CHA Binding Research Articles

Modulation of A 1 adenosine receptor signaling by peroxynitrite

Nitric oxide (NO) is a gaseous free radical involved in many pathophysiological processes. During oxidative stress, NO, its derivatives and adenosine are released. Considering adenosine neuroprotective role in the central nervous system (CNS) and toxicity of NO, we investigated the effect of a NO/peroxynitrite (ONOO −) donor, 3-morpholinosydnonimine (SIN-1), on A 1 adenosine receptor (A 1AR) signaling pathway in rat cortical membranes. Membrane treatment with 0.5 mM SIN-1 for various periods of time (0–240 min) decreased specific binding of the radiolabeled A 1AR agonist, [ 3 H ] N 6-cyclohexyladenosine ([ 3 H ]CHA), in a time-dependent manner, reaching the steady state after 120 min. The inhibitory effect of SIN-1 was concentration-dependent, with an ec 50 value of 0.60±0.30 mM (N=3). Membrane pre-incubation with the superoxide anion (O 2 −) scavenger superoxide dismutase (SOD) followed by SIN-1 addition, abolished SIN-1 inhibition of [ 3 H ]CHA binding. Membrane treatment with 0.5 mM SIN-1 for 120 min caused a significant 2-fold increase of the K D value for [ 3 H ]CHA without changing the B max value. Moreover, pre-incubation of membranes with A 1AR agonists, CHA or N 6-(2-phenylisopropyl)-adenosine (R-PIA) before SIN-1 addition increased the inhibitory effect while the selective A 1AR antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) had no activity. Membrane treatment with SIN-1 decreased receptor-stimulated guanosine 5′- O-(γ[ 35 S ]thio)triphosphate ([ 35 S ]GTPγS) binding in a concentration-dependent manner. This treatment influenced [ 35 S ]GTPγS binding affinity for A 1AR activated G i proteins in cortical membranes. These findings suggest that ONOO − modulates A 1AR signaling pathways by affecting receptor G i protein coupling.

Ribose-modified nucleosides as ligands for adenosine receptors: synthesis, conformational analysis, and biological evaluation of 1'-C-methyl adenosine analogues.

1'-C-Methyl analogues of adenosine and selective adenosine A(1) receptor agonists, such as N-[(1R)-1-methyl-2-phenylethyl]adenosine ((R)-PIA) and N(6)-cyclopentyladenosine, were synthesized to further investigate the subdomain that binds the ribose moiety. Binding affinities of these new compounds at A(1) and A(2A) receptors in rat brain membranes and at A(3) in rat testis membranes were determined and compared. It was found that the 1'-C-methyl modification in adenosine resulted in a decrease of affinity, particularly at A(1) and A(2A) receptors. When this modification was combined with N(6) substitutions with groups that induce high potency and selectivity at A(1) receptors, the high affinity was in part restored and the selectivity was increased. The most potent compound proved to be the 1'-C-methyl analogue of (R)-PIA with a K(i) of 23 nM for the displacement of [(3)H]CHA binding from rat brain A(1) receptors and a > 435-fold selectivity over A(2A) receptors. In functional assays, these compounds inhibited forskolin-stimulated adenylate cyclase with IC(50) values ranging from 0.065 to 3.4 microM, acting as full agonists. Conformational analysis based on vicinal protonminus signproton J-coupling constants and molecular mechanics calculations using the MM2 force field proved that the methyl group on C1' in adenosine has a pronounced impact on the furanose conformation by driving its conformational equilibrium toward the north, gamma+, syn form.

Differential effects of the adenosine A 1 receptor allosteric enhancer PD 81,723 on agonist binding to brain and adipocyte membranes

The benzoylthiophene analog, PD 81,723, has been shown to allosterically enhance agonist binding and functional activation of the mammalian adenosine (ADO) A 1 receptor subtype by putatively maintaining the receptor in a high affinity state. The present studies were conducted to evaluate the ability of PD 81,723 to enhance the binding of [ 3 H ]cyclohexyladenosine ([ 3 H ]CHA) to A 1 receptors of neural (cerebral cortex) and non-neural (adipocyte) origin in three different species; rat, guinea pig and dog. PD 81,723 (0.3–100 μM) produced a concentration-dependent enhancement of [ 3 H ]CHA binding to rat brain A 1 receptors. These effects were also species-dependent with larger enhancements (150–200% of control) observed in guinea pig and dog brain membranes as compared to the rat (120% of control). In contrast, PD 81,723 did not produce any enhancement of [ 3 H ]CHA binding to A 1 receptors in adipocyte membranes from any of the species examined. Additional binding studies were conducted using pharmacological manipulations that have previously been shown to enhance the allosteric effects of PD 81,723. In the presence of 1 mM GTP, the allosteric effects of PD 81,723 (15 μM) were increased in rat, guinea pig and dog brain membranes, however, in adipocyte membranes from each species, no significant alteration in agonist binding was observed. Similarly, the A 1 receptor selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (added to effectively reduce the intrinsic antagonist properties of PD 81,723) was found to enhance the allosteric effects of PD 81,723 (15 μM) in brain, but produce no alteration of agonist binding in adipocyte membranes from each species. Examination of the dissociation kinetics of [ 3 H ]CHA binding from rat brain and adipocyte membranes revealed that PD 81,723 (15 μM) differentially slowed agonist dissociation from brain, but not adipocyte, membranes. Taken together, the present data support the hypothesis that in tissues that are sensitive to PD 81,723, this benzyolthiophene functions to maintain the A 1 receptor in a high-affinity state and that the relative proportions of high-affinity A 1 receptors present in specific tissues may contribute, at least in part, to the apparent differential effects of PD 81,723 on agonist binding. The tissue specific modulation of A 1 receptor function by PD 81,723 also illustrates the possibility that the locus of allosteric modulation by PD 81,723 may be manifest via a specific, but indirect and tissue-dependent, interaction with the A 1 receptor.

Mechanisms of inhibitory effects of zinc and cadmium ions on agonist binding to adenosine A1 receptors in rat brain

The dose-dependent inhibition of zinc and cadmium ions of agonist binding to A1 adenosine receptors in rat brain is prevented by histidine and cysteine, respectively. In the present study, the possible different mechanisms of Zn 2+ and Cd 2+ inhibitions were examined. The effects of Zn 2+ and Cd 2+ on equilibrium binding parameters of the agonists N 6-cyclohexyl-[2,8- 3H]-adenosine ([ 3H]CHA) or chloro- N 6-cyclopentyl-adenosine ([ 3H]CCPA) and the antagonist cyclopentyl-1,3-dipropylxanthine ([ 3H]DPCPX) were compared with those effects of reagents or binding conditions which altered histidyl or cysteinyl residues of the A1 receptor. Zn 2+ pretreatment did not change A1 agonist or antagonist affinity, but did reduce the B max. The inhibitory effects of Zn 2+ pretreatments were also maintained after several membrane washings. Diethylpyrocarbonate, a histidine-specific alkylating reagent, behaved like zinc ions: pretreatment with A1 agonist protected the histidyl residues of the [ 3H]CHA binding site against modification by Zn 2+, while the modification of the protonation state of the nitrogen of the imidazole group of histidines by changing pH indicated that the interactions of Zn 2+ with the histidyl residues were feasible with their unprotonated form. These findings suggest the formation of coordination bonds between Zn 2+ and histidines critical for [ 3H]CHA or [ 3H]DPCPX binding, which may prevent the ligand interaction with the specific sites without modifying the binding kinetics of radioligand to the non-chelated recognition sites. Cd 2+ pretreatment reduced the [ 3H]CCPA affinity, but did not modify the affinity of the antagonist [ 3H]DPCPX, the B max remaining unaffected. As with cadmium effects, the oxidation of the thiol group of cysteine by dithionitrobenzoic acid (DTNB) reduced [ 3H]CCPA affinity without changing the number of binding sites. The reducing reagent dithiothreitol, which alone was unable to modify [ 3H]CCPA binding, overcame the inhibiting effects of both Cd 2+ and DTNB. These findings suggest that cadmium ions may oxidize SH groups of cysteines localized on the A1 receptor molecule or a cysteine localized in the region of G iα subunit involved in the coupling with receptors. This mechanism can justify potential conformational modifications of the receptor molecule producing the decrease in affinity.

Biochemical and pharmacological characterization of adenosine A1 receptors in eel (Anguilla anguilla) brain

N6-cyclohexyl[3H]adenosine ([3H]CHA) was used to label adenosine A1 receptors in membranes prepared from male and female eel whole brain. The A1 receptor agonist [3H]CHA bound saturably, reversibly and with high affinity (Kd = 0.91 ± 0.12 nM; Bmax = 120.36 ± 5.2 fmol mg−1 protein). In equilibrium competition experiments, the adenosine agonists and antagonists all displaced [3H]CHA from high-affinity binding sites with the rank order of potency in displacing, characteristics of an A1 adenosine receptor. Mg2+ dramatically increased the affinity of [3H]CHA without modifying the maximal binding capacity. The specific binding was inhibited by guanosine 5′-triphosphate (Ki = 2.54 ± 0.98 μM). The [3H]CHA binding sites are ubiquitously distributed with a maximum in cerebellum and a minimum in olfactory bulb. No difference was observed between male and female brain. In eel brain, synaptosomes (P2), stimulation of adenosine 3′,5′-monophosphate (cyclic AMP) accumulation with 10−5 M forskolin was markedly reduced (45.5%) by treatment with the adenosine A1 receptor agonist CHA (10−4 M), and the reduction was reversed in presence of the selective A1 receptor antagonist 8-cyclopentyltheophylline (10−5 M). In superfused eel cerebellar synaptosomes, K+ stimulated the release of adenosine in a partially Ca2+-dependent manner. The findings, taken together, suggest the hypothesis that adenosine A1 receptors present in eel brain could modulate synaptic transmission, as A1 receptors do in other vertebrates.

The effects of saponin on the binding and functional properties of the human adenosine A1 receptor.

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.

Effects of neonatal exposure to caffeine on adenosine A 1 receptor ontogeny using autoradiography

The ontogeny of adenosine A 1 receptor density was assessed via autoradiographical analysis of [ 3H]cyclohexyladenosine ([ 3H]CHA) binding in brains of 14–31-day-old rats as a function of exposure to caffeine over postnatal days 2–6. This exposure period was analogous to the period during which human infants are administered caffeine as treatment for apnea of prematurity. [ 3H]CHA binding was greatest in CA1 and CA3 hippocampus in both caffeine-exposed and control rats across all ages. Within the anterior, ventral, lateral and medial regions of the thalamus of unmanipulated rats, [ 3H]CHA binding did not change with age. In caffeine-exposed rats, however, [ 3H]CHA binding increased significantly within these thalamic subregions as the rats aged. In addition, with age in both treatment groups, the molecular and granular layers of the cerebellum and the CA1 and dentate gyrus of the hippocampus displayed increasing [ 3H]CHA density. Furthermore, regardless of age, [ 3H]CHA binding was decreased in the molecular layer of neonatally caffeine-exposed animals as compared to controls. Thus, limited exposure to caffeine within the first postnatal week altered the subsequent expression of adenosine A 1 receptors in most subregions of the thalamus and in the molecular layer of the cerebellum.

Evidence for functionally important adenosine A 2a receptors in the rat hippocampus

Adenosine A 2a receptors are not confined to dopamine-rich areas of the brain, since thermocycling analysis shows that adenosine A 2a receptor mRNA is expressed also in the hippocampus (CA1, CA3 and dentate gyrus) and cerebral cortex. The expression of A 2a mRNA in three main areas of the hippocampus was confirmed by in situ hybridization; A 2a mRNA expression was mainly localized in the pyramidal and granular cells, the same hippocampal regions that showed adenosine A 1 receptor mRNA expression. Receptor autoradiographic studies with [ 3H]CGS 21680 (30 nM), a selective adenosine A 2a receptor agonist, showed specific binding sites in the hippocampus. The density of [ 3H]CGS 21680 binding was greatest in the stratum radiatum of the CA1 area, followed by the stratum oriens of the cornu Ammonis, stratum radiatum of the CA3 area and supra-granular layer of the dentate gyrus. This antomical distribution of [ 3H]CGS 21680 binding was similar to the pattern of [ 3H]CHA binding in the hippocampus. Electrophysiological studies in the Schaffer fibers / CA1 pyramids showed that upon activation of the A 2a receptors with CGS 21680 (10 nM) the ability of the adenosine A 1 receptor agonist, CPA, to inhibit neuronal activity was significantly attenuated. These results show functionally important co-expression of adenosine A 2a and A 1 receptors in the hippocampus. The results also suggest that adenosine A 2a receptor-mediated neuromodulation is not confined to the basal ganglia, but is more widespread throughout the nervous system.

Tolerance to adenosine's accentuation of ethanol-induced motor incoordination in ethanol-tolerant mice.

Our previously published reports have provided data that have supported a functional correlation between ethanol-induced changes in the characteristics of adenosine receptor, adenosine uptake and release in the brain, and ethanol-induced motor incoordination. The present data demonstrated a cross-tolerance between ethanol and adenosine further supporting the hypothesis that brain adenosine modulates the motor impairing effects of ethanol. Mice that received (-)-N6-cyclohexyladenosine (CHA) [0.25 mg/kg/day, intraperitoneally (ip)] for 10 days exhibited marked attenuation (cross-tolerance) to acute ethanol-induced motor incoordination compared with chronic saline (ip) controls. The attenuation of acute ethanol-induced motor incoordination was essentially same in animals that received CHA (25 ng/5 microliters/day for 10 days) by the intracerebroventricular (icv) route as opposed to the controls that chronically received artificial cerebral spinal fluid by the same route. Similarly, tolerance was exhibited to acute CHA (0.125 mg/kg ip and 12.5 ng/5 microliters icv) by animals fed liquid ethanol (19.5 g/kg/24 hr) for 10 days compared with none in the pair-fed sucrose controls. Scatchard plots using cerebellar tissue homogenates from animals given chronic CHA or chronic ethanol indicated no change in Bmax and/or Kd values for CHA binding when compared with CHA binding in tissues from their respective controls. However, a lack of any change in the binding characteristics cannot rule out the involvement of adenosine receptors in the observed cross-tolerance between ethanol and CHA. The results may suggest desensitization of adenosine A1 receptors due to chronic CHA and ethanol as an alternate possible explanation in the development of cross-tolerance between adenosine (CHA) and ethanol.

Long-term changes in gerbil brain neurotransmitter receptors following transient cerebral ischaemia.

1. Receptor autoradiographic and histological techniques were used to investigate long-term changes in the gerbil brain following transient cerebral ischaemia. 2. Transient ischaemia was induced for 3 min and 10 min, and the animals were allowed to survive for 8 months. 3. Histological examination revealed that 3 min ischaemia caused neuronal damage and mild shrinkage only in the hippocampal CA1 sector. Ten minutes of ischaemia produced severe neuronal damage in the striatum and the hippocampal CA1 and CA3 sectors. Considerable shrinkage was seen in the hippocampus; the dentate gyrus, however, was not damaged. 4. Three minutes of ischaemia produced changes in the binding of [3H]-quinuclidinylbenzilate ([3H]-QNB), [3H]-muscimol, and [3H]-MK-801 in various brain regions, as determined autoradiographically. In contrast, [3H]-cyclohexladenosine ([3H]-CHA) and [3H]-PN200-110 ([3H]-isradipine) binding in the brain was unaltered. 5. Ten minutes of ischaemia resulted in a major loss of neurotransmitter receptors, especially in the hippocampus. The substantia nigra showed a significant reduction in [3H]-CHA binding, whereas the striatum, which was morphologically damaged, showed no significant changes in any of the neurotransmitter receptors examined. 6. The results demonstrated that long-term survival after transient cerebral ischaemia produced alterations in neurotransmitter receptors, especially in the hippocampal formation, where considerable shrinkage was noted. These results also suggest that the hippocampal damage was not static, but progressive.

Reduction of A1 adenosine receptors in cortex, hippocampus and cerebellum in ageing mouse brain

Age related changes in A1 adenosine receptor binding were investigated in mouse brain using the selective agonist, [3H]-cyclohexyladenosine ([3H]CHA). In the cortex, hippocampus and cerebellum of aged mice (28 months old), a significant decrease of about 44%, 50% and 12%, respectively, in [3H]CHA binding compared to young animals (3 months old) was observed. According to the Scatchard analysis of the binding data in the cortex, this decrease was due to a receptor density reduction and not to a Kd change. Since the weight and protein content of each tissue tested did not differ significantly between the old and the young animals, our findings may be partly explained by specific reductions of A1 receptors rather than a general cell degeneration in old age.

Antagonism towards endogenous adenosine and inhibition of cGI-PDE in the cardiac effects of amrinone, milrinone and related analogues

1. 1. The effect of amrinone, milrinone and of three milrinone analogues was tested on spontaneous chronotropic and inotropic activity of guinea-pig isolated atria, on the activity of cGMP-inhibited phosphodiesterase (cGI-PDE) from guinea-pig heart and on specific binding of N 6-cyclohexyl[ 3H]adenosine ([ 3H]CHA) to R i adenosine receptors in guinea-pig atria. 2. 2. The K i -values towards [ 3H]CHA binding to R i receptors were linearly related to the EC 50s for the increase in force of contraction but not to the EC 50s for the increase in frequency of the atria. The K i values towards cGI-PDE were linearly related to the EC 50s for the positive chronotropic effect.

Magnesium‐Dependent Enhancement of Endogenous Agonist Binding to A1 Adenosine Receptors: A Complicating Factor in Quantitative Autoradiography

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.

Changes in seizure latency correlate with alterations in A1 adenosine receptor binding during daily repeated pentylentetrazol-induced convulsions in different mouse brain areas

The seizure latency changed during daily pentylentetrazol (PTZ) induced convulsions showing an increase between days 2 and 4, a rapid decrease between days 5 and 10 and a slight increase again between days 11 and 14. At the respective timepoints, [3H]CHA binding, in cortex and cerebellum of PTZ treated animals followed exactly the same pattern, suggesting that the alterations in A1 receptors in these areas may partly determine the PTZ seizure latency curve. On the contrary, the changes of [3H]CHA binding in hippocampus (sustained increase) and striatum (sustained decrease) didn't follow the latency curve pattern. These results suggest that changes in A1 receptor density in specific brain areas may be involved in the modulation of seizure susceptibility.

Autoradiographic changes in brain adenosine A 1 receptors and their coupling to G proteins following seizures in the developing rat

In the central nervous system, adenosine has been shown to be a major regulator of neuronal activity in convulsive disorders, mainly via the A 1 receptor subtype. In a previous work, we have shown that seizures lead to an age-dependent upregulation of cerebral adenosine A 1 sites measured in isolated rat cerebral membranes. However, information concerning regional changes in the receptor density was so far lacking. In the present study, the effects of bicuculline-induced seizures were investigated by quantitative autoradiography of central adenosine A 1 receptors in developing rats and in adults. Animals were sacrificed 30 min after an intraperitoneal injection of either saline or a convulsive dose of bicuculline. Adenosine A 1 receptors in brain section, were labeled by [ 3H]N 6-cyclohexyladenosine (CHA), a potent receptor agonist. Generalized seizures induced a widespread increase in CHA-specific binding, with a marked enhancement in structures that mediate seizure activity, such as substantia nigra, amygdala, septum and hippocampus. Moreover, the addition of guanylyl-5′-imidodiphosphate, a GTP analogue, to the incubation medium reduced CHA binding by the same order of magnitude whether rats were given saline or bicuculline, suggesting that additional adenosine A 1 receptors are also functionally linked to G proteins. The age-related postictal increase in adenosine receptors might contribute to facilitate adenosine anticonvulsant effect, especially in newborns.

Selective alterations in glutamate receptor subtypes after unilateral orbital enucleation

Glutamate is the major excitatory neurotransmitter in the rat visual system. Using quantitative autoradiography the effect of unilateral orbital enucleation on [ 3H]kainate, [ 3H]α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid ([ 3H]AMPA) and [ 3H]glutamate binding to kainate, quisqualate and NMDA receptors respectively has been examined within anatomical components of the visual pathway at 4 time points up to 20 days post-lesion. The time course for the degeneration of retinal projection fibres was assessed in a separate group of animals by quantifying [ 3H]cyclohexyladenosine ([ 3H]CHA) binding to presynaptic adenosine A 1 receptors. Over the first 5 days after orbital enucleation, there were no significant alterations in glutamate or adenosine A 1 receptor binding in visual structures of the visually deprived hemisphere. However, at 10 days post-lesion [ 3H]AMPA binding was significantly reduced (30%) in the visually deprived superior colliculus but unaltered in other visual structures. At this time point there was also a significant reduction (50%) in [ 3H]CHA binding in the visually deprived superior colliculus but not in other retino-recipient nuclei. There were similar changes in [ 3H]AMPA and [ 3H]CHA binding at 20 days post-enucleation. [ 3H]Kainate binding was significantly increased in the visually deprived superior colliculus only at 20 days post-enucleation. Saturation analysis of [ 3H]kainate and [ 3H]AMPA binding at this time point indicated a selective increase in the b max value for the high affinity [ 3H]kainate binding site and a concomitant decrease in the b max value for the high affinity [ 3H]AMPA binding site in the visually deprived superior colliculus. There were, however, no significant alterations in [ 3H]AMPA or [ 3H]kainate binding in other primary projection areas or in secondary visual areas (e.g. visual cortex) at any time point. NMDA sensitive [ 3H]glutamate binding was unaltered in the visually deprived hemisphere up to 20 days post-enucleation. These results suggest an upregulation of kainate receptors in the visually deprived superior colliculus after orbital enucleation and a loss of presynaptic quisqualate receptors on degenerating retinal fibres. The plastic alterations in kainate receptors in the superior colliculus are supportive of electrophysiological data suggesting a physiological role for these sites in mediating excitatory postsynaptic potentials in tectal neurons.

Chronic theophylline treatment in vivo increases high affinity adenosine A1 receptor binding and sensitivity to exogenous adenosine in the in vitro hippocampal slice

The present investigation examined the effects of chronic treatment with the adenosine receptor antagonist theophylline in vivo, on in vitro hippocampal electrophysiology and adenosine A1 receptor binding in the same animals. Adult rats were injected once daily (i.p.) with theophylline for 1 week at 75 mg/kg, followed by an additional week at 100 mg/kg, or with saline for the same 2-week period. Two days following the last injection, hippocampal slices were prepared and population spikes recorded from the pyramidal cell layers of area CA1 were elicited by Schaffer collateral-commissural fiber stimulation. The degree of inhibition caused by superfused adenosine was compared between hippocampal slices from theophylline- and saline-treated rats. Tissue from the contralateral hippocampus was used in [ 3H]cyclohexyladenosine ([ 3H]CHA) receptor binding. Hippocampi from theophylline-treated animals showed a significantly greater number of [ 3H]CHA binding sites (apparent B max; 125% of control, P < 0.05), without a significant change in binding affinity, and were more sensitive than controls to the inhibitory effects of adenosine on the population spike response. These results suggest that chronic adenosine receptor antagonism results in the up-regulation of adenosine A1 receptors which are functional and physiologically relevant in the in vitro hippocampus, and further supports the hypothesis that methylxanthine tolerance is mediated, at least in part, by an increase in adenosine receptor density.

Alterations of A1 adenosine receptors in different mouse brain areas after pentylentetrazol-induced seizures, but not in the epileptic mutant mouse ‘tottering’

Single and repeated Pentylentetrazol (PTZ)-induced convulsions are associated with significant changes of A adensine receptors (detected using the radioligand [ 3H]cyclohexyladenosine, [ 3H]CHA) in 4 different brain areas of the mouse, namely cortex, hippocampus, cerebellum and striatum. In hippocampus and cerebellum, a rapid increase in [ 3H]CHA binding, by 26% and 30% respectively, was observed 1 h after a single PTZ convulsion. In striatum, on the contrary, a significant decrease by 30% in [ 3H]CHA binding was seen, whereas in cortex no significant change could be detected. After daily repeated PTZ convulsions, a significant increase of A1 receptors by 26% appeared also in cortex, while the changes of A1 receptors observed in the other brain areas after a single PTZ convulsion were maintained in almost the same range. All the alterations observed were due to changes of the total number of A1 receptors ( B max) without changes in receptor affinity ( K rmd). A significant increase in the latency of PTZ seizure (time between the PTZ-injection and the beginning of the seizure) was also observed after repeated PTZ-induced convulsions at the time when the changes in A1 adenosine receptors were noted. Considered together, these results provide further evidence for an A1 receptor-mediated modulation of seizure susceptibility and indicate that specific brain areas may play different roles in this modulation. The binding of [ 3H]CHA to membranes from different cortical and subcortical areas of the epileptic mutant mouse ‘tottering’ was not different from that in control animals.

Inhibition of N6‐[3H]Cyclohexyladenosine Binding by Carbamazepine

The mechanism of action of carbamazepine (CBZ) (Tegretol), despite widespread use in the management of partial and tonic-clonic seizures in adults, is not completely understood. In animals, adenosine and adenosine analogues have anticonvulsant effects that may be due to interactions with central A1 adenosine receptors. CBZ (at therapeutically relevant concentrations) inhibits the binding of agonists and antagonists to brain A1 adenosine receptors, but whether as an agonist/antagonist is not clear. The adenosine agonist, N6-[3H]cyclohexyladenosine ([3H]CHA), binds to membranes from rat cortex and hippocampus at two nanomolar binding sites or states. To clarify the actions of carbamazepine at the A1 adenosine receptor, its inhibitory actions were compared with those of known adenosine agonists and xanthine antagonists using 0.1 nM[3H]CHA, in which almost all binding is to the higher affinity state, or 10 nM [3H]CHA, in which there is a substantial contribution of binding from both states. The ratios of the IC50 values (concentration that inhibits specific binding by 50%) at 10 nM [3H]CHA to the IC50 values at 0.1 nM [3H]CHA were 18-31 for the agonists and 4-10 for the xanthine antagonists. CBZ had a ratio of 3. The inhibitory effects of GTP on [3H]CHA binding were less in the presence of the adenosine agonist, 2-chloroadenosine than were inhibitory effects in the presence of the xanthine antagonist theophylline or CBZ in both cortex and hippocampus. These in vitro studies indicate that CBZ is an antagonist at A1 adenosine receptors in cerebral cortical and hippocampal membranes from rat brain. Agonist activity at A1 adenosine receptors would have been compatible with the sedative anticonvulsant effects of CBZ, but these data do not support a role of the anticonvulsant action of carbamazepine on A1 adenosine receptors in cerebral cortex or hippocampus.

Effects of enucleations and visual deprivation on adenosine A 1 and A 2 receptor binding in superior colliculus of rat

Adenosine receptors of A 1 and A 2 subtypes on retino-collicular projections in rat were studied using [ 3H]cyclohexyladenosine ([ 3H]CHA), [ 3 H]N- ethylcarboxamidoadenosine ([ 3H]NECA), and [ 3H]CGS 21680. Unilateral enucleation significantly reduced by 51% [ 3H]CHA binding to A 1 sites, and by 42% the A 1 component of [ 3H]NECA binding in contralateral SC. The binding of [ 3H]CGS 21680 and [ 3H]NECA to A 2 sites was not significantly affected by enucleation procedures. No significant reductions in adenosine receptor binding were observed in SC from rats exposed to a dark environment for three weeks. The reductions in A 1 but not A 2 adenosine receptor binding strongly suggests that A 1 sites are presynaptically located on retinal projections and are vulnerable to deafferentiation-, but not visual deprivation-induced decreases.