Cyclohexyladenosine Binding Research Articles

Evidence for pre- and postsynaptic localization of adenosine A1 receptors in the CA1 region of rat hippocampus: a quantitative autoradiographic study

The cellular localization of adenosine A1 receptors in the CA1 region of the rat hippocampus was investigated using radioligand binding and lesioning methods in a quantitative autoradiographic study. Kainic acid injection reduced [ 3H]cyclohexyladenosine binding in the CA3 region by 40% and in the CA1 region by 30%. Transient cerebral ischemia reduced [ 3H]cyclohexyladenosine binding in the CA1 region by 50%, while no change was measured in the CA3 region. These findings suggest that adenosine A1 receptors are located presynaptically on axon terminals of CA3 pyramidal cells and postsynaptically on the dendrites of CA1 pyramidal cells in the CA1 region of rat hippocampus.

Influence of stereotaxically injected scrapie on neurotransmitter systems of mouse cerebellum

The 22L strain of scrapie was injected stereotaxically into the cerebellum of C57BL/6J mice to determine its effect on several cerebellar neurotransmitter systems during the early clinical stages of the disease. In this model vacuolar lesions are restricted to the cerebellum with no evidence of vacuolization in other brain regions. Although lesions develop throughtout all cell layers of the cerebellum, they are most severe in the granule cell layer. Modest but significant (P < 0.01) reductions in cerebellar weight, glutamate decarboxylase activity, and in the affinity of the N 6-[adenine-2,8- 3H]cyclohexyladenosine binding sites, were observed in scrapie affected mice. The densities of the high- and low-affinity adenosine receptors were unaffected. Adenosine receptors in the cerebellum are highly localized to the axon terminals of the glutamatergic, GABA receptive granule cells. GABA, benzodiazepine, glutamate, and muscarinic cholinergic receptors were significantly altered. In addition, the high-affinity uptake of glutamate, and the activity of choline acetyltransferase were not significantly changed. GABA high-affinity uptake was slightly increased. Even though the granule cell layer of the cerebellum had undergone severe vacuolation, only modest neurotransmitter changes were apparent. Although these results suggest a tenous relationship between scrapie pathology and the integrity of neurotransmitter systems, it is possible that compensatory neurochemical changes in uncompromised neuronal populations may have masked potentially specific neurotransmitter effects.

Persistent upregulation of brain adenosine receptors in response to chronic carbamazepine treatment.

Chronic carbamazepine treatment for a period of 2 weeks caused a highly significant increase in brain adenosine receptors in the rat. The carbamazepine was administered in food pellets in a diet that achieved clinically relevant total plasma concentrations of carbamazepine and its active-10,11-epoxide metabolite. Of the several brain areas examined, the cerebral cortex and hippocampus exhibited the most robust increases in [3H]cyclohexyladenosine (CHA) binding. Increases of 35-40% were observed in these brain regions whereas an 8-10% increase was seen in the cerebellum. The carbamazepine induced increase in brain adenosine receptors in all these areas persisted unabated at 1 and 5 days as well as 2, 4, and 8 weeks following termination of carbamazepine treatment, suggesting a relatively permanent alteration of the adenosine receptor by this drug.

Adenosine antagonist properties of carbamazepine.

The binding of adenosine agonists and antagonists to the adenosine receptor is differentially affected by both temperature and guanine nucleotides. Agonist binding is facilitated at higher temperatures; the reverse is true for adenosine antagonists. In the present study, we demonstrate the feasibility of utilizing the temperature dependency of agonist/antagonist binding to the adenosine receptor in binding inhibition studies. We show that the anticonvulsant drug carbamazepine (CBZ) and several of its structural analogs behave in a manner identical to that of a series of adenosine antagonists; i.e., they are more potent inhibitors of [3H]cyclohexyladenosine (CHA) binding at 8 degrees C as compared to 37 degrees C and are equipotent as inhibitors of [3H]diethylphenylxanthine (DPX) binding at 0 and 30 degrees. We also show that the potency of CBZ and derivatives as inhibitors of [3H]CHA binding is markedly increased in the presence of 10 microM GppNHp, whereas their potency as inhibitors of [3H]DPX binding is unaffected by this guanine nucleotide. These data in conjunction with past studies support the hypothesis that CBZ and its derivatives act as adenosine antagonists at the level of binding interactions at the adenosine receptor.

Effects of stress on [3H]cyclohexyladenosine binding to rat brain membranes.

The investigation of stress-induced changes in neuronal functioning is important to our understanding of mental disorders, stress-induced psychological impairment, and the emotional reactions of fear and anxiety. Data from previous animal studies have demonstrated various pituitary-adrenal responses to stress and also changes in brain neurotransmitters. We are investigating whether stress-induced neuroendocrine and brain monoamine changes are accompanied by concomitant changes in brain neurotransmitter and/or neuromodulator receptors. We have developed a behavioral paradigm of chronic stress which incorporates sustained stress, continuous performance, and disruption of sleep. Animals which are habituated to press a lever to receive food are trained in an active shock escape task. A matched set of animals housed in identical operant chambers but not exposed to footshock are used as comparative controls. [3H]Cyclohexyladenosine ([3H]CHA) (5-7 nM) binding to A1 adenosine receptors in hypothalamic membrane preparations from rats stressed for three days was fifteen percent higher than in matched controls. However, no differences in [3H]CHA binding were found in tissue preparations from frontal cortex, hippocampus, or striatum, when comparing stressed and matched control rats. Plasma corticosterone levels were higher in stressed rats than in matched controls.

Autoradiographic evidence for multiple CNS binding sites for adenosine derivatives

The binding of the adenosine analogues, 5′-N-ethylcarboxamido[ 3H]adenosine and N6-cyclohexyl[ 3H]adenosine, to the rat brain was examined utilizing light-microscopic autoradiographic techniques. While associated with many of the same structures, binding sites for these compounds showed distinct differences in both their patterns of distribution and their capacities to be inhibited by the adenosine analogue, R-phenylisopropyladenosine. Previous studies have shown that, at nanomolar concentrations, cyclohexyladenosine and R-phenylisopropyladenosine bind rather exclusively to the A1 type of adenosine receptors. In contrast, 5′-N-ethylcarboxamidoadenosine has almost equal affinity for A1 and A2 sites. Taking advantage of these characteristics, non-A1 binding sites were resolved by examining 5′-N-ethylcarboxamidoadenosine binding in the presence of micromolar concentrations of unlabeled R-phenylisopropyladenosine. The autoradiographically demonstrated distribution of R-phenylisopropyladenosine-insensitive 5′-N-ethylcarboxamidoadenosine binding sites differed significantly from that of the cyclohexyladenosine binding sites. Such non-A1 binding sites were concentrated in the striatum, nucleus accumbens, medial genicuiate, olfactory tubercle, amygdala and certain thalamic nuclei. In contrast to the distribution of A1 adenosine receptor sites, R-phenylisopropyladenosine-insensitive 5′-N-ethylcarboxamidoadenosine binding was only low to moderate in the hippocampus, cerebellum and superior colliculus, regions which are strongly positive for cyclohexyladenosine binding. The present study provides the first autoradiographic evidence for multiple adenosine binding sites in the brain by demonstrating that the adenosine analogue 5′-N-ethylcarboxamidoadenosine can bind to a site or sites distinct from the A1 adenosine receptor site. The 5′-N-ethylcarboxamidoadenosine binding site which is not displaced by low concentrations of R-phenylisopropyladenosine may correspond to an A2 adenosine receptor site and/or an as yet uncharacterized type of adenosine receptor.

Rapid down regulation of hippocampal adenosine receptors following brief anoxia

Adenosine A 1 receptors, as demonstrated by [ 3H]cyclohexyladenosine (CHA) binding to cryostat sections of the brain, were studied utilizing quantitative autoradiographic techniques. A brief period of global CNS anoxia resulted in the rapid and persistent down regulation of [ 3H]CHA binding sites in the hippocampus but not in the neocortex or striatum. The density of adenosine A 1 receptors in a given brain region has previously been shown to be a critical factor in determining the strength of the inhibitory action of adenosine. Since the down regulation of these sites is correlated temporally with the onset of hyperactivity following transient anoxia, it is suggested that a reduction in the strength of the neuromodulatory action of adenosine contributes to the postanoxic hyperactivity of CA1 pyramidal cells and perhaps to their selective vulnerability.

3H]Cyclohexyladenosine binding in rat brain: A pharmacological analysis using quantitative autoradiography

Quantitative autoradiography was used to characterize the regional binding parameters of [ 3H]cyclohexyladenosine (CHA) and the regional inhibition of specific [ 3H]CHA binding observed with a series of adenosine agonists and antagonists. Under the conditions described [ 3H]CHA binding was best fit by a single, high affinity site model in all areas examined. No major regional differences were observed in the inhibition of [ 3H]CHA binding by the adenosine and xanthine analogs examined. Overall, the rank order of activity throughout all regions was CPA> R-PIA>NECA⪢PACPX⩾2-CADO⩾ S-PIA⪢DPX.

Phylogenetic distribution of [ 3H]cyclohexyladenosine binding sites in nervous tissue

The specific binding of the A 1 adenosine receptor ligand, [ 3H]CHA, was investigated in membrane fractions prepared from brains of eleven vertebrate species and ganglia of four invertebrate species. Substantial amounts of specific [ 3H]CHA binding sites were demonstrated in brain membranes of all vertebrate species examined; however, [ 3H]CHA binding sites were not detectable in nervous tissue of the invertebrate species studied. The densities of [ 3H]CHA binding sites in vertebrate brains increase in higher vertebrates. Moreover, the pharmacological characteristics of the site labeled by [ 3H]CHA in two divergent classes of vertebrates were similar. The broad phylogenetic distribution of A 1 adenosine receptors in primitive as well as advanced vertebrate species suggests a fundamental role for adenosine in neuronal modulation.

1,3-Dipropyl-8-cyclopentylxanthine (DPCPX) inhibition of [ 3H]N-ethylcar☐amidoadenosine (NECA) binding allows the visualization of putative non-A1 adenosine receptors

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.

CHARACTERISTICS OF HIGH AFFINITY AND LOW AFFINITY ADENOSINE BINDING SITES IN HUMAN CEREBRAL CORTEX: 96

Binding characteristics of human cortical membrane fractions were evaluated to test the hypothesis that there are A1 and A2 adenosine binding sites. Binding of chloroadenosine was time dependent, reversible and concentration dependent. The Kd for chloroadenosine was 280 nM with a Bmax of 1.6 pmoles/mg protein. The apparent Kd was estimated to be 0.74 μM for 5'-N-ethylcarbox-amideadenosine, 1 μM cyclohexyladenosine, 13 μM N6-(L-2-phenylisopropyl)adenosine, 84 μM isobutylmethylxanthine and 105 μM theophylline. Hill slope factors ranged from 0.3 to 0.6. The kob was 0.080 min−1, the K1 7.5 × 104 min−1M−1, and the k2 .074 min−1. The Kd calculated from the rate constants was 990 nM. Cyclohexyladenosine binding was concentration dependent and the Kd was 5 nM with a Bmax of 0.35 pmoles/mg protein. Cyclohexyladenosine binding was displaced by N6-(L-2-phenylisopropyl)adenosine (Kd 4 nM), 2-chloroadenosine (Kd 10 nM), 5'-N-ethylcarboxamideadenosine (Kd 6 nM), isobutylmethylxanthine (4 μM) and theophylline (8 μM). Hill slope factors ranged from 0.5 to 0.8.Our data support the existence of two adenosine binding sites in human cortex compatable with low affinity (A2) and high affinity (A1) adenosine receptors.

Cyclohexyladenosine binding in rat striatum

N6-Cyclohexyl-[ 3H]adenosine ([ 3H]CHA) binds specifically to rat brain membranes prepared from the caudate-putamen complex with a K d value of 2.50 ± 0.39nM and B max of458 ± 51fmol/mg protein. Lesioning the nigrostriatal dopaminergic pathway using 6-hydroxydopamine failed to alter [ 3H]CHA binding characteristics. Intrastriatal kainate lesions reduced the binding capacity of [ 3H]CHA by 28% though this was not statistically significant (0.1 < P > 0.05). In kainate-lesioned striata, however, 2-deoxyglucose uptake was reduced by only 39%.

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.

An A 1-adenosine receptor, characterized by [ 3H]cyclohexyladenosine binding, mediates the depression of evoked potentials in a rat hippocampal slice preparation

In slices of rat hippocampus, adenosine and several adenosine derivatives depressed evoked neuronal responses to afferent stimulation. The nanomolar potency of adenosine derivatives and their relative effectiveness indicate that the depression of evoked potentials is mediated via an A 1-adenosine receptor. A remarkable similarity was found between the relative potencies of nucleoside derivatives with respect to their electrophysiological effects and to their inhibition of high affinity [ 3H]cyclohexyladenosine ([ 3H]CHA) binding to rat brain membranes. We conclude that the [ 3H]CHA binding site in rat brain membranes represents a physiological adenosine receptor of the A 1-type.