Adenosine Binding Sites Research Articles

Characterization of Adenosine A 2 Receptors in Bovine Retinal Pigment Epithelial Membranes

The pharmacological characteristics of adenosine A 2 receptors are described for membranes prepared from bovine retinal pigmented epithelial (RPE) cells. RPE cells were isolated after removal of retina, lysed by freeze-thawing, and membranes separated from cytoplasmic components. A single population of adenosine binding sites is present in RPE membranes, as determined from saturation analysis and competition binding assays. From Scatchard plots, this single class of binding sites exhibited low affinity for adenosine receptor agonists. These low affinity sites were labeled by [ 3H]-N-ethylcarboxamidoadenosine (NECA) or [ 3H]-CGS 21680 and K ds of 423 and 5·3 μM were determined for each radioligand, respectively. NECA-mediated stimulation of adenylate cyclase demonstrated that these binding sites represent adenosine receptors. No high affinity A 2a binding sites were detected in RPE membranes by either saturation studies, or by competition with adenosine A 1-selective agonists which only displaced radioligand binding at high micromolecular concentrations. The low affinity A 2 receptor on RPE differs from the high affinity A 2a receptor characterized in bovine retinal membranes, but may be similar or identical to the lower affinity A 2b receptor detected in retinal membranes as well as other tissues.

Failure to stimulate melatonin synthesis in the rat pineal gland with an adenosine analog

Recent studies indicate the presence of adenosine binding sites in rat pinealocytes and an effect of their activation on pineal serotonin metabolism. However, controversial data exist, and reports suggest that the role of adenosine in pineal physiology is complex. On this basis, we evaluated the effects of an adenosine analog (N-ethyl-carboxamido-adenosine: NECA) on in vitro and in vivo melatonin production in the rat pineal gland. In the in vitro protocol, pineals were incubated with NECA (0.5 mM, 1 mM, or 2.5 mM) or isoproterenol (ISO: 10 −6 M) for 4 hr. In the in vivo experiments, animals were given NECA (1 mg/kg IP), ISO (0.5 mg/kg IP) or 1 ml saline diluent and sacrificed 2 hr later. The samples were assayed for pineal N-acetyltransferase (NAT) activity and melatonin concentrations. ISO caused the expected marked rise in NAT activity and melatonin levels in both protocols. NECA was ineffective in causing any modification of the parameters measured. We conclude that the adenosine analog NECA may not be involved in the activation of melatonin production. These data contrast with others in which NECA administration resulted in an increase in an increase in melatonin levels. The participation, if any, of the purinergic system in the physiology of the pineal gland is still far from being characterized.

Characterization of human striatal A2-adenosine receptors using radioligand binding and photoaffinity labeling.

The adenosine agonist [3H]CGS21680 (2-[4-[[2-carboxyethyl]phenyl]ethylamino]-5'- N-ethylcarboxamidoadenosine) bound to A2 receptors in human striatal membranes with a Kd of 17.8 +/- 1.1 nM and a Bmax of 313 +/- 10 fmol/mg protein. The addition of 100 microM GTP diminished both the affinity of agonist radioligand for A2 adenosine binding sites and the total binding, resulting in Kd and Bmax values of 28.6 +/- 1.0 nM and 185 +/- 22 fmol/mg of protein. Adenosine ligands competed for [3H]CGS21680 with the expected potency order. The adenosine antagonist [3H]XAC (8-[4-[[[[(2-aminoethyl)-amino]carbonyl]methyl] oxy]phenyl]-1,3-dipropylxanthine), although A1-selective in the rat, binds to human striatal A2 receptors with high affinity. 25 nM CPX (8-cyclopentyl-1,3-dipropylxanthine), an A1-selective antagonist, was added to the incubation medium and effectively eliminated 91% of [3H]XAC (1 nM) binding to human A1 receptors, yet preserved 90% of binding to A2 receptors. [3H]XAC exhibited saturable, specific binding (50% of total) to A2 sites with a Kd of 2.98 +/- 0.54 nM and a Bmax of 0.71 +/- 0.23 pmol/mg protein (25 degrees C, non-specific binding defined with 100 microM NECA). The potency order for antagonists against 1 nM [3H]XAC was CGS15943A greater than XAC approximately PD115,119 greater than PAPA-XAC greater than CPX greater than HTQZ approximately XCC approximately CP-66,713 greater than theophylline approximately caffeine, indicative of an A2-type binding site. A2a-receptors were found to be present in the human cortex, albeit at a much lower density than in the striatum. Photoaffinity labeling using 125I-PAPA-APEC revealed a molecular weight of 45K, but proteolytic cleavage was observed, resulting in fragments of MW 43K and 37K. In the absence of proteolytic inhibitors the 37K fragment, which still bound 125I-PAPA-APEC, was predominant.

Carbamazepine distinguishes between adenosine receptors that mediate different second messenger responses

The mechanism of the therapeutic and prophylactic effects of carbamazepine (CBZ) in affective psychoses is unknown but may in part be related to the potent competitive interaction of CBZ with adenosine-binding sites in the brain. The anticonvulsant and sedative properties of CBZ are reminiscent of the effects evoked by adenosine-agonists and contrast sharply with the opposite actions of adenosine-antagonists like caffeine. However, indirect evidence suggests an antagonist- rather than an agonist-like activity of CBZ at adenosine-receptors. We have used various model systems, in which adenosine receptor subtypes mediate different second messenger-responses, to investigate this apparent paradox. CBZ was found to antagonize the A 1-receptor-mediated inhibition of cyclic AMP accumulation in cultured astroblasts and in GH 3-cells. Furthermore, CBZ also inhibits the adenosine-induced increase in the level of cyclic AMP in cultured astroblasts, which is mediated by low-affinity A 2b-receptors. In contrasts, CBZ does not block the inhibition elicited by adenosine-agonists of the agonist-induced increased formation of inositolphosphates in human neutrophils, which is mediated by high-affinity A 2a-receptors. The specific antagonism by CBZ of A 1- but not of high-affinity A 2a-receptors was further supported by binding experiments using rat brain membranes. These results suggest that the paradox of CBZ's antagonistic effects at adenosine-receptors might be at least partially reconciled by a selective antagonistic action of CBZ at A 1 receptors but not at high-affinity A 2a-receptors.

Assignment of the nucleotide binding sites and the mechanism of substrate inhibition of Escherichia coli adenylate kinase

Site-directed mutagenesis of key amino acids of adenylate kinase has been used to suggest a new model for the location of the AMP and ATP binding sites. Phe-86 and Tyr-133, which are in close contact with the inhibitor Ap5A according to previous crystallographic results, have been independently changed to tryptophan and other amino acids. The Phe-86----Trp mutant had a 3- to 6-fold change in the Km for ATP and a 44-fold increase in the Km for AMP with a simultaneous loss of AMP substrate inhibition. Thus Phe-86 is probably in close contact with bound AMP. The Tyr-133----Trp mutant showed no large effects on enzyme kinetics and suggests that the previous assignment of Ap5A occupying natural adenosine binding sites is probably incorrect. A temperature-sensitive Leu-107----Gln mutant showed a 6-fold decrease in the Km for ATP and no effect on AMP binding, suggesting that this amino acid is near the ATP binding site. Changes in the fluorescence of single tryptophan-containing mutant enzymes provided specific information about AMP and ATP binding. The fluorescence results are consistent with the kinetic studies, and also suggest that AMP substrate inhibition is caused by the formation of an abortive complex that prevents the release of product.

Chronic carbamazepine down-regulates adenosine A2 receptors: studies with the putative selective adenosine antagonists PD115,199 and PD116,948

Carbamazepine (CBZ), an anticonvulsant with psychotropic and anti-pain properties, has been reported to displace ligands at adenosine binding sites. This paper describes biochemical and behavioural studies in rodents comparing CBZ to the adenosine agonists L-phenylisopropyl-adenosine (L-PIA) and N-ethylcarboxamido-adenosine (NECA), the new antagonists PD116,948 and PD115,199 which are also relatively A1 and A2 specific respectively, and the mixed antagonists theophylline and caffeine, attempting to determine functional correlates of the binding studies. Changes in cAMP synthesis and behavioural syndromes produced by the drugs, alone and in combination, were monitored. Classification of the observed effects in terms of A1 and A2 activity was complex, probably due to functional interactions between A1 and A2 subtypes. Nevertheless, it was found that chronic CBZ administration (0.25% in food for 3 days, followed by 0.5% for 11 days) produced a pattern of interaction identical to that of PD115,199 (10-100 mg/kg IP). Thus, both treatments attenuated the behavioural syndrome produced by L-PIA (0.1 or 0.5 mg/kg SC), but did not affect that produced by NECA (0.03 mg/kg SC). CBZ mildly increased hypoactivity after clonidine (0.2 mg/kg IP) which was used as a control. By contrast, the A1 antagonist PD116,948 (0.1-10 mg/kg IP) antagonised both behavioural syndromes. Similarly in the biochemical experiments both chronic CBZ and PD115,199 (10-100 microM) reduced stimulation of cAMP synthesis by L-PIA (confirming that this is mediated by A2 receptors), while only basal cAMP synthesis was affected by PD116,948 (10 microM) and theophylline (60 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

The ability of denbufylline to inhibit cyclic nucleotide phosphodiesterase and its affinity for adenosine receptors and the adenosine re‐uptake site

1. Denbufylline has been examined for its ability to inhibit cyclic nucleotide phosphodiesterase isoenzymes from rat cardiac ventricle and cerebrum, as well as for its affinity for adenosine A1 and A2 receptors and the re-uptake site. For comparison, SK&F 94120, theophylline and 3-isobutyl-1-methyl-xanthine (IBMX) were examined as phosphodiesterase inhibitors whilst N6-cyclohexyladenosine, R(-)-N6-(2-phenylisopropyl)-adenosine, 5'-N-ethylcarboxamido-adenosine, 2-nitrobenzylthioinosine, theophylline and IBMX were examined for their affinity for adenosine binding sites. 2. This investigation confirmed the presence of four phosphodiesterase activities in rat cardiac ventricle; in rat cerebrum only three were present. 3. Denbufylline selective inhibited one form of Ca2+-independent, low Km cyclic AMP phosphodiesterase. The form inhibited was one of two present in cardiac ventricle and the sole one in cerebrum. This form was not inhibited by cyclic GMP. The inotropic agent SK&F 94120 selectively inhibited the form of cyclic AMP phosphodiesterase which was inhibited by cyclic GMP present in cardiac ventricle. Theophylline and IBMX were relatively non-selective phosphodiesterase inhibitors. 4. Denbufylline was a less potent inhibitor of ligand binding to adenosine receptors than of cyclic AMP phosphodiesterase. This contrasted with theophylline, which had a higher affinity for adenosine receptors, and IBMX which showed no marked selectivity. Denbufylline, theophylline and IBMX all had a low affinity for the adenosine re-uptake site. 5. Denbufylline is being developed as an agent for the therapy of multi-infarct dementia. The selective inhibition of a particular low Km cyclic AMP phosphodiesterase may account for the activity of this compound.

5'-<italic>N</italic>-Ethylcarboxamide[<sup>3</sup>H] Adenosine Binding Sites of Mouse Mastocytoma P815 Cell Membranes: Characterization and Solubilization<xref ref-type="fn" rid="fn1"><sup>1</sup></xref>

Mouse mastocytoma P815 cell membranes were found to possess adenosine binding sites as assessed by using the adenosine agonist [3H]5'-N-ethylcarboxamideadenosine (NECA). The Kd and Bmax for the [3H]NECA binding at 0 degrees C were 380 nM and 17 pmol/mg of protein, respectively. The rank order of potency for inhibition of [3H]NECA binding was NECA greater than 5'-N-cyclopropylcarboxamideadenosine greater than 2-chloroadenosine greater than 2',5'-dideoxyadenosine greater than isobutylmethylxanthine greater than theophylline greater than N6-[(R)-1-methyl-2-phenylethyl]adenosine = N6-[(S)-1-methyl-2- phenylethyl]adenosine. Thermodynamic analyses of the adenosine receptor agonist and antagonist binding showed that all such ligands displayed negative values of both enthalpy and entropy which suggested that the driving force for the binding was enthalpic. [3H]NECA binding sites of P815 cell membranes were solubilized with sodium cholate and retaining the same ligand-binding characteristics as those of the membrane-bound form. By gel filtration on a Sepharose CL-6B column, the adenosine binding site was estimated to have a Stokes radius of approximately 6.7 nm.

5′-<italic>N</italic>-Ethylcarboxamido[<sup>3</sup>H] Adenosine Binding Sites of Mouse P815 Mastocytoma Cell Membranes: Solubilization and Partial Purification by Affinity Chromatography

A 5′-N-ethylcarboxamido[3H]adenosine ([3H]NECA) binding site of mouse mastocytoma P815 cell membranes has been purified ∼old by affinity chromatography. This adenosine binding site, which has a similar specificity to that of the A2 adenosine receptor, was adsorbed on NECA-linked Sepharose 6B and eluted with NECA. The adsorption of the [3H]NECA binding site to the affinity matrix was specifically blocked by NECA. The [3H]-NECA binding site bound on the affinity matrix was also specifically eluted by NECA. This affinity matrix adsorbed ∼90% of the digitonin-solubilized [3H]NECA binding activity applied, and after the gel was washed, 30–50% of the adsorbed binding activity could be eluted with 500 μM NECA with specific binding activity of 50–70 pmol/mg of protein. The affinity-purified [3H]NECA binding site retained the same ligand binding specificities as the original membrane preparation. The results indicate that the NECA-Sepharose 6B should provide a powerful tool for the eventual purification of [3H]NECA binding sites of P815 cell membranes.

Rat adipose tissue adenosine sensitivity and adenosine content after subcutaneous administration of N6-(phenylisopropyl)adenosine.

Injections of N6-(phenylisopropyl)adenosine, a nonmetabolizable adenosine A1 receptor agonist, desensitized rat adipocytes to the drug in 20 h. Lipolysis stimulated by 2 mumol/l DL-isoproterenol was inhibited markedly less by N6-(phenylisopropyl)adenosine in adipocytes isolated from treated than control animals (P less than 0.01). Glucose uptake was more responsive to N6-(phenylisopropyl)adenosine in adipocytes from control than treated animals (P less than 0.02). Adenosine content was the same in adipose tissue of control and treated animals. The number of adenosine binding sites was not significantly lower in treated compared with control animals (1580 +/- 279 and 1988 +/- 575 fmol/mg protein; mean +/- SEM). There was no change in receptor affinity (Kd = 10 nmol/l in both groups). There was no decrease in the amounts of the inhibitory guanine nucleotide binding protein (Gi) alpha subunits as studied by pertussis toxin catalyzed ADP-ribosylation. It is concluded that desensitization to N6-(phenylisopropyl)adenosine can be observed without changes in the adenosine receptor status or decrease in the amount of inhibitory guanine nucleotide binding protein and that adipose tissue adenosine content is not changed by the agonist treatment.

Adenosine binding sites of rat pheochromocytoma PC 12 cell membranes: partial characterization and solubilization.

Rat pheochromocytoma PC 12 cell membranes were shown to possess A2-like adenosine binding sites as assessed by using 5'-N-ethylcarboxamide[3H]adenosine [( 3H]NECA). Specific [3H]NECA binding to PC 12 cell membrane at 0 degrees C was saturable and showed a monophasic saturation profile. In contrast, [3H]NECA binding to PC 12 cell membrane at 30 degrees C exhibited a biphasic profile suggesting the presence of two specific binding site. The rank order of potency for inhibition of [3H]NECA binding at 0 degrees C was NECA greater than 2-chloroadenosine greater than 2',5'-dideoxyadenosine greater than isobutylmethylxanthine much greater than phenylisopropyladenosine. These adenosine binding sites were solubilized with sodium cholate and the solubilized portion retained the same ligand binding characteristics as those of the membrane-bound form. Gel filtration experiments indicated an apparent Stokes radius of 6.7 nm for these adenosine binding sites/detergent complexes.

Characterization of adenosine binding sites in bovine testicular tissue using 8-cyclopentyl-1,3-[ 3H]dipropylxanthine

The existence of specific adenosine binding sites in bovine testicular tissue was evaluated using the novel antagonist radioligand 8-cyclopentyl-1,3-[ 3H]dipropylxanthine ([ 3H]DPCPX). Saturation analysis revealed specific binding that was saturable at ∼ 1 nM. Scatchard analysis indicated a single class of binding sites with K D = 0.26 nM and a B max = 0.37 pmol/mg protein. Affinity profiles suggest an A 1 subtype recognition site that is different from the classical A 1 adenosine receptor. The results presented should prove useful in subsequent studies concerning heterogeneity among adenosine receptors and also aid in discerning the role of adenosine in reproduction.

Characterization of the human T lymphocyte adenosine receptor: comparison of normal and systemic lupus erythematosus cells

To determine whether a defect in the T cell response to adenosine exists at the level of the adenosine receptor in systemic lupus erythematosus (SLE) we measured the binding affinity and maximum binding of T cell membranes from both normal and SLE T cells by utilizing radiolabeled adenosine ligands. Normal T lymphocyte membranes possess a single class of [3H]5-N-ethylcarboxamide adenosine binding sites with a Kd of 0.61 microM, a Bmax of 23.5 pmol/mg protein, and a Hill coefficient of 0.98, which indicates the presence of noncooperative sites. In contrast, T cell membranes do not bind significant amounts of either [3H]cyclohexyladenosine or [3H]phenylisopropyladenosine. These data indicate that T lymphocyte membranes have only A2, and not A1, adenosine receptors. Similarly, T cells from both active and inactive SLE subjects also express only A2 receptors with a Kd of 0.93 microM, a Bmax of 20.4 pmol/mg protein, and a Hill coefficient of 0.85, which is consistent with the presence of noncooperative sites. There is no difference in the on-rate, affinity, or density of T cell A2 receptors from active SLE patients, inactive SLE patients, or healthy controls. We conclude that T lymphocytes from both healthy and SLE subjects express A2, but not A1, receptors. Thus, the inability of SLE T cells to respond to adenosine does not reflect a decreased density of A2 (stimulatory) receptors, diminished A2 receptor binding, or an increased affinity or number of A1 (inhibitory) adenosine receptors. These observations support the conclusion that the defect in the T cell cAMP-dependent pathway may occur at a point distal to the adenosine receptor.

Differential effects of adenosine analogs on amygdala, hippocampus, and caudate nucleus kindled seizures.

This study explored the anticonvulsant effects of adenosine analogs at the focus of seizures kindled from various brain structures. Chemitrodes were implanted in the amygdala (AM), hippocampus (HIPP), or caudate nucleus (CN) of Long-Evans rats and electrically stimulated once daily until fully generalized seizures appeared (i.e., kindled). Once kindled, various doses (0.001-0.5 microgram/0.5 microliter) of the adenosine analogs, L-phenylisopropyladenosine (L-PIA), N-ethylcarboximidoadenosine (NECA) or vehicle were injected into the seizure focus 5 min prior to electrical stimulation. The afterdischarge (AD) and behavioral seizure stages were measured. L-PIA had potent anticonvulsant effects when injected directly into the kindled seizure focus in the AM, HIPP, or CN. NECA effects were statistically significant only in CN-kindled seizures. The regional differences in efficacy of the two adenosine analogs suggest that L-PIA, an A1 adenosine subtype agonist, may exert its effects through A1 adenosine receptors in the AM, HIPP, and CN, where A1 binding has been demonstrated, whereas NECA, an A2 adenosine receptor agonist, may only be maximally effective in the CN where A2 adenosine binding sites are located.

Aminergic systems in Alzheimer's disease and Parkinson's disease.

Biochemical markers for serotoninergic and catecholaminergic neurons in frontal and temporal poles were examined post mortem in brains of patients with Alzheimer's disease, Parkinson's disease, and the two combined. Binding of [3H] citalopram to serotoninergic uptake sites and levels of serotonin were decreased by 40 to 50% in brains of persons in each disease category. In contrast, significant reductions of catecholaminergic markers were not detected. In all three disease groups, the choline acetyltransferase activity was reduced by 50 to 60%. Binding sites for adenosine (A1), muscarinic cholinergic, phencyclidine, beta-adrenergic, and calcium antagonist receptors were unchanged. We conclude that substantial damage to serotoninergic neurons occurs in persons with Parkinson's and Alzheimer's diseases.

Proconvulsant effects of theophylline on hippocampal afterdischarges

We tested the effects of the adenosine antagonist, theophylline, on electrically induced afterdischarges in the hippocampus of rats. Theophylline did not significantly alter the threshold for afterdischarge initiation or the duration of the initial afterdischarge. However, theophylline greatly prolonged the duration of secondary afterdischarges, and permitted generalization to motor seizures. Because theophylline antagonizes adenosine, and the hippocampus is a region rich in adenosine binding sites, we interpret these results to mean that endogenous adenosine has little action on focal seizure initiation, but a strong action to suppress secondary seizures and to inhibit seizure generalization.

Synaptic and extrasynaptic localization of adenosine binding sites in the rat hippocampus

In vitro binding sites for [ 125I]iodohydroxyphenylisopropyladenosine, an A1 adenosine agonist, were visualized in the CA1 area of the rat hippocampus by electron microscopical autoradiography. By fixing hippocampal slices after incubation in paraformaldehyde and osmium tetroxyde, the specifically bound radioactive ligands were preferentially retained and cross-linked to the tissue. Autoradiographic silver grains were localized by a statistical evaluation according to the ‘50% probability circle analysis’ and by measuring the distance of the grains from neighbouring membrane structures. A significant association of silver grains, indicating the presence of Al adenosine receptors, was found at synaptic complexes and in addition at extrasynaptic sites on dendritic membranes. This suggests that modulation of nerve cell activity by adenosine involves synaptic as well as non-synaptic mechanisms.

Adenosine kinase from human erythrocytes: kinetic studies and characterization of adenosine binding sites.

The reaction catalyzed by adenosine kinase purified from human erythrocytes proceeds via a classical ordered sequential mechanism in which adenosine is the first substrate to bind to and AMP is the last product to dissociate from the enzyme. However, the interpretation of the steady-state kinetic data is complicated by the finding that while AMP acts as a classical product inhibitor at concentrations greater than 5 mM, at lower concentrations AMP can act as an apparent activator of the enzyme under certain conditions. This apparent activation by AMP is proposed to be due to AMP allowing the enzyme mechanism to proceed via an alternative reaction pathway that avoids substrate inhibition by adenosine. Quantitative studies of the protection of the enzyme afforded by adenosine against both spontaneous and 5,5'-dithiobis(2-nitrobenzoic acid)-mediated oxidation of thiol groups yielded "protection" constants (equivalent to enzyme-adenosine dissociation constant) of 12.8 microM and 12.6 microM, respectively, values that are more than an order of magnitude greater than the dissociation constant (Kia = 0.53 microM) for the "catalytic" enzyme-adenosine complex. These results suggest that adenosine kinase has at least two adenosine binding sites, one at the catalytic center and another quite distinct site at which binding of adenosine protects the reactive thiol group(s). This "protection" site appears to be separate from the nucleoside triphosphate binding site, and it also appears to be the site that is responsible for the substrate inhibition caused by adenosine.

Muscarinic, benzodiazepine, gaba, chloride channel and other binding sites in frontal cortex in hepatic coma in man

1. 1. Alterations in several neurotransmitter systems in brain have been implicated in the pathophysiology of hepatic coma (HC). Studies on human autopsy material are few. We investigated H-quinuclidlnylbenzllate (QNB), 3H-spiperone, 3H-imipramine, 3H-PN-200-110, 3naloxone, 3H-Flunitrazepam, 3H-muscimol, 35S-t-butylbicyclophosphothionate and 3H-cyclohexyladenosine binding sites In frontal cortex from seven patients with HC and five controls. 2. 2. The density of 3H-QNB binding sites was significantly decreased and the affinity slightly increased in HC. The functional significance of these selective changes in muscarinic receptor binding sites Is unclear. Further studies evaluating cholinergic function In HC are Indicated. 3. 3. Acute studies in animals point to an Increase In GABA and BZ binding sites in HC. The present results show that the BZ/GABA-receptor-chloride-Ionophore complex is unchanged in HC in man. 4. 4. Serotonergic (5HT-2), adenosine (A-l), imipramlne (5HT uptake sites), opiate (naloxone) and calcium channel antagonist binding sites are unchanged in HC.

Characterization, distribution, and ontogenesis of adenosine binding sites in cat visual cortex

In vitro autoradiographic techniques were used to characterize binding sites for 3H-cyclohexyladenosine (CHA) and 3H-5'-N-ethylcarboxamidoadenosine (NECA) in cat and kitten visual cortex. 3H-CHA binding sites in adult cat have a Bmax of 1,363 fmol/mg protein and a Kd of 6.8 nM. Displacement experiments indicate that 3H-CHA binds to an adenosine receptor similar to the A1-adenosine receptor described by other investigators. 3H-NECA binding sites in adult cat have a Bmax of 518 fmol/mg protein and a Kd of 15.4 nM. Displacement experiments do not allow us to identify this binding site unambiguously. Bmax values increase during postnatal development for both binding sites, peaking in adulthood for 3H-CHA and at 30 d for 3H-NECA. Kd values show neither consistent nor significant differences during postnatal development for either binding site. 3H-CHA and 3H-NECA binding sites are concentrated in layers 1-3 and upper layer 5 in the visual cortex of adult cats. These laminar patterns, however, change during postnatal development, showing the densest binding in the deep cortical layers (5 and 6) in kittens younger than 30 d of age and a fairly homogeneous binding in older kittens before achieving the adult distribution.