Adenosine A1 Binding Sites Research Articles

Role of brainstem adenosine A1 receptors in the cardiovascular response to hypothalamic defence area stimulation in the anaesthetized rat.

1. The role of centrally located adenosine A1 receptors in the cardiovascular changes associated with the hypothalamic defence response has been investigated by in vitro autoradiography and the intraventricular application of an A1 receptor antagonist. 2. 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX), a highly selective adenosine A1 antagonist and its vehicle, ethanol, were administered directly into the posterior portion of the fourth ventricle of alpha-chloralose anaesthetized, paralysed and artificially ventilated rats. 3. DPCPX (0.01 to 0.3 mg kg-1) caused a dose-dependent decrease in the magnitude of the evoked pressor response (from -13 to -23 mmHg) elicited on hypothalamic defence area stimulation at a dose 10 fold lower than that required to produce an equivalent effect following systemic administration whilst ethanol, the vehicle, had no effect. 4. In vitro autoradiography revealed a heterogeneous distribution of adenosine A1 binding sites in the lower brainstem of rats. Image analysis showed the ventrolateral medulla to have the highest density of A1 receptors. Intermediate levels of binding were seen in caudal regions of the nucleus tractus solitarii and the hypoglossal nucleus. 5. These data imply that a proportion of the cardiovascular response to hypothalamic defence area stimulation are produced by the activation of adenosine A1 receptors localized close to the surface of, or adjacent to, the fourth ventricle in the immediate vicinity of the injection site.

Characterization and Localization of Adenosine A2 Receptors in Bovine Rod Outer Segments

Previous work from this laboratory has shown that retinal adenosine A2 binding sites are localized over outer and inner segments of photoreceptors in rabbit and mouse retinal sections. In the present study, adenosine receptor binding has been characterized and localized in membranes from bovine rod outer segments (ROS). Saturation studies with varying concentrations (10-150 nM) of 5'-(N-[2,8-3H]ethylcarboxamido)adenosine ([3H]NECA) and 100 micrograms of ROS membrane protein show a single site with a KD of 103 nM and a Bmax of 1.3 pM/mg of protein. Cold Scatchards, which used nonradiolabeled NECA (concentrations ranging from 10 nM to 250 microM) in competition with a fixed amount of [3H]NECA (30 nM), demonstrated the presence of a low-affinity site (KD, 50 microM) in addition to the high-affinity site. To confirm the presence of A2a binding sites, saturation analyses with 2-p-(2-[3H]-carboxyethyl)phenylamino-5'-N-ethylcarboxamido adenosine (0-80 nM) also revealed a single population of high-affinity A2a receptors (KD, 9.4 nM). The binding sites labeled by [3H]NECA appear to be A2 receptor sites because binding was displaced by increasing concentrations of 5'-(N-methylcarboxamido)adenosine and 2-chloroadenosine. ROS were fractionated into plasma and disk membranes for localization studies. Receptor binding assays, used to determine specific binding, showed that the greatest concentration of A2 receptors was on the plasma membranes. Therefore, adenosine A2 receptors are in a position to respond to changes in the concentration of extracellular adenosine, which may exhibit a circadian rhythm.

Postischemic alteration of muscarinic acetylcholine and adenosine A1 binding sites in gerbil brain

We studied the alterations in the binding of muscarinic cholinergic and adenosine A1 receptors following transient cerebral ischemia in Mongolian gerbils and examined the effects of the novel vinca alkaloid derivative vinconate and pentobarbital against the alterations in the binding of these receptors. Animals were allowed to survive for 5 h and 7 days after 10 min of cerebral ischemia induced by bilateral occlusion of common carotid arteries. [3H]Quinuclidinyl benzilate (QNB) and [3H]cyclohexyladenosine (CHA) were used to label muscarinic cholinergic and adenosine A1 receptors, respectively. The [3H]QNB and [3H]CHA bindings showed no significant alteration in the gerbil brain 5 h after ischemia. However, these bindings in the striatum, the hippocampal CA1 sector, and the hippocampal CA3 sector revealed a significant reduction 7 days after ischemia. The [3H]CHA binding also showed a significant decline in the dentate molecular layer 7 days after ischemia. Intraperitoneal application of vinconate (100 and 300 mg/kg) 10 min and pentobarbital (40 mg/kg) 30 min before ischemia showed a mild reduction in the [3H]CHA binding in the brain 5 h after ischemia. Especially, the reduction was found in the hippocampal CA1 sector and the dentate molecular layer. However, the [3H]QNB binding revealed no significant alteration in the brain 5 h after ischemia. Seven days after ischemia, both drugs prevented a marked reduction in the [3H]CHA binding in the striatum, but not in the hippocampal CA1 sector, the hippocampal CA3 sector, and the dentate molecular layer. By contrast, vinconate and pentobarbital failed to prevent the reduction in the [3H]QNB binding in the striatum. Morphological study indicated that vinconate and pentobarbital ameliorated the neuronal damage to the striatum, but not the hippocampal damage 7 days after ischemia. This histological finding was relatively consistent with the alteration in the [3H]CHA binding. These receptor autoradiographic and histological data suggest that vinconate and pentobarbital can protect the brain from both cellular and functional consequences of ischemia. These findings are of interest in relation to the mechanisms of ischemic brain damage.

Ontogeny of gene expression of adenosine A2 receptor in the striatum: early localization in the patch compartment.

The ontogeny of adenosine A2 receptor mRNA and adenosine A2 binding sites distributions was studied by in situ hybridization histochemistry and receptor autoradiography in pre- and post-natal rat striatum, postnatal dog striatum, and a human fetus striatum and compared to that of dopamine D1 and mu opiate receptors. The early postnatal striatum demonstrated heterogeneous distributions of adenosine A2 receptor mRNA and adenosine A2 binding sites with patches of dense labeling corresponding to dopamine D1 and mu opiate receptors enriched zones. This patchy pattern evolved to the homogeneous distribution observed in the adult. The higher intensity of adenosine A2 receptor mRNA enriched patches correspond at the microscopical level to a higher density of labeled neurons in the patches areas and also to a higher level of expression per labeled patches neuron than in the matrix ones. This demonstrates for the first time that differences in patch/matrix receptor density is at least partly linked to different levels of receptor gene expression.

The effect of soluflazine on adenosine receptors in the rat brain

Soluflazine, a potent adenosine transport Inhibitor, was intracerebroventricularly administered to rats via ALZET mini osmotic pumps (4nmole, 0.5 L/hr) for 14 days and the effect on adenosine receptors was determined in specific brain areas. Soluflazine decreased adenosine Al radioligand binding in the hippocampus as measured by [3H]R-PIA, and lowered adenosine A2 binding sites in the striatum, as estimated by the “NECA minus R-PIA” assay. Previous work from our lab has shown the ability of diazepam and triazolam to decrease adenosine binding in the same brain areas. The data show that a specific adenosine transport Inhibitor produces the same effect on adenosine receptors as benzodiazepines, and suggest a role for adenosine In the CNS effects of benzodiazepines.

Adenosine A2 receptors: Selective localization in the human basal ganglia and alterations with disease

Adenosine A2 receptors were labeled and visualized by autoradiography in tissue sections of the human brain using the A2-selective agonist ligand[ 3H](2-p-(2-car☐yethyl)phenylamino)-5′-N-car☐amidoadenosine (CGS 21680). The binding of this ligand was of high affinity, reversible and was blocked by adenosine A2 agents. Autoradiographic mapping of adenosine A2 sites revealed them to be exclusively restricted to the caudate nucleus, putamen, nucleus accumbens, olfactory tubercle and the lateral segment of the globus pallidus. The densities of adenosine A2 receptors in other brain areas did not differ from background levels. This selective localization prompted us to study the consequences of neurodegenerative diseases such as Parkinson's disease and Huntington's chorea on the densities and localization of these sites in the basal ganglia. In Parkinson's disease the density of adenosine A2 binding sites was comparable to that seen in control cases. In contrast, density values of A2 sites were dramatically decreased, compared to control values, in the basal ganglia of patients with Huntington's chorea. Similar losses of A2 receptors were observed in the guinea-pig striatum after local application of quinolinic acid while lesioning of the dopaminergic neurons was without effect. All these results taken together suggest that adenosine A2 receptors are localized on striatal output neurons which degenerate in Huntington's chorea.

Characterization of the adenosine receptor in porcine coronary arteries

1. Relaxant responses of ring preparations from porcine ventricular coronary arteries to adenosine and various stable adenosine analogues were investigated in vitro. 2. The adenosine analogues did not produce contraction but elicited almost complete relaxation of coronary arteries preconstricted with 3 microM prostaglandin F2 alpha (PGF2 alpha), even after removal of the endothelium. 3. The order of potency, was 5'-N-ethylcarboxamide-adenosine (NECA) greater than 2-(2-phenylethylamino)5'-N-ethylcarboxamide-adenosine (2-PEA-NECA) greater than 2-phenylamino-adenosine (CV-1808) greater than N6-[R(-)-1-phenyl-2-propyl]adenosine (R-PIA) greater than N6-[S(+)-1-phenyl-2-propyl]adenosine (S-PIA) greater than N6-cyclopentyl-adenosine (CPA) greater than adenosine greater than ATP offDP which suggested the presence of adenosine A2-receptor subtypes. 4. There was an excellent correlation between the calculated pD2 values on coronary arteries and the pKD values at adenosine A2 binding sites, whereas no correlation was obtained when the pD2 values were compared to the pKD values at adenosine A1-binding sites on membranes from porcine striata. 5. The relaxant effects of adenosine and its analogues were competitively antagonized by 8-(p-sulphophenyl)-theophylline (8-SPT), producing pA2 values similar to the respective pKD value of the antagonist at adenosine A2 binding sites. 6. It is suggested that the porcine coronary artery possesses adenosine A2 receptors which seem to be similar to the adenosine A2 binding site in pig striatum, whereas no evidence was obtained for the presence of adenosine A1 receptors.

Affinity labeling of adenosine A1 binding sites.

The adenosine A1 receptors of sheep brain membranes have been identified by the specific binding of radiolabeled cyclohexyl[3H]adenosine ([3H]CHA). Pretreatment of membranes with periodate-oxidized CHA causes a dose- and time-dependent decrease in the number of binding sites. No decrease occurs when membranes are pretreated with CHA. Binding of [3H]CHA to the remaining sites occurs with the same characteristics as binding to the untreated receptor population.

GABA and benzodiazepine receptors in the gerbil brain after transient ischemia: demonstration by quantitative receptor autoradiography.

Quantitative receptor autoradiography was used to measure the binding of gamma-aminobutyric acid (GABA) and benzodiazepine receptors after ischemia by means of transient occlusion of bilateral common carotid arteries in the gerbil. [3H]Muscimol was used to label the GABAA receptors and [3H]flunitrazepam to label central type benzodiazepine receptors. In the superolateral convexities of the frontal cortices, [3H]muscimol binding was increased in 60% of the animals killed 3 days after ischemia, and decreased in 67% of the animals killed 27 days after ischemia. Twenty-seven days after ischemia, [3H]flunitrazepam binding in the substantia nigra pars reticulata increased to 252% of the control, though the increase in [3H]muscimol binding was not significant. In the dorsolateral region of the caudate putamen, marked neuronal necrosis and depletion of both [3H]muscimol and [3H]flunitrazepam binding sites were observed 27 days after ischemia, the ventromedial region being left intact. In spite of the depletion of pyramidal cells in the CA1 region of the hippocampus, both [3H]muscimol and [3H]flunitrazepam binding sites were preserved 27 days after ischemia. Since our previous study revealed that adenosine A1 binding sites were depleted in the CA1 subfield of the hippocampus after ischemia correlating with neuronal damage, GABAA and benzodiazepine receptors may not be distributed predominantly on the pyramidal cells in the CA1 region.

Solubilization of adenosine A1 binding sites from sheep cortex

[(3)H]N(6)-cyclohexyladenosine binds with high affinity to sheep brain membranes with a drug specificity indicating an association with A(1) adenosine receptors. The [(3)H]N(6)-cyclohexyladenosine binding site has been solubilized with sodium cholate being the only detergent able to maintain specific binding after solubilization. After solubilization, the kinetics and drug specificity of binding are virtually identical with those obtained in the intact membranes, indicating a conservation of the binding site after removal of the receptor from its lipid environment. Gel filtration experiments indicated an apparent molecular weight of 400,000 for the receptor-detergent complex and a Stokes radius of 6.2 nm.