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

Hyperalgesic priming, a sexually dimorphic model of transition to chronic pain, is expressed as prolongation of prostaglandin E2-induced hyperalgesia by the activation of an additional pathway including an autocrine mechanism at the plasma membrane. The autocrine mechanism involves the transport of cyclic adenosine monophosphate (AMP) to the extracellular space, and its conversion to AMP and adenosine, by ecto-5'phosphodiesterase and ecto-5'nucleotidase, respectively. The end product, adenosine, activates A1 receptors, producing delayed onset prolongation of prostaglandin E2 hyperalgesia. We tested the hypothesis that the previously reported, estrogen-dependent, sexual dimorphism observed in the induction of priming is present in the mechanisms involved in its expression, as a regulatory effect on ecto-5'nucleotidase by estrogen receptor α (EsRα), in female rats. In the primed paw AMP hyperalgesia was dependent on conversion to adenosine, being prevented by ecto-5'nucleotidase inhibitor α,β-methyleneadenosine 5'-diphosphate sodium salt and A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine. To investigate an interaction between EsRα and ecto-5'nucleotidase, we treated primed female rats with oligodeoxynucleotide antisense or mismatch against EsRα messenger RNA. Whereas in rats treated with antisense AMP-induced hyperalgesia was abolished, the A1 receptor agonist N. This study presents evidence of an estrogen-dependent mechanism of expression of chronic pain in female rats, supporting the suggestion that differential targets must be considered when establishing protocols for the treatment of painful conditions in men and women.

Topics: 5'-Nucleotidase; Adenosine; Adenosine A1 Receptor Antagonists; Adenosine Monophosphate; Animals; Chronic Pain; Dinoprostone; Disease Models, Animal; DNA, Antisense; Estrogen Receptor alpha; Female; Gene Expression Regulation; Hyperalgesia; Male; Pain Threshold; Rats; Rats, Sprague-Dawley; Ryanodine; Sex Factors; Time Factors; Xanthines

Adenosine agonists or low doses of morphine exert anti-convulsant effects in different models of seizures. On the other hand, a tight interaction has been reported between morphine and adenosine in various paradigms. This study investigated the effect of the interaction of adenosine and morphine on seizure susceptibility in the intravenous mouse model of pentylenetetrazole (PTZ)-induced clonic seizures. The researchers used acute systemic administration of morphine, N(6)-cyclohexyladenosine (CHA) (a selective A1 receptor agonist), naltrexone (an opioid receptor antagonist) and 8-Cyclopentyl-1,3-dimethylxanthine (8-CPT) (a selective A1 receptor antagonist). Acute administration of morphine (0.25, 0.5 and 1 mg/kg) or CHA (0.25, 0.5, 1, 2 and 4 mg/kg) raised the threshold of seizures induced by PTZ. Non-effective dose of 8-CPT (2 mg/kg) inhibited the anticonvulsant effects of CHA (0.5 and 1 mg/kg). Combination of sub-effective doses of morphine (0.125 mg/kg) and CHA (0.125 mg/kg) increased clonic seizure latency showing the additive effect of morphine and CHA. The enhanced latency induced by combination of low doses of morphine and CHA completely reversed by 8-CPT (2 mg/kg) or naltrexone (1 mg/kg). Moreover, 8-CPT (2 mg/kg) inhibited anticonvulsant effects of morphine (0.25 and 0.5 mg/kg) and naltrexone (1 mg/kg) inhibited anticonvulsant effects of CHA (0.25, 0.5 and 1 mg/kg). Combination of low doses of 8-CPT (1 mg/kg) and naltrexone (0.5 mg/kg) inhibited the anticonvulsant effect of CHA (0.5 and 1 mg/kg). In conclusion, adenosine and morphine exhibit an additive effect on the enhancement of the pentylenetetrazole-induced seizure threshold in mice, probably through A1 or μ receptors.

Topics: Adenosine; Animals; Convulsants; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Interactions; Male; Mice; Morphine; Naltrexone; Narcotic Antagonists; Pentylenetetrazole; Seizures; Xanthines

Stress and psychiatric illness have been associated with a dysregulation of glutamatergic neurotransmission. Recently, positive allosteric modulators (PAMs) of the metabotropic glutamate 2 (mGlu₂) receptor have been found to exert antidepressant-like activity in rats performing under a differential reinforcement of low rate (DRL) 72-s schedule. An autoreceptor role at glutamatergic synapses is the most salient physiological role played by the mGlu₂ receptor. Adenosine A₁ receptors play a heteroreceptor role at many of the same forebrain synapses where mGlu₂ autoreceptors are found. Agonists and/or PAMs of mGlu₂ receptors act similarly to adenosine A₁ receptor agonists with respect to a wide range of electrophysiological, biochemical, and behavioral responses mediated by limbic circuitry thought to play a role in the pathophysiology of neuropsychiatric disease and to mediate therapeutic drug effects. Therefore, the role of adenosine A₁ receptor activation on rat DRL 72-s behavior was explored to provide preclinical evidence consistent or inconsistent with potential antidepressant effects. The adenosine A₁ receptor agonist N⁶-cyclohexyladenosine (CHA) increased the reinforcement rate, decreased the response rate, and induced a rightward shift in inter-response time distributions in a dose-dependent fashion similar to most known antidepressant drugs. The adenosine A₁ receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) blocked these antidepressant-like effects. These novel observations with CHA and DPCPX suggest that activation of adenosine A₁ receptors could contribute to antidepressant effects, in addition to previous preclinical reports of anxiolytic and antipsychotic effects. By implication, targeting a dysregulated glutamatergic system may be an important principle in discovering novel antidepressant agents that may also possess anti-impulsive activity.

Topics: Adenosine; Adenosine A1 Receptor Agonists; Adenosine A1 Receptor Antagonists; Animals; Antidepressive Agents; Autoreceptors; Behavior, Animal; Glutamic Acid; Male; Rats; Rats, Sprague-Dawley; Receptor, Adenosine A1; Receptors, Metabotropic Glutamate; Reinforcement Schedule; Reinforcement, Psychology; Synapses; Synaptic Transmission; Xanthines

Emergence of slow EEG rhythms within the delta frequency band following an ischemic insult of the brain has long been considered a marker of irreversible anatomical damage. Here we investigated whether ischemic adenosine release and subsequent functional inhibition via the adenosine A(1) receptor (A(1)R) contributes to post-ischemic delta activity.. Rats were subjected to episodes of non-injuring transient global cerebral ischemia (GCI) under chloral hydrate anesthesia.. We found that a GCI lasting only 10s was enough to induce a brief discharge of rhythmic delta activity (RDA) with a peak frequency just below 1 Hz quantified as an increase by twofold of the 0.5-1.5 Hz spectral power. This post-ischemic RDA did not occur following administration of the A(1)R antagonist 8-cyclopentyl-1,3-dipropylxanthine. Nevertheless, a similar RDA could be induced in rats not subjected to GCI, by systemic administration of the A(1)R agonist N(6)-cyclopentyladenosine.. Our data suggest that A(1)R activation at levels that occur following cerebral ischemia underlies the transient post-ischemic RDA.. It is likely that the functional, thus potentially reversible, synaptic disconnection by A(1)R activation promotes slow oscillations in the cortical networks. This should be accounted for in the interpretation of early post-ischemic EEG delta activity.

Topics: Adenosine; Adenosine A1 Receptor Antagonists; Animals; Delta Rhythm; Disease Models, Animal; Electrocardiography; Electroencephalography; Heart Rate; Ischemic Attack, Transient; Male; Rats; Rats, Wistar; Receptor, Adenosine A1; Statistics, Nonparametric; Theophylline; Xanthines

Adenosine is an inhibitory modulator of neuronal transmission, including GABAergic transmission in the hypothalamus. It is known that the local GABAergic inputs tonically inhibit the hypothalamic paraventricular neurons projecting to the rostral ventrolateral medulla (RVLM; PVN-RVLM neurons) which regulate sympathetic outflow. In this study, we examined the effects of adenosine on GABAergic synaptic transmission in the PVN-RVLM neurons using whole cell patch-clamp combined with the retrograde labeling technique. Adenosine (100 μM) reversibly decreased the frequency of miniature IPSCs (from 3.41 ± 0.75 to 2.19 ± 0.49 Hz) in a concentration-dependent manner (IC₅₀ = 1.0 μM) without affecting the amplitude and the decay time constant of miniature IPSCs. Adenosine increased the paired-pulse ratio of evoked IPSCs from 1.19 ± 0.05 to 2.28 ± 0.09 (P<0.001). The effects of adenosine was mimicked by a selective A₁ receptor agonist (CHA, 10 μM), and blocked by a selective A₁ receptor antagonist (DPCPX, 2 μM), but not by a selective A₂ receptor antagonist (DMPX, 10 μM). In conclusion, the results showed that adenosine inhibits synaptic GABA release via presynaptic A₁ receptors in the PVN-RVLM neurons, indicating a potential of adenosine A₁ receptors in regulating sympathetic tone in normal and disease states.

Topics: Adenosine; Adenosine A1 Receptor Agonists; Adenosine A1 Receptor Antagonists; Adenosine A2 Receptor Antagonists; Afferent Pathways; Animals; Biophysics; Electric Stimulation; gamma-Aminobutyric Acid; Inhibitory Postsynaptic Potentials; Male; Medulla Oblongata; Neural Inhibition; Neurons; Paraventricular Hypothalamic Nucleus; Patch-Clamp Techniques; Rats; Rats, Sprague-Dawley; Receptor, Adenosine A1; Theobromine; Xanthines

Modulation of glutamatergic neurotransmission by metabotropic glutamate2/3 (mGlu2/3) receptor agonists effectively treats seemingly diverse neuropsychiatric illness such as generalized anxiety disorder and schizophrenia. Activation of adenosine A(1) heteroceptors, like mGlu2 autoreceptors, decreases glutamate release in the medial prefrontal cortex (mPFC) and other limbic brain regions. Previously, we have reported electrophysiological, neurochemical and behavioral evidence for interactions between the 5-hydroxytryptamine(2A) (5-HT(2A)) and mGlu2/3 receptors in the mPFC. The present studies were designed to investigate the effects in rats of adenosine A(1) receptor activation/blockade on a behavior modulated by 5-HT(2A) receptor activation/blockade in the mPFC: head shakes induced in the rat by phenethylamine hallucinogens. An adenosine A(1) receptor agonist, N(6)-cyclohexyladenosine (CHA) suppressed head shakes induced by activation of 5-HT(2A) receptors with the phenethylamine hallucinogen (+/-)-2,5-dimethoxy-4-iodoamphetamine hydrochloride (DOI). An adenosine A1 receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), enhanced DOI-induced head shakes and blocked the suppressant action of an adenosine A(1) receptor agonist on DOI-induced head shakes. Thus, the pattern of activity for an agonist and antagonist at the adenosine A1 receptor with respect to modulating DOI-induced head shakes is similar to the pattern observed with mGlu2/3 receptor agonists and antagonists. These novel observations with an adenosine A(1) receptor agonist suggest that this pharmacological action could contribute to antipsychotic effects in addition to thymoleptic effects.

Topics: Adenosine; Adenosine A1 Receptor Agonists; Adenosine A1 Receptor Antagonists; Amphetamines; Animals; Dose-Response Relationship, Drug; Glutamic Acid; Hallucinogens; Head Movements; Injections, Intraperitoneal; Locomotion; Male; Motor Activity; Prefrontal Cortex; Rats; Rats, Sprague-Dawley; Receptor, Adenosine A1; Serotonin Receptor Agonists; Xanthines

Agonists at A(1) receptors and antagonists at A(2A) receptors are known to be neuroprotective against excitotoxicity. We set out to clarify the mechanisms involved by studying interactions between adenosine receptor ligands and endogenous glutamate in cultures of rat cerebellar granule neurons (CGNs). Glutamate and the selective agonist N-methyl-D: -aspartate (NMDA), applied to CGNs at 9 div (days in vitro), both induced cell death in a concentration-dependent manner, which was attenuated by treatment with the NMDA receptor antagonists dizocilpine, D: -2-amino-5-phosphono-pentanoic acid (D: -AP5) or kynurenic acid (KYA), but not by the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Glutamate toxicity was reduced in the presence of all of the following: cyclosporin A (CsA), a blocker of the membrane permeability transition pore, the caspase-3 inhibitor, benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethylketone (Z-DEVD-fmk), the poly (ADP-ribose) polymerase (PARP-1) inhibitor 3,4-dihydro-5-[4-(1-piperidinyl)butoxyl]-1(2H)-isoquinolinone (DPQ), and nicotinamide. This is indicative of involvement of both apoptotic and necrotic processes. The A(1) receptor agonist, N (6)-cyclopentyladenosine (CPA), and the A(2A) receptor antagonist 4-(2-[7-amino-2-[2-furyl][1,2,4]triazolo[2,3-a][1,3,5]triazo-5-yl-amino]ethyl)phenol (ZM241385) afforded significant protection, while the A(1) receptor blocker 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and the A(2A) receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxyamidoadenosine (CGS21680) had no effect. These results confirm that glutamate-induced neurotoxicity in CGNs is mainly via the NMDA receptor, but show that a form of cell death which exhibits aspects of both apoptosis and necrosis is involved. The protective activity of A(1) receptor activation or A(2A) receptor blockade occurs against this mixed profile of cell death, and appears not to involve the selective inhibition of classical apoptotic or necrotic cascades.

Topics: Adenosine; Adenosine A1 Receptor Antagonists; Adenosine A2 Receptor Antagonists; Animals; Apoptosis; Cell Death; Cell Survival; Cells, Cultured; Cerebellum; Cytarabine; Glutamic Acid; Kynurenic Acid; Necrosis; Neurons; Rats; Rats, Sprague-Dawley; Xanthines

It was previously shown that the acute administration of zinc chloride elicits an antidepressant-like effect in the mouse forced swimming test (FST). We have also shown that the activation of adenosine A(1) and A(2A) receptors produces an antidepressant-like effect in FST. Thus, this study investigated the involvement of adenosine receptors in the antidepressant-like effect of zinc in the FST. The antidepressant-like effect of ZnCl(2) (30 mg/kg, i.p.) in the FST was prevented by the pretreatment of animals with caffeine (3 mg/kg, i.p., a non-selective adenosine receptor antagonist), DPCPX (2 mg/kg, i.p., a selective adenosine A(1) receptor antagonist) or ZM241385 (1 mg/kg, i.p., a selective adenosine A(2A) receptor antagonist), administered at doses that per se produced no anti-immobility effect. Moreover, the treatment of mice with CHA (0.05 mg/kg, i.p., a selective adenosine A(1) receptor agonist), DPMA (0.1 mg/kg, i.p., a selective adenosine A(2A) receptor agonist) or dipyridamole (0.1 microg/site, i.c.v., an adenosine transporter inhibitor) was able to potentiate the action of sub-effective doses of ZnCl(2). Taken together, the results suggest that the antidepressant-like effect of zinc in the mouse FST might involve a direct or indirect activation of adenosine A(1) and A(2A) receptors.

Topics: Adenosine; Adenosine A1 Receptor Agonists; Adenosine A2 Receptor Agonists; Aniline Compounds; Animals; Antidepressive Agents; Caffeine; Central Nervous System Stimulants; Dipyridamole; Female; Male; Mice; Motor Activity; Nucleoside Transport Proteins; Receptor, Adenosine A1; Receptor, Adenosine A2A; Swimming; Triazines; Triazoles; Xanthines; Zinc

Adenosine is a defensive metabolite that is critical to anoxic neuronal survival in the freshwater turtle. Channel arrest of the N-methyl-d-aspartate receptor (NMDAR) is a hallmark of the turtle's remarkable anoxia tolerance and adenosine A1 receptor (A1R)-mediated depression of normoxic NMDAR activity is well documented. However, experiments examining the role of A1Rs in regulating NMDAR activity during anoxia have yielded inconsistent results. The aim of this study was to examine the role of A1Rs in the normoxic and anoxic regulation of turtle brain NMDAR activity. Whole-cell NMDAR currents were recorded for up to 2 h from turtle cortical pyramidal neurons exposed to pharmacological A1R or Gi protein modulation during normoxia (95% O(2)/5% CO2) and anoxia (95% N2/5% CO2). NMDAR currents were unchanged during normoxia and decreased 51+/-4% following anoxic exposure. Normoxic agonism of A1Rs with adenosine or N6-cyclopentyladenosine (CPA) decreased NMDAR currents 57+/-11% and 59+/-6%, respectively. The A1R antagonist 8-cyclopentyl-1,3-dimethylxanthine (DPCPX) had no effect on normoxic NMDAR currents and prevented the adenosine and CPA-mediated decreases in NMDAR activity. DPCPX partially reduced the anoxic decrease at 20 but not 40 min of treatment. The Gi protein inhibitor pertussis toxin (PTX) prevented both the CPA and anoxia-mediated decreases in NMDAR currents and calcium chelation or blockade of mitochondrial ATP-sensitive K+ channels also prevented the CPA-mediated decreases. Our results suggest that the long-term anoxic decrease in NMDAR activity is activated by a PTX-sensitive mechanism that is independent of A1R activity.

Topics: Adenosine; Analysis of Variance; Animals; Anorexia; Anti-Arrhythmia Agents; Cerebral Cortex; Chelating Agents; Decanoic Acids; Drug Interactions; Egtazic Acid; Hydroxy Acids; In Vitro Techniques; Membrane Potentials; Neurons; Patch-Clamp Techniques; Pertussis Toxin; Receptor, Adenosine A1; Receptors, N-Methyl-D-Aspartate; Time Factors; Turtles; Xanthines

Adenosine can regulate synaptic transmission through modulation of the action of other neurotransmitters. The influence of adenosine on VIP enhancement of synaptic transmission in hippocampal slices was investigated. Facilitation of fEPSP slope by 1 nM VIP (23.3+/-1.3%) was turned into an inhibition (-12.1+/-3.4%) when extracellular endogenous adenosine was removed using adenosine deaminase (ADA, 1U/ml). Blockade of adenosine A(1) receptors with 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 10 nM) or of A(2A) receptors with ZM241385 (20 nM) attenuated the effect of VIP. When both DPCPX and ZM241385 were present the effect of VIP was abolished. In the presence of ADA, selective A(1) receptor activation with N(6)-cyclopentyladenosine (CPA, 15 nM) or A(2A) receptor-activation with CGS21680 (10 nM) partially readmitted the excitatory effect of VIP on fEPSPs. In contrast, facilitation of PS amplitude by 1 nM VIP (19.1+/-1.2%) was attenuated in the presence of ADA or DPCPX but was not changed by ZM241385. CPA, in the presence of ADA, fully restored the effect of VIP on PS amplitude. In conclusion, VIP facilitation of synaptic transmission to hippocampal pyramidal cell dendrites is dependent on both A(1) and A(2A) receptor activation by endogenous adenosine. VIP effects on PS amplitude are only dependent on A(1) adenosine receptor activation. This differential sensitivity to adenosine modulation might be due to the different VIP circuits contributing to VIP effects on pyramidal cell dendrites and pyramidal cell bodies.

Topics: Adenosine; Adenosine Deaminase; Analysis of Variance; Animals; Drug Interactions; Electric Stimulation; Excitatory Postsynaptic Potentials; Hippocampus; In Vitro Techniques; Male; Membrane Potentials; Neuroprotective Agents; Patch-Clamp Techniques; Pyrimidines; Rats; Rats, Wistar; Receptor, Adenosine A1; Receptor, Adenosine A2A; Synaptic Transmission; Triazines; Triazoles; Vasoactive Intestinal Peptide; Xanthines

We used the patch-clamp technique to examine the effect of adenosine on the basolateral K channels in the thick ascending limb (TAL) of the rat kidney. A 50-pS inwardly rectifying K channel was detected in the basolateral membrane, and the channel activity was decreased by hyperpolarization. Application of adenosine (10 microM) increased the activity of basolateral 50 pS K channels, defined by NP(o), from 0.21 to 0.41. The effect of adenosine on the 50 pS K channels was mimicked by cyclohexyladenosine (CHA), which increased channel activity by a dose-dependent manner. However, inhibition of the A1 adenosine receptor with 8-cyclopentyl-1, 3-dipropylxanthine (DPCPX) failed to block the effect of CHA. In contrast, application of 8-(3-chlorostyryl) caffeine (CSC), an A2 adenosine antagonist, abolished the stimulatory effect of CHA. The possibility that the effect of adenosine and adenosine analog on the basolateral 50 pS K channel was the result of activation of the A2 adenosine receptor was also suggested by the observation that application of CGS-21680, a selected A(2A) adenosine receptor agonist, increased the channel activity. Also, inhibition of PKA with N-[2-(methylamino)ethyl]-5-isoquinoline sulfonamide-2HC1 abolished the stimulatory effect of CHA on the basolateral 50 pS K channel. Moreover, addition of the membrane-permeable cAMP analog increases the activity of 50 pS K channels. We conclude that adenosine activates the 50 pS K channel in the basolateral membrane of the TAL and the stimulatory effect is mainly mediated by a PKA-dependent pathway via the A2 adenosine receptor in the TAL.

Topics: Adenosine; Adenosine A2 Receptor Antagonists; Adenylyl Cyclases; Algorithms; Animals; Bucladesine; Caffeine; Dose-Response Relationship, Drug; Enzyme Activation; Female; Kidney; Male; Patch-Clamp Techniques; Phenethylamines; Potassium Channels; Rats; Rats, Sprague-Dawley; Receptors, Adenosine A2; Stimulation, Chemical; Xanthines

Adenosine A1 receptors are ubiquitous mediators of presynaptic inhibition of neurotransmission in the central nervous system, yet the signalling pathway linking A1 receptor activation and decreased neurotransmitter release remains poorly resolved. We tested the contribution of c-Jun N-terminal kinase (JNK) to adenosine A1 receptor-mediated depression of field excitatory postsynaptic potentials (fEPSPs) in area CA1 of the rat hippocampus. We found that inhibition of JNK with SP600125 or JNK inhibitor V, but not an inactive analogue, attenuated the depression of fEPSPs induced by adenosine, hypoxia, and the A1 receptor agonist N(6)-cyclopentyladenosine (CPA). In contrast, the JNK inhibitor SP600125 did not inhibit GABA(B)-mediated synaptic depression. In support of our electrophysiological findings, Western blot analysis showed that A1 receptor stimulation resulted in a transient increase in JNK phosphorylation in the membrane fraction of hippocampal lysates. The total amount of JNK in the membrane fraction was unchanged by CPA treatment. The increase in phosphorylated JNK induced by A1 receptor stimulation was blocked by the A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), indicating that A1 receptors specifically activate JNK in the hippocampus. Together with functional data indicating that JNK inhibition decreased CPA-induced paired pulse facilitation, these results suggest that JNK activation is necessary for adenosine A1 receptor-mediated synaptic depression occurring at a presynaptic locus The adenosine A1 receptor-JNK signalling pathway may represent a novel mechanism underlying inhibition of neurotransmitter release in the CNS.

Topics: Adenosine; Adenosine A1 Receptor Agonists; Adenosine A1 Receptor Antagonists; Animals; Animals, Newborn; Baclofen; Drug Interactions; Electric Stimulation; Enzyme Inhibitors; Excitatory Postsynaptic Potentials; GABA Agonists; Gene Expression; Hippocampus; Hypoxia; In Vitro Techniques; JNK Mitogen-Activated Protein Kinases; Long-Term Synaptic Depression; Rats; Rats, Sprague-Dawley; Receptor, Adenosine A1; Time Factors; Xanthines

The adenosine A(1) receptor (A(1)R)-adenylyl cyclase (AC) pathway was studied in post-mortem human frontal and occipital cortex from Pick's disease (PiD) cases and age-matched nondemented controls. In frontal cortex, the main brain area affected in PiD, A(1)Rs, determined by radioligand binding, Western blotting and real-time PCR assays, were significantly increased in PiD samples, suggesting up-regulation of this receptor. AC activity was determined in basal and stimulated conditions via stimulatory guanine nucleotide binding proteins (Gs) using GTP, or directly with forskolin. Basal AC activity was reduced in brains from PiD cases. This agrees with the decrease in AC type I (AC I) level detected by Western blotting. However, inhibition of forskolin-stimulated AC activity by a selective A(1)R agonist was significantly increased in brains from PiD. In occipital cortex, adenosine A(1)R numbers were similar in control and PiD cases, and no significant differences were found in A(1)R-mediated AC inhibition. These results show that the adenosine A(1)R-AC transduction pathway is specifically up-regulated and sensitized in frontal cortex brain in PiD.

Topics: Adenosine; Adenosine A1 Receptor Agonists; Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Aged; Brain; Cadaver; Cell Membrane; Colforsin; Female; Frontal Lobe; Humans; Male; Occipital Lobe; Pick Disease of the Brain; Receptor, Adenosine A1; RNA, Messenger; Up-Regulation; Xanthines

We have previously demonstrated that cerebellar adenosine modulates ethanol ataxia. Using Rotorod method, we investigated the role of cerebellar GABA(A) receptors in the adenosinergic modulation of ethanol ataxia in mice. Direct cerebellar microinfusion of GABA(A) agonist, muscimol (2.5, 5 and 10 ng) and antagonist, bicuculline (50, 100 and 200 ng), via permanently implanted guide cannulas, produced a marked and dose-dependent accentuation and attenuation, respectively, of ethanol (2g/kg; IP) ataxia. The accentuation of ethanol ataxia by intracerebellar muscimol was through GABA(A) receptor because intracerebellar pretreatment with bicuculline virtually abolished muscimol effect. Intracerebellar microinfusion of adenosine A(1) agonist, N(6)-cyclohexyladenosine (CHA: 4 ng), and antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX: 100 ng) markedly accentuated and attenuated, respectively, ethanol ataxia consistent with our previously published data. Intracerebellar microinfusion of CHA (4 ng) or DPCPX (100 ng) markedly enhanced and reduced, respectively, muscimol (10 ng)-induced accentuation of ethanol ataxia suggesting co-modulation of ethanol ataxia by cerebellar adenosinergic A(1) and GABA(A) receptors. Similarly, intracerebellar bicuculline (200 ng) pretreatment not only prevented CHA-induced accentuation of ethanol ataxia, but caused further decrease in ethanol ataxia. No change in the normal coordination was observed when microinfusion of the highest dose of muscimol, bicuculline, DPCPX or CHA alone or in combination was followed by saline injection instead of ethanol. The results of the present study suggest a functional similarity between GABA(A) and adenosine A(1) receptors even though both receptor types are known to couple to different signaling system and their location is on the opposite ends of the cerebellar granule cells, axons and axonal terminals (i.e., GABA(A) at the granule cells and adenosine A(1) on axons and axonal terminals of the granule cells) and act as co-modulators of ethanol ataxia.

Topics: Acute Disease; Adenosine; Adenosine A1 Receptor Agonists; Adenosine A1 Receptor Antagonists; Alcohol-Induced Disorders, Nervous System; Animals; Central Nervous System Depressants; Cerebellar Ataxia; Cerebellum; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Interactions; Ethanol; GABA Agonists; GABA Antagonists; gamma-Aminobutyric Acid; Male; Mice; Mice, Inbred ICR; Presynaptic Terminals; Receptor, Adenosine A1; Receptors, GABA-A; Synaptic Transmission; Xanthines

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

Topics: Adenosine; Adenosine Deaminase; Animals; Binding, Competitive; Cell Membrane; CHO Cells; Cricetinae; Cricetulus; Guanylyl Imidodiphosphate; Humans; Kinetics; Ligands; Protein Denaturation; Receptor, Adenosine A1; Temperature; Transfection; Urea; Xanthines

The purine nucleoside, adenosine, has been implicated in the regulation of circadian phase in the hamster. In the current report, we present pharmacological evidence supporting the involvement of adenosine A1 receptors in the regulation of the response of the circadian clock to light in mice. Systemic injection of the selective adenosine A1 receptor agonist, N(6)-cyclohexyladenosine (CHA; 0.3 mg/kg) resulted in a 49% reduction (P<0.05) in the magnitude of light-induced phase delays. The inhibitory effect of CHA on light-induced phase delays was dose dependent over a range of 0.1 to 5 mg/kg with an apparent EC(50) of 0.3 mg/kg. Prior administration of the selective adenosine A1 receptor antagonist dipropylcyclopentylxanthine (DPCPX; 1 mg/kg) completely blocked the effect of CHA on photic phase delays. Finally, CHA significantly attenuated light-induced phospho-extracellular signal-related kinases (ERK) immunoreactivity in the mouse suprachiasmatic nucleus (SCN), consistent with a mode of action involving events that occur early in the signaling cascade through which photic information is conveyed to the circadian clock. These data indicate that the role of adenosine in the regulation of circadian phase is similar in mice and hamsters.

Topics: Adenosine; Animals; Circadian Rhythm; Dose-Response Relationship, Drug; Immunohistochemistry; Light; Male; Mice; Mice, Inbred C57BL; Mitogen-Activated Protein Kinase 1; Motor Activity; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Receptors, Purinergic P1; Suprachiasmatic Nucleus; Xanthines

Adenosine has been implicated as a modulator of retinohypothalamic neurotransmission in the suprachiasmatic nucleus (SCN), the seat of the light-entrainable circadian clock in mammals. Intracellular recordings were made from SCN neurons in slices of hamster hypothalamus using the in situ whole-cell patch clamp method. A monosynaptic, glutamatergic, excitatory postsynaptic current (EPSC) was evoked by stimulation of the optic nerve. The EPSC was blocked by bath application of the adenosine A(1) receptor agonist cyclohexyladenosine (CHA) in a dose-dependent manner with a half-maximal concentration of 1.7 microM. The block of EPSC amplitude by CHA was antagonized by concurrent application of the adenosine A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). The adenosine A(2A) receptor agonist CGS21680 was ineffective in attenuating the EPSC at concentrations up to 50 microM. Trains of four consecutive stimuli at 25 ms intervals usually depressed the EPSC amplitude. However, after application of CHA, consecutive responses displayed facilitation of EPSC amplitude. The induction of facilitation by CHA suggested a presynaptic mechanism of action. After application of CHA, the frequency of spontaneous EPSCs declined substantially, while their amplitude distribution was unchanged or slightly reduced, again suggesting a mainly presynaptic site of action for CHA. Application of glutamate by brief pressure ejection evoked a long-lasting inward current that was unaffected by CHA at concentrations sufficient to reduce the evoked EPSC amplitude substantially (1 to 5 microM), suggesting that postsynaptic glutamate receptor-gated currents were unaffected by the drug. Taken together, these observations indicate that CHA inhibits optic nerve-evoked EPSCs in SCN neurons by a predominantly presynaptic mechanism.

Topics: Adenosine; Animals; Antihypertensive Agents; Circadian Rhythm; Cricetinae; Excitatory Postsynaptic Potentials; Male; Mesocricetus; Neural Inhibition; Phenethylamines; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Receptors, Presynaptic; Receptors, Purinergic P1; Retina; Suprachiasmatic Nucleus; Synaptic Transmission; Visual Pathways; Xanthines

There are conflicting reports in the literature regarding the adenosine receptor that mediates the increase in sodium transport in the A6 cell. In this study we used specific A1 and A2 adenosine receptor agonists and antagonists, as well as two different subclones of the A6 cell, to determine which adenosine receptor mediates the increase in sodium transport. In the A6S2 subclone, basolateral and apical N6-cyclohexyladenosine (CHA), a selective A1 receptor agonist, stimulated sodium transport at a threshold concentration <10(-7) M, whereas CGS-21680, a selective A2 receptor agonist, had a threshold concentration that was at least 10(-5) M. The A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) was found to have a nonspecific effect on CHA-stimulated sodium transport, whereas the A2 receptor antagonist 8-(3-chlorostyryl)caffeine (CSC) had no effect. As with the A6S2 subclone, basolateral and apical CHA stimulated sodium transport at a nanomolar concentration in the A6C1 subclone and the threshold concentration for CGS-21680 was in the high micromolar range. Concurrent with the increase in 1 receptor in different subclones of the A6 cell, including a subclone capable of anion secretion.

Topics: Adenosine; Animals; Biological Transport; Cell Line; Cell Membrane; Dimethyl Sulfoxide; Epithelial Cells; Intracellular Membranes; Kidney; Phenethylamines; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Receptors, Purinergic P1; Sodium; Solvents; Xanthines; Xenopus laevis

In this study the role of ATP-sensitive K(+) channels (K(ATP) channels) in the A(1) receptor mediated presynaptic inhibitory modulation of acetylcholine release was investigated in the rat hippocampus. N(6)-Cyclohexyladenosine (CHA), the selective A(1)-adenosine receptor agonist, reduced concentration-dependently the stimulation-evoked (2 Hz, 1 ms, 240 shocks) [3H]acetylcholine ([3H]ACh) release, from in vitro superfused hippocampal slices preloaded with [3H]choline, an effect prevented by the selective A(1) receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). By themselves, neither K(ATP) channel openers, i.e. diazoxide, pinacidil and cromakalim, nor glibenclamide and glipizide, the inhibitors of K(ATP) channels, exerted a significant effect on the resting and evoked release of [3H]ACh. Glibenclamide and glipizide (10-100 microM) completely prevented the inhibitory effect of 0.1 microM CHA and shifted the concentration response curve of CHA to the right. 4-Aminopyridine (10-100 microM), the non-selective potassium channel blocker, increased the evoked release of [3H]ACh, but in the presence of 4-aminopyridine, the inhibitory effect of CHA (0.1 microM) still persisted. Oxotremorine, the M(2) muscarinic receptor agonist, decreased the stimulation-evoked release of [3H]ACh, but its effect was not reversed by glibenclamide. 1,3-Diethyl-8-phenylxanthine (DPX), the selective A(1)-antagonist, effectively displaced [3H]DPCPX in binding experiments, while in the case of glibenclamide and glipizide, only slight displacement was observed. In summary, our results suggest that K(ATP) channels are functionally coupled to A(1) receptors present on cholinergic terminals of the hippocampus, and glibenclamide and glipizide, by interacting with K(ATP) channels, relieve this inhibitory neuromodulation.

Topics: 4-Aminopyridine; Acetylcholine; Adenosine; Animals; ATP-Binding Cassette Transporters; Binding, Competitive; Brain Ischemia; Electric Stimulation; Hippocampus; In Vitro Techniques; KATP Channels; Male; Muscarinic Antagonists; Oxotremorine; Potassium Channel Blockers; Potassium Channels; Potassium Channels, Inwardly Rectifying; Rats; Rats, Wistar; Receptors, Presynaptic; Receptors, Purinergic P1; Xanthines

Circadian rhythms are synchronized to the environmental light-dark cycle by daily, light-induced adjustments in the phase of a biological clock located in the suprachiasmatic nucleus. Ambient light alters the phase of the clock via a direct, glutamatergic projection from retinal ganglion cells. We investigated the hypothesis that adenosine A1 receptors modulate the phase adjusting effect of light on the circadian clock. Systemic administration of the selective adenosine A1 receptor agonist, N6-cyclohexyladenosine (CHA), significantly (p<0.05) attenuated light-induced phase delays and advances of the circadian activity rhythm. Selective agonists for the adenosine A2A and adenosine A3 receptors were without effect. The inhibitory effect of CHA on light-induced phase advances was dose-dependent (0.025-1.0 mg/kg, ED(50)=0.3 mg/kg), and this effect was blocked in a dose-dependent (0.005-1.0 mg/kg) manner by the adenosine A1 receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). Injection of CHA (10 microM) into the region of the suprachiasmatic nucleus significantly attenuated light-induced phase advances, and this effect was also blocked by DPCPX (100 microM). The results suggest that adenosine A1 receptors located in the region of the suprachiasmatic nucleus regulate the response of the circadian clock to the phase-adjusting effects of light.

Topics: Adenosine; Animals; Circadian Rhythm; Cricetinae; Dose-Response Relationship, Drug; Light; Male; Motor Activity; Receptors, Purinergic P1; Suprachiasmatic Nucleus; Xanthines

Cannabinoids are known to impair motor function in humans and laboratory animals. We have demonstrated an accentuation of cannabinoid (CP55,940)-induced motor incoordination in mice by the adenosine A(1) receptor-selective agonist N(6)-cyclohexyladenosine (CHA) (4 ng) using an intracerebellar (ICB) microinjection method. This effect was mediated by the A(1) receptor because pre-treatment with ICB 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) (100 ng), an adenosine A(1) receptor selective antagonist, completely abolished the accentuation. Furthermore, ICB pre-treatment with DPCPX (100 ng) before ICB CP55,940 (15 microg) attenuated the motor incoordination suggesting a modulation by an endogenous adenosine A(1) system. ICB microinjection of CHA or DPCPX prior to ICB vehicle had no effect on normal motor coordination. ICB microinjection of dipyridamole (25 microg), an adenosine transport inhibitor, significantly accentuated the motor incoordination by ICB CP55,940 (15 microg), providing further support for the involvement of endogenous adenosine in the action of CP55,940. Tolerance to the motor incoordinating effect of ICB CP55,940 was demonstrated following 3 days of i.p. CP55,940 (0.1, 1 or 2 mg/kg every 12 or 24 h; total of six or three injections, respectively). Interestingly, animals which exhibited tolerance to ICB CP55,940 also demonstrated tolerance to the accentuating effect of ICB CHA suggesting cross-tolerance between adenosine agonists and cannabinoids. Cross-tolerance was also demonstrated following 3 days of i.p. CHA (0.25 or 1 mg/kg every 24 h; total of three injections) as further evidence of the modulatory role of the cerebellar adenosine system in the acute manifestation of CP55,940-induced motor incoordination. The involvement of cerebellar adenosine and the A(1) receptor in cannabinoid actions is circumstantially supported by previous evidence that CB(1) receptors and A(1) receptors are both localized on cerebellar granule cell parallel fiber terminals and basket cell neurons where they serve to inhibit the release of neurotransmitters.

Topics: Adenosine; Analgesics; Animals; Cannabinoids; Cerebellar Ataxia; Cerebellum; Cyclohexanols; Dipyridamole; Dose-Response Relationship, Drug; Drug Interactions; Drug Tolerance; Male; Marijuana Abuse; Mice; Neurons; Phosphodiesterase Inhibitors; Psychomotor Performance; Receptors, Cannabinoid; Receptors, Drug; Receptors, Purinergic P1; Signal Transduction; Tritium; Xanthines

The effects of angiotensin (ANG) III and ANG IV on pentylenetetrazol (PTZ) seizure susceptibility--threshold and kindling in mice--as well as the influence of adenosine A(1) receptor agents (agonist and antagonist) on these effects were studied. It was found that ANG III and ANG IV increased dose-dependently the PTZ seizure threshold and decreased the seizure intensity in PTZ kindled mice. Cyclohexyladenosine (CHA), an adenosine A(1) receptor agonist, potentiated the effects of ANG III and ANG IV on the seizure threshold and kindling, whereas DPCPX (an A(1) receptor antagonist) reversed peptide-induced effects on the PTZ kindling. Taken together, ANG III and ANG IV decrease the PTZ seizure susceptibility. We could suggest that these effects are realized in part through interaction with adenosine A(1) receptors.

Topics: Adenosine; Angiotensin II; Angiotensin III; Angiotensins; Animals; Brain; Convulsants; Disease Susceptibility; Dose-Response Relationship, Drug; Drug Interactions; Epilepsy; Kindling, Neurologic; Male; Mice; Mice, Inbred ICR; Pentylenetetrazole; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Receptors, Purinergic P1; Seizures; 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

Effects of different doses of adenosine receptor agonists and antagonists on naloxone-induced jumping and diarrhea in morphine-dependent mice were studied. The adenosine A1 receptor agonists, N6-cyclohexyladenosine (CHA: 0.1, 0.25 and 0.5 mg kg(-1)) and R-isomer of N6-phenylisopropyladenosine (R-PIA: 0.1, 0.3 and 1 mg kg(-1)), decreased jumping and diarrhea induced by naloxone in morphine-dependent mice. The adenosine A1 receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX: 0.3-9 mg kg(-1)), increased jumping but decreased diarrhea. The adenosine A2 receptor agonist, 5'-(N-cyclopropyl)-carboxamidoadenosine (CPCA), decreased jumping and diarrhea. However, the adenosine A2 receptor antagonist, 3,7-dimethyl-1-propargylxanthine (DMPX: 0.5 and 1 mg kg(-1)), did not elicit any response in this respect. DPCPX (0.3 and 3 mg kg(-1)), decreased the inhibition of jumping and diarrhea induced by CHA (0.5 mg kg(-1)), while DMPX (0.5 and 1 mg kg(-1)), decreased the inhibition of diarrhea induced by CPCA (0.1 mg kg(-1)). It is concluded that jumping induced by naloxone in morphine-dependent mice may be modified by the adenosine A receptor mechanism(s) and diarrhea induced by the opioid receptor antagonist could be mediated by the adenosine A1 and A2 receptors.

Topics: Adenosine; Analysis of Variance; Animals; Behavior, Animal; Diarrhea; Male; Mice; Mice, Inbred Strains; Morphine; Morphine Dependence; Naloxone; Narcotic Antagonists; Narcotics; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Substance Withdrawal Syndrome; Theobromine; Xanthines

We tested the possibility that extracellular adenosine concentration varies with tissue temperature by measuring the tonic adenosinergic inhibition of excitatory synaptic transmission at different temperatures in the in vitro rat hippocampus. Application of the A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) enhanced population excitatory postsynaptic potentials (EPSPs) by antagonizing tonic adenosinergic inhibition; this effect was greatest at 25 degrees C, and was progressively reduced at 35 and 37.5 degrees C. These results demonstrate that tonic adenosinergic inhibition is inversely related to temperature. In a second experiment, an exogenous A1 agonist, N6-cyclohexyladenosine (CHA), was applied to slices to inhibit evoked EPSPs. CHA inhibition of EPSPs was greater at 35 than at 25 degrees C, demonstrating that the reduced adenosinergic inhibition at higher temperatures is not a result of reduced A1 receptor function.

Topics: Adenosine; Animals; Excitatory Postsynaptic Potentials; Hippocampus; In Vitro Techniques; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Rats; Rats, Sprague-Dawley; Synaptic Transmission; Temperature; Xanthines

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

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

1. Adenosine is a depressant in the central nervous system with pre- and postsynaptic effects. In the present study, intracellular recording techniques were applied to investigate the modulatory effects of adenosine on projection neurons in the lateral rat amygdala (LA), maintained as slices in vitro. 2. Adenosine reversibly reduced the amplitude of a fast inhibitory postsynaptic potential (IPSP) that was evoked by electrical stimulation of the external capsule and pharmacologically isolated by applying an N-methyl-D-aspartate and non-N-methyl-D-aspartate receptor antagonist, DL-(-)-2-amino-5-methyl-4-isoxazolepropionic acid and 6, 7-Dinitroquinoxaline-2,3-dione, respectively, and the gamma-aminobutyric acidB (GABAB) receptor antagonist CGP 35348. The postsynaptic potential that remained was abolished by locally applying bicuculline. 3. Adenosine reduced the amplitude of the fast IPSP on average by 40.3%. It had no significant effect on responses to exogenously applied GABA, on membrane potential or on input resistance, suggesting that the site of action was at presynaptic inhibitory interneurons in the LA. 4. The response to adenosine was mimicked by the selective adenosine A1 receptor agonist N6-cyclohexyladenosine and blocked by the selective adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine. 5. Neuronal responsiveness in the amygdala is largely controlled by inhibitory processes. Adenosine can presynaptically downregulate inhibitory postsynaptic responses and could exert dampening effects likely by depression of both excitatory and inhibitory neurotransmitter release.

Topics: Adenosine; Amygdala; Animals; Bicuculline; Electric Stimulation; Excitatory Postsynaptic Potentials; Female; GABA Antagonists; gamma-Aminobutyric Acid; In Vitro Techniques; Male; Membrane Potentials; Neurons; Organophosphorus Compounds; Patch-Clamp Techniques; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Rats; Rats, Long-Evans; Receptors, Purinergic P1; Synaptic Transmission; Xanthines

Presynaptic adenosine receptors inhibit transmitter release at many synapses and are known to exist on retinotectal terminals. In this paper we show that adenosine decreases retinotectal field potentials by approximately 30% and investigate the mechanism. First, as judged by the effects of specific calcium channel blockers, retinotectal transmission was mediated almost exclusively by N-type calcium channels, which are known to be modulated by adenosine A1 receptors. Transmission was completely blocked by either omega-Conotoxin GVIA (-100%, N-type blocker) or omega-Conotoxin MVIIC (-99%, N-, P- and Q-type blocker) and was not significantly affected by omega-Agatoxin IVA [+1.7 +/- 9. 3% (SE), P-,Q-type blocker], but was augmented slightly by nifedipine(+9.3 +/- 2.1%, L-type blocker). Second, the adenosine inhibition was presynaptic, as indicated by a 43% increase in paired-pulse facilitation. Third, the selective A1 agonist cyclohexyl adenosine (CHA) at 50 nM caused a 21% decrease in amplitude and the selective A2 agonist N6-[2-(3, 5-dimethoxyphenyl)-2-(2-methylphenyl)-ethyl]adenosine (DPMA) at 100 nM caused a 24% increase. Fourth, the selective A1 antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) alone produced an increase in the field potential, suggesting a tonic inhibition mediated by endogenous adenosine. Fifth, pertussis toxin eliminated adenosine inhibition implicating Gi or Go protein coupling. Sixth, C-kinase activation eliminated the A1-mediated inhibition. In regenerating projections, adenosine also caused a decrease in transmission (-30 +/- 12%), but after induction of long-term potentiation (LTP) via trains of stimuli or via treatment with the phosphatase inhibitor okadaic acid, the adenosine response was converted to an augmentation. Because LTP is associated with C-kinase activation, this is consistent with C-kinase uncoupling the A1 receptor from inhibiting N-type Ca2+ channels. This uncovers the A2-mediated augmentation as demonstrated in normals with DPMA. Such an effect could account in part for the LTP of immature synapses and the change from rapidly fatiguing to robust synaptic transmission.

Topics: Adenosine; Animals; Calcium; Enzyme Activation; Evoked Potentials, Visual; Goldfish; Long-Term Potentiation; Nerve Crush; Nerve Regeneration; Nifedipine; omega-Agatoxin IVA; omega-Conotoxin GVIA; omega-Conotoxins; Optic Nerve; Optic Nerve Injuries; Peptides; Pertussis Toxin; Protein Kinase C; Receptors, Purinergic P1; Retina; Spider Venoms; Superior Colliculi; Synaptic Transmission; Virulence Factors, Bordetella; Visual Pathways; Xanthines

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

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

The role of adenosine and ATP-sensitive potassium channels (KATP) in the mechanism of ischemic preconditioning (IPC)-induced protection against the post-ischemic endothelial dysfunction was studied. Langendorff-perfused guinea-pig hearts were subjected either to 40 min of global ischemia and 40 min reperfusion or were preconditioned prior to the ischemia/reperfusion with three cycles of either 5 min ischemia/5 min reperfusion (IPC) or 5 min infusion/5 min wash-out of adenosine, adenosine A1 receptor agonist, N6-cyclohexyladenosine (CHA) or KATP opener, pinacidil. The magnitude of coronary flow reduction caused by NO-synthase inhibitor, Nomega-nitro-l-arginine methyl ester (l-NAME), served as an index of a basal endothelium-dependent vasodilator tone. Coronary overflows produced by a bolus of acetylcholine (ACh) and sodium nitroprusside (SNP) were used as measures of agonist-induced endothelium-dependent and endothelium-independent vascular function, respectively. The coronary flow, LVDP, ACh response and l-NAME response were reduced by 8, 32, 41 and 54%, respectively, while SNP response was not changed in the hearts subjected to ischemia/reperfusion. ACh response was fully restored, l-NAME response was partially restored, and SNP response was not affected in the hearts subjected to IPC. The post-ischemic recoveries of coronary flow and LVDP were not improved by IPC. The protective effect of IPC on the ACh response was mimicked by adenosine, CHA, and pinacidil. The protective effect of IPC, CHA and pinacidil was abolished by KATP antagonist, glibenclamide. The IPC protection was affected neither by a non-specific adenosine antagonist, 8-p-sulfophenyltheophylline, nor by a specific adenosine A1 receptor antagonist, 8-cyclopentyl-1, 3-dipropylxanthine (DPCPX). Our data indicate that: (1) IPC affords endothelial protection in the mechanism that involves activation of KATP, but not adenosine A1 receptors; (2) exogenous adenosine and A1 receptor agonist afford the protection, which might be of a potential clinical significance; (3) the endothelial dysfunction is not involved in the mechanism of myocardial stunning in guinea-pig hearts.

Topics: Acetylcholine; Adenosine; Adenosine Triphosphate; Animals; Endothelium, Vascular; Enzyme Inhibitors; Female; Guinea Pigs; Heart; Hemodynamics; In Vitro Techniques; Ischemic Preconditioning, Myocardial; Male; NG-Nitroarginine Methyl Ester; Nitroprusside; Pinacidil; Potassium Channel Blockers; Potassium Channels; Vasodilator Agents; Xanthines

Freshly isolated rat juxtaglomerular cells (JGC) were superfused to study renin secretion rate (RSR) at the cellular level. Effluates from the superfusion chamber collected in 20-min intervals showed a time-dependent decline in RSR from 85.5 +/- 32 to 4.0 +/- 2.4 ng ANG I. ml-1. h-1. mg protein-1. min-1 within 100 min of collection (mean +/- SE, n = no. of JGC preparations/superfusion chambers = 9/18). Addition of adenosine deaminase type II (ADA II, 3 U/1.4 mg protein) to the superfusion medium increased RSR more than fourfold to 402 +/- 100 ng in the first collection period, which dropped to 237.5 +/- 67 ng ANG I. ml-1. h-1. mg protein-1. min-1 (n = 9/18) within 100 min. This ADA II effect was rapid in onset and fully reversible. When the purified ADA type VII, with a 40-fold higher specific activity, was added to the superfusate, RSR was increased only by 96 +/- 17.8% compared with controls. This ADA VII (5 U/30 microgram) effect could be mimicked by the selective adenosine A1-receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 10(-6) mol/l). Since albumin stimulated RSR in a concentration-dependent fashion, to an extent similar to that of ADA II, we assume that the ADA II effect was largely unspecific in nature. We conclude that 1) superfusion of isolated JGC from rats is suitable for investigations of renin secretion at the cellular level, 2) the increase in RSR by ADA II appears to be only in part due to deamination of endogenously generated adenosine, and 3) albumin in the superfusate induces a similar stimulatory effect as ADA II.

Topics: Adenosine; Adenosine Deaminase; Animals; Cell Separation; Isoenzymes; Juxtaglomerular Apparatus; Male; Perfusion; Rats; Rats, Sprague-Dawley; Renin; Serum Albumin; Time Factors; Xanthines

Topics: Adenosine; Animals; Blood Pressure; Body Temperature; Female; Fever; Male; Oxygen Consumption; Purinergic P1 Receptor Antagonists; Pyrogens; Rabbits; Xanthines

The possible cardioprotective roles of adenosine A1 and A3 receptors were investigated in a cardiac myocyte model of injury. The adenosine A3 receptor is a novel cardiac receptor capable of mediating potentially important cardioprotective functions. Prolonged hypoxia with glucose deprivation was used to simulate ischemia and to induce injury in cardiac ventricular myocytes cultured from chick embryos 14 days in ovo. When present during the prolonged hypoxia, the adenosine A3 agonists N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide (IB-MECA) and 2-chloro-N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide (CI-IB-MECA) caused a dose-dependent reduction in the extent of hypoxia-induced injury as manifested by a decrease in the amount of creatine kinase released and the percentage of myocytes killed. The adenosine A1 agonists 2-chloro-N6-cyclopentyladenosine (CCPA), N6-cyclohexyladenosine, and adenosine amine congener were also able to cause a decrease in the extent of myocyte injury. The A1 receptor-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine blocked the cardioprotective effect of the A1 but not of the A3 agonists. Conversely, the selective A3 antagonists MRS-1191 and MRS-1097 blocked the protection induced by CI-IB-MECA but had minimal effect on that caused by CCPA. Thus the cardioprotective effects of A1 and A3 agonists were mediated by their respective receptors. This study defines a novel cardioprotective function of the cardiac A3 receptor and provides conclusive evidence that activation of both A1 and A3 receptors during hypoxia can attenuate myocyte injury.

Topics: Adenosine; Animals; Cardiotonic Agents; Cell Hypoxia; Cells, Cultured; Chick Embryo; Dihydropyridines; Heart; Heart Ventricles; Myocardial Ischemia; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Receptor, Adenosine A3; Receptors, Purinergic P1; Xanthines

The effect of oral treatment with caffeine, in doses that are known to produce marked adaptive effects, was investigated on A1 and A2A receptors in the mouse brain. Caffeine (0.1, 0.3 or 1 g/l) was added to the drinking water and the animals were sacrificed after a 14-day treatment period. Ligand binding to A1 receptors was studied, using quantitative autoradiography, with the agonist [3H]cyclohexyladenosine (CHA) and the antagonist [3H]1,3-dipropyl-8-cyclopentyl xanthine (DPCPX). Caffeine did not remain in the sections during the autoradiography experiments. Caffeine treatment (1 g/l, but not 0.1 or 0.3 g/l) tended to increase [3H]CHA binding to the CA3 subfield of the hippocampus, but in no other region studied. There was no change in the number of A1 receptors since [3H]DPCPX binding to the CA3, cerebral and cerebellar cortex was not influenced by caffeine treatment. There was similarly no change in the ability of CHA to displace [3H]DPCPX binding, suggesting that there are no major changes in the proportion of A1 receptors that are coupled to G-proteins. mRNA for the A1 receptor, measured by in situ hybridization, did not differ significantly between caffeine-treated and control mice in the structures examined. Thus, higher doses of caffeine can cause an increase in A1 agonist binding without a corresponding change in A1 mRNA or in A1 antagonist binding, suggesting that the adaptive changes seen upon prolonged caffeine treatment may be in sites different from A1 receptors. Caffeine (1 g/l) increased A2A receptors in the striatum measured as binding of the agonist [3H]CGS 21680 suggesting that up-regulation of A2A receptors may be an adaptive effect of caffeine intake.

Topics: Adenosine; Animals; Antihypertensive Agents; Autoradiography; Brain Chemistry; Caffeine; Central Nervous System Stimulants; Cerebellum; Cerebral Cortex; Corpus Striatum; Hippocampus; In Situ Hybridization; Male; Mice; Mice, Inbred Strains; Phenethylamines; Radioligand Assay; Receptors, Purinergic P1; RNA, Messenger; Time Factors; Tritium; Xanthines

Previous studies from our laboratory have provided strong evidence that brain adenosine modulates acute ethanol (i.p.)-induced motor incoordination (MI) through receptor mediated mechanism(s). Recently, we have reported the involvement of the striatum in ethanol-induced MI as well as the striatal adenosinergic modulation of the ethanol-induced motor deficit. The present study was thus designed to further characterize the modulatory effect of striatal adenosine on ethanol-induced MI and to look for its functional correlation with chloride flux within the rat striatum. Intrastriatal microinfusion of adenosine A1 receptor agonist N6-cyclohexyladenosine (CHA) and antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), significantly accentuated and attenuated, respectively, the motor incoordinating effect of ethanol while having no effect on the normal motor coordination in saline-treated control animals. These data confirmed the role of striatal adenosine in ethanol-induced MI. The selectivity of interactions between adenosine A1 agonist and antagonist and ethanol was further confirmed by the study in which neither intrastriatal CHA nor DPCPX significantly altered the MI induced by sodium pentobarbital. Previously, we have shown that intrastriatal Ro15-4513 not only significantly attenuated ethanol-induced MI but also blocked its accentuation by intrastriatal CHA. It is well known that Ro15-4513 antagonizes many, but not all, CNS effects of ethanol by blocking the ethanol potentiation of GABA-stimulated uptake of chloride. Therefore, experiments using striatal microsac preparations were carried out to investigate the possible modulation of chloride conductance by CHA and its relationship to ethanol. High concentrations of CHA (10 and 100 nM) increased the total chloride uptake by the striatal microsacs. Corresponding to the ethanol-adenosine interaction observed behaviorally, a much lower concentration (1 nM) of CHA, being ineffective itself, significantly enhanced the stimulatory action of ethanol on chloride uptake. This effect was blocked by either Ro15-4513 (100 nM) or DPCPX (10 nM). The modulatory effect of GABA and/or ethanol on chloride influx was also evaluated, and the results supported the appropriateness to use striatal microsac preparations in the present study. Overall, the data suggested a functional interaction between ethanol and striatal adenosine and further supported the hypothesis that striatal adenosine might, in part, modulate ethanol-ind

Topics: Adenosine; Affinity Labels; Animals; Azides; Benzodiazepines; Central Nervous System Depressants; Cerebrospinal Fluid; Chlorides; Corpus Striatum; Ethanol; GABA Modulators; Male; Microdialysis; Motor Activity; Pentobarbital; Radioisotopes; Rats; Rats, Sprague-Dawley; Xanthines

The binding of the adenosine A2A receptor selective agonist 2-[4-(2-p-carboxyethyl)phenylamino] -5'-N-ethylcarboxamidoadenosine (CGS 21680) to the rat hippocampal and cerebral cortical membranes was studied and compared with that to striatal membranes. [3H] CGS 21680, in the concentration range tested (0.2-200 nM), bound to a single site with a Kd of 58 nM and a Bmax of 353 fmol/mg protein in the hippocampus, and with a Kd of 58 nM and a Bmax of 264 fmol/mg protein in the cortex; in the striatum, the single high-affinity [3H] CGS 21680 binding site had a Kd of 17 nM and a Bmax of 419 fmol/mg protein. Both guanylylimidodiphosphate (100 microM) and Na+ (100 mM) reduced the affinity of [3H] CGS 21680 binding in the striatum by half and virtually abolished [3H] CGS 21680 binding in the hippocampus and cortex. The displacement curves of [3H] CGS 21680 binding with 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), N6-cyclohexyladenosine (CHA), 5'-N-ethylcarboxamidoadenosine (NECA) and 2-chloroadenosine (CADO) were biphasic in the hippocampus and cortex as well as in the striatum. The predominant [3H]CGS 21680 binding site in the striatum (80%) had a pharmacological profile compatible with A2A receptors and was also present in the hippocampus and cortex, representing 10-25% of [3H]CGS 21680 binding. The predominant [3H]CGS 21680 binding site in the hippocampus and cortex had a pharmacological profile distinct from A2A receptors: the relative potency order of adenosine antagonists DPCPX, 1,3-dipropyl- 8-¿4-[(2-aminoethyl)amino]carbonylmethyl- oxyphenyl¿ xanthine (XAC), 8-(3-chlorostyryl)caffeine (CSC), and (E)-1,3-dipropyl-8-(3,4-dimethoxystyryl)- methylxanthine (KF 17,837) as displacers of [3H] CGS 21680 (5 nM) binding in the hippocampus and cerebral cortex was DPCPX > XAC >> CSC approximately KF 17,837, and the relative potency order of adenosine agonists CHA, NECA, CADO, 2-[(2-aminoethylamino)carbonylethylphenylethylamino]-5'-N- ethylcarboxamidoadenosine (APEC), and 2-phenylaminoadenosine (CV 1808) was CHA approximately NECA > or = CADO > APEC approximately CV1808 > CGS 21680. In the presence of DPCPX (20 nM), [3H] CGS 21680 (0.2-200 nM) bound to a site (A2A-like) with a Kd of 20 nM and a Bmax of 56fmol/mg protein in the hippocampus and with a Kd of 22 nM and a Bmax of 63fmol/mg protein in the cortex. In the presence of CSC (200 nM), [3H]CGS 21680(0.2-200 nM) bound to a second high-affinity site with a Kd of 97 nM and a Bmax of 255 fmol/mg protein in the hippocampus

Topics: 2-Chloroadenosine; Adenosine; Animals; Binding, Competitive; Cerebral Cortex; Corpus Striatum; Dose-Response Relationship, Drug; Hippocampus; Male; Phenethylamines; Purinergic P1 Receptor Agonists; Rats; Rats, Sprague-Dawley; Structure-Activity Relationship; Xanthines

1. Experiments with adenosine deaminase suggest that adenosine is present in membrane preparations from CHO cells bearing adenosine A1 receptors. 2. Pretreatment of the membranes (ca 0.6 mg protein ml-1) with the permeabilizing agent saponin (100 micrograms ml-1) or addition of saponin (10 micrograms ml-1) to the membranes (0.02-0.08 mg protein ml-1) in the assay, generates homogeneous low affinity agonist binding curves in the presence of GTP and an increased function, assessed by agonist stimulation of [35S]-GTP gamma S binding. The affinity constants for the binding of an agonist and an antagonist are not affected by this saponin treatment. Saponin facilitates the interaction of guanine nucleotides with receptor G-protein complexes, possibly by removing a permeability barrier to access of G-proteins by GTP. However, adenosine is still present in the binding assays after saponin treatment. 3. The agonist binding properties of the human A1 receptor have been characterized. In saponin pretreated membranes, 80-90% of the A1 receptors are capable of forming agonist-receptor-G protein complexes in the absence of GTP. These complexes have a 300-600 fold higher affinity than uncoupled receptors for N6-cyclohexyladenosine. 4. A very slow component is observed in the association and dissociation kinetics of the agonist [3H]-N6-cyclohexyladenosine ([3H]-CHA) and in the association but not dissociation kinetics of the antagonist [3H]-8-cyclopentyl-1,3-dipropylxanthine ([3H]-DPCPX). The slow association component of [3H]-DPCPX is essentially absent when incubations are carried out in the presence of GTP. The slow dissociation component of [3H]-CHA binding is rapidly disrupted by GTP. 5. It is hypothesized that long-lasting adenosine-receptor-G protein complexes are present in the CHO membrane preparations. The existence of these complexes, resistant to the action of adenosine deaminase but sensitive to GTP, may rationalize the observed kinetics and the increase in 3H-antagonist binding produced by GTP which has been observed in essentially all studies of A1 receptors and has been ascribed previously to precoupling of A1 receptors to G-proteins in the absence of agonists.

Topics: Adenosine; Animals; Cell Membrane; Cell Membrane Permeability; CHO Cells; Cricetinae; GTP-Binding Proteins; Guanosine Triphosphate; Kinetics; Protein Binding; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Radioligand Assay; Receptors, Purinergic P1; Saponins; Xanthines

1. This study examined effects of adenosine and selective adenosine A1 and A2 receptor agonists on glucose metabolism in rat isolated working hearts perfused under aerobic conditions and during reperfusion after 35 min of global no-flow ischaemia. 2. Hearts were perfused with a modified Krebs-Henseleit buffer containing 1.25 mM Ca2+, 11 mM glucose, 1.2 mM palmitate and insulin (100 muu ml-1), and paced at 280 beats min-1. Rates of glycolysis and glucose oxidation were measured from the quantitative production of 3H2O and 14CO2, respectively, from [5-3H/U-14C]-glucose. 3. Under aerobic conditions, adenosine (100 microM) and the adenosine A1 receptor agonist, N6-cyclohexyladenosine (CHA, 0.05 microM), inhibited glycolysis but had no effect on either glucose oxidation or mechanical function (as assessed by heart rate systolic pressure product). The improved coupling of glycolysis to glucose oxidation reduced the calculated rate of proton production from glucose metabolism. The adenosine A1 receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX 0.3 microM) did not alter glycolysis or glucose oxidation per se but completely antagonized the adenosine- and CHA-induced inhibition of glycolysis and proton production. 4. During aerobic reperfusion following ischaemia, CHA (0.05 microM) again inhibited glycolysis and proton production from glucose metabolism and had no effect on glucose oxidation. CHA also significantly enhanced the recovery of mechanical function. In contrast, the selective adenosine A2a receptor agonist, CGS-21680 (1.0 microM), exerted no metabolic or mechanical effects. Similar profiles of action were seen if these agonists were present during ischaemia and throughout reperfusion or when they were present only during reperfusion. 5. DPCPX (0.3 microM), added at reperfusion, antagonized the CHA-induced improvement in mechanical function. It also significantly depressed the recovery of mechanical function per se during reperfusion. Both the metabolic and mechanical effects of adenosine (100 microM) were antagonized by the nonselective A1/A2 antagonist, 8-sulphophenyltheophylline (100 microM). 6. These data demonstrate that inhibition of glycolysis and improved recovery of mechanical function during reperfusion of rat isolated hearts are mediated by an adenosine A1 receptor mechanism. Improved coupling of glycolysis and glucose oxidation during reperfusion may contribute to the enhanced recovery of mechanical function by decreasing proton pr

Topics: Adenosine; Animals; Energy Metabolism; Glycolysis; Heart; Male; Myocardial Ischemia; Myocardial Reperfusion; Purinergic P1 Receptor Agonists; Rats; Rats, Sprague-Dawley; 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 of rats with R-PIA 'in vivo' desensitized adenosine A1 receptor-mediated inhibition of adenylyl cyclase in brain plasma membranes and increased basal and forskolin-stimulated adenylyl cyclase. The adenosine A1 receptor agonist CHA (cyclohexyl adenosine) inhibited forskolin-stimulated adenylyl cyclase in synaptic plasma membranes from control rats but failed to do so in membranes isolated from rats treated with R-PIA. This loss of response was accompanied with a significant decrease in both, total number of adenosine A1 receptors and steady-state level of alpha-Gi in synaptic plasma membranes. An increase in the steady-state level of alpha-Gs in synaptic plasma membranes was also observed by R-PIA treatment. Concurrently, a significant increase of adenosine A1 receptors was observed in microsomes and coated vesicles. These results demonstrate adenosine A1 receptor desensitization in rat brain by 'in vivo' treatment with R-PIA and suggest a role for coated vesicles in the internalization of G-protein coupled receptors.

Topics: Adenosine; Adenylyl Cyclase Inhibitors; Animals; Brain; Coated Vesicles; Colforsin; Enzyme Activation; Guanosine Triphosphate; Immunoblotting; Intracellular Membranes; Male; Microsomes; Purinergic P1 Receptor Agonists; Radioligand Assay; Rats; Rats, Wistar; Receptors, Purinergic P1; Vasodilator Agents; Xanthines

The ability of adenosine agonists to modulate the electrically evoked release of acetylcholine (ACh) from [3H]choline preloaded guinea-pig superior cervical ganglia (SCG) was investigated. The adenosine A1-receptor selective agonist N6-cyclohexyladenosine (CHA) and 2-chloroadenosine (2-CADO) inhibited the evoked transmitter release, the effect being reversed by the A1-receptor selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), and by sulmazole (SUL), which blocks both the A1-receptor and the adenylate cyclase inhibitory regulator Gi. In whole ganglia, CHA decreased both the basal and the forskolin (FSK)-stimulated cyclic AMP synthesis. The latter effect was again prevented by the A1 antagonist DPCPX. These results are compatible with the existence, in the guinea-pig SCG, of adenosine A1-receptors, part of which are located on the presynaptic nerve terminals mediating an inhibition of ACh release.

Topics: Acetylcholine; Adenosine; Animals; Colforsin; Cyclic AMP; Depression, Chemical; Electric Stimulation; Guinea Pigs; Imidazoles; Phosphodiesterase Inhibitors; Presynaptic Terminals; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Superior Cervical Ganglion; Sympathetic Nervous System; Xanthines

1. Adenoregulin is an amphilic peptide isolated from skin mucus of the tree frog, Phyllomedusa bicolor. Synthetic adenoregulin enhanced the binding of agonists to several G-protein-coupled receptors in rat brain membranes. 2. The maximal enhancement of agonist binding, and in parentheses, the concentration of adenoregulin affording maximal enhancement were as follows: 60% (20 microM) for A1-adenosine receptors, 30% (100 microM) for A2a-adenosine receptors, 20% (2 microM) for alpha 2-adrenergic receptors, and 30% (10 microM) for 5HT1A receptors. High affinity agonist binding for A1-, alpha 2-, and 5HT1A-receptors was virtually abolished by GTP gamma S in the presence of adenoregulin, but was only partially abolished in its absence. Magnesium ions increased the binding of agonists to receptors and reduced the enhancement elicited by adenoregulin. 3. The effect of adenoregulin on binding of N6-cyclohexyladenosine ([3H]CHA) to A1-receptors was relatively slow and was irreversible. Adenoregulin increased the Bmax value for [3H]CHA binding sites, and the proportion of high affinity states, and slowed the rate of [3H]CHA dissociation. Binding of the A1-selective antagonist, [3H]DPCPX, was maximally enhanced by only 13% at 2 microM adenoregulin. Basal and A1-adenosine receptor-stimulated binding of [35S]GTP gamma S were maximally enhanced 45% and 23%, respectively, by 50 microM adenoregulin. In CHAPS-solubilized membranes from rat cortex, the binding of both [3H]CHA and [3H]DPCPX were enhanced by adenoregulin. Binding of [3H]CHA to membranes from DDT1 MF-2 cells was maximally enhanced 17% at 20 microM adenoregulin. In intact DDT1 MF-2 cells, 20 microM adenoregulin did not potentiate the inhibition of cyclic AMP accumulation mediated via the adenosine A1 receptor. 4. It is proposed that adenoregulin enhances agonist binding through a mechanism involving enhancement of guanyl nucleotide exchange at G-proteins, resulting in a conversion of receptors into a high affinity state complexed with guanyl nucleotide-free G-protein.

Topics: Adenosine; Adrenergic alpha-Agonists; Amphibian Proteins; Animals; Antimicrobial Cationic Peptides; Brain; Cell Line; Cell Membrane; Cyclic AMP; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Kinetics; Magnesium Chloride; Peptides; Purinergic P1 Receptor Agonists; Ranidae; Rats; Receptors, Purinergic P1; Sodium Chloride; Sulfur Radioisotopes; Thiophenes; Tritium; Xanthines

In the present study some characteristics of long-term potentiation (LTP) in the hippocampal CA1 region were examined under different conditions of transmitter release. Adenosine A1 agonist/antagonists, or in some instances changes in the extracellular calcium/magnesium ratio, were used to alter release probability. The overall LTP time course (onset latency, growth phase, and subsequent decay for both the non-NMDA and NMDA receptor-mediated EPSPs) following a brief tetanus was essentially the same over an almost 10-fold variation in release probability (measured as change in field EPSP magnitude). The major difference observed was a faster initial decay of LTP evoked at low levels of release probability, possibly related to impaired induction conditions. It was also observed that LTP induced at one level of release probability occluded that induced at a lower (or higher) level, and that changes in release probability induced by adenosine agonist/antagonists affected potentiated and "naive" EPSPs to an equal extent. Taken together, these data do not provide support for the notion of different locations for LTP expression at different conditions of release probability. The results are also more compatible with the notion of a single, rather than several, expression mechanism(s) within the first hour of LTP in the hippocampal CA1 region.

Topics: Adenosine; Animals; Calcium; Electric Stimulation; Electrophysiology; Guinea Pigs; Hippocampus; Long-Term Potentiation; Magnesium; N-Methylaspartate; Neurotransmitter Agents; Presynaptic Terminals; Probability; Reaction Time; Xanthines

This report describes the effects of midazolam and beta-carboline-3-carboxylate ethyl ester (beta-CCE) on extracellular concentrations of dopamine in the nucleus accumbens of freely moving rats measured by in vivo microdialysis. The two compounds had opposite effects, midazolam (0.075 and 0.15 mg/kg i.v.) dose dependently decreasing, and beta-CCE (3 and 10 mg/kg i.p.) dose dependently increasing, dialysate concentrations of dopamine. Flumazenil (6 micrograms/kg i.v.) did not affect the efflux of dopamine but it prevented the effects of both midazolam and beta-CCE on dopamine efflux. N6-Cyclohexyladenosine (0.1, and 1 mg/kg i.p.), a selective adenosine A1 agonist, dose dependently increased the efflux of dopamine. This effect was blocked by 8-cyclopentyl-1,3-dipropylxanthine (25 mg/kg i.p.), a selective adenosine A1 receptor antagonist, a dose which given alone did not affect dopamine efflux; responses to midazolam were not affected. 3,7-Dimethyl-1-propargylxanthine (1 and 3 mg/kg i.p.), a selective adenosine A2 receptor antagonist, did not mimic the effects of beta-CCE. The results suggest that midazolam and beta-CCE modulate dopamine release in the nucleus accumbens by an action at the benzodiazepine binding site associated with the GABAA receptor complex.

Topics: Adenosine; Animals; Carbolines; Dopamine; Dose-Response Relationship, Drug; Flumazenil; Ligands; Male; Microdialysis; Midazolam; Nucleus Accumbens; Purinergic P1 Receptor Antagonists; Rats; Rats, Sprague-Dawley; Receptors, GABA-A; Tetrodotoxin; Theobromine; Xanthines

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

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

1. We have used a rabbit isolated ear, buffered-perfused preparation to investigate the effects of adenosine analogues on the vasodilatation to the potassium channel opener, levcromakalim (the active (-)-enantiomer of cromakalim). We have examined the effects of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), a selective adenosine A1 antagonist, on vasodilatation to levcromakalim under hypoxic conditions and also following inhibition of nitric oxide synthesis. 2. Levcromakalim relaxed preconstricted preparations with an EC50 = 369 +/- 48 nM and maximum relaxation of tone (Rmax) = 81.0 +/- 3.2%. In the presence of 1 microM N6-cyclohexyladenosine (CHA) a selective adenosine A1 agonist, there was a significant (P < 0.01) leftward shift in the concentration-response curve with an EC50 = 194 +/- 54 nM and Rmax = 93.2 +/- 2.0%. Conversely, the presence of CHA did not influence vasodilatation to either pinacidil or sodium nitroprusside. 3. Hypoxia also significantly (P < 0.001) increased the vasodilator potency of levcromakalim (EC50 = 134 +/- 22 nM), and this enhancement was completely reversed (EC50 = 380 +/- 107 nM, P < 0.01) by pretreatment of the preparations with 5 microM DPCPX, a selective A1 adenosine antagonist. However, under normoxic conditions DPCPX did not influence vasodilatation to levcromakalim. 4. Inhibition of nitric oxide synthesis with 100 microM NG-nitro-L-arginine methyl ester (L-NAME) caused a significant (P < 0.001) leftward shift in the concentration-response curve to levcromakalim (EC50 = 73.0 +/- 7.6 nM). Pretreatment of preparations with DPCPX partially reversed the increase in potency found in the absence of nitric oxide synthesis (EC50 = 153 +/- 18 nM, P < 0.001). 5. We have shown that an adenosine Al agonist may increase the potency of levcromakalim indicating that adenosine receptor activation may augment the vasodilator activity of levcromakalim. That responses to levcromakalim but not those to pinacidil were affected by CHA points to further differences in the pharmacology of these potassium channel openers. The reversal by the adenosine Al antagonist of the hypoxic-potentiation of vasodilatation to levcromakalim, and also augmentation following inhibition of nitric oxide synthesis, suggests that under these conditions there is an endogenous release of adenosine which may enhance responses to levcromakalim. The findings of this study suggest that levcromakalim may selectively dilate vessels where there is elevated adenosine release.

Topics: Adenosine; Animals; Arginine; Benzopyrans; Blood Pressure; Cromakalim; Drug Synergism; Ear, External; Guanidines; Hypoxia; In Vitro Techniques; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitroprusside; Pinacidil; Pyrroles; Rabbits; Regional Blood Flow; Vasodilation; Vasodilator Agents; Xanthines

The effect of chronic administration of selective adenosine A1 receptor agonists and antagonists on the outcome of cerebral ischemia is entirely unknown. Therefore, we have investigated the impact of such regimens on the hippocampal adenosine A1 receptor density, and on the recovery from 10 min forebrain ischemia in gerbils. While acutely administered N6-cyclopentyladenosine (CPA) given at 0.02 mg/kg resulted only in a significant reduction of mortality, at 1 mg/kg it improved both survival and neuronal preservation in the hippocampal CA1 region. Acute treatment with 1,3-dipropyl-8-cyclopentylxanthine (CPX) significantly worsened the outcome and enhanced neuronal destruction. The effects of chronic administration of these drugs (15 days followed by 1 drug-free day) were opposite. Thus, although chronic CPA at 0.02 mg/kg did not have any effect at all, at 1 mg/kg both survival and neuronal preservation were significantly poorer than in controls, while chronic CPX resulted in a significant improvement of both measures. These results were not accompanied by adenosine A1 receptor up- or downregulation. Our study indicates that highly selective adenosine analogues may have therapeutic potential in treatment of cerebral ischemia/stroke and possibly other neurodegenerative disorders as well.

Topics: Adenosine; Animals; Body Temperature; Brain Ischemia; Down-Regulation; Female; Gerbillinae; Hippocampus; Prosencephalon; Purinergic P1 Receptor Antagonists; Receptors, Purinergic P1; Up-Regulation; Xanthines

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

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

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

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

Although adenosine is known to affect renal function through stimulating adenosine receptors, little is known about A1 receptors in human glomeruli. Thus, we attempted to identify the adenosine A1 receptor-cyclic AMP (cAMP) system in human glomeruli. Normal renal cortical tissues were obtained at nephrectomy of patients with renal cell carcinoma. Glomeruli were isolated using a graded sieving method or dissected manually under a stereomicroscope. Radioligand binding assay using 2-chloro-N-[3H] cyclopentyl adenosine ([3H]CCPA, an A1 agonist ligand) was performed at 30 degrees C for 90 minutes. Cyclic AMP (cAMP) produced in glomeruli was measured after incubation with different concentrations of N6-cyclohexyladenosine (CHA; A1 agonist) and a phosphodiesterase inhibitor. The specific binding was saturated within 60 minutes and reversible by adding 1 mM of theophylline. Scatchard plot analysis revealed a single class of binding site (Kd = 1.78 +/- 0.21 nM, Bmax = 271.7 +/- 35.8 fmol/mg protein). The specific binding was inhibited dose-dependently by various agents in an order suggesting A1 receptor specificity. CHA inhibited the production of cAMP in microdissected human glomeruli. This inhibitory effect was antagonized by 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; A1 antagonist). This is the first study revealing the presence of the A1 receptor-cAMP system in human glomeruli using a radioligand binding assay method and by measuring the cAMP production.

Topics: Adenosine; Binding, Competitive; Cyclic AMP; Humans; In Vitro Techniques; Kidney Glomerulus; Kinetics; Radioligand Assay; Receptors, Purinergic; Xanthines

The effects of the adenosine A1 agonist N6-cyclohexyladenosine (CHA) on MPTP-induced dopamine (DA) depletion in the striatum of C57BL/6 mice were studied. Twenty hours after a single injection of MPTP (30 mg/kg, s.c.), the toxin caused 62% depletion of striatal DA. CHA (0.2-3 mg/kg, s.c.), when given together with MPTP, prevented the toxin-induced DA depletion in a dose-dependent manner. This protective action was apparently mediated by the A1 receptors, because this effect was selectively antagonized by pretreating the animals with the A1 antagonist 8-cyclopentyl-1,3-dipropylxanthine (25 mg/kg, i.p.) but not with the A2 antagonist 1,3-dipropyl-7-methylxanthine (25 mg/kg, i.p.). When CHA (3 mg/kg) was injected 5 h after MPTP administration, at which point striatal DA levels were already reduced significantly, a rapid and complete recovery of the striatal DA levels occurred. These neurochemical data suggest that the A1 agonist CHA is potentially useful as a neuroprotective agent against MPTP-induced toxicity.

Topics: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; 1-Methyl-4-phenylpyridinium; Adenosine; Animals; Corpus Striatum; Dopamine; Kinetics; Male; Mice; Mice, Inbred C57BL; Receptors, Purinergic; Time Factors; Xanthines

Binding to adenosine A1 receptors and the status of their coupling to G proteins were studied in the hippocampus and parahippocampal gyrus of Alzheimer individuals and age-matched controls. The binding to A1 receptors was compared with binding to the N-methyl-D-aspartate receptor complex channel-associated sites (labeled with (+)-[3H]5-methyl-10,11-dihydro-5H- dibenzo[a,d]cyclohepten-5,10-imine maleate). In vitro quantitative autoradiography demonstrated a similar anatomical distribution of A1 receptors labeled either with an agonist ((-)-[3H]phenylisopropyladenosine) or antagonist ([3H]8-cyclopentyl-1,3-dipropylxanthine) in the brains of elderly controls. In Alzheimer patients, significant decreases in the density of both agonist and antagonist binding sites were found in the molecular layer of the dentate gyrus. Decreased A1 agonist binding was also observed in the CA1 stratum oriens and outer layers of the parahippocampal gyrus, while reduced antagonist binding was found in the subiculum and CA3 region. Reduced density of the N-methyl-D-aspartate receptor channel sites was found in the CA1 region and parahippocampal gyrus. The reductions in binding to adenosine A1 and N-methyl-D-aspartate receptors were due to a decrease in the density of binding sites (Bmax), and not changes in receptor affinity (KD). In both elderly control and Alzheimer subjects, GTP substantially reduced the density of A1 agonist binding sites with a concomitant increase in the KD values, whereas antagonist binding was unaffected by GTP. The results suggest that adenosine A1 receptor agonists and antagonists recognize overlapping populations of binding sites. Reduced density of A1 receptors in the molecular layer of the dentate gyrus most probably reflects damage of the perforant path input in Alzheimer's disease, while altered binding in the CA1 and CA3 regions is probably due to loss of intrinsic neurons. Similar effects of GTP on binding to A1 receptors in control and Alzheimer individuals suggest lack of alterations in coupling of A1 receptors to G proteins in Alzheimer's disease, thus supporting the notion of normal receptor coupling to their effector systems in Alzheimer's disease.

Topics: Adenosine; Aged; Aged, 80 and over; Alzheimer Disease; Autoradiography; Dizocilpine Maleate; Female; GTP-Binding Proteins; Hippocampus; Humans; Kinetics; Male; Organ Specificity; Pyramidal Tracts; Receptors, N-Methyl-D-Aspartate; Receptors, Purinergic; Tritium; Xanthines

We have previously reported that in several renal cell types, adenosine receptor agonists inhibit adenylyl cyclase and activate phospholipase C via a pertussis toxin-sensitive G protein. In the present study, in 28A cells, both of these adenosine receptor-mediated responses were inhibited by 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), a highly selective A1 adenosine receptor antagonist. The binding characteristics of the adenosine A1 receptor in the 28A renal cell line were studied using the radiolabeled antagonist [3H]DPCPX to determine whether two separate binding sites could account for these responses. Saturation binding of [3H]DPCPX to 28A cell membranes revealed a single class of A1 binding sites with an apparent Kd value of 1.4 nM and maximal binding capacity of 64 fmol/mg protein. Competition experiments with a variety of adenosine agonists gave biphasic displacement curves with a pharmacological profile characteristic of A1 receptors. Comparison of [3H]DPCPX competition binding data from 28A cell membranes with rabbit brain membranes, a tissue with well-characterized A1 receptors, reveals that the A1 receptor population in 28A cells has similar agonist binding affinities to the receptor population in brain but has a considerably lower density. Addition of guanosine 5'-triphosphate (100 microM) to 28A cell membranes caused the competition curves to shift from biphasic to monophasic, indicating that the A1 receptors exist in two interconvertible affinity states because of their coupling to G proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenosine; Adenylate Cyclase Toxin; Animals; Binding, Competitive; Brain; Calcium; Cell Line; Cell Membrane; Cyclic AMP; Kidney Medulla; Kidney Tubules, Collecting; Pertussis Toxin; Rabbits; Receptors, Purinergic; Virulence Factors, Bordetella; Xanthines

The effects of adenosine in the nonischemic heart have been shown to be mediated via its binding to extracellular adenosine A1 and A2 receptors located predominantly on myocytes and endothelial cells, respectively. We tested the hypothesis that the beneficial effect of adenosine on postischemic myocardial function is mediated via an adenosine A1 receptor mechanism. Isolated rat hearts perfused at constant pressure (85 cmH2O) were subjected to 30 min of global no-flow ischemia (37 degrees C) and 45 min of reperfusion. Hearts treated with adenosine (100 microM) and the adenosine A1 receptor agonist N6-cyclohexyladenosine (CHA; 0.25 microM) recovered 72 +/- 4 and 70 +/- 4% of preischemic left ventricular developed pressures (LVDP), respectively, after 45 min of reperfusion compared with untreated hearts (54 +/- 3% of preischemic LVDP). Adenosine and CHA hearts exhibited greater myocardial ATP contents than control hearts after 10 min of ischemia, but there were no differences in tissue ATP levels after 30 min of ischemia. In contrast, hearts treated with the adenosine A2 receptor agonist phenylaminoadenosine (0.25 microM) failed to demonstrate improved postischemic function (52 +/- 5%). The addition of the A1-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine blocked the cardioprotective effect of adenosine (57 +/- 4%). These results suggest that adenosine enhances postischemic myocardial function via an A1 receptor mechanism.

Topics: Adenine Nucleotides; Adenosine; Animals; Hemodynamics; In Vitro Techniques; Male; Myocardial Ischemia; Myocardial Reperfusion; Myocardium; Rats; Rats, Wistar; Receptors, Purinergic; Xanthines

In a previous study we showed that in vivo treatment with pertussis toxin could inhibit some, but not all, effects of adenosine in the rat hippocampus. In this study we investigated the effect of pertussis toxin on the binding of adenosine analogues to A1 receptors in rat brain. Intraventricular injection of pertussis toxin (10 micrograms into the lateral ventricle) did not affect A1 receptor binding in any brain region studied, as evaluated by autoradiography. In vitro treatment of brain sections (10 microns) with pertussis toxin for 5 h, under conditions when greater than 80% of the G proteins were ADP ribosylated, did not alter radioligand binding to adenosine A1 receptors. GTP (10 microM) virtually abolished the high-affinity agonist binding to the A1 receptor. On the other hand, in solubilized cortical membrane preparations, pertussis toxin pretreatment induced a complete shift of the A1 receptors to the low-affinity state. This suggests that the ability of pertussis toxin to affect G proteins coupled to A1 receptors in brain depends not only on the distribution of the toxin but also on the configuration of receptors and G proteins.

Topics: Adenosine; Adenosine Diphosphate Ribose; Animals; Autoradiography; Brain; Ethylmaleimide; Guanosine Diphosphate; Magnesium Chloride; Male; Pertussis Toxin; Radioligand Assay; Rats; Rats, Inbred Strains; Receptors, Purinergic; Solubility; Virulence Factors, Bordetella; Xanthines

Much evidence has accumulated supporting the hypothesis that the purine nucleoside adenosine may indeed function as a neuromodulator in the mammalian retina, but to date no reports have directly illustrated a physiological role for this nucleoside. In other regions of the CNS, adenosine agonists decrease transmitter release, whereas antagonists increase release. A similar role for adenosine in the retina is now apparent. The cholinergic amacrine cells of the rabbit retina were labeled with [3H]choline, and the effects of enzymatic adenosine degradation or adenosine antagonists on the light-evoked efflux of acetylcholine were evaluated. When endogenous adenosine was degraded by addition of adenosine deaminase, the light-evoked release of radioactivity derived from [3H]choline was significantly increased compared with control values. A similar response was observed when rabbit eyecups were superfused with a selective adenosine A1 receptor antagonist. The effect elicited by adenosine deaminase could be almost completely reversed by addition of cyclopentyladenosine, a highly selective A1 receptor agonist. These effects were observed in either the presence or the absence of picrotoxin. The results demonstrate a modulation of retinal physiology by adenosine.

Topics: Acetylcholine; Adenosine; Animals; Binding, Competitive; Light; Phenylisopropyladenosine; Rabbits; Retina; Xanthines

Quantitative autoradiography was used to investigate the effects of Mg2+ on agonist and antagonist binding to A1 receptors in rat striatum. A1 receptors were labelled with the selective agonist N6-[3H]cyclohexyladenosine ([3H]CHA) or the selective antagonist 1,3-[3H]dipropyl-8-cyclopentylxanthine ([3H]DPCPX). Mg2+ had no significant effect on equilibrium binding constants for [3H]CHA [control: KD (95% confidence interval) of 0.34 (0.15-0.80) nM and Bmax of 267 +/- 8 fmol/mg of gray matter; with 10 mM Mg2+: KD of 0.8 (0.13-4.9) nM and Bmax of 313 +/- 8.9 fmol/mg of gray matter] or [3H]DPCPX [control: KD of 0.54 (0.30-0.99) nM and Bmax of 256 +/- 2.3 fmol/mg of gray matter; with 10 mM Mg2+: KD of 1.54 (0.2-11.0) nM and Bmax of 269 +/- 35.7 fmol/mg of gray matter]. In contrast, Mg2+ slowed the apparent association rate for both ligands; this was observed as a shift from a one-component to a two-component model for [3H]DPCPX. Mg2+ also affected the dissociation rates of both ligands; for [3H]CHA, dissociation in the presence of Mg2+ was not detected. Mg2+ produced a concentration-dependent inhibition of [3H]CHA binding only prior to equilibrium. HPLC was performed on untreated sections, sections preincubated with adenosine deaminase (ADA), and sections preincubated with ADA and incubated with ADA in the absence or presence of Mg2+. Adenosine was found in measurable quantities under all conditions, and the concentration was not influenced by Mg2+ or by the inclusion of GTP in the preincubation medium. From these data, we conclude the following: (a) adenosine is present and may be produced continuously in brain sections; (b) ADA is not capable of completely eliminating the produced adenosine; (c) Mg2+ apparently does not influence adenosine production or elimination; (d) A1 receptor-guanine nucleotide binding protein coupling is maximal in this preparation; and (e) Mg2+ decreases the dissociation rate of bound endogenous adenosine from A1 receptors, thus limiting the access of [3H]CHA and [3H]DPCPX to the receptors. Thus, enhancement of endogenous adenosine binding to A1 receptors by Mg2+ is a complicating factor in receptor autoradiography and may be so in other preparations as well.

Topics: Adenine Nucleotides; Adenosine; Animals; Autoradiography; Corpus Striatum; Magnesium; Male; Rats; Rats, Inbred Strains; Receptors, Purinergic; Xanthines

Studies on factors modulating the binding of agonist ligands to A1 adenosine receptors in rat forebrain membranes revealed that the reduction of [3H]cyclohexyladenosine [( 3H]CHA) binding, observed after removing Mg2+ by pretreatment with ethylene-dinitrilo-tetraacetic acid (EDTA), was restored by the polyamine, spermine (1mM). Parallel electrophysiological experiments performed on rat hippocampal slices in Mg(2+)-free medium indicated that spermine also led to a recovery of the depressive effect of 1 microM adenosine on stimulus train-evoked neuronal Ca2+ influx. These observations suggest that the polyamine, spermine is, like Mg2+, able to control the physiological adenosine-mediated modulation of synaptic transmission by changing the affinity state of A1 receptors.

Topics: Adenosine; Animals; Brain Chemistry; Edetic Acid; Electrophysiology; Hippocampus; In Vitro Techniques; Kinetics; Magnesium; Male; Rats; Rats, Inbred Strains; Receptors, Purinergic; Spermine; Synaptic Transmission; Xanthines

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

The cells of origin of the perforant pathway are destroyed in Alzheimer's disease (AD). In rat the adenosine A1-receptors are specifically localized on the perforant path terminals in the molecular layer of the dentate gyrus. In the present study the density of A1-receptors in the hippocampus of Alzheimer's disease (AD) patients (n = 9) and non-dement controls (n = 3) has been investigated autoradiographically with [3H]8-cyclopentyl-1,3-dipropylxanthine ([3H]CPDPX) as the ligand probe. In AD hippocampi binding of [3H]CPDPX was greatly reduced in the outer two thirds of the dentate gyrus molecular layer, likely due to the degeneration of the perforant path. Binding of [3H]CPDPX was not significantly altered in other parts of the AD hippocampus, e.g. the CA1 and the CA3, in spite of a pronounced cellular pathology and reduced N-methyl-D-aspartate (NMDA) receptor densities, assessed as strychnine insensitive [3H]glycine autoradiography. This contrasts with the presumed localization on dendrites of pyramidal neurons of A1 receptors within the CA1 and the CA3.

Topics: Adenosine; Aged; Aged, 80 and over; Alzheimer Disease; Autoradiography; Female; Hippocampus; Humans; Male; Middle Aged; Neural Pathways; Receptors, Glycine; Receptors, N-Methyl-D-Aspartate; Receptors, Neurotransmitter; Receptors, Purinergic; Xanthines

We have previously shown that adenosine, acting at an A1 receptor, contracts the smooth muscle of virgin guinea pig uterus (M. A. Smith, I. L. O. Buxton, and D. P. Westfall. J. Pharmacol, Exp. Ther. 247: 1059-1063, 1988) and is not coupled to the expected inhibition of adenylate cyclase (M. A. Smith, J. L. Silverstein, D. P. Westfall, and I. L. O. Buxton. Cell. Signal. 1: 357-365, 1989). To probe the importance of contractile actions of adenosine in uterine smooth muscle and to further characterize the signal transduction pathway involved in A1-receptor action, we have studied the adenosine receptor and its coupling in pregnant guinea pig myometrium. Adenosine agonist and antagonist radioligands bind to saturable sites of the A1 subtype homogeneously distributed in the smooth muscle of pregnant guinea pig uterus. Agonist competition of antagonist radioligand binding in both the absence and presence of guanine nucleotide reveals high and low agonist affinity states of the receptor. Pretreatment of tissues with pertussis toxin (PTx) shifts the high-affinity sites to a lower affinity but does not affect low-affinity sites, whereas agonist competition in the presence of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) is indistinguishable from the control, which is consistent with coupling of A1 receptors to both PTx-sensitive and PTx-insensitive GTP-binding proteins. Adenosine receptor inhibition of adenylate cyclase activity is prevented after pretreatment of the tissue with PTx, whereas increased inositol phosphate production is not. The data presented here are consistent with coupling of the A1 receptor to dual effectors in the pregnant state of the smooth muscle. The unique action of an A1 receptor to contract mammalian smooth muscle and the appearance, only in the pregnant state, of coupling to adenylate cyclase inhibition suggest a role for adenosine in parturition biology.

Topics: Adenosine; Adenylate Cyclase Toxin; Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Animals; Female; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guinea Pigs; Inositol Phosphates; Muscle, Smooth; Myometrium; Pertussis Toxin; Pregnancy; Receptors, Purinergic; Type C Phospholipases; Virulence Factors, Bordetella; Xanthines

In order to examine the possibility of an interaction between adenosine and angiotensin II (A II) in the control of the renal microcirculation, we studied the effects of agonists and antagonists of both substances by means of in vivo microscopy in the split hydronephrotic rat kidney. In a first series of experiments (n = 6), local application of the A II receptor antagonist saralasin (10(-6) mol.liter-1 abolished the vasoconstriction and the reduction of glomerular blood flow induced by the A1-adenosine receptor agonist N6-cyclohexyladenosine (CHA, local concentration 10(-7) mol.liter-1). Without saralasin (second series, n = 6), CHA reduced glomerular blood flow and decreased vessel diameters as previously reported from our laboratory. In a third series of experiments (n = 6), A II significantly reduced vessel diameters and glomerular blood flow both alone and during blockage of the A1-adenosine receptor by the selective antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 10(-5) mol.liter-1). In additional experiments, we excluded nonspecific receptor effects of saralasin and confirmed the inhibitory action of DPCPX on the adenosine-induced vasoconstriction. We suppose that adenosine needs a functioning A II receptor system for its vasoconstrictor action, whereas A II can induce a nonadenosine-dependent vasoconstriction.

Topics: Adenosine; Angiotensin II; Animals; Drug Interactions; Female; Kidney Glomerulus; Microcirculation; Rats; Rats, Inbred Strains; Receptors, Angiotensin; Saralasin; Vasoconstriction; Xanthines

Experiments were performed in anesthetized rats to study the effect of the selective adenosine1 (A1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (CPX) on tubuloglomerular feedback (TGF) responses assessed as the maximum change of stop-flow pressure (PSF). Compared with control, PSF responses were reduced during luminal application of CPX at 10(-4) and 10(-5)M (-4.9 +/- 0.44 vs. + 0.9 +/- 0.42 mmHg and -6.8 +/- 0.69 vs. -1.4 +/- 0.7 mmHg, respectively), during peritubular administration of CPX at 10(-4)M (-6.2 +/- 0.44 vs. -2.8 +/- 0.42 mmHg), and during infusion of CPX at 10(-4) M into the lumen of a neighboring nephron (-5.6 +/- 0.6 vs. -1.98 +/- 0.51 mmHg). Selectivity of CPX was tested by studying its effect on the PSF reduction produced by the A1-receptor agonist N6-cyclohexyladenosine (CHA). CHA at 10(-5)M reduced PSF when infused into the peritubular blood (-11.8 +/- 3.7 mmHg), and this effect was blunted by luminal application of CPX (-1.5 +/- 0.6 mmHg). CHA also reduced PSF when infused into a neighboring nephron, and this effect was blunted by infusing CPX at 10(-4)M into the same neighboring nephron, a different neighboring nephron, or a peritubular capillary. These results are consistent with the concept that activation of A1-receptors on vascular cells of the afferent arterioles participates in the mediation of TGF responses.

Topics: Adenosine; Adrenergic alpha-Antagonists; Animals; Capillaries; Feedback; Injections; Kidney Glomerulus; Kidney Tubules; Male; Pressure; Rats; Rats, Inbred Strains; Xanthines

The effects of 2 weeks' treatment with theophylline (20 mg/kg i.p.) on the binding to adenosine A1-receptors was studied by autoradiography using the agonist [3H]N6-cyclohexyladenosine ([3H]CHA) and the antagonist [3H]8-cyclopentyl-1,3-dipropylxanthine ([3H]DPCPX) as ligands. A significant increase (10%) in [3H]CHA binding was measured only in the frontoparietal cortex. However, if the brain sections were incubated in the presence of 5 microM guanosine-5'-triphosphate (GTP), which by itself decreased binding by between 15 and 80% depending on the region, the increase in the frontoparietal cortex was larger (30%) and significant increases of the same magnitude were also seen in several other structures, e.g. the caudate putamen and the central gray matter of the midbrain. In some regions, for example the hippocampus and the cerebellar cortex, small or no increases were seen. GTP 100 microM practically eliminated [3H]CHA binding in both control and treated animals, suggesting that these receptors are all coupled to G-proteins. The binding of [3H]DPCPX was increased significantly only in the frontoparietal and striate cortex (5-10%). These results suggest that the theophylline treatment had little on the total receptor number but may have altered the coupling between A1-receptors and regulatory GTP-binding proteins.

Topics: Adenosine; Animals; Autoradiography; Brain; GTP-Binding Proteins; Male; Rats; Rats, Inbred Strains; Receptors, Purinergic; Theophylline; Xanthines

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

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

Adenosine has been proposed to act within the juxtaglomerular apparatus (JGA) as a mediator of the inhibition of renin secretion produced by a high NaCl concentration at the macula densa. To test this hypothesis, we studied the effects of the adenosine1 (A1)-receptor blocker 8-cyclopentyl-1,3-dipropylxanthine (CPX) on renin release from single isolated rabbit JGAs with macula densa perfused. The A1-receptor agonist, N6-cyclohexyladenosine (CHA), applied in the bathing solution at 10(-7) M, was found to inhibit renin secretion, an effect that was completely blocked by adding CPX (10(-5) M) to the bath. Applied to the lumen, 10(-5) M CPX produced a modest stimulation of renin secretion rates suppressed by a high NaCl concentration at the macula densa (P less than 0.05). The effect of changing luminal NaCl concentration on renin secretion rate was examined in the presence of CPX (10(-7) and 10(-5) M) in the bathing solution and in vehicle control experiments. The control response to increasing luminal NaCl concentration was a marked suppression of renin secretion, that was maintained as long as luminal NaCl concentration was high and was promptly reversible when concentration was lowered. CPX did not alter renin release when luminal NaCl was low, but diminished the reduction caused by high NaCl (P less than 0.01). It is concluded that A1-receptors are located within the JGA, and that A1-receptor activation inhibits renin release. A high NaCl concentration at the macula densa appears to influence A1-receptor activation, but a low NaCl concentration does not. The findings support participation of adenosine in macula densa control of renin secretion.

Topics: Adenosine; Animals; In Vitro Techniques; Juxtaglomerular Apparatus; Kinetics; Loop of Henle; Perfusion; Rabbits; Receptors, Purinergic; Renin; Sodium Chloride; Xanthines

The effect of increasing doses of GTP on agonist and antagonist binding to adenosine A1-receptors in different regions of rat brain was studied by autoradiography. A high concentration of GTP (100 microM) practically eliminated the binding of the agonist [3H]N6-cyclohexyladenosine in all regions. However, there were regional differences in the effects of low concentrations of GTP (0.1-10 microM). In some regions, for example the hippocampus, all concentrations of GTP decreased [3H]N6-cyclohexyladenosine binding, by decreasing the Bmax. In other structures, e.g. the superior colliculus, there was a biphasic response to GTP. Concentrations of 0.1-3 microM increased agonist binding, apparently due to a decrease in KD, whereas higher concentrations also decreased binding in these regions. The effects of GTP were mimicked by the stable GTP analogue guanosine-5'-O-(3-thiotriphosphate). GTP (0.5-100 microM) increased the binding of the antagonist [3H]8-cyclopentyl-1,3-dipropylxanthine in all regions, but most markedly in those where GTP had a biphasic effect on agonist binding. Decreasing the levels of endogenous adenosine by increasing the concentration of adenosine deaminase and adding the 5'-nucleotidase inhibitor alpha-beta-methylene adenosine-5'-diphosphate gave an increase in [3H]8-cyclopentyl-1,3-dipropylxanthine binding and diminished the response to GTP. In sections treated with adenosine deaminase and alpha-beta-methylene adenosine-5'-diphosphate, GTP steadily decreased [3H]N6-cyclohexyladenosine binding in all regions. Thus, the GTP-induced increase in both agonist and antagonist binding may be due to a displacement of endogenous adenosine. In the presence of 1 mM EDTA, GTP had a monophasic effect on the binding of [3H]N6-cyclohexyladenosine in all regions. In the presence of 2 mM MgCl2 a biphasic response to GTP was seen in all regions. In EDTA washed sections, the effect of MgCl2 on [3H]N6-cyclohexyladenosine binding was more pronounced in the superior colliculus, where we had observed a biphasic response to GTP. The results suggest that there are regional differences in the effects of GTP on adenosine A1-receptor binding in rat brain, that reflect regional differences in the magnesium-dependent binding of endogenous adenosine, which is bound to the receptor by tight binding, is very difficult to remove, and easily interferes with radioligand binding in in vitro experiments. There may be regional differences in the sensitivity of A1-receptor-G-prote

Topics: Adenosine; Animals; Autoradiography; Brain; Guanine Nucleotides; Guanosine Triphosphate; Male; Purinergic Antagonists; Rats; Rats, Inbred Strains; Receptors, Purinergic; Xanthines

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

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