Polyamine and Redox Modulation of [3H]MK‐801 Binding to N‐Methyl‐d‐aspartate Receptors in the Spinal Cord and Cerebral Cortex

Abstract: The pharmacology of N‐methyl‐d ‐aspartate (NMDA) receptors shows regional differences in affinity for various agonists and antagonists. We have investigated the modulatory mechanisms acting via the polyamine, redox and proton sites in the cerebral cortex and the spinal cord of adult, male rats using [³H]MK‐801 binding. The affinity for glycine‐independent spermine stimulation was one magnitude higher in cerebrocortical than in spinal cord membranes while the affinity for the spermine antagonist arcaine was similar. Spermine abolished the inhibiting effect of low pH in both regions. Thus, the difference in the polyamine site between the two regions seems to be restricted to agonist binding. The proportion of high affinity/total ifenprodil binding was ≈35% both in the spinal cord and the cerebral cortex, suggesting similar relative amounts of the NMDA receptor subunit 2B. The affinity of ifenprodil to the high affinity site was however significantly higher in the cerebral cortex. Redox modulatory agents had similar effects in the two regions but spermine fully counteracted the inhibiting effect of 0.2 mM 5,5′‐dithio‐bis(2‐nitrobenzoic acid) (DTNB) in the cerebral cortex while there was only a partial effect in the spinal cord. These data show that the regional pharmacological heterogeneity involves several of the mechanisms regulating the function of the NMDA receptor. The data also indicate that the NMDA receptor subunit 2B is much more common in spinal cord than previously suggested.     

Polyamine and redox modulation of [3H]MK-801 binding to N-methyl-D-aspartate receptors in the spinal cord and cerebral cortex.

The pharmacology of N-methyl-D-aspartate (NMDA) receptors shows regional differences in affinity for various agonists and antagonists. We have investigated the modulatory mechanisms acting via the polyamine, redox and proton sites in the cerebral cortex and the spinal cord of adult, male rats using [3H]MK-801 binding. The affinity for glycine-independent spermine stimulation was one magnitude higher in cerebrocortical than in spinal cord membranes while the affinity for the spermine antagonist arcaine was similar. Spermine abolished the inhibiting effect of low pH in both regions. Thus, the difference in the polyamine site between the two regions seems to be restricted to agonist binding. The proportion of high affinity/total ifenprodil binding was approximately 35% both in the spinal cord and the cerebral cortex, suggesting similar relative amounts of the NMDA receptor subunit 2B. The affinity of ifenprodil to the high affinity site was however significantly higher in the cerebral cortex. Redox modulatory agents had similar effects in the two regions but spermine fully counteracted the inhibiting effect of 0.2 mM 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) in the cerebral cortex while there was only a partial effect in the spinal cord. These data show that the regional pharmacological heterogeneity involves several of the mechanisms regulating the function of the NMDA receptor. The data also indicate that the NMDA receptor subunit 2B is much more common in spinal cord than previously suggested.     

Phencyclidine analogues inhibit NMDA-stimulated [3H]GABA release from cultured cortex neurons

The inhibition of [³H]TCP binding to rat cortical membranes by substances of the PCP/σ-opiate type was compared with effects of the same compounds on N-methyl-D-aspartate (NMDA) responses in cultured mouse cortex neurons. A new technique of excitatory amino acid-induced [³H]GABA (γ-amino-butyric acid) release in a continuous superfusion model was used for the purpose. A similar rank order of potencies was found for the substances whether as inhibitors of [³H]TCP binding (MK 801 > PCP > ketamine-cyclazocine) or as antagonists of NMDA (20 μM)-stimulated [³H]GABA release in the cultured neurons (phencyclidine = MK 801 > ketamine > cyclazocine).     

The crude extract from the sea anemone, Bunodosoma caissarum elicits convulsions in mice: possible involvement of the glutamatergic system

The crude extract from the sea anemone, Bunodosoma caissarum caused dose-dependent convulsions by i.c.v. route in mice. The involvement of the glutamatergic system in the convulsions was investigated. MK-801 and ketamine, non-competitive NMDA receptor antagonists, prolonged the latencies for convulsion onset. AP-5, a competitive NMDA receptor antagonist, reduced the number of animals convulsing and also increased the latency for convulsion onset. 7-Chlorokynurenic acid, an antagonist of the glycine site on the NMDA receptor, reduced the incidence of convulsions. GMP, a nucleotide known to antagonize some NMDA actions, reduced the incidence and the severity of convulsions and prolonged the latency for their onset. Riluzole, a neuroprotective and anticonvulsant agent, blocked the appearance of convulsions. In vitro, the crude extract inhibited [³H]glutamate binding to cerebral cortical membranes and enhanced [³H]glutamate release from cortical synaptosomes. Heating the crude extract to 100 °C for 30 min or preincubating it with sphingomyelin, abolished its effect on glutamate release, but did not alter its ability to induce convulsions and to inhibit glutamate binding. However, the convulsant action was inhibited when the crude extract was submitted to trypsin treatment. Our data suggest that the convulsions elicited by the crude extract are not due to the presence of cytolysin and are not related to an increase in glutamate release, but seem to be dependent on the interaction between a peptide component of the extract and NMDA receptors.     

Blockade of 5-HT7 receptors reduces tactile allodynia in the rat

This study assessed the role of systemic and spinal 5-HT₇ receptors on rats submitted to spinal nerve injury. In addition, the 5-HT₇ receptors level in dorsal root ganglion and spinal cord was also determined. Tactile allodynia was induced by L5/L6 spinal nerve ligation. Systemic (0.01-10 mg/kg) or spinal (0.3-30 μg) administration of the selective 5-HT₇ receptor antagonist SB-269970 but not vehicle reduced in a dose-dependent manner established tactile allodynia. This effect was maintained for about 6 h. SB-269970 was more potent and effective by the spinal administration route than through systemic injection. Spinal nerve ligation reduced expression of 5-HT₇ receptors in the ipsilateral but not contralateral dorsal root ganglia. Moreover, 5-HT₇ receptor levels were lower in the ipsilateral dorsal spinal cord of neuropathic rats compared to naïve and sham rats. No changes in the receptor levels were observed in the contralateral dorsal spinal cord and in both regions of the ventral spinal cord. Data suggest that spinal 5-HT₇ receptors play a pronociceptive role in neuropathic rats. Results also indicate that spinal nerve injury leads to a reduced 5-HT₇ receptors level in pain processing-related areas which may result from its nociceptive role in this model. Data suggest that selective 5-HT₇ receptor antagonists may function as analgesics in nerve injury pain states.     

N-methyl-D-aspartate receptors,synaptic plasticity,and Alzheimer's disease

The NMDA (N-methyl-D -aspartate) excitatory amino acid receptor mediates a potent excitatory response in many regions of the CNS and plays a key role in excitotoxicity and synaptic plasticity. Results from radioligand-binding studies indicate that NMDA agonists and antagonists preferentially bind to differing populations of NMDA receptors. The distribution of NMDA receptors as labeled by the agonist L-[³H]glutamate differs from that obtained with the radiolabeled antagonist [³H]3-((±2-carboxypiperazine-4-yl)-propyl-1-phosphonic acid ([³H]CPP)). In addition, NMDA agonists and antagonists display complementary regional variations in their ability to displace L-[³H]glutamate binding. At least some of the differences between agonist and antagonist-binding properties can be accounted for by suggesting that glycine converts the NMDA receptor from an antagonist-preferring state into an agonist-preferring state. Since NMDA and other excitatory amino acid receptor functions are necessary for synaptic function, certain forms of synaptic plasticity, and learning, we have evaluated their status in Alzheimer's disease (AD). Using radioligand-binding techniques, we find that NMDA receptors are generally preserved in the course of AD pathology in the human hippocampus in spite of partial cell loss. Kainate receptors, on the other hand, display a radistribution within the hippocampus. This redistribution corresponds to an apparent synaptic reorganization owing to the loss of input from the entorhinal cortex. The response of both the NMDA and kainate receptor systems may be adaptive plastic responses that attempt to maintain hippocampal function that is compromised by AD. However, these mechanisms may also render the hippocampus more vulnerable to damage following excessive neuronal activation.     

Binding of the antiretroviral drug, d-aspartate-β-hydroxamate on the NMDA receptor

d-Aspartate-β-hydroxamate (d-A β H) exhibits antiretroviral properties in vitro and in vivo. It has glutamate agonist properties at the N-methyl-d-aspartate (NMDA) receptor in neuronal cell cultures. This study characterizes its binding properties to the NMDA receptor by measuring its stimulating effect on N-(1-(2-thienyl)[³H]cyclohexyl)piperidine ([³H]TCP) binding to the ionic channel in rat brain membranes. d-A β H stimulated [³H]TCP binding in a dose-dependent manner but to a lower extent than glutamate, suggesting only partial glutamate agonist properties. In the presence of antagonists of the different effector sites of the NMDA receptor the affinity of d-A β H was competitively decreased by CGS-19755 and 7-chlorokynurenate and unaffected by arcaine. Among several d-A β H analogues VHS.125 behaved as a full NMDA agonist, but l- or d-glutamate γ-monohydroxamate (d-GH or l-GH) were without effect. This study shows that d-A β H has potential neurotoxic effects due to its direct interaction with the NMDA receptor and that analogues such as d-GH or l-GH may rather be used in humans.     

The glutamate story

Glutamatergic synaptic transmission in the mammalian central nervous system was slowly established over a period of some 20 years, dating from the 1950s. Realisation that glutamate and like amino acids (collectively known as excitatory amino acids (EAA)) mediated their excitatory actions via multiple receptors preceded establishment of these receptors as synaptic transmitter receptors. EAA receptors were initially classified as N-methyl-D-aspartate (NMDA) and non-NMDA receptors, the latter subdivided into quisqualate (later AMPA) and kainate receptors after agonists that appeared to activate these receptors preferentially, and by their sensitivity to a range of differentially acting antagonists developed progressively during the 1970s. NMDA receptors were definitively shown to be synaptic receptors on spinal neurones by the sensitivity of certain excitatory pathways in the spinal cord to a range of specific NMDA receptor antagonists. Importantly, specific NMDA receptor antagonists appeared to be less effective at synapses in higher centres. In contrast, antagonists that also blocked non-NMDA as well as NMDA receptors were almost universally effective at blocking synaptic excitation within the brain and spinal cord, establishing both the existence and ubiquity of non-NMDA synaptic receptor systems throughout the CNS. In the early 1980s, NMDA receptors were shown to be involved in several central synaptic pathways, acting in concert with non-NMDA receptors under conditions where a protracted excitatory postsynaptic potential was effected in response to intense stimulation of presynaptic fibres. Such activation of NMDA receptors together with non-NMDA receptors led to the phenomenon of long-term potentiation (LTP), associated with lasting changes in synaptic efficacy (synaptic plasticity) and considered to be an important process in memory and learning. During the 1980s, it was shown that certain glutamate receptors in the brain mediated biochemical changes that were not susceptible to NMDA or non-NMDA receptor antagonists. This dichotomy was resolved in the early 1990s by the techniques of molecular biology, which identified two families of glutamate-binding receptor proteins (ionotropic (iGlu) and metabotropic (mGlu) receptors). Development of antagonists binding to specific protein subunits is currently enabling precise identification of discrete iGlu or mGlu receptor subtypes that participate in a range of central synaptic processes, including synaptic plasticity.     

Binding of the antiretroviral drug, -aspartate-β-hydroxamate on the NMDA receptor

-Aspartate-β-hydroxamate ( -A β H) exhibits antiretroviral properties in vitro and in vivo. It has glutamate agonist properties at the N-methyl- -aspartate (NMDA) receptor in neuronal cell cultures. This study characterizes its binding properties to the NMDA receptor by measuring its stimulating effect on N-(1-(2-thienyl)[³H]cyclohexyl)piperidine ([³H]TCP) binding to the ionic channel in rat brain membranes. -A β H stimulated [³H]TCP binding in a dose-dependent manner but to a lower extent than glutamate, suggesting only partial glutamate agonist properties. In the presence of antagonists of the different effector sites of the NMDA receptor the affinity of -A β H was competitively decreased by CGS-19755 and 7-chlorokynurenate and unaffected by arcaine. Among several -A β H analogues VHS.125 behaved as a full NMDA agonist, but - or -glutamate γ-monohydroxamate ( -GH or -GH) were without effect. This study shows that -A β H has potential neurotoxic effects due to its direct interaction with the NMDA receptor and that analogues such as -GH or -GH may rather be used in humans.     

Sensitive and rapid behavioral differentiation ofN-methyl-d-aspartate receptor antagonists

Behavioral effects of PCP-type noncompetitive antagonists ofN-methyl-d-aspartate (NMDA) receptors overlap with those of a host of other centrally acting compounds. In the present experiment, locomotor activity and performance on an inverted screen test in untrained mice were used to differentiate PCP-type non-competitive NMDA antagonists from other drug classes. These uncompetitive NMDA antagonists [PCP, dizocilpine, (–)-MK-801, TCP, (+)-SKF 10,047, dextrorphan, ketamine] produced dose-related increases in locomotor activity and the percentage of mice falling off an inverted, elevated wire mesh screen. Both effects demonstrated stereoselectivity, occurred at comparable dose levels, and were within the range of doses producing other biological effects (e.g., anticonvulsant). The potencies of these drugs for producing behavioral effects were positively correlated with affinities for PCP ([³H]MK-801) but not ([³H]SKF 10,047) receptors. Although muscarinic antagonists (benactyzine, atropine) produced effects in the same direction, locomotor stimulation was small and occurred at lower doses than those inducing screen failures. Competitive NMDA antagonists (LY 274614, LY 233536, CPP, NPC 12626), receptor ligands (DTG, dextromethorphan), postsynaptic dopamine agonists (quinpirole, SKF 38393) and antagonists (haloperidol, SCH 39166), and some depressant compounds (morphine, diazepam) increased failures on the screen test but decreased locomotor activity. Ligands of the polyamine regulatory site of the NMDA receptor (ifenprodil, SL 82.0715-10) and the AMPA receptor antagonist NBQX decreased locomotor activity without increasing screen failures. An antagonist of the strychnine-insensitive glycine receptor (7-chlorokynurenic acid) did not affect performance on either test. Psychomotor stimulants (cocaine and methamphetamine) stimulated locomotor activity without affecting screen performance. The only false positives occurred with barbiturates (pentobarbital, phenobarbital). Nonetheless, the present procedure demonstrates excellent sensitivity and power for rapid discrimination of uncompetitive NMDA antagonists.     

Long-term desipramine or chlorpromazine treatment and rat brain N-methyl-D-aspartate receptor.

The effects of tricyclic antidepressants and antipsychotic drugs on the NMDA receptor in rat brain were investigated. Both types of drugs, in the micromolar concentration range and with Hill coefficients greater than unity, inhibited the binding of [3H]TCP to rat brain membranes. Tricyclic antidepressants containing secondary amine on the aliphatic side chain were more potent than the corresponding tertiary amine derivatives. Antipsychotic drugs containing large side chains were much less active than chlorpromazine. Neither tricyclic antidepressants nor antipsychotic drugs had an inhibitory effect on [3H]CPP binding to the glutamate site of the NMDA complex. Chronic administration of desipramine or chlorpromazine did not change the binding parameters of [3H]TCP to rat cerebral cortex membranes. It is possible that these psychoactive drugs bind to a site adjacent to the [3H]TCP binding site within the ionic channel of the NMDA receptor complex.     

Characterization of the binding of radioligands to the N-methyl-D-aspartate,phencyclidine, and glycine receptors in buffy coat membranes

Rapid vacuum filtration assays were used to quantitate radioligand binding to phencyclidine (PCP), N-methyl-D-aspartate (NMDA), and strychnine-insensitive glycine receptors in a simple buffy coat preparation of rat cortical membranes. K_D and B_max values for [³H]glycine binding were very similar to those previously reported by workers who used centrifugation for the separation of free and bound [³H]glycine. We also found that this preparation had a high percentage of NMDA-displaceable L-[³H]glutamate binding sites, which demonstrated a pharmacology very similar to that previously observed in more purified synaptic plasma membranes. Hill analysis of the displacement curves indicated that glutamate bound to a single class of sites, but that NMDA and NMDA antagonists may interact with this site in a negatively cooperative fashion. This preparation was also found to be suitable for the study of NMDA and glycine receptor regulation of the associated ion channel, as these effectors, alone and in combination, increased the affinity with which [³H]TCP bound to the PCP receptor believed to be located within the ion channel. Thus, the ability to measure radioligand binding to these three sites in the same simple membrane preparation should greatly facilitate the study of the interaction between them.     

Pharmacological characterization of the Raji lymphoblast platelet-activating factor receptor

Previous studies in our laboratory have described the presence of high-affinity plateletactivating factor (PAF) binding sites on the B lymphoblast cell line, Raji. In the present study, PAF receptor antagonists were used to examine the specificity of [³H]PAF binding and PAF-induced intracellular calcium mobilization in this human lymphocyte cell line. In binding studies conducted at 4°C, PAF receptor antagonists competed with [³H]PAF for binding to Raji lymphocytes with the following rank order of potency: Ro 19-3704 ～ CV-6209 > CV-3988 > BN52021 > alprazolam. This order of potency is similar to those described for binding studies in platelets and neutrophils. The PAF receptor antagonists CV-6209 and alprazolam inhibited the PAF-induced calcium changes and PAF binding with similar potencies, indicating that the high-affinity PAF binding sites are functional receptors coupled to calcium mobilization. The calcium channel antagonists verapamil, diltizem, and nimodipine inhibited Raji lymphoblast PAF binding with similar potencies, but had no effect on PAF-induced intracellular calcium changes, suggesting the possibility that these compounds are not true competitive antagonists. These studies provide evidence that the Raji lymphoblast PAF receptor is pharmacologically similar to PAF receptors found on platelets and neutrophils, and thus could be of use as a model system to study the PAF receptor.     

Phencyclidine receptors and N-methyl-D-aspartate antagonism: electrophysiologic data correlates with known behaviours

Using cortical wedges and isolated frog spinal cords, the potency of a series of psychoactive phencyclidine (PCP) and sigma receptor ligands as antagonists of N-methyl-D-aspartate (NMDA) has been compared with their potency in neurochemical and behavioural studies. Phencyclidine receptor, but not sigma or kappa, ligands were selective antagonists of NMDA on both preparations. Combination studies suggested that dissociative anaesthetics and sigma benzomorphans act at the same site. The relative potencies of the drugs as NMDA antagonists correlated well with their potency in PCP receptor binding studies in vitro and in PCP discrimination studies in vivo.     

Glycine regulation of the N-methyl-D-aspartate receptor-gated ion channel in hippocampal membranes

The N-methyl-D-aspartate receptor-gated ion channel (NMDA channel) is regulated by glycine. To examine the interaction of glycine and NMDA receptor ligands on NMDA channel function, we used a biochemical marker of channel opening, [3H]N-(1-[thienyl]cyclohexyl)piperidine (TCP). We quantified [3H]glycine,L-[3H]glutamate, and TCP binding in an identical membrane preparation. This allowed direct comparison of NMDA and glycine receptor occupancy and channel activation. Glycine increased the association and dissociation rates of NMDA-dependent TCP binding to hippocampal membranes, without altering the Kd or Bmax for TCP binding. Structurally similar amino acids mimicked the action of glycine, with D-isomers being more potent than L-isomers. The potency of glycine in regulating TCP binding matched that for displacing [3H]glycine. Glycine stimulation of TCP binding required the presence of NMDA agonists and was inhibited by the NMDA antagonist D-2-amino-5-phosphonovaleric acid. Glycine stimulation of NMDA-dependent TCP binding was not associated with an increase in agonist binding to the NMDA receptor. Likewise, NMDA stimulation of glycine-dependent TCP binding was not associated with an increase in the binding of glycine to the glycine receptor. These findings permit the following conclusions: 1) glycine stimulates TCP binding solely by increasing the access of TCP to its site in the NMDA channel; 2) TCP binding can be used to quantify glycine regulation of the NMDA channel; 3) a stereospecific glycine receptor, as part of the NMDA receptor-channel complex, regulates NMDA-evoked channel opening by a mechanism not involving increased agonist binding to the NMDA receptor. Thus, it appears that the mechanism of glycine and NMDA receptor regulation of the NMDA channel is analogous to that of a two-key lock; both receptors, by independent and mutually required mechanisms, alter channel conformation to allow ion passage.     

Modulation of the NMDA receptor by D-serine in the cortex and the spinal cord, in vitro

We present a comparative study of the modulation of the N-methyl-D-aspartate (NMDA) receptor at the strychnine-insensitive glycine site in the spinal cord and in the cortex. The excitatory effect of NMDA was potentiated by D-serine (a glycine mimetic) in the hemisected rat spinal cord. The non-competitive NMDA antagonists 7-chlorokynurenic acid (7-Cl KYNA; 10 microM) and 3-amino-1-hydroxypyrrolid-2-one (HA-966; 100 or 200 microM) antagonized the effect of NMDA in the spinal cord and cortical wedge preparation. The antagonism was reversed by the addition of D-serine. This effect was strychnine-insensitive and hence not related to the inhibitory glycine receptor known to be present in the spinal cord. Our results suggest strongly that glycine positively modulates the NMDA system not only at a supraspinal level but also at the spinal level. As the positive modulation of NMDA responses by D-serine was also seen in the presence of tetrodotoxin, we conclude that the NMDA/glycine complex is (also) located on motoneurones in addition to the known glycine-mediated inhibitory system.     

Ketobemidone plus (RS)-3-dimethylamino-1,1-diphenylbut-1-ene (A29) is More Potent at NMDA Receptors than Ketobemidone Alone: Evidence for A29 as a Non-Competitive NMDA Receptor Antagonist

Abstract: The opioid, ketobemidone, has previously been shown to be a non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist. In Denmark, ketobemidone is available in a formulation which contains ketobemidone and a spasmolytic compound, (RS)-3-dimethylamino-l,l-diphenylbut-l-ene, hydrochloride (A29), in a one to five ratio. Using in vitro receptor binding techniques and an in vitro electrophysiological preparation consisting of rat cerebral cortex, we have characterized the interaction between A29 and the different glutamale receptor subtypes. A29 selectively inhibited binding of the non-competitive NMDA receptor antagonist ³H-MK-801 with a K_i value 16 ± 4.5 μM, but was inactive in assays measuring affinities for other glutamate receptors. In agreement with the binding studies, A29 was found to selectively inhibit responses to NMDA in the rat cortical wedge preparation, whereas responses to kainate and AMPA were unaffected. Analysis of dose response curves showed A29 to be a NMDA receptor antagonist with an IC₅₀ value of 100 μM versus responses to 10 μM NMDA. The inhibitory effects of ketobemidone and A29 on responses to 10 uM NMDA were additive. These data show that the combination of A29 and ketobemidone exert more potent antagonism at the NMDA receptor than does ketobemidone alone.     

Quantitative autoradiographic localization of NMDA receptors in rat brain using [3H]CPP: comparison with [3H]TCP binding sites

The regional distribution of N-methyl-D-aspartate (NMDA) receptors in rat brain using the selective NMDA receptor ligand [3H]3-[+/-)-2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) has been quantitated by in vitro autoradiography. [3H]CCP binding was highest in the CA1 region of the hippocampus. Relatively high levels of binding were also observed in the cerebral cortex, while moderate binding was obtained in the thalamus, striatum and granule cell layer of the cerebellum. Concurrent studies examining the phencyclidine receptor ligand [3H]1-[1-(2-thienyl)cyclohexyl]-piperidine (TCP), revealed a similar pattern of binding that correlated well with the localization of [3H]CPP-labeled NMDA receptors (r = 0.88, P less than 0.01).     

Relationship between alpha 2-adrenergic receptor binding sites and the functional receptors inhibiting norepinephrine release in rat cerebral cortex

The properties of alpha 2-adrenergic receptor binding sites and the receptors inhibiting [3H]norepinephrine (3H-NE) release from slices of cerebral cortex were compared. [3H]RX 781094, an alpha 2-adrenergic receptor antagonist radioligand, labeled a single class of binding sites in membranes at 37 degrees with the pharmacological specificity expected of alpha 2-adrenergic receptors. 5'-Guanylimidodiphosphate (Gpp(NH)p) and NaCl caused small increases in the potencies of antagonists at the 3H-RX binding sites but decreased the potencies of agonists 4-22-fold. Antagonists blocked the inhibition of potassium-evoked 3H-NE release caused by exogenous NE with potencies similar to those in competing for 3H-RX binding sites. Partial receptor inactivation with N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) was used to determine whether there was a receptor reserve. EEDQ dose-dependently decreased both the density of 3H-RX binding sites and the maximal inhibition of 3H-NE release by different agonists. For most agonists, KA values calculated after the receptor inactivation did not differ significantly from EC50 values; however, for epinephrine a small receptor reserve was apparent. The proportion of 3H-RX binding sites lost was similar to the proportion of functional receptors lost after EEDQ treatment. The functional KA values for agonists correlated most closely with KD values for the low affinity binding state observed in the presence of Gpp(NH)p and NaCl. However, both epinephrine and NE still showed two-site binding curves under these conditions, and it was the high affinity subpopulation of sites observed in the presence of Gpp(NH)p and NaCl which resembled most closely the functional KA values. These data suggest that 3H-RX labels binding sites with properties similar to the alpha 2-adrenergic receptors inhibiting 3H-NE release in cerebral cortex. There is little or no receptor reserve for this effect, and there appears to be a binding state for the natural catecholamine agonists which has an affinity lower than that for mediating this functional response.