Autoradiographic localization of calcium channel antagonist receptors in rat brain with [H]nitrendipine

In vitro autoradiographic techniques have been used to localize [³H]nitrendipine binding sites in the rat brain. The superficial cerebral cortex, the ventral, lateral and posterior nuclei of the thalamus, the molecular layer of the dentate gyrus, the substantia nigra and the external plexiform layer of the olfactory bulb, all contain high densities of silver grains. The level of binding sites are greatly reduced in areas low in synaptic connections. The corpus callosum, the alveus hippocampi and the dorsal commissure of the fornix all lack specific grains, as does the lateral olfactory tract of theolfactory bulb. Specific silver grains are not found in the habenula or the hypothalamus. Grains are not associated with blood vessels profiles. The discrete localizations of [³H]nitrendipine bindings sites suggest a specific synaptic role.     

Normalization of subtype-specific muscarinic receptor binding in the denervated hippocampus by septodiagonal band grafts

Unilateral fimbria-fornix lesions were made by aspiration in female Sprague-Dawley rats. In a group of these rats, fetal septal tissue was transplanted into the lesion cavity. Lesion of the fimbria-fornix resulted in a reduction of cholinergic input to the hippocampal formation as indicated by the loss of acetylcholinesterase (AChE)-positive staining in all ipsilateral hippocampal laminae and a loss of [3H]hemicholinium-3 binding to cholinergic terminals in the strata oriens (82% reduction) and radiatum (77% reduction) of areas CA2 and CA3 and in the molecular layer of the dentate gyrus (83% reduction). In contrast, the density of muscarinic receptor binding ([3H]QNB) increased in the strata oriens (80% increase) and radiatum (70% increase) in areas CA2-CA4. This was shown to be due to an actual increase in receptor number (Bmax) and not to a change in affinity (KD). Analysis of muscarinic receptor subtypes indicated that the increase in receptor binding in the stratum radiatum was of the M-1 subtype ([3H]-pirenzepine) and in the stratum oriens was of the M-2 subtype ([3H]QNB + 100 nM pirenzepine). In the host hippocampus after fetal septal graft, the staining for AChE, the binding of [3H]hemicholinium-3, and the binding of muscarinic receptors (both the M-1 and M-2 receptor subtypes) were all comparable to nonlesioned control values. These data indicate that the fetal septal grafts have reinnervated the host hippocampus and have made synaptic contact with host cells in a manner capable of regulating postsynaptic muscarinic receptors.     

A radiohistochemical measure of [3H]TCP binding to the activated NMDA-receptor-gated ion channel in rat brain.

The N-methyl-D-aspartate (NMDA) subtype of excitatory amino acid receptor is linked to an ion channel that is blocked by the phencyclidine analog N-(1-[thienyl]cyclohexyl)piperidine (TCP). Previous studies have shown that NMDA and glycine act together to increase the access of [3H]TCP to its binding site, presumably by increasing channel opening; NMDA/glycine-enhanced [3H]TCP binding performed under non-equilibrium conditions thereby serves as a dynamic molecular marker of channel activation. In this study we tested whether NMDA and glycine regulate [3H]TCP binding in slide-mounted brain sections. Striking activation of the NMDA ion channel was observed in neo- and allocortex; dentate gyrus and strata radiatum and oriens of CA1 and CA3 of hippocampal formation; and certain amygdaloid nuclei (basomedial, basolateral, and cortical). Other nuclei of the amygdala (central, posterolateral, and medial), basal ganglia, and numerous regions within the diencephalon, brainstem and cerebellum showed relatively little activation of the NMDA ion channel. Our demonstration of enriched NMDA/glycine-stimulated [3H]TCP binding in stratum radiatum of hippocampal region CA1 but not in cerebellar granule cell layer correlates with electrophysiologic studies that showed NMDA channel ion flux in CA1 but not in cerebellar granule cell layer in adult rats. These data demonstrate that NMDA and glycine regulation of [3H]TCP binding can be quantified with a radiohistochemical method to provide a regional measure of the activated NMDA-receptor-gated ion channel. This technique is a powerful tool for functional analysis of the NMDA receptor/channel complex in both physiologic and pathologic states.     

Regulation of neurotensin-containing neurons in the rat striatum. Effects of unilateral striatal lesions with quinolinic acid and ibotenic acid on neurotensin content and its binding site density

Recently, we reported bilateral increases in striatal neurotensin (NT) levels following unilateral 6-hydroxydopamine lesion of the nigrostriatal dopaminergic pathway. In the present study, the effect of unilateral striatal lesions with quinolinic acid (QA, 300 nmol) or ibotenic acid (IBO, 130 nmol) on striatal NT levels and binding site densities were analyzed in order to investigate other possible regulations of NT systems. QA and IBO injection decreased γ-aminobutyric acid (GABA) levels and [¹²⁵I]iodosulpride (a specific D2 receptor antagonist) binding site densities in the lesioned striatum, indicating degeneration of striatal intrinsic neurons. Striatal dopaminergic terminals were not altered by QA as shown by the lack of changes in [³H]dihydrotetrabenazine ([³H]TBZOH, a specific ligand of the vesicular monoamine transporter) binding site densities. Moreover, QA lesion induced an increase in NT levels and a decrease in NT binding sites in the lesioned striatum without any change in the contralateral structure. In contrast to QA, IBO might destroy a certain proportion of dopaminergic terminals in the lesioned striatum, as shown by a 54% decrease in [³H]TBZOH binding. Furthermore, IBO lesion enhanced striatal NT levels bilaterally, while NT binding sites decreased in the lesioned striatum and increased in the contralateral side. The present results suggest that not only dopaminergic neurons but also striatal intrinsic neurons may control NT systems in the striatum.     

Autoradiographic evidence that NMDA receptor-coupled channels are located postsynaptically and not presynaptically in the perforant path-dentate granule cell system of the rat hippocampal formation

In vitro quantitative autoradiography with [3H]MK-801 was used to determine Kd and Bmax values for the NMDA receptor-coupled channel in subregions of the rat hippocampal formation. A single form of the channel with an apparent Kd in the 15-20 nM range was found for [3H]MK-801 binding in the presence of both 1 microM glutamate and 1 microM glycine. Specific binding was highest in the molecular layer of the dentate gyrus, followed by CA1 stratum radiatum and CA1 stratum oriens. Fewer binding sites were observed in the hilus of the dentate gyrus, cerebral cortex, CA1 stratum pyramidale, CA3 subregion (stratum oriens, stratum pyramidale, stratum radiatum), and thalamus. Selective destruction of dentate granule cells by colchicine microinjections reduced the amount of specific [3H]MK-801 binding by half in the molecular layer of the dentate, compared to intact tissue. [3H]MK-801 binding did not change in other hippocampal subregions as a consequence of colchicine injection. Electrolytic entorhinal cortical lesions produced no changes in regional MK-801 binding site density in any of the regions under study. To address the tissue shrinkage following entorhinal cortex lesions, detailed analysis of the binding site density per fixed (16 microns) length of granule cell dendrite, and of the aggregate density across the entire molecular layer revealed no change in the number of MK-801 binding sites per unit length of dendrite in the molecular layer of the dentate gyrus. These findings indicate that NMDA receptor-coupled channels are confined to a postsynaptic location in the perforant path-dentate granule cell system of the adult rat.     

Failure to detect changes in AMPA receptor binding after long-term potentiation

Recent experimental evidence suggests that changes in(RS)--amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/quisqualate receptor mediated currents account for the increase in synaptic responses known as long-term potentiation (LTP). These changes could occur in several parameters including receptor density, binding affinity or in the kinetics and conductance properties of the receptor channel. In this study we tested if LTP modifies the affinity or overall number of binding sites. Hippocampal slices were stimulated electrically in at least 12 locations in the stratum radiatum of CA1 in order to induce potentiation in a maximal number of Schaffer-collateral axons. Sections were then prepared from the middle of the slices, incubated with [³H]AMPA or [³H]CNQX (6-cyano-7-nitroquinoxaline-2,3-dione) and processed for autoradiography. To correct for variability among sections, binding in CA1 was expressed relative to that in the molecular layer of the dentate gyrus, which did not receive potentiation. No differences were observed in the absolute or relative amount of binding except for a small decrease of about 5% in [³H]AMPA binding that was not statistically significant. Although the percentage of synapses which were potentiated by our stimulation procedure cannot be determined with certainty, it is unlikely that an increase in the number or affinity of AMPA receptors could explain the 50% LTP effect typically observed in slices.     

Localization and quantification of the dopamine transporter: comparison of [H]WIN 35,428 and [I]RTI-55

Transport into the presynaptic terminal by the dopamine transporter is the primary mechanism for removing dopamine from the synaptic cleft. This transporter is a specific marker for dopamine terminals and is a primary site for CNS actions of cocaine. Several radioligands have been developed for analysis of the dopamine transporter. The ligands vary in affinity and specificity, leading to differences in reported transporter density in brain regions. We compared two of the most commonly used ligands, [³H]WIN 35,428 and [¹²⁵I]RTI-55, analyzing the localization and density of sites in the rat brain using serial sections and quantitative autoradiography. Citalopram at 50 nmol/1 was used to block [¹²⁵I]RTI-55 binding to serotonin transport sites. Transporter density was highest in the striatum and both ligands labeled equivalent numbers of sites, with lateral to medial and anterior to posterior gradients. In most areas the density of sites measured with the two ligands was similar. However, [¹²⁵I]RTI-55 binding was significantly higher than [³H]WIN 35,428 binding in the substantia nigra zona compacta, ventral tegmental area, subthalamic nucleus and a number of other subcortical nuclear groups while [³H]WIN 35,428 binding was higher in lateral striatum and in olfactory tubercle. These differences could reflect different forms of the transporter, perhaps due to post-translational modifications, and they may provide a basis for differential pharmacological regulation of transporter function in discrete brain regions and disease states.     

Regional distribution of [H]imipramine binding in rat brain

[³H]imipramine binding was measured in 23 microdissected areas of the rat brain and compared to published values for the endogenous levels of serotonin, noradrenaline and dopamine in the same areas.

The density of [³H]imipramine binding sites appears to be highly correlated with the distribution of endogenous serotonin especially where the serotonin is located mainly in nerve terminals. A weak but still significant correlation also exists with the distribution of endogenous noradrenaline whereas no such correlation could be detected for endogenous dopamine.     

Effects of subacute lead exposure on [H]MK-801 binding in hippocampus and cerebral cortex in the adult rat

We used the NMDA receptor non-competitive antagonist, [³H]MK-801, as a ligand for an autoradiographic study to determine the effects of lead on NMDA receptor in the rat brain. Adult male rats were administered lead acetate, 100 mg/kg, or sodium acetate, 36 mg/kg (control), by i.p. for 7 days. High lead levels were detected in blood (41.1 μg/dl) and in brain (16.7–29.4 μg/g). Concentrations of lead in brain regions were not significantly different. The [³H]MK-801 binding was heterogeneously distributed throughout the rat brain with the following order of binding densities: hippocampal formation>cortex>caudate-putamen>thalamus>brainstem. Lead exposure produced a significant decrease in [³H]MK-801 binding to the NMDA receptor in the hippocampal formation including CA2 stratum radiatum, CA3 stratum radiatum, hilus dentate gyrus and presubiculum, and in the cerebral cortex including agranular insular, cingulate, entorhinal, orbital, parietal and perirhinal areas. The hippocampal formation is known as a critical neural structure for learning and memory processes, whereas, cortical and subcortical regions have been demonstrated to be involved in the modulation of complex behavioral processes. The NMDA receptor has been demonstrated to play a key role in synaptic plasticity underlying learning and memory. Lead-induced alterations of NMDA receptors in the hippocampal formation and cortical areas may play a role in lead-induced neurotoxicity.     

Changes of GABA(A) receptor binding and subunit mRNA level in rat brain by infusion of subtoxic dose of MK-801

In the present study, we have investigated the effects of prolonged inhibition of NMDA receptor by infusion of subtoxic dose of MK-801 to examine the modulation of GABA_A receptor binding and GABA_A receptor subunit mRNA level in rat brain. It has been reported that NMDA-selective glutamate receptor stimulation alters GABA_A receptor pharmacology in cerebellar granule neurons in vitro by altering the levels of selective subunit. However, we have investigated the effect of NMDA antagonist, MK-801, on GABA_A receptor binding characteristics in discrete brain regions by using autoradiographic and in situ hybridization techniques. The GABA_A receptor bindings were analyzed by quantitative autoradiography using [³H]muscimol, [³H]flunitrazepam, and [³⁵S]TBPS in rat brain slices. Rats were infused with MK-801 (1 pmol/10 μl per h, i.c.v.) for 7 days, through pre-implanted cannula by osmotic minipumps (Alzet, model 2ML). The levels of [³H]muscimol binding were highly elevated in almost all of brain regions including cortex, caudate putamen, thalamus, hippocampus, and cerebellum. However, the [³H]flunitrazepam binding and [³⁵S]TBPS binding were increased only in specific regions; the former level was increased in parts of the cortex, thalamus, and hippocampus, while the latter binding sites were only slightly elevated in parts of thalamus. The levels of β2-subunit were elevated in the frontal cortex, thalamus, hippocampus, brainstem, and cerebellar granule layers while the levels of β3-subunit were significantly decreased in the cortex, hippocampus, and cerebellar granule layers in MK-801-infused rats. The levels of 6- and δ-subunits, which are highly localized in the cerebellum, were increased in the cerebellar granule layer after MK-801 treatment. These results show that the prolonged suppression of NMDA receptor function by MK-801-infusion strongly elevates [³H]muscimol binding throughout the brain, increases regional [³H]flunitrazepam and [³⁵S]TBPS binding, and alters GABA_A receptor subunit mRNA levels in different directions. The chronic MK-801 treatment has differential effect on various GABA_A receptor subunits, which suggests involvement of differential regulatory mechanisms in interaction of NMDA receptor with the GABA receptors.     

Hippocampal and cortical opioid receptor binding: Changes related to the hibernation state

In vitro opioid receptor binding in the dorsal hippocampal formation and parietal cortex was surveyed in ground squirrels (Citellus lateralis) in the contrasting physiological states of hibernation and euthermia (i.e. not hibernating). Computer-assisted autoradiographic analysis of coronal sections incubated with [³H]dihydromorphine (DMH; 4 nM) revealed statistically significant reductions in specific opioid binding associated with hibernation. In the dorsal hippocampal formation of hibernating animals, binding in the striatum radiatum of CA3, hilus of the dentate gyrus and molecular layer of the dentate gyrus exhibited decreases up to 34% compared to euthermic animals. The stratum radiatum of CA3 exhibited the smallest decrease overall. DHM binding in parietal cortex displayed significant hibernation-related reductions, although they were not uniformly observed across all lamine at the 3 different brain levels examined. These experiments present evidence of changes in brain opioid binding related to the mammalian state of hibernation. The results suggest that changes in opioid receptor binding during hibernation may contribute to the earlier reported⁵ apparent failure of morphine physical dependence to develop during hibernation.     

Decreased [H]hemicholinium binding to high-affinity choline uptake sites in aged rat brain

The binding of [³H]hemicholinium ([³H]HCh-3) to sodium-dependent high-affinity choline uptake sites provides a useful neuroanatomical and functional marker of the cholinergic system. We examined the autoradiographic distribution of [³H]HCh-3 binding sites in the forebrain of young (4–6 months) and old (32 months) rats. There was a widespread reduction of [³H]HCh-3 binding site density in the aged rat brain. This loss presented regional differences with maximal reduction in the medial and posterior striatum (55%) and in the dentate gyrus (47%), in limbic areas such as basolateral amygdala, tubercle olfactorium and piriform cortex the autoradiographic signal was about 25–30% lower. In aged hippocampus and cerebral cortex the density of [³H]HCh-3 binding sites was about 40% lower, the difference between young and senescent animals being less evident in the medial septum and basal nucleus. No significant alterations were observed in interpeduncular nucleus from old rats. These data are in agreement with the functional results obtained by measuring other cholinergic parameters in the aged rat and confirm the vulnerability of cholinergic system during aging     

Regional [H]acetylcholine and [H]nicotine binding in developing mouse brain

The postnatal development of nicotine-like binding sites in the cortex, hippocampus, midbrain and cerebellum of 3-, 7-, 12-, 17- and 30-day-old mice was studied. Two different nicotinic cholinergic ligands, namely [³H]acetylcholine ([³H]ACh) and [³H]nicotine ([³H]NIC) were used to detect the nicotine-like binding sites in in vitro binding assays. The postnatal development of the binding sites of [³H]NIC increased gradually with age in all brain regions studied. The [³H]ACh binding, on the other hand, showed a marked peak on day 12 in the cerebellum and midbrain but did not change notably with age in the hippocampus and cortex, except for a slight temporary increase in the cortex on day 7. The time-course for the appearance of nicotinic binding sites as observed with [³H]ACh was found to be rather similar to that earlier described for [³H]alpha-bungarotoxin binding sites, whereas that for [³H]NIC differed from that described for other nicotinic ligands.     

Quantitative autoradiography of the dopamine uptake complex in rat brain using [H]GBR 12935: binding characteristics

The dopamine uptake complex was examined in the rat central nervous system using [³H]GBR 12935 and in vitro quantitative autoradiography to determine all binding data. [³H]GBR 12935 labels two unique binding sites, the dopamine uptake complex and a piperazine acceptor site. These two sites differ in their pharmacologic properties, anatomical distributions, densities, and response to lesions. Using appropriate binding conditions, [³H]GBR 12935 can be used to specifically label the dopamine uptake complex. [³H]GBR 12935 labeled a single binding site with characteristics of the dopamine uptake complex when mazindol (25 μM) was used as a blank. The specific binding and autoradiographic appearance of [³H]GBR 12935 to the dopamine uptake complex was improved by including trans-flupentixol (0.75 μM) to displace binding to a previously desrribed piperazine acceptor site, recently determined to be a site on cytochrome P450IID1. Binding was saturable and reversible to the dopamine uptake complex. The equilibrium dissociation constant (1.4 ± 0.7nM), maximal number of binding sites (6.0 ± 1.3pmol/mg protein), and Hill coefficient (1.1 ± 0.1) of [³H]GBR 12935 in rat striatum using mazindol to define non-specific binding was not significantly altered by the inclusion of trans-flupentixol (0.75 μM). Using GBR 12909 as a blank produced a greater maximal number of binding sites (8.4 ± 2.3pmol/mg protein), but no significant difference in the equilibrium dissociation constant (1.6 ± 0.3nM) or Hill coefficient (1.1 ± 0.1). A series of drugs that bind to the dopamine uptake complex displaced [³H]GBR 12935 in a rank order consistent with other binding and behavioral studies of this complex. The rank order of these drugs was GBR 12909 > mazindol > nomifensine > benztropine > desipramine > amphetamine > dopamine; all these drugs displayed a Hill coefficient near one and were best modeled as a single site. Cocaine and WIN 35, 428 (a cocaine congener) were unique in their competition for [³H]GBR 12935 binding, displaying biphasic curves, low Hill coefficients, and were best modeled as two site fits. Lesioning of the dopaminergic median forebrain bundle resulted in a dramatic loss of the dopamine uptake complex in the striatum, nucleus accumbens, olfactory tubercle, and substantia nigra. Other dopaminergic projection areas were decreased to a lesser extent. Striatal ibotenate lesions did not decrease the density of the dopamine uptake complex, despite a large decrease in the dopamine D₁ receptor. [³H]GBR 12935 can be used as an effective ligand to label the dopamine uptake complex for quantitative autoradiographic studies. It offers a number of advantages over previous autoradiographic assays for this complex including high specificity (> 95% specific binding in rat striatum), high sensitivity (detection of mazindol displaceable sites in the cerebral cortex), low background (comparable to film background), and low cost. This assay also supports the existence of two binding sites for cocaine on the dopamine uptake complex. The exact nature and differences between these two cocaine sites remains to be determined.     

Alterations in [3H]kainate and N-methyl-D-aspartate-sensitive L-[3H]-glutamate binding in the rat hippocampal formation following fimbria-fornix lesions.

Following lesions of the fimbria-fornix, there is a time-dependent increase in interictal spikes and seizure susceptibility. This may result from sprouting of local excitatory and inhibitory circuits in response to the loss of subcortical and commissural innervation of the hippocampal formation. We used receptor autoradiography to examine the density of N-methyl-D-aspartate (NMDA)-sensitive L-[3H]glutamate and [3H]-kainate (KA) binding sites in the hippocampal formation at 5 days, 3 months, and 1 year following bilateral aspiration lesions of the fimbria-fornix. At 5 days post-lesion, the CA3 and CA1 strata radiatum and oriens displayed a decrease (20-42%, P less than 0.01) in NMDA-sensitive L-[3H]glutamate binding. The initial decrease was followed by a moderate recovery at later time points but was still evident at 1 year postlesion. This may reflect a lesion-induced turnover of synaptic complexes, down-regulation of postsynaptic receptors, or loss of presynaptic receptors. Five days following fimbria-fornix lesion there was also a decrease (13-15%, P less than 0.05) in [3H]KA binding in CA3 strata radiatum and pyramidale. However, at 3 months postlesion KA receptor density was elevated by 29-33% (P less than 0.01) in the outer molecular layer of the dentate gyrus with no significant change in binding to the inner molecular layer. By 1 year postlesion, the density of [3H]KA binding sites was not significantly different from that observed in control animals of the same age. The increase in KA receptor density in the outer molecular layer 3 months after fimbria-fornix lesion may reflect sprouting of the perforant path input or mossy fibers to this region and contribute to the increase in interictal spikes and seizures susceptibility.     

Localization in rat brain of binding sites for parkinsonian toxin MPTP: similarities with [H]parygyline binding to monoamine oxidase

A high-affinity binding site exists in rat brain for the parkinsonian toxin 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP). The pharmacological specificity of this binding site suggests that it may coprrespond to monoamine oxidase (MAO). We have used quantitative autoradiography to map in detail the anatomical distribution of the [³H]MPTP binding site in rat brain and compared it with the anatomical distribution of MAO as determined by in vitro autoradiography with [³H]paragyline. Under the conditions of the assay, [³H]pargyline labeled the type B form of MAO. There were strong similarities in the anatomical distribution of [³H]MPTP and [³H]pargyline, with high levels of both binding sites occuring in the arcuate nucleus, the locus coeruleus, the dorsal raphe nucleus and all circumventricular organs. Low levels of both binding sites were found in the substantia nigra and the caudate-putamen. These results provide additional evidence that the high-affinity binding site for MPTP is MAO. The parkinsonian actions of MPTP might result from metabolites produced by MAO.     

Striatal dopaminergic terminals in type 1 and type 2 alcoholics measured with [H]dihydrotetrabenazine and human whole hemisphere autoradiography

A number of studies have pointed to the importance of dopamine system in the context of alcoholism. Previous studies have shown lower dopamine transporter levels on late-onset Cloninger type 1 alcoholics. However, whether this lower level is due to a lower level of dopamine transporter protein or a lower level of dopaminergic nerve terminals remains unclear. The aim of this study was to compare putative alterations of dopaminergic terminals in caudate, putamen and nucleus accumbens of type 1 and type 2 alcoholics and healthy controls by using [³H]dihydrotetrabenazine as a radioligand in postmortem human whole hemisphere autoradiography. We compared the present results with the findings of our earlier studies on the dopamine transporter in these same subjects, demonstrating that alcoholics do not differ significantly from controls in striatal [³H]dihydrotetrabenazine binding. Although type 1 alcoholics have been reported to have up to 36% lower striatal dopamine transporter levels than controls, the results suggest that the density of their dopaminergic nerve terminals is not altered.     

Brain slices in radioligand binding assays: Quantification of opiate, benzodiazepines and beta-adrenergic ([H] CGP-12177) receptors

1. We have characterized and quantified specific binding of [³H]-flunitrazepam (FNZ: (benzodiazepine), [³H]-naloxone (NAL: (opiate) and [³h]cgp-12177(CGP: (beta-adrenergic) to thick slices (230–400 μm) of mouse and rat brain.

2. The binding sites are stereospecific, saturable and of high affinity. In all cases, the binding of the ligands is readily reversible and demonstrates the appropriate drug specificity.

3. In mouse brain [³H]-NAL binding is elevated by chronic treatment with naloxone (via capsules).

4. We have been unsuccessful in quantifying beta adrenoreceptors with the archetypal ligand [³H]-dihydroalprenolol (DHA). However, the use of [³H]-CGP 12177 enabled us to detect high-affinity beta adrenoreceptors in brain slices.

5. [³H]-CGP also permits the demonstration of rapid and reversible agonist-induced down-regulation (internalization) of beta binding sites.

6. We have been successful in quantifying beta adrenergic sites in single pineal glands of rat and hamster.     

Cobalt ion enhancement of 2-chloro[H]adenosine binding to a novel class of adenosine receptors in brain: antagonism by calcium

We have recently reported the identification of a novel class of micromolar-affinity adenosine binding sites in rat brain membranes using the adenosine agonist 2-chloro[³H]adenosine (Cl[³H]Ado). These binding sites are distinguishable from the A1 and A2 adenosine receptors by a number of pharmacological criteria, and we have designated this new class of binding sites as the A3 adenosine binding sites. In the present study, the effects of a wide range of divalent and trivalent cations on micromolar Cl[³H]Ado binding to brain membranes were examined. Co²⁺, Ni²⁺ and La³⁺ markedly stimulated specific Cl[³H]Ado binding by 45–150% above control when tested at concentrations of 1–10 mM. Ca²⁺ had no significant effect on binding except at high concentrations where it depressed binding slightly. Ca²⁺, however, completely prevented the stimulation of Cl[³H]Ado binding by Co²⁺. These findings further distinguish the A3 class of adenosine binding sites from the previously characterized adenosine receptors and suggest that the A3 binding sites are associated with calcium systems in brain.