonist also increased peak power in some frequency bands. The results are consistent with the view that A1 adenosine receptors mediate a generally depressant effect on neuronal activity in most brain regions, but may increase activity in areas with low resting rates of firing. The modest elevation of wave power by CPX indicates a limited control by resting endogenous adenosine, which is greatest in areas of highest activity, consistent with adenosine release being related to neuronal activity.

Topics: Adenosine; Animals; Electroencephalography; Male; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Rats; Rats, Wistar; Xanthines

Cell motility is an essential component of tumor progression and metastasis. A number of factors, both autocrine and paracrine, have been found to influence cell motility. In the present study, adenosine and adenine nucleotides directly stimulated chemotaxis of A2058 melanoma cells in the absence of exogenous factors. Three adenosine receptor agonists stimulated motility in the melanoma cells and two adenosine receptor antagonists strongly inhibited the chemotactic response to both adenosine and AMP. The chemotactic stimulation by adenosine and AMP was pertussis toxin sensitive. Otherwise unresponsive Chinese hamster ovary cells which were transfected with the adenosine A1 receptor cDNA acquired the direct, pertussis toxin sensitive, chemotactic response to adenosine, and this response was inhibited by adenosine receptor antagonists. These findings demonstrate that adenosine and adenine nucleotides are capable of stimulating chemotaxis of tumor cells mediated through an adenosine receptor, probably of the A1 subtype. The possibility of antimetastatic therapies based on inhibition of adenosine receptor activity is raised.

Topics: Adenosine; Adenosine Monophosphate; Animals; Chemotaxis; CHO Cells; Cricetinae; Dose-Response Relationship, Drug; Humans; Melanoma, Experimental; Pertussis Toxin; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Receptors, Purinergic P1; Transfection; Virulence Factors, Bordetella; Xanthines

We showed previously that exposure of cerebellar granule cells to the A1 adenosine receptor (A1AR)-selective agonist, cyclopentyladenosine, decreases A1AR density and G protein coupling corresponding to blunted agonist-induced adenylyl cyclase (EC 4.6.1.1) inhibition. We have now determined that A1AR-mediated adenylyl cyclase inhibition was desensitized in a homologous manner. Carbachol- and baclofen-induced inhibition of adenylyl cyclase was unaffected by 48-h exposure to 10 microM cyclopentyladenosine. Expression of G protein alpha-subunits was not affected dramatically by agonist exposure. The fraction of sequestered A1AR was increased significantly at 4, 24, and 48 h of cyclopentyladenosine exposure (35, 57, and 81% increase over control, respectively). The time course of agonist-induced A1AR sequestration was slower than that reported for other G protein-coupled receptors. Incubation with the adenosine receptor antagonist, 8-p-sulfophenyltheophylline or adenosine deaminase did not alter sequestration significantly. Neither steady-state A1AR mRNA levels nor transcript stability was affected by 48-h agonist exposure. We determined that A1AR half-life in cerebellar granule cells is 20.9 h, which is considerably longer than that reported for several other G protein-coupled receptors. The slow time course of A1AR sequestration and the stability of the corresponding mRNA may be a reflection of the tonic inhibitory tone exerted by adenosine in brain.

Topics: Adenosine; Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Animals; Cells, Cultured; Cerebellum; Down-Regulation; Gene Expression; GTP-Binding Protein alpha Subunits, Gs; Neurons; Purinergic P1 Receptor Agonists; Rats; Rats, Sprague-Dawley; Receptors, Purinergic P1; RNA, Messenger; Theophylline; Tritium; Xanthines

The nucleus accumbens (NAc) is a site mediating the rewarding properties of drugs of abuse, such as cocaine, amphetamine, opiates, nicotine, and alcohol (Wise and Bozarth, 1987; Koob, 1992; Samson andHarris, 1992; Woolverton and Johnson, 1992; Self and Nestler, 1995; Pontieri et al., 1996). Acute cocaine has been shown to decrease excitatory synaptic transmission mediated by the cortical afferents to the NAc (Nicola et al., 1996), but the effects of long-term cocaine treatment and withdrawal have not been explored. Here, we report that long-term (1 week) withdrawal from chronic cocaine reduced the potency of adenosine to presynaptically inhibit glutamate (Glu) release by activating adenosine A1 receptors. Adenosine A1 receptors were not desensitized, because the potency of the metabolically stable adenosine analog N6-cyclopentyl-adenosine was unchanged after chronic cocaine withdrawal. When adenosine transporters were blocked, the potency of adenosine to inhibit Glu release from naive and cocaine-withdrawn NAc slices was similar. These results suggest that one of the long-term consequences of cocaine withdrawal is an augmented uptake of adenosine. This long-lasting change expressed at the presynaptic excitatory inputs to the medium spiny output neurons in the NAc may help identify new therapeutic targets for the treatment of drug abuse.

Topics: 4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone; Adenosine; Affinity Labels; Animals; Cocaine; Dipyridamole; Dopamine Uptake Inhibitors; Excitatory Postsynaptic Potentials; Glutamic Acid; Hippocampus; Male; Nucleus Accumbens; Phosphodiesterase Inhibitors; Presynaptic Terminals; Purinergic P1 Receptor Agonists; Rats; Rats, Sprague-Dawley; Stimulation, Chemical; Substance Withdrawal Syndrome; Substance-Related Disorders; Theophylline; Thioinosine; Vasodilator Agents; Xanthines

Adenosine in the extracellular space modulates stimulated glucose transport in striated muscle. In the heart and in adipocytes, adenosine potentiates insulin-stimulated glucose transport. There is controversy regarding the effect of adenosine in skeletal muscle, with reports of both an inhibitory effect and no effect, on insulin-stimulated glucose transport. We found that, in rat epitrochlearis and soleus muscles, removing adenosine with adenosine deaminase or blocking its action with the adenosine receptor blocker CPDPX markedly reduces the responsiveness of glucose transport to stimulation by 1) insulin alone, 2) contractions alone, and 3) insulin and contractions in combination. Measurement of the increase in GLUT4 at the cell surface in response to a maximally effective insulin stimulus in the epitrochlearis muscle, using the exofacial label ATB-[3H]BMPA, showed that adenosine deaminase treatment markedly reduces cell-surface GLUT4 labeling. The reduction in cell-surface GLUT4 labeling was similar in magnitude to the decrease in maximally insulin-stimulated glucose transport activity in adenosine deaminase-treated muscles. These results show that adenosine potentiates insulin- and contraction-stimulated glucose transport in skeletal muscle by enhancing the increase in GLUT4 at the cell surface and raise the possibility that decreased adenosine production or action could play a causative role in insulin resistance.

Topics: Adenosine; Adenosine Deaminase; Adenylyl Cyclase Inhibitors; Animals; Biological Transport; Cyclic AMP; Drug Synergism; Glucose; Glucose Transporter Type 4; Insulin; Male; Monosaccharide Transport Proteins; Muscle Contraction; Muscle Proteins; Muscle, Skeletal; Rats; Rats, Wistar; Receptors, Purinergic P1; Xanthines

We tested the hypothesis that adenosine excites nociceptive primary afferents innervating the knee joint. Neuronal recordings were made from fine nerve filaments innervating the knee joint in rats anaesthetized with pentobarbitone. Drugs were injected close-arterially (i.a.) or into the articular space (i.art.). We studied normal and chronically inflamed arthritic joints, the latter 14-21 days after a single intra-articular injection of Freund's Complete Adjuvant, performed under halothane anaesthesia. Adenosine injected i.a. caused delayed (approximately 10 s) excitation of the majority of polymodal C-fibre afferents, and had similar effects when injected directly into the joint. Adenosine triphosphate (ATP) had biphasic effects on discharge, a fast (<1 s) excitation was followed by a delayed increase similar to that seen with adenosine. The adenosine A1 receptor agonists N6-cyclopentyladenosine (CPA) and N-[(1S,trans)-2-hydroxypentyl] adenosine (GR79236) also excited the C-fibre afferents. The A1 antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) antagonized the responses evoked by adenosine, CPA, and the delayed increase seen after ATP, indicating that excitation of the nociceptive afferents was mediated via adenosine A1 receptors. Adenosine and ATP evoked delayed excitatory effects of similar magnitude, regardless of whether or not the knee joint was chronically inflamed. The increased basal discharge observed in arthritic joints was unaffected by DPCPX, which implies that the increase in spontaneous activity associated with arthritis is unlikely to involve tonically released adenosine. The results support the hypothesis that adenosine excites primary afferent nociceptive nerve terminals in the rat knee joint, an effect mediated by adenosine A1 receptors. ATP, adenosine, and A1 receptors may play a role in generating the peripheral nociceptive (pain) signal.

Topics: Action Potentials; Adenosine; Adenosine Triphosphate; Analgesics; Animals; Arthritis, Experimental; Hypolipidemic Agents; Knee Joint; Male; Neurons, Afferent; Nociceptors; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Rats; Rats, Wistar; Receptors, Purinergic P1; Xanthines

Adenosine plays an important role in postischemic dysfunction of cardiac sympathetic nerves because exogenously infused adenosine produces and adenosine deaminase prevents "neural stunning." We examined whether adenosine acts via a specific receptor mechanism to produce neural stunning. Anesthetized dogs were treated with propranolol to attenuate increases in coronary flow due to adrenergic stimulation of myocardial metabolism. A 15-min occlusion of the left anterior descending coronary artery (LAD) attenuated subsequent LAD coronary vasoconstriction to bilateral sympathetic stimulation during reperfusion by 75% (P < 0.05). Coronary infusion of the adenosine-receptor antagonist 8-p-sulfophenyltheophylline (nonspecific), 8-cyclopentyl-1,3-dipropylxanthine (A1 specific), or 3,7-dimethyl-1-propagylxanthine (A2 specific) during LAD occlusion prevented the attenuation of sympathetic coronary constriction. In separate experiments, either the specific adenosine agonist N6-cyclopentyl-adenosine (A1 specific) or CGS-21680 (A2 specific) or a combination of both agonists was infused into the LAD for 15 min. Neither agonist alone attenuated subsequent sympathetic coronary constriction. In contrast, 15 min after the combined administration of both agonists, sympathetic vasoconstriction was reduced. We conclude that adenosine is capable of attenuating neurogenic coronary constriction through a receptor-mediated mechanism. Activation of more than one receptor subtype is necessary to produce neural stunning.

Topics: Adenosine; Animals; Coronary Vessels; Dogs; Female; Male; Myocardial Stunning; Phenethylamines; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Receptors, Purinergic P1; Sympathetic Nervous System; Theobromine; Theophylline; Vasoconstriction; Xanthines

1. The effects of adenosine, the adenosine analogue, 2-chloroadenosine (2-CADO), the specific adenosine A1 receptor agonist, N6-cyclopentyladenosine (CPA) and A2 receptor agonist 5'-(N-cyclopropyl) carboxamidoadenosine (CPCA), were examined against seizures induced by acute administration of pentylenetetrazole (PTZ), 60 mg kg-1, and PTZ kindled seizures, in rats. 2. Adenosine 1000 mg kg-1, i.p., 5 min pretreatment and CPA 10 mg kg-1 i.p., 60 min pretreatment, showed significant protection against acute PTZ-induced seizures while, CPCA up to 10 mg kg-1 was ineffective. The adenosine analogue 2-CADO in a dose of 5 mg kg-1 was only partially protective and on increasing the dose to 10 mg kg-1, this protection was lost. 3. Theophylline, a non specific adenosine receptor antagonist at 50 mg kg-1 and the specific adenosine A1 receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), at 1 mg kg-1, if administered before the maximally protective doses of adenosine and CPA, completely reversed the protection afforded by them against PTZ seizures. While, pretreatment with the adenosine A2 receptor antagonist, 3,7-dimethyl-1-propargylxanthine (DMPX), failed to reverse the protection. 4. Adenosine and the adenosine A1 receptor agonist in doses that protected against seizures after acute PTZ administration, offered only incomplete protection when tested against PTZ kindled seizures. 5. The effects of adenosine and adenosine receptor agonists on mean arterial pressure, heart rate and rectal temperature were studied, to rule out the possibility of their systemic effects mediating the protection of PTZ seizures. All these agents produced a fall in mean arterial pressure, heart rate and hypothermia in the doses exhibiting an anticonvulsant response. While the effect on blood pressure and heart rate was immediate i.e. seen within 5 min and, maintained throughout the observation period, the development of hypothermia lagged behind the onset of hypotension and bradycardia. However, there was no correlation between haemodynamic and hypothermic response and the anticonvulsant effect. 6. The results indicate that the adenosine mediated anticonvulsant effect is via stimulation of A1 receptors. Hypotension and hypothermia do not appear to contribute to the protection observed with adenosine and the adenosine A1 receptor agonists.

Topics: Adenosine; Animals; Blood Pressure; Body Temperature; Heart Rate; Male; Pentylenetetrazole; Rats; Rats, Wistar; Receptors, Purinergic P1; Seizures; Xanthines

The Y-maze was used to assess spontaneous alternation behaviour in mice to examine possible interactions between the N-methyl-D-aspartate receptor channel blocker dizocilpine and purine receptor agonists and antagonists. Scopolamine reduced spontaneous alternation. Dizocilpine also produced a dose-dependent reduction in alternation scores, which was accompanied by an increase in locomotion. The selective A1 adenosine receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (CPX) had no effect when administered alone, or in combination with scopolamine. However, when co-administered with dizocilpine, CPX reversed both the deficit in alternation behaviour and also the increase in locomotion induced by dizocilpine. The A1 selective agonist N6-cyclopentyladenosine (CPA) had no effect on either locomotion or alternation scores when administered alone, but in combination with scopolamine, CPA attenuated the scopolamine-induced deficit. CPA had no significant effect on the dizocilpine-induced deficit. The A2 selective agonist N6-[2-(3, 5-dimethoxyphenyl)-2(2-methylphenyl)-ethyl]adenosine (DPMA), had no effect on spontaneous alternation when administered alone, but did cause a depression of locomotion. DPMA had no significant effect when co-administered with scopolamine, but reversed the deficit in spontaneous alternation, and the increase in locomotion induced by dizocilpine. The A2 selective antagonist 3,7-dimethyl-1-propargylxanthine (DMPX) had no effect when given alone or in combination with scopolamine, but when co-administered with dizocilpine, DMPX reversed the reduction in spontaneous alternation caused by dizocilpine. It is concluded that dizocilpine has a detrimental effect on spontaneous alternation which is mediated partly by A1 and A2 adenosine receptors.

Topics: Adenosine; Animals; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Male; Maze Learning; Memory; Mice; Muscarinic Antagonists; Receptors, Purinergic P1; Scopolamine; Xanthines

Adenosine A1 receptors induce an inhibition of adenylyl cyclase via G-proteins of the Gi/o family. In addition, simultaneous stimulation of A1 receptors and of receptor-mediated activation of phospholipase C (PLC) results in a synergistic potentiation of PLC activity. Evidence has accumulated that Gbetagamma subunits mediate this potentiating effect. However, an A1 receptor-mediated increase in extracellular glutamate was suggested to be responsible for the potentiating effect in mouse astrocyte cultures. We have investigated the synergistic activation of PLC by adenosine A1 and alpha1 adrenergic receptors in primary cultures of astrocytes derived from different regions of the newborn rat brain. It is reported here that (1) adenosine A1 receptor mRNA as well as receptor protein is present in astrocytes from all brain regions, (2) A1 receptor-mediated inhibition of adenylyl cyclase is of similar extent in all astrocyte cultures, (3) the A1 receptor-mediated potentiation of PLC activity requires higher concentrations of agonist than adenylyl cyclase inhibition and is dependent on the expression level of A1 receptor, and (4) the potentiating effect on PLC activity is unrelated to extracellular glutamate. Taken together, our data support the notion that betagamma subunits are the relevant signal transducers for A1 receptor-mediated PLC activation in rat astrocytes. Because of the lower affinity of betagamma, as compared with alpha subunits, more betagamma subunits are required for PLC activation. Therefore, only in cultures with higher levels of adenosine A1 receptors is the release of betagamma subunits via Gi/o activation sufficient to stimulate PLC. It is concluded that variation of the expression level of adenosine A1 receptors may be an important regulatory mechanism to control PLC activation via this receptor.

Topics: Adenosine; Adenylate Cyclase Toxin; Adrenergic alpha-Agonists; Animals; Astrocytes; Brain; Cells, Cultured; Cyclic AMP; Enzyme Activation; Excitatory Amino Acid Antagonists; Extracellular Space; Glutamates; Inositol Phosphates; Intercellular Signaling Peptides and Proteins; Peptides; Rats; Receptors, Adrenergic, alpha; Receptors, Purinergic P1; RNA, Messenger; Type C Phospholipases; Virulence Factors, Bordetella; Wasp Venoms; Xanthines

Experiments on hippocampal slices were carried out in order to find out whether the release of noradrenaline in the hippocampus can be modulated through P2-receptors. The slices were preincubated with [3H]-noradrenaline, superfused with medium containing desipramine (1 microM), and stimulated electrically, in most experiments by 4 pulses/100 Hz. The adenosine A1-receptor agonist N6-cyclopentyl-adenosine (CPA) and the nucleotides ATP, adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S) and adenosine-5'-O-(2-thiodiphosphate) (ADP beta S) decreased the evoked overflow of tritium by up to 55%. The adenosine A2a-agonist 2-p-(2-carboxyethyl)-phenethylamino-5'-N-ethylcarboxamido-adenosin e (CGS 21680; 0.003-0.3 microM) caused no change. The concentration-response curve of CPA was shifted to the right by the A1-antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 3 nM) but not by the P2-receptor antagonists cibacron blue 3GA (30 microM) and reactive blue 2 (30 microM); the apparent pKB value of DPCPX against CPA was 9.0. In contrast, the concentration-response curve of ATP was shifted to the right by DPCPX (3 nM), apparent pKB 8.7, as well as by cibacron blue 3GA (30 microM), apparent pKB 5.2, and reactive blue 2 (30 microM), apparent pKB 5.6; the antagonist effects of DPCPX and cibacron blue 3GA were additive in a manner compatible with the blockade of two separate receptors for ATP. The same pattern was obtained with ATP gamma S: its concentration-response curve was shifted to the right by DPCPX as well as by cibacron blue 3GA and reactive blue 2. Suramin (300 microM) antagonized neither the effect of ATP nor that of ATP gamma S. The 5'-nucleotidase inhibitor alpha, beta-methylene-ADP (100 microM) did not change the effect of ATP. Only cibacron blue 3GA (30 microM) but not reactive blue 2 (30 microM), given alone, consistently caused a small increase of the evoked overflow of tritium. Hippocampal slices degraded exogenous ATP, and this degradation was reduced by cibacron blue 3GA (30 microM), reactive blue 2 (30 microM) and suramin (300 microM). The results indicate that the noradrenergic terminal axons of the rat hippocampus possess P2-receptors in addition to the known A1-adenosine receptors. The presynaptic P2-receptors mediate an inhibition of noradrenaline release, are activated by nucleotides but not nucleosides, and are blocked by cibacron blue 3GA and reactive blue 2. ATP and ATP gamma S act at both the A1- and the P2-receptors. An autoreceptor function

Topics: Adenosine; Adenosine Diphosphate; Adenosine Triphosphate; Animals; Electric Stimulation; Hippocampus; In Vitro Techniques; Isotope Labeling; Male; Norepinephrine; Phenethylamines; Rats; Rats, Wistar; Receptors, Purinergic P2; Thionucleotides; Triazines; Tritium; Xanthines; Yohimbine

To determine why methotrexate (MTX) exacerbates rheumatoid nodules in some patients, despite the effective suppression of synovial inflammation.. Phorbol myristate acetate (PMA)-induced differentiation of monocytes into multinucleated giant cells was used as an in vitro model to study the effects of adenosine on nodulosis.. MTX at 200-2,000 nM or the adenosine A1 agonist N5-cyclopentyl adenosine (CPA) (10(-12) to 10(-9) M) or the A2 antagonist 3,7-dimethyl-1-propargylxanthine markedly enhanced giant cell formation, whereas the adenosine A1 antagonist 8-cyclopentyl-dipropylxanthine completely reversed these effects. PMA, CPA, and MTX induced adenosine release by cultured monocytes at concentrations consistent with those associated with predominantly A1 effects. Furthermore, surface expression of A1 receptors was found to remain unchanged on the differentiating cells throughout the culture period.. Agents that inhibit adenosine A1 receptors might be useful in the treatment of MTX-induced rheumatoid nodulosis, while still potentiating the A2-mediated antiinflammatory effects of MTX on synovitis.

Topics: Adenosine; Antirheumatic Agents; Arthritis, Rheumatoid; Cells, Cultured; Humans; Methotrexate; Monocytes; Receptors, Purinergic P1; Rheumatoid Nodule; Tetradecanoylphorbol Acetate; Theobromine; 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

It has been claimed that blockade of receptors for N-methyl-D-aspartate (NMDA) can enhance adenosine receptor function on single neurones. Previous work has also indicated that the NMDA channel blocker dizocilpine, and the A1 selective agonist N6-cyclopentyladenosine (CPA) both had anxiolytic profiles in the elevated plus-maze. The anxiolytic effect of dizocilpine was accompanied by an increase in locomotor activity. In the present study, the elevated plus-maze has been used to determine whether dizocilpine's effects on behaviour are mediated through activation of adenosine receptors. When co-administered with dizocilpine (0.05 mg/kg), CPA (0.05 mg/kg) reduced the anxiolytic and locomotor effects of dizocilpine. The A1 selective antagonist 1,3-dipropyl-8-cyclopentylxanthine (CPX, 0.05 mg/kg) had no effect when administered alone. When co-administered with dizocilpine, CPX reversed the anxiolytic and increased locomotor effects induced by dizocilpine. The A2 receptor selective agonist N6-[2-(3,5-dimethoxyphenyl)-2(2-methylphenyl)ethyladenosine (DPMA) (1 mg/kg) reversed both the anxiolytic effect and the increased locomotion induced by dizocilpine, while the A2 selective antagonist 3,7-dimethyl-1-propargylxanthine (DMPX) (1 mg/kg) did not. It is concluded that at least part of the anxiolytic and locomotor stimulant properties of dizocilpine may be explained by the release of endogenous adenosine acting at A1, but not A2 receptors.

Topics: Adenosine; Animals; Dizocilpine Maleate; Drug Interactions; Excitatory Amino Acid Antagonists; Male; Mice; Motor Activity; Receptors, N-Methyl-D-Aspartate; Receptors, Purinergic P1; 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

The synthesis and biological evaluation of N6, C8-disubstituted derivatives of adenosine as potential partial agonists for adenosine receptors is described. Via three routes, two series of compounds were prepared, viz., N6-cyclopentyladenosine derivatives 3a-e and C8-(cyclopentylamino)adenosine analogs 3e and 9a-d, respectively. The X-ray structure determination of one of these compounds, N6-ethyl-8(cyclopentylamino)adenosine (9b), was carried out (orthorhombic, space group P2(1)2(1)2(1) (No. 19) with a = 11.039(3), b = 8.708(2), and c = 24.815(12) angstrom, Z=4,R1=0.0974,R2(W) = 0.2455). Due to intramolecular hydrogen bonding, the ribose moiety of this compound is in an anti conformation. The compounds were tested in vitro in radioligand binding studies, yielding their affinities for A1 and A2a adenosine receptors. All compounds appeared A1 selective, with affinities in the high nanomolar, low micromolar range. On A1 receptors the so-called GTP shift was also determined, i.e., the ratio between the affinities measured in the presence and absence of 1 mM GTP. All GTP shifts (values between 1.1 and 3.8) were lower than the GTP shift for CPA (6.0). This GTP shift appeared indicative for partial agonism in vivo, since the N6-cyclopentyladenosine derivatives showed lower intrinsic activities than the prototypic full agonist N6-cyclopentyladenosine on the decrease in heart rate in conscious, normotensive rats.

Topics: Adenosine; Animals; Antihypertensive Agents; Binding, Competitive; Blood Pressure; Brain; Crystallography, X-Ray; Guanosine Triphosphate; Heart Rate; Magnetic Resonance Spectroscopy; Models, Molecular; Molecular Structure; Phenethylamines; Protein Binding; Purinergic P1 Receptor Agonists; Rats; Rats, Wistar; Receptors, Purinergic P1; Xanthines

Adenosine A1 receptors are known to be widely distributed in various regions of the brain. A2A receptors are enriched in the dopamine-rich areas of the brain, but are also present in rat cortex. Electrically stimulated, perfused rat cortical slices were used to examine the influence of interactions between A1 and A2A receptors on the release of acetylcholine (ACh) from cortical cholinergic nerves. The A1-selective agonist, N6-cyclopentyladenosine (CPA) caused a dose-dependent inhibition of ACh release, which was attenuated in the added presence of the A1-selective antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 1 microM). The inhibitory effects of CPA were unaltered in the added presence of the A2-selective antagonist (E)-8-(3,4-dimethyloxystyryl)-1,3-dipropyl-7-methylxanthine (KF 17837; 1 microM). The A2A-selective agonist 2-[p-(carboxyethyl)-phenethylamino]-5'-N-ethylcarboxamidoadenosine (CGS 21680), over the concentration range 1 nM to 10 microM, did not significantly alter ACh release when given alone or in the presence of DPCPX or KF 17837. These data suggest that the A(2A) receptors previously identified in rat cortex are not functionally coupled to modulation of ACh release in this tissue. This does not exclude that these receptors may regulate the release of other neurotransmitters.

Topics: 2-Chloroadenosine; Acetylcholine; Adenosine; Animals; Cerebral Cortex; Electric Stimulation; In Vitro Techniques; Male; Phenethylamines; Rats; Rats, Sprague-Dawley; Receptors, Purinergic P1; Xanthines

The adenosine analogue N6-cyclopentyladenosine (CPA), acting via postjunctional A1 receptors, has been shown to enhance contractions of the rat vas deferens induced by adenosine 5'-triphosphate (ATP), the sympathetic cotransmitter in this tissue. The aim of the present study was to examine the ability of CPA to enhance contractions induced by other contractile agents. CPA (0.01-0.3 microM) enhanced contractions induced by exogenous ATP (10 microM), 5-hydroxytryptamine (5-HT) (3 microM), tyramine (10 microM), 2-methyl-5-hydroxytryptamine (2-Me-5-HT) (10 microM) and KCl (35 mM) and this enhancement was blocked by an A1-selective concentration (3 nM) of 1, 3-dipropyl-8-cyclopentylxanthine (DPCPX). CPA failed to enhance contractions induced by exogenous noradrenaline (NA) (1 microM or 10 microM), bradykinin (0.1 microM), phenylephrine (3 microM) or carbachol (10 microM). The contractions induced by ATP (10 microM), 5-HT (3 microM), 2-Me-5-HT (10 microM) and KCl (35 mM) were unaffected by tetrodotoxin (1 microM) as well as by desensitisation of the P2x-purinoceptors with the ATP analogue adenosine 5'-(alpha, beta-methylene) triphosphonate. The contractions induced by tyramine (10 microM) and 2-Me-5-HT (10 microM) were blocked by prazosin (100 nM) or by imipramine (1 microM). Ketanserin (10 nM) antagonised the response to 5-HT giving a dose-ratio of 12.9 corresponding to an apparent pA2 of 9.1. In conclusion, the A1-mediated effect was clearly selective for certain contractile agents and not due to a non-specific increase in contractility of the tissue. CPA enhanced contractions induced by both ATP and indirect sympathomimetics which release endogenous NA, and this enhancement of the two sympathetic cotransmitters may have a functional significance, and demonstrates the complexity of the neuromodulatory effects of adenosine in the rat vas deferens.

Topics: Adenosine; Adenosine Triphosphate; Adrenergic alpha-Antagonists; Animals; Bradykinin; Carbachol; Dose-Response Relationship, Drug; Drug Interactions; Drug Synergism; Male; Muscle Contraction; Muscle, Smooth; Norepinephrine; Phenylephrine; Potassium Chloride; Purinergic P1 Receptor Agonists; Rats; Rats, Wistar; Serotonin; Tyramine; Vas Deferens; 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

1. The effects of analogues of adenosine and ATP on noradrenaline release elicited by electrical stimulation (5 Hz, 2700 pulses) were studied in superfused preparations of rat tail artery. The effects of purinoceptor antagonists, of adenosine deaminase and of adenosine uptake blockade were also examined. Noradrenaline was measured by h.p.l.c. electrochemical detection. 2. The A1-adenosine receptor agonist, N6-cyclopentyladenosine (CPA; 0.1-100 nM) reduced, whereas the A2A-receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS 21680; 3-30 nM) increased evoked noradrenaline overflow. These effects were antagonized by the A1-adenosine receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 20 nM) and the A2-adenosine receptor antagonist, 3,7-dimethyl-1-propargylxanthine (DMPX; 100 nM), respectively. The P2Y-purinoceptor agonist, 2-methylthio-ATP (1-100 microM) reduced noradrenaline overflow, an effect prevented by the P2-purinoceptor antagonist, cibacron blue 3GA (100 microM) and suramin (100 microM). 3. Adenosine deaminase (2 u ml-1), DMPX (100 nM) and inhibition of adenosine uptake with S-(p-nitrobenzyl)-6-thioinosine (NBTI; 50 nM) decreased evoked noradrenaline overflow. DPCPX alone did not change noradrenaline overflow but prevented the inhibition caused by NBTI. The P2Y-purinoceptor antagonist, cibacron blue 3GA (100 microM) increased evoked noradrenaline overflow as did suramin, a non-selective P2-antagonist. 4. It is concluded that, in rat tail artery, inhibitory (A1 and P2Y) and facilitatory (A2A) purinoceptors are present and modulate noradrenaline release evoked by electrical stimulation. Endogenous purines tonically modulate noradrenaline release through activation of inhibitory P2Y and facilitatory A2A purinoceptors, whereas a tonic activation of inhibitory A1 purinoceptors seems to be prevented by adenosine uptake.

Topics: Adenosine; Adenosine Deaminase; Adenosine Triphosphate; Animals; Arteries; Male; Norepinephrine; Phenethylamines; Rats; Rats, Wistar; Receptors, Purinergic; Tail; Thioinosine; Thionucleotides; Xanthines

Endogenous adenosine in the brain may inhibit central sympathetic tone and thereby restrain renin release, a mechanism that may be particularly important when sympathetic activity is enhanced. The purpose of our study was to test the hypothesis that the adenosine receptor antagonist caffeine increases renin release in part by disabling the central nervous system (CNS) adenosine brake on renin release. This hypothesis was tested by conducting three protocols in anesthetized rats. In the first protocol, intracerebroventricular (i.c.v.) infusions of caffeine (10 micrograms/kg/min) did not alter either bradycardic responses to intravenous (i.v.) infusion of N6-cyclopentyladenosine (CPA, A1-receptor agonist) or depressor responses to i.v. infusions of CGS21680 (A1-receptor agonist). However, i.c.v. caffeine did block bradycardic responses to i.c.v. boluses of CPA and depressor responses to i.c.v. boluses of CGS21680, thus demonstrating that i.c.v. caffeine at the dose used blocks CNS but not peripheral adenosine receptors. In the second protocol, hydralazine (1 and 10 mg/kg, administered intraperitoneally) significantly enhanced both the renal secretion of renin and the renal spillover of norepinephrine (NE), thus confirming that hydralazine can increase renin release by unloading arterial baroreceptors and increasing sympathetic tone to the kidneys. In the third protocol, the effects of i.c.v. caffeine (10 micrograms/kg/min) on hydralazine-induced (1 and 10 mg/kg, administered intraperitoneally) changes in renal secretion of renin and renal NE spillover were investigated. In this protocol, i.c.v. caffeine did not alter baseline values for either the renal secretion of renin or NE. In contrast, i.c.v. caffeine significantly (p = 0.03) enhanced the increase in renal renin secretion induced by 1 and 10 mg/kg hydralazine (for 1 mg/kg hydralazine delta of 6.4 +/- 46.7 and 142.4 +/- 142.9 renin activity/min/kg body weight in control and caffeine-treated animals, respectively; for 10 mg/kg hydralazine, delta 227.8 +/- 73.9 and 600.8 +/- 168.9 renin activity/min/kg body weight in control and caffeine-treated animals, respectively). The enhanced renin-secretion response to hydralazine in caffeine-treated rats was accompanied by augmented hydralazine-induced increase in renal NE spillover (p = 0.035). These data strongly support the hypothesis of a CNS adenosine brake on renin release that is disabled by caffeine.

Topics: Adenosine; Animals; Caffeine; Central Nervous System Stimulants; Hydralazine; Injections, Intraventricular; Kidney; Male; Norepinephrine; Purinergic P1 Receptor Antagonists; Rats; Rats, Sprague-Dawley; Renin; 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

Chronic treatment with the adenosine receptor antagonist caffeine evokes an up-regulation of A1 adenosine receptors and increased coupling of the receptor to G proteins in rat brain membranes. However, chronic agonist exposure has not been explored. Primary cultures of cerebellar granule cells were exposed chronically to A1 adenosine receptor agonists and antagonists. Exposure to the A1 adenosine receptor agonist N6-cyclopentyladenosine resulted in (1) a time- and concentration-dependent reduction in the density of receptors labeled by 1,3-[3H]dipropyl-8-cyclopentylxanthine, (2) an enhanced ability of guanyl nucleotides to decrease the fraction of A1 adenosine receptor sites displaying high affinity for 2-chloroadenosine, and (3) a functional uncoupling of receptors from adenylyl cyclase (EC 4.6.1.1). The adenosine antagonists caffeine and 8-p-sulfophenyltheophylline produced alterations in A1 adenosine receptor homeostasis that were antipodal to those associated with agonist treatment. Antagonist exposure (1) increased the density of A1 adenosine receptors in cerebellar granule cell membranes, (2) blunted the effect of guanyl nucleotides on receptor coupling to G proteins, and (3) increased the functional coupling of receptors to adenylyl cyclase inhibition. Forskolin treatment of cerebellar granule cells did not affect receptor density, suggesting that cyclic AMP is not involved in the regulation of A1 adenosine receptor expression.

Topics: 2-Chloroadenosine; 4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone; Adenosine; Adenylyl Cyclases; Animals; Binding, Competitive; Cell Membrane; Cerebellum; GTP-Binding Proteins; Guanylyl Imidodiphosphate; Kinetics; Neurons; Phenylisopropyladenosine; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Rats; Rats, Sprague-Dawley; Receptors, Purinergic P1; Theophylline; 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 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

1. We looked for P2-purinoceptors modulating noradrenaline release in rat heart atria. Segments of the atria were preincubated with [3H]-noradrenaline and then superfused with medium containing desipramine (1 microM) and yohimbine (1 microM) and stimulated electrically, by 30 pulses/1 Hz unless stated otherwise. 2. The adenosine A1-receptor agonist, N6-cyclopentyl-adenosine (CPA; EC50 9.7 nM) and the nucleotides, ATP (EC50 6.6 microM) and adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S; EC50 4.8 microM), decreased the evoked overflow of tritium. The adenosine A2a-agonist, 2-p-(2-carbonylethyl)-phenethylamino-5'-N-ethylcarboxamido-a denosine (CGS-21680; 0.03-0.3 microM) and the P2x-purinoceptor agonist beta, gamma-methylene-L-ATP (30 microM) caused no change. 3. The concentration-response curve of CPA was shifted to the right by the adenosine A1-receptor antagonist, 8-cyclopentyl-1,3-dipropyl-xanthine (DPCPX; 3 nM; apparent pKB value 9.7) but hardly affected by the P2-purinoceptor antagonist, cibacron blue 3GA (30 microM). In contrast, the concentration-response curves of ATP and ATP gamma S were shifted to the right by DPCPX (3 nM; apparent pKB values 9.3 and 9.4, respectively) as well as by cibacron blue 3GA (30 microM; apparent pKB values 5.0 and 5.1, respectively). Combined administration of DPCPX and cibacron blue 3GA caused a much greater shift of the concentration-response curve of ATP than either antagonist alone. The concentration-response curve of ATP was not changed by indomethacin, atropine or the 5'-nucleotidase blocker alpha, beta-methylene-ADP. 4. Cibacron blue 3GA (30 microM) increased the evoked overflow of tritium by about 70%. The increase was smaller when the slices were stimulated by 9 pulses/O00 Hz instead of 30 pulses/I Hz.5. The results indicate that the postganglionic sympathetic axons in rat atria possess P2-purinoceptors in addition to the known adenosine Al-receptor. Both mediate inhibition of noradrenaline release. Some adenine nucleotides such as ATP and ATP gamma S act at both receptors. The presynaptic P2-purinoceptor seems to be activated by an endogenous ligand, presumably ATP, under the condition of these experiments. This is the first evidence for presynaptic P2-purinoceptors at cardiac postganglionic sympathetic axons.

Topics: Adenosine; Adenosine Diphosphate; Adenosine Triphosphate; Animals; Antihypertensive Agents; Atropine; Desipramine; Dose-Response Relationship, Drug; Electric Stimulation; Heart Atria; Indomethacin; Male; Norepinephrine; Phenethylamines; Protein Synthesis Inhibitors; Purinergic P1 Receptor Antagonists; Purinergic P2 Receptor Antagonists; Rats; Rats, Wistar; Receptors, Purinergic P1; Receptors, Purinergic P2; Suramin; Triazines; Xanthines; Yohimbine

The electrostatic properties of adenosine-based agonists and xanthine-based antagonists for the adenosine A1 receptor were used to assess various proposals for their relative orientation in the unknown binding site. The electrostatic properties were calculated from distributed multipole representations of SCF wavefunctions. A range of methods of assessing the electrostatic similarity of the ligands were used in the comparison. One of the methods, comparing the sign of the potential around the two molecules, gave inconclusive results. The other approaches, however, provided a mutually complementary and consistent picture of the electrostatic similarity and dissimilarity of the molecules in the three proposed relative orientations. This was significantly different from the results obtained previously with MOPAC AM1 point charges. In the standard model overlay, where the aromatic nitrogen atoms of both agonists and antagonists are in the same position relative to the binding site, the electrostatic potentials are so dissimilar that binding to the same receptor site is highly unlikely. Overlaying the N6-region of adenosine with that near C8 of theophylline (the N6-C8 model) produces the greatest similarity in electrostatic properties for these ligands. However, N6-cyclopentyladenosine (CPA) and 1,3-dipropyl-8-cyclopentyl-xanthine (DPCPX) show greater electrostatic similarity when the aromatic rings are superimposed according to the flipped model, in which the xanthine ring is rotated around its horizontal axis. This difference is mainly attributed to the change in conformation of N6-substituted adenosines and could result in a different orientation for theophylline and DPCPX within the receptor binding site. However, it is more likely that DPCPX also binds according to the N6-C8 model, as this model gives the best steric overlay and would be favoured by the lipophilic forces, provided that the binding site residues could accommodate the different electrostatic properties in the N6/N7-region. Finally, we have shown that Distributed Multipole Analysis (DMA) offers a new, feasible tool for the medicinal chemist, because it provides the use of reliable electrostatic models to determine plausible relative binding orientations.

Topics: Adenosine; Computer Graphics; Drug Design; Electrochemistry; Kinetics; Ligands; Models, Molecular; Molecular Conformation; Molecular Structure; Purinergic P1 Receptor Antagonists; Receptors, Purinergic P1; Theophylline; Xanthines

1. The effects of adenosine A1 receptor stimulation on basal and histamine-stimulated levels of intracellular free calcium ion concentration ([Ca2+]i) have been investigated in primary astrocyte cultures derived from neonatal rat forebrains. 2. Histamine (0.1 microM-1 mM) caused rapid, concentration-dependent increases in [Ca2+]i over basal levels in single type-2 astrocytes in the presence of extracellular calcium. A maximum mean increase of 1,468 +/- 94 nM over basal levels was recorded in 90% of type-2 cells treated with 1 mM histamine (n = 49). The percentage of type-2 cells exhibiting calcium increases in response to histamine appeared to vary in a concentration-dependent manner. However, the application of 1 mM histamine to type-1 astrocytes had less effect, eliciting a mean increase in [Ca2+]i of 805 +/- 197 nM over basal levels in only 30% of the cells observed (n = 24). 3. In the presence of extracellular calcium, the A1 receptor-selective agonist, N6-cyclopentyladenosine (CPA, 10 microM), caused a maximum mean increase in [Ca2+]i of 1,110 +/- 181 nM over basal levels in 30% of type-2 astrocytes observed (n = 53). The size of this response was concentration-dependent; however, the percentage of type-2 cells exhibiting calcium increases in response to CPA did not appear to vary in a concentration-dependent manner. A mean calcium increase of 605 +/- 89 nM over basal levels was also recorded in 23% of type-1 astrocytes treated with 10 microM CPA (n = 30). 4. In the absence of extracellular calcium, in medium containing 0.1 mM EGTA, a mean increase in [Ca2+]i of 504 +/- 67 nM over basal levels was recorded in 41% of type-2 astrocytes observed (n = 41) after stimulation with 1 microM CPA. However, in the presence of extracellular calcium, pretreatment with the A1 receptor-selective antagonist, 8-cyclopentyl-1,3-dipropylxanthine, for 5-10 min before stimulation with 1 microM CPA, completely antagonized the response in 100% of the cells observed. 5. In type-2 astrocytes, prestimulation with 10 nM CPA significantly increased the size of the calcium response produced by 0.1 microM histamine and the percentage of responding cells. Treatment with 0.1 microM histamine alone caused a mean calcium increase of 268 +/- 34 nM in 41% of the cells observed (n = 34). After treatment with 10 nM CPA, mean calcium increase of 543 +/- 97 nM was recorded in 100% of the cells observed (n = 33). 6. These data indicate that adenosine Al receptors couple to intracellular cal

Topics: Adenosine; Animals; Animals, Newborn; Astrocytes; Calcium; Cells, Cultured; Histamine; Histamine Agonists; Image Processing, Computer-Assisted; Rats; Rats, Wistar; Receptors, Histamine H1; Receptors, Purinergic P1; 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

This study aimed at characterizing the influence of endothelium on noradrenaline release from the canine pulmonary artery. Tritium overflow from intact or endothelium-free vessels preloaded with 0.2 mumol.l-1 3H-noradrenaline was evoked by electrical stimulation (1 Hz, during 5 min) or potassium (25-100 mmol.l-1). The fractional release of tritium evoked by electrical stimulation was increased by removing the endothelium [from 1.7 (1.2; 2.4) to 2.7(2.3; 3.2) x 10(-5).pulse-1, n = 10; P < 0.05]. Neither NG-nitro-L-arginine methyl ester (L-NAME) (up to 300 mumol.l-1) nor indomethacin (up to 30 mumol.l-1), nor endothelin-1 (up to 30 nmol.l-1), nor suramin (up to 300 mumol.l-1) changed tritium release evoked by electrical stimulation. In contrast, the selective A1-adenosine antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) (3.3-33 nmol.l-1) concentration-dependently increased, and the selective A1-adenosine agonist N6-cyclopentyladenosine (CPA) (3.3-100 nmol.l-1) concentration-dependently decreased the evoked release of noradrenaline. Since the effects of DPCPX were observed in endothelium-intact tissues only, it may be concluded that adenosine secreted by the endothelium activates prejunctional release-inhibiting A1-receptors. Tetraethylammonium (TEA) (3.3-33 mmol.l-1) enhanced tritium overflow evoked by electrical stimulation more in endothelium-free than in endothelium-intact vessels, indicating that some K(+)-channel opener is involved in the inhibitory role of endothelium on noradrenaline release. Since it had been previously shown that A1-adenosine receptors are coupled to K(+)-channels, it is suggested that adenosine may inhibit noradrenaline release through the opening of K(+)-channels. In conclusion, the results show that in the canine pulmonary artery, adenosine is a good candidate for the endothelium-dependent inhibitory factor which is responsible for the reduction of noradrenaline release evoked by electrical stimulation.

Topics: Adenosine; Animals; Dogs; Electric Stimulation; Endothelin-1; Endothelium, Vascular; Female; Isotope Labeling; Male; Neuromuscular Junction; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Norepinephrine; Potassium; Potassium Channels; Pulmonary Artery; Purinergic P1 Receptor Antagonists; Suramin; Tetraethylammonium; Tetraethylammonium Compounds; Tritium; Xanthines

We investigated the effects of adenosine receptor antagonists, caffeine, theophylline, 8-phenyltheophylline, and 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), in a light/dark test in mice. All antagonists decreased the time spent in the light zone in this test, which suggested that these compounds have anxiogenic effects. The anxiogenic effects of theophylline were reduced by pretreatment with CGS 21680, an A2-selective agonist, but not by N6-cyclopentyladenosine (CPA), an A1-selective agonist. However, the antagonism of the theophylline-induced anxiogenic effects by CGS21680 was only observed in the time spent in the light zone, and DPCPX-induced anxiogenic effects were neither reversed by CGS 21680 nor by CPA. Finally, it is notable that xanthine-derived adenosine antagonists tested here commonly showed anxiogenic effects in the light/dark test in mice. It is suggested that there is a minor contribution of adenosine receptors to these effects, although theophylline-induced anxiogenic effects were antagonized by an A2 receptor agonist.

Topics: Adenosine; Animals; Antihypertensive Agents; Anxiety; Behavior, Animal; Caffeine; Darkness; Drug Interactions; Light; Male; Mice; Phenethylamines; Purinergic P1 Receptor Antagonists; Structure-Activity Relationship; Theophylline; 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

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

The effect of chronic administration of the adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA) and the adenosine A1 antagonist 8-cyclopentyl-1,3-dipropylxanthine (CPX) on N-methyl-D-aspartate (NMDA)-evoked seizures was studied in C57BL/6 mice (20/group). Animals were injected i.p. for 9 days with either 1.0 mg/kg CPA or 1.0 mg/kg CPX followed by 2 injection-free days (the washout period) and subsequent administration of a single dose of 60 mg/kg NMDA. As in our previous study, this dose of NMDA caused clonic/tonic seizures resulting in high (60%) mortality within 3 h after injection of the drug. Despite insignificant changes in seizure latency, chronic pretreatment with CPA increased the incidence of clonic/tonic episodes and end-point mortality. Conversely; chronic exposure to CPX completely eliminated clonic/tonic episodes, significantly increased average survival time, and reduced end-point mortality (P < 0.05). The results indicate that chronic treatment with adenosine A1 receptor antagonist may protect against NMDA-evoked seizures to the same degree as previously observed following a single, acute exposure to CPA. Since the density of adenosine receptor binding sites was unchanged after chronic treatment with either CPX or CPA, it is likely that the mechanism behind the observed protection may rest at the level of second messenger systems coupled to adenosine A1 receptors.

Topics: Adenosine; Animals; Binding Sites; Injections, Subcutaneous; Male; Mice; Mice, Inbred C57BL; Motor Activity; N-Methylaspartate; Receptors, Purinergic P1; Seizures; Xanthines

1. The presence of A1 adenosine receptors in CHO-K1 cells transfected with the human brain A1 sequence was confirmed by ligand binding studies using 8-cyclopentyl-[3H] 1,3-dipropylxanthine ([3H]-DPCPX). 2. Alterations in intracellular calcium ([Ca2+]i) were measured with the calcium-sensitive dye, fura-2. 3. N6-cyclopentyladenosine (CPA), the selective A1 agonist, and 5'-N-ethylcarboxaminoadenosine (NECA), a relatively non-selective adenosine receptor agonist, elicited rapid, biphasic increases in [Ca2+]i which involved both mobilisation from intracellular stores and calcium entry. 4. The calcium response to CPA was significantly inhibited by the selective A1 antagonist DPCPX. The non-selective adenosine receptor, xanthine amino congener (XAC), was less potent. 5. The calcium response to CPA was completely prevented by pretreatment of the cells with pertussis toxin implicating the involvement of Gi in the receptor-mediated response. 6. In summary, we present evidence for the coupling of transfected human brain A1 adenosine receptors in CHO-K1 cells to mobilisation of [Ca2+]i via a pertussis toxin-sensitive G protein.

Topics: Adenosine; Animals; Brain; Calcium; CHO Cells; Cricetinae; GTP-Binding Proteins; Humans; Pertussis Toxin; Receptors, Purinergic P1; Transfection; Virulence Factors, Bordetella; Xanthines

Adenosine receptors on enteric nerves mediate inhibitory responses to adenosine and its analogs and contribute to the overall excitability of enteric nerves. In characterizing these receptors, the response of the electrically stimulated guinea pig ileum longitudinal muscle-myenteric plexus preparation to receptor-selective analogs of adenosine was investigated and the antagonism of such activity by selective antagonists quantitated. The A1-selective agonist N6-cyclopentyladenosine, the nonselective agonist 5'-N-ethylcarboxamidoadenosine and the 2-substituted uronamides, 2-[p-(carboxyethyl)-phenethylamino]-5'-N-ethylcarboxamidoadenosine and 2-[-(4-fluorophenyl)-ethoxy]-5'-N-ethylcarboxamidoadenosine, both relatively A2-selective agonists, inhibited field-stimulated responses of the ileum with the potency rank order: N6-cyclopentyladenosine > 5'-N-ethylcarboxamidoadenosine >> 2-[p-(carboxyethyl)-phenethylamino]-5'-N-ethylcarboxamidoadenosine approximately 2-[-(4-fluorophenyl)-ethoxy]-5'-N-ethylcarboxamidoadenosine. Antagonism of these responses by receptor-selective antagonists was quantitated using the Schild technique and, for 1,3-dipropyl-8-cyclopentylxanthine, the A1-selective antagonist, demonstrated simple competitive interaction with the responses to N6-cyclopentyladenosine yielding a linear Schild isobole with unit slope. In contrast, responses to the uronamides could not be antagonized in a simple competitive manner. The potency order of the selective agonists is compatible with the presence on enteric nerve endings of an A1 receptor but does not support the presence of the A2 subtype. Moreover, these data demonstrate that the putatively A2-selective adenosine analogs 2-[p-(carboxyethyl)-phenethylamino]-5'-N-ethylcarboxamidoadenosine and 2-[-(4-fluorophenyl)-ethoxy]-5'-N-ethylcarboxamidoadenosine interact with 1,3-dipropyl-8-cyclopentylxanthine at enteric nerve adenosine receptors in a manner which is not compatible with simple competitive interactions.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenosine; Animals; Female; Guinea Pigs; In Vitro Techniques; Male; Myenteric Plexus; Neurotransmitter Agents; Phenethylamines; Purinergic Antagonists; Receptors, Purinergic; Xanthines

The effect of the adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA) and antagonist 8-cyclopentyl-1,3-dipropylxanthine (CPX) on N-methyl-D-aspartate (NMDA)-evoked seizures was studied in C57BL/6 mice (20/group). Animals were injected i.p. either with CPA (0.5, 1, 2 mg/kg) or CPX (1, 2 mg/kg) 15 min prior to administration of NMDA (30, 60, 125 mg/kg). Administration of NMDA alone resulted in a complete locomotor arrest at 30 mg/kg, while clonic/tonic seizures and progressively increasing mortality were seen at higher doses. Prior administration of CPA resulted either in a delay of seizure onset and unchanged mortality (0.5 mg/kg CPA, 60 mg/kg NMDA) or in elimination of tonic episodes and a significant reduction in postictal mortality (1, 2 mg/kg CPA; 60, 125 mg/kg NMDA). Pretreatment with CPX at either 1 or 2 mg/kg eliminated locomotor depression in animals injected with NMDA at 30 mg/kg. At 60 mg/kg NMDA, the effect of CPX administration resulted in mortality equivalent to that seen with 125 mg/kg NMDA administered alone. The results indicate that A1 receptor agonists may protect against NMDA-evoked seizures and that the adenosine A1 receptor may be directly involved in these actions.

Topics: Adenosine; Animals; Drug Interactions; Injections, Intraperitoneal; Male; Mice; Mice, Inbred C57BL; Motor Activity; N-Methylaspartate; Seizures; Survival Rate; Temperature; 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

Adenosine, a locally released and rapidly metabolized nucleoside, protects the heart from damage during ischemia by reducing oxygen demand and increasing oxygen supply. The aminothiophene derivative (2-amino-4,5-dimethylthien-3-yl)[3-(trifluoromethyl)phenyl]-met hanone (PD 81,723) has been shown to act as an allosteric enhancer of the adenosine A1 receptor in brain membranes and thyroid cells. The present study investigates the effects of PD 81,723 in spontaneously contracting right atria and electrically stimulated left atria isolated from Sprague-Dawley rats. N6-cyclopentyladenosine (CPA), an adenosine A1 receptor agonist, produced concentration-dependent inhibition of heart rate in right atria and contractile parameters in left atria. In the right atrium, 5 microM of PD 81,723 significantly shifted the concentration-response curves for CPA to the left, both in the absence and presence of a nonselective adenosine receptor antagonist, 8-(p-sulfophenyl)theophylline (8-SPT, 10 microM). In the left atrium, PD 81,723 also shifted the concentration-response curves for CPA to the left, but only in the presence of 8-SPT. Potentiation of CPA-induced negative chronotropic and inotropic responses with PD 81,723, although not significant, was also observed in the presence of a selective adenosine A1 receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 1 nM). These results demonstrate that PD 81,723 enhances the direct negative chronotropic and inotropic effects of adenosine A1 receptor activation in rat atria.

Topics: Adenosine; Allosteric Regulation; Animals; Atrial Function; Depression, Chemical; Drug Synergism; Heart; Heart Atria; Heart Rate; Male; Myocardial Contraction; Rats; Rats, Sprague-Dawley; Receptors, Purinergic; Theophylline; Thiophenes; 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

The vasodilator activity of adenosine has been associated with selective stimulation of A2 receptors. In the present study, the vasorelaxant (VR) activity of an A1-selective agonist, CPA (cyclopentyladenosine), was examined in isolated porcine coronary arterial rings precontracted with prostaglandin F2 alpha and compared to the A2-selective agonist DPMA (N6-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)-ethyl] adenosine). DPMA, approximately 13-fold selective for the rat brain A2 receptor, relaxed isolated coronary arterial rings with an EC50 of 0.59 +/- 0.19 microM (n = 23) whereas CPA, 2200-fold selective for the rat brain A1 receptor, was approximately 5-fold less potent with an EC50 of 3.18 +/- 0.6 microM (n = 11). At low concentrations (10-300 nM) CPA caused vasoconstriction, indicative of the A1-selective nature of this agonist. CGS 15943 (100 nM), a nonselective adenosine antagonist, attenuated the VR activity of DPMA and CPA, causing a 9- and 12-fold rightward shift of the dose-response curves, respectively, whereas 8-cyclopentyl-1,3-dipropylxanthine (20 nM), a highly A1-selective blocker, had no such effect. Both adenosine antagonists abolished the vasoconstrictor response of CPA at low concentrations. The contributions of the cyclic GMP pathway to adenosine-induced VR was assessed using an inhibitor of endothelium-dependent relaxing factor (L-nitroarginine). L-nitroarginine had no effect on the EC50 for CPA-induced VR and, marginally, but not significantly, attenuated DPMA effects. Moreover, no elevation in cyclic GMP levels could be observed in tissues stimulated with CPA or DPMA.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenosine; Animals; Brain; Coronary Vessels; Cyclic GMP; In Vitro Techniques; Nitric Oxide; Potassium; Potassium Channels; Rats; Receptors, Purinergic; Swine; Vasodilator Agents; Xanthines

To investigate whether endogenous purinoceptor agonists affect the sympathetic neurotransmission in the rat isolated iris, and to classify the purinoceptors modulating exocytotic [3H]-noradrenaline release, we have determined the effect of adenosine receptor antagonists on, and the relative potency of selected agonists in modulating, the field stimulation-evoked (3 Hz, 2 min) [3H]-noradrenaline overflow. In addition, the apparent affinity constants of 8-phenyltheophylline (8-PT) and 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) in antagonizing the prejunctional effects of purinoceptor agonists were estimated. The relatively A1-selective DPCPX 10 and 100 nmol/l increased the evoked [3H]-noradrenaline overflow by about 25%-35% indicating a minor inhibition of evoked release by endogenous purinoceptor agonists probably via an A1 adenosine receptor. Whereas the A1/A2-antagonist 8-PT failed to increase the evoked [3H]-noradrenaline overflow in the absence of exogenous agonists (without or with dipyridamole 1 mumol/l present), the relatively A2-selective antagonist CP-66,713 (4-amino-8-chloro-1-phenyl(1,2,4)triazolo(4,3-a)quinoxaline) 100 nmol/l decreased it by 20%-30% in the absence and continuous presence of DPCPX. This may be compatible with a minor A2-mediated facilitation by an endogenous purinoceptor agonist. All exogenous agonists tested (except UTP 100 mumol/1) inhibited the evoked [3H]-noradrenaline overflow. The relative order of agonist potency (IC40, concentration in mumol/l for inhibition of evoked release by 40%) was CPA (N6-(cyclopentyl)adenosine, 0.004) greater than R-PIA (R(-)N6-(2-phenylisopropyl)adenosine, 0.066) = CHA (N6-(cyclohexyl)adenosine, 0.082) greater than NECA (N5-(ethyl-carboxamido)adenosine 0.44) greater than ADO (adenosine, 4.1). ATP was nearly equipotent with ADO. Maximum inhibition was 70%-80% and similar for all agonists.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenosine; Animals; Electric Stimulation; In Vitro Techniques; Iris; Male; Norepinephrine; Pyrazines; Rats; Rats, Inbred Strains; Receptors, Purinergic; Synaptic Transmission; Theophylline; 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 purpose of the present study was to develop a pharmacological method for determining in the rat in vivo whether endogenous adenosine participates in a given process via activation of A1 adenosine receptors. In anesthetized rats, A1 receptors were activated by infusing the highly selective A1 receptor agonist N6-cyclopentyladenosine, and A2 receptors were stimulated by infusing the highly selective A2 receptor agonist CGS21680C. The bradycardic response to N6-cyclopentyladenosine and the hypotensive response to CGS21680C were used to assess A1 receptor and A2 receptor activation, respectively. After control responses to these purinergic agonists were elicited, animals were given infusions for several hours of either vehicle or one of six dosage levels of FK453 (a potent, selective, nonxanthine A1 receptor antagonist), one of three dosage levels of FR113452 (the S-enantiomer of FK453) or one of seven dosage levels of DPCPX (a potent, selective, xanthine A1 receptor antagonist). Antagonists were infused for > 4 hr, and at various times during the infusions, bradycardic and hypotensive responses to N6-cyclopentyladenosine and CGS21680C, respectively, were reassessed. Both FK453 and DPCPX were highly potent A1 receptor antagonists in vivo, and complete inhibition of bradycardic responses to N6-cyclopentyladenosine were obtained with 3 and 1 micrograms/kg/min, respectively. FR113452 was a very weak antagonist and only slightly reduced bradycardic responses to N6-cyclopentyladenosine at 100 micrograms/kg/min. In vivo FK453 and DPCPX were > 300 and 1000 times selective for the A1 receptor, respectively, compared with the A2 receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenosine; Animals; Blood Pressure; Dose-Response Relationship, Drug; Heart Rate; Male; Phenethylamines; Purinergic Antagonists; Pyrazoles; Pyridines; Rats; Rats, Sprague-Dawley; Receptors, Purinergic; Stereoisomerism; Xanthines

The amino acid sequence of the canine adenosine A1 receptor and the atomic coordinates of a structurally related protein, bacteriorhodopsin, were combined to generate a three-dimensional model for the adenosine A1 receptor. This model consists of seven amphipathic alpha-helices, forming a pore that has a rather distinct partition between hydrophobic and hydrophilic regions. Subsequently, a highly potent and selective ligand, N6-cyclopentyladenosine, was docked into this cavity. A binding site is proposed that takes into account the conformational characteristics of the ligand, obtained from indirect modeling studies by the 'active analog approach'. Moreover, it involves two histidine residues that were shown to be important for ligand coordination from chemical modification studies. Finally, the deduced binding site was used to model other potent ligands that could all be accommodated consistent with earlier biochemical and pharmacological findings.

Topics: Adenosine; Amino Acid Sequence; Animals; Bacteriorhodopsins; Dogs; GTP-Binding Proteins; Histidine; Ligands; Models, Chemical; Models, Molecular; Molecular Sequence Data; Protein Binding; Receptors, Purinergic; Xanthines

The effects of adenosinergic antagonists caffeine and DPCPX, and of the adenosinergic agonists L-PIA, CPA and CGS 21680 were investigated on fully and partially reversible hypoxia-induced electrophysiological changes in rat hippocampal slices. The influence of a high potassium solution and of the N-methyl-D-aspartate antagonist dizocilpine (MK 801) was also tested. The latency to obtain a 50% decrease in the amplitude of the CA1 population spike (CA1 PS) during a short- (5-10 min) lasting hypoxic period was significantly increased (P less than 0.01) by slice perfusion with caffeine (50 microM), DPCPX (0.2 microM), and by increasing (from 3 to 4 mM) the potassium concentration in the medium bathing the hippocampal slices. The latency was significantly decreased (P less than 0.01) by slice perfusion with L-PIA (0.2 microM) and CPA (0.05 microM). It was not significantly modified by CGS 21680 (5 microM). The incidence of reappearance of the CA1 PS during reoxygenation after long- (45 min) lasting hypoxia was significantly increased (P less than 0.05) by slice perfusion with MK 801 (50 microM), while it was not significantly affected by slice perfusion with caffeine (50 microM) or DPCPX (0.2 microM) or L-PIA (0.2 microM) or CPA (0.05 microM) or CGS 21680 (5 microM). The results indicate a prevalent involvement of the A1 adenosine receptors in the early mechanisms underlying hypoxia-induced reversible changes. Adenosine seems to have a limited role in the late mechanisms occurring after a long-lasting hypoxic period.

Topics: Adenosine; Animals; Caffeine; Dizocilpine Maleate; Electrophysiology; Hippocampus; Hypoxia; In Vitro Techniques; Male; Membrane Potentials; Phenethylamines; Phenylisopropyladenosine; Potassium; Purinergic Antagonists; Rats; Rats, Inbred Strains; Synapses; Xanthines

A human brain hippocampus cDNA library was screened by hybridization with a dog A1 adenosine receptor cDNA probe. Sequencing of the resulting clones identified a 978 residue open reading frame encoding a 326 amino acid polypeptide showing 95.7% similarity with the dog A1 adenosine receptor. Individual clones of stably transfected CHO cells expressing the human A1 receptor were obtained and tested for their response to the A1 agonist CPA [N6-cyclopentyladenosine] in the presence of forskolin. One clone was further characterized with respect to membrane binding of various adenosine agonists and antagonists. The rank order of affinities observed was typical of an A1 adenosine receptor. A Kd value of 2.28 nM was determined using [3H]DPCPX [dipropylcyclopentyl-xanthine], an A1 selective antagonist.

Topics: Adenosine; Amino Acid Sequence; Animals; Base Sequence; CHO Cells; Cloning, Molecular; Colforsin; Cricetinae; DNA; DNA Probes; Dogs; Hippocampus; Humans; Molecular Sequence Data; Nucleic Acid Hybridization; Receptors, Purinergic; Sequence Homology, Nucleic Acid; Transfection; 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

Microinjections of selective agonists for adenosine receptor subtypes were made into the caudal NTS of rats. CGS 21680, a selective A2 receptor agonist, elicited pronounced, dose-related decreases in mean arterial blood pressure (ED50 = .021 nmols/rat). Conversely, CPA, a selective A1 receptor agonist, elicited potent dose-related increases in mean arterial blood pressure (ED50 = 0.185 nmols/rat). Additionally, the depressor responses elicited by the A2 agonist and the pressor responses elicited by the A1 agonist were completely and selectively blocked, respectively, by the selective A2 antagonist, CGS 15943A, and the selective A1 antagonist, DPCPX. These data indicate that selective activation of brainstem adenosine receptors in vivo may elicit distinct and opposing response patterns.

Topics: Adenosine; Animals; Antihypertensive Agents; Blood Pressure; Brain Stem; Dose-Response Relationship, Drug; Male; Phenethylamines; Quinazolines; Rats; Rats, Inbred Strains; Receptors, Purinergic; Triazoles; Xanthines

The effects of N6-cyclopentyladenosine (CPA), a highly selective agonist for adenosine A1 receptors, on retention of one-trial inhibitory avoidance behavior were examined in mice. Water-deprived animals were trained to avoid drinking by pairing foot-shock with licks from a water spout. Retention was measured as the suppression of drinking (latency to drink) 48 h following training. Administration of CPA (0.15-2.25 mumol/kg) 30 min prior to training produced a dose-dependent impairment in memory of the original avoidance task. The CPA-elicited deficits in retention performance were blocked by pretreatment with 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), a selective A1 receptor antagonist; DPCPX (15 mumol/kg) administration alone had no effect on retention performance. These findings suggest that selective activation of a presumably central population of A1 receptors may impair retention performance and influence information processing.

Topics: Adenosine; Animals; Avoidance Learning; Drug Interactions; Electroshock; Male; Memory; Mice; Receptors, Purinergic; Time Factors; Water Deprivation; Xanthines

The role of adenosine systems in the initiation and termination of seizures was examined using a unique marker for limbic seizures--maximal dentate activation (MDA). At 10 mg/kg 2-chloroadenosine shortened the duration of MDA, a measure of seizure terminating processes. The selective A1 agonist, cyclopentyladenosine, at 3 mg/kg, blocked the increase in duration of MDA, while the A1 antagonist, 1,3-dipropyl-8-cyclopentylxanthine (0.05 mg/kg) had the opposite effect. None of the compounds tested altered the time to onset of MDA, a measure of processes that initiate seizures. Therefore, modulation of A1 adenosine systems appears to alter seizure termination much more than seizure initiation.

Topics: 2-Chloroadenosine; Adenosine; Animals; Electric Stimulation; Hippocampus; Limbic System; Male; Rats; Rats, Inbred Strains; Receptors, Purinergic; Seizures; Time Factors; Xanthines

8-Cyclopentyl-1,3-dipropylxanthine (PD 116,948) is a very potent, very A1-selective adenosine antagonist, with a Ki of 0.46 nM in 3H-CHA binding to A1 receptors in rat whole brain membranes and 340 nM in 3H-NECA binding to A2 receptors in rat striatal membranes. Its 740-fold A1-selectivity is the highest reported for an adenosine antagonist. 3H-PD 116,948 (117 Ci/mmol) was prepared by reduction of the diallyl analog. 3H-PD 116,948 bound to a single site in rat whole brain membranes, with a Bmax of 46 pmol/g wet weight and Kd of 0.42 nM. Nonspecific binding was extremely low, amounting to about 3% of total binding under standard conditions and less than 1% when higher tissue concentrations were used. Affinities of compounds for inhibition of 3H-PD 116,948 binding were highly consistent with an A1 adenosine receptor. Antagonists were equally potent in 3H-PD 116,948 binding and in 3H-CHA binding, while agonists were consistently about 12-fold more potent in 3H-CHA binding. Hill coefficients were 1.0 for antagonists and about 0.65 for agonists. 3H-PD 116,948 should be a useful antagonist ligand for adenosine A1 receptors.

Topics: Adenosine; Animals; Brain; Cell Membrane; Female; Kinetics; Nucleosides; Rats; Xanthines