n(6)-cyclopentyladenosine and 8-cyclopentyl-1-3-dimethylxanthine

Drugs of abuse produce psychomotor stimulation as one of their characteristic behavioral effects. Single administration of opiates stimulates motor activity via effects on gamma-aminobutyric acid (GABA) and dopamine transmission in the ventral tegmental area (VTA). Adenosine A(1) receptor agonists inhibit VTA GABAergic and dopaminergic transmission and are predicted to alter the behavioral effects of opiates. This study examined the effects of intra-VTA administration of selective adenosine A(1) receptor agonist N(6)-cyclopentyladenosine (CPA) and antagonist 8-cyclopentyl-1, 3-dimethylxanthine (CPT) on morphine-induced motor stimulation in C57BL/6 mice. It also examined the effects of CPA on morphine's activation of VTA neurons projecting to limbic and cortical regions including the nucleus accumbens (NAc), anterior cingulate cortex (ACg) and prelimbic cortex (PrL) via quantitation of immediate-early gene c-Fos protein in these regions. Mice received subcutaneous morphine and intra-VTA administration of CPA and then automated motor activity was measured. Morphine treatment induced both motor activity and Fos immunoreactivity in the NAc, ACg and PrL suggesting that behavioral stimulation is produced by neural activation in these regions. Intra-VTA CPA administration produced a dose-dependent inhibition of morphine-induced motor stimulation and blocked c-Fos induction in all three regions. Intra-VTA CPT treatment had no effects on motor activity or on morphine-induced motor stimulation. VTA adenosine A(1) agonist inhibition of morphine's effects on motor activity and of neural activation of VTA projections suggests that these neurons and their regulation are critical to morphine's stimulant effects. Adenosine A(1) receptor agonists and purinergic modulators may represent useful treatment approaches for blocking the behavioral effects of opiates.

Topics: Adenosine; Adenosine A1 Receptor Agonists; Animals; Biomarkers; Cerebral Cortex; Dose-Response Relationship, Drug; Gene Expression Regulation; Limbic System; Male; Mice; Mice, Inbred C57BL; Morphine; Motor Activity; Neurons; Nucleus Accumbens; Proto-Oncogene Proteins c-fos; Theophylline; Ventral Tegmental Area

Adenosine (Ado) mediates vasoconstriction via A(1)-Ado receptors and vasodilation via A(2)-Ado receptors in the kidney. It interacts with angiotensin II (Ang II), which is important for renal hemodynamics and tubuloglomerular feedback (TGF). The aim was to investigate the function of Ado receptors in the Ado-Ang II interaction in mouse microperfused, afferent arterioles. Ado (10(-11)-10(-4) mol/l) caused a biphasic response: arteriolar diameters were reduced (-7%) at Ado 10(-11)-10(-9) mol/l and returned to control values at higher concentrations. Treatment with Ang II (10(-10) mol/l) transformed the response into a concentration-dependent constriction. N(6)-cyclopentyladenosine (A(1)-Ado receptor agonist) reduced diameters (12% at 10(-6) mol/l). Application of CGS21680 (10(-12)-10(-4) mol/l, A(2A) receptor agonist) increased the diameter by 13%. Pretreatment with ZM241385 (A(2A)-Ado receptor antagonist) alone or in combination with MRS1706 (A(2B)-Ado receptor antagonist) resulted in a pure constriction upon Ado, whereas 8-cyclopentyltheophylline (CPT) (A(1)-Ado receptor antagonist) inhibited the constrictor response. Afferent arterioles of mice lacking A(1)-Ado receptor did not show constriction upon Ado. Treatment with Ado (10(-8) mol/l) increased the response upon Ang II, which was blocked by CPT. Ado (10(-5) mol/l) did not influence the Ang II response, but an additional blockade of A(2)-Ado receptors enhanced it. The action of Ado on constrictor A(1)-Ado receptors and dilatory A(2)-Ado receptors modulates the interaction with Ang II. Both directions of Ado-Ang II interaction, which predominantly leads to an amplification of the contractile response, are important for the operation of the TGF.

Topics: Adenosine; Adenosine A1 Receptor Agonists; Adenosine A1 Receptor Antagonists; Adenosine A2 Receptor Agonists; Adenosine A2 Receptor Antagonists; Angiotensin II; Animals; Arterioles; Feedback; Female; Kidney Glomerulus; Kidney Tubules; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Phenethylamines; Purines; Receptor, Adenosine A1; Receptors, Adenosine A2; Theophylline; Triazines; Triazoles; Vasoconstriction; Vasodilation

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

1. The cardiovascular effects of the adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA) and the adenosine A2A receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS 21680) were investigated in rats implanted with telemetry transmitters for the measurement of blood pressure and heart rate. 2. Intraperitoneal (i.p.) injections of the adenosine A1 receptor agonist CPA led to dose-dependent decreases in both blood pressure and heart rate. These effects of 0.3 mg kg(-1) CPA were antagonized by i.p. injections of the adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dimethyl-xanthine (CPT), but not by i.p. injections of the adenosine A2A receptor antagonist 3-(3-hydroxypropyl)-8-(m-methoxystyryl)-7-methyl-1-propargylxanthine phosphate disodium salt (MSX-3). Injections (i.p.) of the peripherally acting nonselective adenosine antagonist 8-sulfophenyltheophylline (8-SPT) and the purported nonselective adenosine antagonist caffeine also antagonized the cardiovascular effects of CPA. 3. The adenosine A2A agonist CGS 21680 given i.p. produced a dose-dependent decrease in blood pressure and an increase in heart rate. These effects of 0.5 mg kg(-1) CGS 21680 were antagonized by i.p. injections of the adenosine A2A receptor antagonist MSX-3, but not by i.p. injections of the antagonists CPT, 8-SPT or caffeine. 4. Central administration (intracerebral ventricular) of CGS 21680 produced an increase in heart rate, but no change in blood pressure. MSX-3 given i.p. antagonized the effects of the central injection of CGS 21680. 5. These results suggest that adenosine A1 receptor agonists produce decreases in blood pressure and heart rate that are mediated by A1 receptors in the periphery, with little or no contribution of central adenosine A1 receptors to those effects. 6. The heart rate increasing effect of adenosine A2A agonists appears to be mediated by adenosine A2A receptors in the central nervous system. The blood pressure decreasing effect of adenosine A2A agonists is most probably mediated in the periphery.

Topics: Adenosine; Adenosine A1 Receptor Agonists; Adenosine A1 Receptor Antagonists; Adenosine A2 Receptor Agonists; Adenosine A2 Receptor Antagonists; Animals; Blood Pressure; Caffeine; Heart Rate; Injections, Intraperitoneal; Male; Phenethylamines; Rats; Rats, Sprague-Dawley; Theophylline; Xanthines

Using a splanchnic nerve-spinal cord preparation in vitro, we have previously demonstrated that tonic sympathetic activity is generated from the thoracic spinal cord. Here, we sought to determine if adenosine receptors play a role in modulating this spinally generated sympathetic activity. Various adenosine analogs were applied. N6-Cyclopentyladenosine (CPA, adenosine A1 receptor agonist) and 5'-N-ethylcarboxamidoadenosine (NECA, adenosine A1/A2 receptor agonist) reduced, while N6-[2-(4-aminophenyl)ethyl]adenosine (APNEA, non-selective adenosine A3 receptor agonist) did not alter sympathetic activity. The inhibitory effect of CPA or NECA on sympathetic activity was reversed by 8-cyclopentyltheophylline (CPT, adenosine A1 receptor antagonist) or abolished by CPT pretreatment. In the presence of 3,7-dimethyl-1-propargylxanthine (DMPX, adenosine A2 receptor antagonist), sympathetic activity was still reduced by CPA or NECA. Sympathetic activities were not changed by applications of the more selective adenosine A2 or A3 receptor agonists or antagonists, including 4-[2-[[6-amino-9-(N-ethyl-beta-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid (CGS21680), 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM241385), 2-chloro-N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (Chloro-IB-MECA), and 3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1,4-(+/-)-dihydropyridine-3,5-dicarboxylate (MRS1191). These findings exclude a possible involvement of A2 or A3 receptors in sympathetic regulation at the spinal levels. Interestingly, CPT alone did not affect sympathetic activity, suggesting that adenosine A1 receptors are endogenously quiescent under our experimental conditions. We conclude that intraspinal adenosine A1 receptors may down-regulate sympathetic outflow and serve as a part of the scheme for neuroprotection.

Topics: Adenosine; Adenosine A1 Receptor Agonists; Adenosine A1 Receptor Antagonists; Adenosine A2 Receptor Agonists; Adenosine A2 Receptor Antagonists; Adenosine A3 Receptor Agonists; Adenosine A3 Receptor Antagonists; Adenosine-5'-(N-ethylcarboxamide); Animals; Dihydropyridines; Rats; Rats, Sprague-Dawley; Receptor, Adenosine A1; Spinal Cord; Splanchnic Nerves; Sympathetic Nervous System; Synaptic Transmission; Theobromine; Theophylline; Triazines; Triazoles

The adenosine A(1) receptor is involved in spinal cord antinociception. As its role at supraspinal sites is not well known, we studied the systemic effects of its agonist N-cyclopentyl-adenosine (CPA) in single motor units from adult-spinalized, intact and sham-spinalized rats. CPA was not effective after spinalization, but it was very effective in intact animals (ID50: 92+/-1.3 microg/kg, noxious pinch) and over 10-fold more potent in sham-spinalized animals (ID50 of 8.3+/-1 microg/kg). Wind-up was also inhibited by CPA. We also studied the effect of CPA in the immature spinal cord preparation, where CPA dose-dependently inhibited responses to low (IC50s: 9+/-0.7 and 7.7+/-1.3 nM) and high intensity stimulation (IC50s: 4.9+/-0.5 and 12.1+/-2 nM). We conclude that the integrity of the spinal cord is crucial for the antinociceptive activity of systemic CPA in adult rats but not in immature rats, not yet influenced by a completely developed supraspinal control.

Topics: Adenosine; Adenosine A1 Receptor Agonists; Adenosine A1 Receptor Antagonists; Analgesics; Animals; Blood Pressure; Dose-Response Relationship, Drug; Electric Stimulation; Evoked Potentials, Motor; Female; Hyperalgesia; In Vitro Techniques; Inflammation; Male; Rats; Rats, Wistar; Receptor, Adenosine A1; Reflex, Monosynaptic; Spinal Cord; Spinal Nerve Roots; Stress, Mechanical; Theophylline

The voltage-gated Ca(2+) channels responsible for synaptic transmission at CA3-CA1 synapses are mainly P/Q- and N-types. It has been shown that tonic inhibition of transmission due to activation of adenosine A(1) receptors occurs at this synapse. We have recently developed a technique to monitor synaptically released glutamate which is based on synaptically induced glial depolarisation. Using this technique, we have examined the effects of different voltage-gated Ca(2+) channel blockers on glutamate release. Under conditions in which the adenosine A(1) receptor was not blocked, omega-AgaIVA (a P/Q-type voltage-gated Ca(2+) channel blocker) suppressed synaptically induced glial depolarisation to a greater extent than omega-CgTxGVIA (an N-type voltage-gated Ca(2+) channel blocker) did. In contrast, in the presence of an adenosine A(1) receptor antagonist, omega-AgaIVA was less effective at suppressing synaptically induced glial depolarisation than omega-CgTxGVIA. These results indicate that, in the absence of adenosine A(1) receptor-mediated tonic inhibition, the contribution of N-type is much greater than that of P-type, and that N-types are the primary target of tonic inhibition in normal conditions in which adenosine A(1) receptor-mediated tonic inhibition is present.

Topics: Adenosine; Adenosine A1 Receptor Agonists; Adenosine A1 Receptor Antagonists; Animals; Calcium Channel Blockers; Calcium Channels, N-Type; Dose-Response Relationship, Drug; Glutamic Acid; Hippocampus; In Vitro Techniques; Male; omega-Agatoxin IVA; omega-Conotoxin GVIA; Rats; Rats, Wistar; Receptor, Adenosine A1; Synapses; Synaptic Transmission; Theophylline

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

At the calyx of Held synapse in brainstem slices of 5- to 7-day-old (P5-7) rats, adenosine, or the type 1 adenosine (A1) receptor agonist N6-cyclopentyladenosine (CPA), inhibited excitatory postsynaptic currents (EPSCs) without affecting the amplitude of miniature EPSCs. The A1 receptor antagonist 8-cyclopentyltheophylline (CPT) had no effect on the amplitude of EPSCs evoked at a low frequency, but significantly reduced the magnitude of synaptic depression caused by repetitive stimulation at 10 Hz, suggesting that endogenous adenosine is involved in the regulation of transmitter release. Adenosine inhibited presynaptic Ca(2+) currents (IpCa) recorded directly from calyceal terminals, but had no effect on presynaptic K+ currents. When EPSCs were evoked by IpCa during simultaneous pre- and postsynaptic recordings, the magnitude of the adenosine-induced inhibition of IpCa fully explained that of EPSCs, suggesting that the presynaptic Ca(2+) channel is the main target of A1 receptors. Whereas the N-type Ca(2+) channel blocker omega-conotoxin attenuated EPSCs, it had no effect on the magnitude of adenosine-induced inhibition of EPSCs. During postnatal development, in parallel with a decrease in the A1 receptor immunoreactivity at the calyceal terminal, the inhibitory effect of adenosine became weaker. We conclude that presynaptic A1 receptors at the immature calyx of Held synapse play a regulatory role in transmitter release during high frequency transmission, by inhibiting multiple types of presynaptic Ca(2+) channels.

Topics: Adenosine; Animals; Auditory Pathways; Baclofen; Blotting, Western; Brain Stem; Calcium; Calcium Channels; Dose-Response Relationship, Drug; Excitatory Postsynaptic Potentials; GABA-B Receptor Agonists; Gene Expression Regulation, Developmental; Immunohistochemistry; Neural Inhibition; omega-Conotoxin GVIA; Patch-Clamp Techniques; Potassium; Potassium Channels; Presynaptic Terminals; Rats; Rats, Wistar; Receptor, Adenosine A1; Receptors, Presynaptic; Synaptophysin; Tetrodotoxin; Theophylline

Adenosine, by acting on adenosine A1 and A2A receptors, is known to antagonistically modulate dopaminergic neurotransmission. We have recently reported that nonselective adenosine receptor antagonists (caffeine and 3,7-dimethyl-1-propargylxanthine) can partially substitute for the discriminative-stimulus effects of methamphetamine. In the present study, by using more selective compounds, we investigated the involvement of A1 and A2A receptors in the adenosinergic modulation of the discriminative-stimulus effects of both cocaine and methamphetamine. The effects of the A1 receptor agonist N6-cyclopentyladenosine (CPA; 0.01-0.1 mg/kg) and antagonist 8-cyclopentyl-1,3-dimethylxanthine (CPT; 1.3-23.7 mg/kg) and the A2A receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine hydrochloride (CGS 21680; 0.03-0.18 mg/kg) and antagonist 3-(3-hydroxypropyl)-8-(3-methoxystyryl)-7-methyl-1-propargylxanthin phosphate disodium salt (MSX-3; 1-56 mg/kg) were evaluated in rats trained to discriminate either 1 mg/kg methamphetamine or 10 mg/kg cocaine from saline under a fixed-ratio 10 schedule of food presentation. The A1 and A2A receptor antagonists (CPT and MSX-3) both produced high levels of drug-lever selection when substituted for either methamphetamine or cocaine and significantly shifted dose-response curves of both psychostimulants to the left. Unexpectedly, the A2A receptor agonist CGS 21680 also produced drug-appropriate responding (although at lower levels) when substituted for the cocaine-training stimulus, and both CGS 21680 and the A1 receptor agonist CPA significantly shifted the cocaine dose-response curve to the left. In contrast, both agonists did not produce significant levels of drug-lever selection when substituted for the methamphetamine-training stimulus and failed to shift the methamphetamine dose-response curve. Therefore, adenosine A1 and A2A receptors appear to play important but differential roles in the modulation of the discriminative-stimulus effects of methamphetamine and cocaine.

Topics: Adenosine; Adenosine A1 Receptor Agonists; Adenosine A1 Receptor Antagonists; Adenosine A2 Receptor Agonists; Adenosine A2 Receptor Antagonists; Animals; Central Nervous System Stimulants; Cocaine; Conditioning, Operant; Discrimination Learning; Discrimination, Psychological; Dose-Response Relationship, Drug; Generalization, Psychological; Male; Methamphetamine; Phenethylamines; Rats; Rats, Sprague-Dawley; Receptor, Adenosine A1; Receptor, Adenosine A2A; Theophylline; Xanthines

Adenosine and adenosine A1 receptor agonists are often, but not always, protective against metabolic insults. The effects of an A1 agonist and antagonist on neuronal death were determined in cortical cell cultures. The A1 agonist cyclohexyladenosine did not attenuate neuronal death induced by oxygen-glucose deprivation, but did attenuate death caused by glucose deprivation or NMDA. Extracellular adenosine levels during oxygen-glucose deprivation were significantly higher than those during glucose deprivation or NMDA exposure. The A1 antagonist 8-cyclopentyltheophylline increased death induced by oxygen-glucose deprivation, but not that caused by glucose deprivation or NMDA exposure. Thus, while activation of A1 receptors can provide neuroprotection, the protective effect appears to become saturated by high levels of endogenous extracellular adenosine during oxygen-glucose deprivation.

Topics: Adenosine; Animals; Cell Culture Techniques; Cell Death; Cerebral Cortex; Excitatory Amino Acid Agonists; Extracellular Space; Glucose; Hypoxia; Mice; N-Methylaspartate; Neurons; Neuroprotective Agents; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Receptors, Purinergic P1; Theophylline

CA3 pyramidal neurons in the rat hippocampus show selective vulnerability to the intracerebroventricular injection of kainic acid (KA). However, the mechanism of this selective neuronal vulnerability remains unclear. In this study, we examined the contribution of endogenous adenosine, a potent inhibitory neuromodulator, to the differences in the neuronal vulnerability of the hippocampus, using microtubule-associated protein (MAP)-2, phosphorylated c-Jun, and major histocompatibility complex (MHC) class II immunoreactivities as markers for neuronal cell loss, neuronal apoptosis and glial activation, respectively. Pretreatment with 8-cyclopenthyltheophylline (CPT), an A1 adenosine receptor antagonist, significantly exacerbated KA-induced neuronal cell loss in both the CA1 and CA3. Although c-Jun phosphorylation, a critical step in neuronal apoptosis, was not detected in the vehicle-injected rat hippocampus, c-Jun phosphorylation was induced in the CA3 by the injection of KA alone. Pretreatment with CPT induced c-Jun phosphorylation in both the CA1 and CA3. MHC class II antigen was also detected in the regions of c-Jun phosphorylation. Coadministration of N6-cyclopenthyladenosine (CHA), an A1 adenosine receptor agonist, attenuated the neuronal cell loss in the CA1 and CA3 with or without pretreatment with CPT. These results strongly suggest that endogenous adenosine has neuroprotective effects against excitotoxin-induced neurodegeneration in the CA1 through its A1 receptors.

Topics: Adenosine; Animals; Apoptosis; Disease Models, Animal; Excitatory Amino Acid Agonists; Glial Fibrillary Acidic Protein; Hippocampus; Histocompatibility Antigens Class II; Injections, Intraventricular; Kainic Acid; Macrophage-1 Antigen; Male; Microtubule-Associated Proteins; Nerve Degeneration; Neuroglia; Neurons; Phosphorylation; Proto-Oncogene Proteins c-jun; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Rats; Rats, Wistar; Receptors, Purinergic P1; Theophylline

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 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

The P1 purinoceptor subtype mediating the negative inotropic responses of guinea-pig left atria and the negative chronotropic responses of beating right atria were characterized. Guinea-pig isolated paced left atria (2Hz, 5ms, threshold voltage+50%) and spontaneously beating right atria were set up in Krebs-bicarbonate solution and isometric tension and rate of contraction, respectively, were recorded. Concentration-response curves for the reduction of tension and rate, respectively, by adenosine receptor agonists, N6-cyclopentyladenosine (CPA), the R- and S- stereoisomers of N6-(2-phenylisopropyl) adenosine (R-PIA and S-PIA), 5'-(N-carboxamido) adenosine (NECA) and 2-p-((carboxyethyl)-phenethylamino)-5'-(N-carboxamido) adenosine (CGS21680) were obtained. The orders of potency on the left atria (CPA = NECA > R-PIA > S-PIA > CGS21680) and right atria (CPA = R-PIA > S-PIA > CGS21680) were consistent with the responses being mediated via A1 receptors. Antagonism of the responses to CPA or R-PIA by 8-cyclo-1,3-dimethylxanthine (CPT) was examined by a full Schild analysis. Concentration-response curves for CPA or R-PIA were obtained in the absence or presence of five or six concentrations (10(-7)-10(-5) or 3 x 10(-5)M) of CPT. The shift in the concentration-response by CPT was expressed as the concentration-ratio (CR) and plotted as -log(CR-1) against log molar concentration of CPT (Schild plot). pA2 values were calculated from the intercept on the concentration axis and by application of the equation; pA2 = log(antagonist concentration) -log (CR-1). The Schild plots had unity slopes indicating competitive antagonism and the pA2 values derived therefrom indicated that the responses were mediated via A1-receptor. Closer inspection of the Schild plots, however, showed that at the higher concentrations of CPT there was a limit to the displacement of the concentration-response curves of the left and right atria to CPA and of the left atria to R-PIA. There were also significant differences in the apparent pA2 values calculated from the equation, when different concentrations of antagonist were examined. These results indicated that at higher concentrations of agonist there may be a component of the response that is resistant to antagonism by CPT. Whether this is related to the proposal that cardiac responses are mediated via A3 receptors is discussed.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Guinea Pigs; Heart Atria; Heart Rate; In Vitro Techniques; Male; Myocardial Contraction; Phenethylamines; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Receptors, Purinergic P1; Theophylline

1. The effects of adenosine receptor agonists were examined on isolated rings of guinea-pig pulmonary artery under normoxic and hypoxic conditions. The rings were denuded of endothelium and tissues were precontracted with phenylephrine (3 x 10(-6) M) before constructing cumulative concentration-response curves to the agonists. 2. 5'-(N-ethylcarboxamido)adenosine (NECA) caused concentration-dependent contractions of the pulmonary artery which were not different between hypoxia and normoxia. The contractions were converted to a relaxation in the presence of the cyclooxygenase inhibitor, indomethacin, and again these were unaffected by hypoxia. 3. Examination of a range of agonists under normoxic conditions in the presence of indomethacin revealed relaxations, except for the A2a receptor-selective agonist, CGS 21680. The vasorelaxation was therefore A2b receptor-mediated. 4. In hypoxia, however, in the presence of indomethacin, vasoconstriction occurred to R(-)-N(6)-(2-phenylisopropyl)adenosine (R-PIA) and, to a greater extent, to Nb-cyclopentyladenosine (CPA). In the absence of indomethacin, the constriction by CPA during hypoxia was significantly greater. 5. The indomethacin-resistant contraction by CPA was abolished by the A1 receptor antagonist, 8-cyclopentyltheophylline (CPT, 3 x 10(-6) M). 6. This study has demonstrated cyclooxygenase-dependent and-independent vasoconstrictions to adenosine agonists in guinea-pig pulmonary artery under hypoxic conditions. The cyclooxygenase-independent contraction is mediated via A1 receptors. 7. These results suggest that endogenous adenosine released in the pulmonary circulation under hypoxic conditions will cause vasoconstriction and may contribute to the pulmonary hypertension associated with acute respiratory failure.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Guinea Pigs; Hypoxia; In Vitro Techniques; Indomethacin; Male; Muscle Contraction; Muscle Relaxation; Muscle, Smooth, Vascular; Phenethylamines; Phenylisopropyladenosine; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Receptors, Purinergic P1; Theophylline; Vasoconstriction; Vasodilation

Adenosine is a CNS depressant with both pre- and postsynaptic actions. Presynaptically, adenosine decreases neurotransmitter release in the hippocampus but only at excitatory terminals. In the thalamus, however, we show that, in addition to its actions at excitatory synapses, adenosine strongly suppresses monosynaptic inhibitory currents both in relay cells of the thalamic ventrobasal complex (VB) and in inhibitory neurons of the nucleus reticularis thalami (nRt). A concomitant increase in transmission failures and results coefficient of variation analysis are both consistent with a presynaptic mechanism. Pharmacological manipulations support an A1 receptor-mediated process. Slow thalamic oscillations induced in vitro by extracellular stimulation and recorded with extracellular multiunit electrodes in VB and nRt are dampened by adenosine without affecting their periodicity. We conclude that adenosine can presynaptically down-regulate inhibitory postsynaptic responses in thalamus and exert robust antioscillatory effects, likely by synergistic depression of both excitatory and inhibitory neurotransmitter release.

Topics: Adenosine; Animals; Bicuculline; Electric Conductivity; Excitatory Amino Acid Antagonists; Female; GABA Antagonists; gamma-Aminobutyric Acid; Glutamic Acid; Male; Quinoxalines; Rats; Receptors, Purinergic P1; Synapses; Thalamus; Theophylline

1. The purpose of this investigation was to develop a pharmacokinetic-pharmacodynamic model for the interaction between an adenosine A1 receptor agonist and antagonist in vivo. The adenosine A1 receptor agonist, N6-cyclopentyladenosine (CPA) and the antagonist, 8-cyclopentyltheophylline (CPT) were used as model drugs. The CPA-induced reduction in mean arterial pressure and heart rate were used as measurements of effect. 2. Four groups of eight rats each received 200 micrograms kg-1 of CPA i.v. in 5 min during a steady-state infusion of CPT at a rate of 0, 57, 114 or 228 micrograms kg-1 h-1. The haemodynamic parameters were continuously measured and frequent blood samples were taken to determine the pharmacokinetics of the drugs. 3. CPT had no influence on the pharmacokinetics of CPA and the baseline values of the haemodynamic variables. Furthermore, no clear antagonism by CPT was observed of the CPA-induced reduction in mean arterial pressure. However, CPT antagonized the effect on heart rate, and with increasing CPT concentrations, a parallel shift of the CPA concentration-effect relationship to the right was observed. 4. An agonist-antagonist interaction model was used to characterize the interaction quantitatively. On the basis of this model, the pharmacodynamic parameters of both CPA and CPT could be estimated. For CPA the values were (mean +/- s.e.): Emax = 198 +/- 11 b.p.m., EC50 = 2.1 +/- 0.7 ng ml-1, Hill factor = 2.3 +/- 0.6 and for CPT: EC50 = 3.7 +/- 0.3 ng ml-1 and Hill factor = 3.1 +/- 0.1. 5. It is concluded that the competitive agonist-antagonist interaction model may be of value to characterize quantitatively the pharmacodynamic interactions between adenosine A1 receptor ligands in vivo.

Topics: Adenosine; Animals; Blood Pressure; Blood Proteins; Heart Rate; Male; Models, Chemical; Protein Binding; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Rats; Rats, Wistar; Receptors, Purinergic P1; Theophylline

Methamphetamine (METH)-induced neurotoxicity to nigrostriatal dopaminergic neurons in experimental animals appears to have a glutamatergic component because blockade of N-methyl-D-aspartate receptors prevents the neuropathologic consequences. Because adenosine affords neuroprotection against various forms of glutamate-mediated neuronal damage, the present studies were performed to investigate whether adenosine plays a protective role in METH-induced toxicity. METH-induced decrements in neostriatal dopamine content and tyrosine hydroxylase activity in mice were potentiated by concurrent treatment with caffeine, a nonselective adenosine antagonist that blocks both A1 and A2 adenosine receptors. In contrast, chronic treatment of mice with caffeine through their drinking water for 4 weeks, which increased the number of adenosine A1 receptors in the neostriatum and frontal cortex, followed by drug washout, prevented the neurochemical changes produced by the treatment of mice with METH treatment. In contrast, this treatment did not prevent 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine-induced dopaminergic neurotoxicity. Furthermore, concurrent administration of cyclopentyladenosine, an adenosine A1 receptor agonist, attenuated the METH-induced neurochemical changes. This protection by cyclopentyladenosine was blocked by cyclopentyltheophylline, an A1 receptor antagonist. These results indicate that activation of A1 receptors can protect against METH-induced neurotoxicity in mice.

Topics: Adenosine; Animals; Caffeine; Corpus Striatum; Dopamine; Male; Methamphetamine; Mice; MPTP Poisoning; Receptors, Purinergic P1; Theophylline; Tyrosine 3-Monooxygenase

The intraperitoneal injection of d-amphetamine (5 mg/kg i.p.), preceded (10 min before) by intrastriatal injection of an adenosine A2 receptor agonist (CGS 21680, 5-10 micrograms) or followed (5 min later) by an intrastriatal adenosine A1 receptor agonist (N6-cyclopentyladenosine, CPA, 30 micrograms), induced ipsilateral rotations in rats. The opposite effect (contralateral rotations) was observed with adenosine receptor antagonists (A2 antagonist, 3,7-dimethyl-1-propargylxanthine, DMPX, 10 micrograms; A1 antagonist, 8-cyclopentyl-1,3-dimethylxanthine, CPT, 2.5 micrograms). These results confirm that both adenosine A2 and A1 receptors modulate striatal dopaminergic neurotransmission.

Topics: Adenosine; Animals; Behavior, Animal; Corpus Striatum; Dextroamphetamine; Dopamine; Injections, Intraperitoneal; Male; Phenethylamines; Purinergic P1 Receptor Antagonists; Rats; Rats, Wistar; Receptors, Purinergic P1; Synaptic Transmission; Theobromine; Theophylline

Adenosine is a potent modulator of various physiological functions. Although adenosine receptors have been demonstrated in the retina, little is known about their functional role. This study determined the effects of relatively selective adenosine agonists on K+ depolarization-induced release of dopamine and retinal arteriolar tone. For dopamine release studies, bovine retinas were isolated and endogenous synaptosomal stores were loaded with [3H] dopamine. Retinas were then transferred to a superfusion chamber and the spontaneous and K+ depolarization-induced release of dopamine were determined. Cyclopentyladenosine (CPA) did not significantly alter the spontaneous release of dopamine; however, CPA produced a dose-related inhibition of K+ depolarization-evoked release of dopamine. This CPA-induced suppression of dopamine release was reversed by pretreatment with the adenosine A1 antagonist cyclopentyltheophylline. In retinal vasculature studies, adenosine and its agonists injected intravitreally dilated retinal arterioles and venules, in newborn pigs, with a potency profile indicative of mediation by A2 adenosine receptors. Intravitreal injections of drugs inhibiting the metabolism of endogenous adenosine also induced an arteriolar vasodilation which was inhibited by co-administration of an adenosine receptor antagonist. Intravitreally administered adenosine antagonists also attenuated the vasodilative response to both systemic hypoxia and systemic hypotension in the newborn pig, indicating that endogenously produced adenosine is important in retinal blood flow regulation.

Topics: Adenosine; Animals; Cattle; Dopamine; Dose-Response Relationship, Drug; Muscle, Smooth, Vascular; Receptors, Purinergic P1; Retina; Retinal Vessels; Swine; Theophylline; Vasodilation

This report describes a rapid and sensitive analysis for the simultaneous detection of the adenosine A1 receptor ligands N6-cyclopentyladenosine (CPA) and 8-cyclopentyltheophylline (CPT) in rat blood. The method involved alkaline extraction of the compounds and internal standard N6-cyclohexyladenosine (CHA) with ethyl acetate, followed by isocratic reversed-phase high-performance liquid chromatography on a 3-microns MicroSphere C18 column with UV detection at 269 nm. The mobile phase consisted of a mixture of 10 mM acetate buffer (pH 4.0)-methanol-acetonitrile (56:40:4, v/v/v) with a flow-rate of 0.50 ml/min. The total run time was ca. 19 min. For CPA and CPT extraction yields were greater than 77 and 66% in the concentration range of 0.010-0.75 microgram/ml and 0.025-15 micrograms/ml, respectively, with intra- and inter-assay variations less than 9%. In 100 microliter blood samples the corresponding limits of detection were 3.3 and 6.2 ng/ml (signal-to-noise ratio = 3). CPA was found to be degraded in rat blood in vitro with a half-life of 24 min at 37 degrees C. The utility of the analytical method was established by analyzing blood samples from rats which had received an intravenous administration of 200 micrograms/kg CPA or 12 mg/kg CPT. Due to its rapidity and sensitivity this method is concluded to be particularly useful in pharmacokinetic studies with CPA and CPT.

Topics: Adenosine; Animals; Chromatography, High Pressure Liquid; Drug Stability; Hydrogen-Ion Concentration; Kinetics; Ligands; Male; Purinergic P1 Receptor Antagonists; Rats; Rats, Wistar; Receptors, Purinergic P1; Theophylline

Spatial memory acquisition in Morris water maze was tested in C57BL/6 mice. Animals were injected once daily with different doses of either N6-cyclopentyladenosine (CPA) or 8-cyclopentyl-1,3-dipropylxanthine (CPX). Drugs were administered for 9 days either concurrently with water maze testing (drugs injected 1 h after each trial), or prior to the entire block of trials. In the latter case, 1 day without injections preceded water maze experiments. Chronic administration of CPA resulted in a significant, dose-dependent reduction of target latencies, rapid development of spatial preference, and the absence of animals unable to perform the task. CPX treated animals did not show significant performance changes, and failed to develop spatial preference. Locomotor disturbances were not the cause of the observed effects. Our results indicate that chronic treatment with agents acting at adenosine A1 receptors results in behavioral effects that are significantly different from those observed following their acute administration. Therefore, particular caution is required in development of adenosine-based strategies targeted at neurodegenerative or cognitive disorders in which chronic treatment is advocated.

Topics: Adenosine; Animals; Dose-Response Relationship, Drug; Injections, Intraperitoneal; Learning; Male; Memory; Mice; Mice, Inbred C57BL; Motor Activity; Spatial Behavior; Theophylline

Glutamate has been shown to play an important role in delayed neuronal cell death occurring due to ischemia. Attenuation of synaptically released glutamate can be accomplished by modulators such as adenosine and baclofen. This study focused on the ability of adenosine to attenuate the excitotoxicity secondary to glutamate receptor activation in vitro after exposure to potassium cyanide (KCN) in hippocampal neuronal cell cultures. For this study, hippocampal cell cultures were obtained from 1-day-old rats and trypan blue staining was used for assessment of cell viability. It was found that the N-methyl-D-aspartate-specific antagonist MK801 (10 microM) attenuated neuronal cell death resulting from exposure to 1 mM KCN for 60 minutes. Adenosine (10 to 1000 microM) decreased neuronal cell death secondary to the same concentration of KCN in a dose-dependent manner. This same neuroprotective effect is mimicked by the adenosine A1-specific receptor agonist N6-cyclopentyladenosine (10 microM). The A1-specific receptor antagonist 8-cyclopentyl-1,3-dimethylxanthine (10 to 1000 nM) blocked the neuroprotective effect of adenosine in a dose-dependent manner. Therefore, neuronal cell death produced by KCN in the experimental model described was mediated at least in part by glutamate. This neuronal cell death was attenuated by adenosine via the A1-specific mechanism.

Topics: Adenosine; Animals; Cell Death; Cells, Cultured; Dizocilpine Maleate; Hippocampus; Neurons; Osmolar Concentration; Potassium Cyanide; Purinergic Antagonists; Theobromine; Theophylline

Whole-cell patch-clamp was used to investigate synaptic transmission in hippocampal slices. Excitatory post-synaptic currents (EPSCs) were facilitated by low (less than or equal to 1 microM) adenosine (Ado) concentrations, while high concentrations had well-known inhibitory effects on the EPSC. When added on the background of preapplied Ado, methylxanthines caused a large potentiation of EPSCs. At saturation, the enhanced EPSC could exceed the control almost by an order of magnitude. Pertussis toxin strongly impaired the ability of Ado to block EPSCs but did not augment the facilitatory effect. The two components of the EPSC mediated by N-methyl-D-aspartate (NMDA) and non-NMDA receptors were facilitated simultaneously and in equal proportions.

Topics: Adenosine; Animals; Dose-Response Relationship, Drug; Drug Synergism; Evoked Potentials; Hippocampus; In Vitro Techniques; N-Methylaspartate; Pyramidal Tracts; Rats; Rats, Inbred Strains; Synapses; Synaptic Transmission; Theophylline

Interactions between ATP and adenosine on the formation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and mobilization of intracellular calcium were investigated in the smooth muscle cell line DDT1 MF-2. Activation of adenosine A1 receptors with adenosine or cyclopentyladenosine (CPA) or of nucleotide receptors with ATP increased both Ins(1,4,5)P3 formation and intracellular calcium concentrations. The A1 receptor-induced Ins(1,4,5)P3 formation (EC50 10 nM) was antagonized by the A1 antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and by pretreatment of the cells with pertussis toxin (PTX). ATP-stimulated Ins(1,4,5)P3 formation (EC50 21 microM) was attenuated, but still present, after PTX treatment. ATP and CPA had supraadditive effects on Ins(1,4,5)P3 accumulation and CPA increased ATP-induced Ins(1,4,5)P3 accumulation in a concentration-dependent manner with an EC50 of 3 nM, a concentration which per se had little or no effect on Ins(1,4,5)P3 accumulation. ATP (EC50 4 microM) and CPA (EC50 4 nM) both increased intracellular calcium levels. The effect of ATP was partially sensitive to PTX treatment, whereas the effect of CPA was blocked both by PTX and by DPCPX. Concentrations of ATP and CPA that by themselves were insufficient to raise intracellular calcium were able to do so when combined. The synergy between ATP and CPA on the mobilization of intracellular calcium was abolished after treatment of cells with PTX or when DPCPX was included in the experiment. Since ATP was metabolized by ecto-enzymes to ADP, AMP, and adenosine, we also examined whether adenosine formed from ATP could enhance the ATP effects on Ins(1,4,5)P3 accumulation. Indeed, the addition of the A1 receptor antagonist DPCPX or removal of endogenous adenosine by inclusion of adenosine deaminase in the experimental medium significantly attenuated the ATP response, and the two treatments did not have additive effects. The present study thus demonstrates that in a clonal cell line two types of receptors increase phospholipase C activity, but via different pathways; nucleotide receptors appeared to act via partially PTX-insensitive, and A1 receptors via PTX-sensitive G-proteins. ATP and CPA are not only able per se to induce formation of Ins(1,4,5)P3 and mobilize intracellular calcium, but they also act synergistically. Finally, it is demonstrated that endogenous adenosine, possibly formed from the rapid breakdown of ATP, can significantly enhance some ATP effects.

Topics: Adenosine; Adenosine Deaminase; Adenosine Triphosphate; Animals; Calcium; Cell Line; Colforsin; Cyclic AMP; Dose-Response Relationship, Drug; Inositol 1,4,5-Trisphosphate; Kinetics; Leiomyosarcoma; Muscle, Smooth; Pertussis Toxin; Purinergic Antagonists; Receptors, Purinergic; Theophylline; Type C Phospholipases; Virulence Factors, Bordetella