On the Specificity of 125I-α-Bungarotoxin Binding to Axonal Membranes

Abstract: ¹²⁵I-α-Bungarotoxin (α-BGT) was used to characterize the binding sites for cholinergic ligands in lobster walking leg nerve membranes. The toxin binding component has been visualized histochemically on the external surfaces of intact axons and isolated axonal membrane fragments. Binding of α-BGT to nerve membrane preparations was demonstrated to be saturable and highly reversible ( K _D^app± 1.7 ± 0.32 × 10^-7 M; B _max± 249 ± 46 pmol/mg protein) at pH 7.8, 10 mM-Tris buffer. Binding showed a marked sensitivity to ionic strength that was attributable to the competitive effects of inorganic cations (particularly Ca²⁺ and Mg²⁺) in the medium. ¹²⁵I-α-BGT binding could be inhibited by cholinergic drugs (atropine ≅ d -tubocurarine > nicotine > carbamylcholine ≅ choline) and local anesthetics (procaine > tetracaine = lidocaine), but was unaffected by other neuroactive compounds tested (e.g., tetrodotoxin, 4-aminopyridine, quinuclidinyl benzilate, octopamine, bicuculline, haloperidol, ouabain). The pharmacological sensitivity of toxin binding resembles that of nicotine binding to axonal membranes, but differs significantly from nicotinic cholinergic receptors described in neuromuscular junctions, fish electric organs, sympathetic ganglia, and the CNS. The possible physiological relevance of the axonal cholinergic binding component and its relationship to α-BGT binding sites in other tissues are discussed.     

Autoradiographic Distribution and Characteristics of High- and Low-Affinity Polyamine-Sensitive [3H]Ifenprodil Sites in the Rat Brain: Possible Relationship to NMDAR2B Receptors and Calmodulin

Abstract: We have studied the regional distribution and characteristics of polyamine-sensitive [³H]ifenprodil binding sites by quantitative autoradiography in the rat brain. In forebrain areas ifenprodil displaced [³H]ifenprodil (40 nM) in a biphasic manner with IC₅₀ values ranging from 42 to 352 nM and 401 to 974 µM. In hindbrain regions, including the cerebellum, ifenprodil displacement curves were monophasic with IC₅₀ values in the high micromolar range. Wiping studies using forebrain slices (containing both high- and low-affinity sites) or cerebellar slices (containing only the low-affinity site) showed that high- and low-affinity ifenprodil sites are sensitive to spermine and spermidine, to the aminoglycoside antibiotics neomycin, gentamicin, and kanamycin, and to zinc. Two calmodulin antagonists, W7 and calmidazolium, also displaced [³H]ifenprodil from both sites. Other calmodulin antagonists, including trifluoperazine, prenylamine, and chlorpromazine, selectively displaced [³H]ifenprodil from its low-affinity site in hindbrain and forebrain regions. High-affinity [³H]ifenprodil sites, defined either by ifenprodil displacement curves or by [³H]ifenprodil binding in the presence of 1 mM trifluoperazine, were concentrated in the cortex, hippocampus, striatum, and thalamus with little or no labeling of hindbrain or cerebellar regions. This distribution matches that of NMDAR2B mRNA, supporting data showing that ifenprodil has a preferential action at NMDA receptors containing this subunit. Low-affinity [³H]ifenprodil sites have a more ubiquitous distribution but are especially concentrated in the molecular layer of the cerebellum. [³H]Ifenprodil was found to bind to calmodulin-agarose with very low affinity (IC₅₀ of ifenprodil = 516 µM). This binding was displaced by calmodulin antagonists and by polyamines, with a potency that matched their displacement of [³H]ifenprodil from its low-affinity site in brain sections. However, the localization of the low-affinity [³H]ifenprodil site does not strictly correspond to that of calmodulin, and its identity remains to be further characterized. The restricted localization of high-affinity [³H]ifenprodil binding sites to regions rich in NMDAR2B subunit mRNA may explain the atypical nature of this NMDA antagonist.     

A Thermodynamic Study of 5-[3H]Hydroxytryptamine Binding to Human Cortex Membranes

Kinetic and equilibrium measurements of [3H]-serotonin (5-hydroxytryptamine) binding to human frontal cortex membranes have been made between 4 and 30 degrees C. The effects of spiperone and ascorbate on binding have also been determined. Under the conditions used, binding was saturable and reversible. Affinity constants derived from kinetic and equilibrium data were comparable. Serotonin binding to several sites had substantial enthalpic as well as entropic components.     

Comparison of 125I-Angiotensin III and 125I-Angiotensin II Binding to Rat Brain Membranes

The binding of 125I-angiotensin III (125I-ANG III) to rat brain membranes was examined and compared with that of 125I-angiotensin II (125I-ANG II). Degradation of each ligand, as monitored by HPLC, was effectively inhibited using fragments of ANG III and ANG II known to have little affinity for angiotensin binding sites. Three classes of 125I-ANG III-binding sites were observed based on affinity (KD = 0.13, 1.83, and 10.16 nM) and capacity (Bmax = 1.30, 18.41, and 67.2 fmol/mg protein, respectively). Two classes of 125I-ANG II-binding sites of high affinity (KD = 0.11 and 1.76 nM) and low capacity (Bmax = 1.03 and 18.86 fmol/mg protein, respectively) were also identified. Cross-displacement studies confirmed that the two highest-affinity 125I-ANG III-binding sites and the 125I-ANG II-binding sites were the same. On the other hand, the binding of 125I-ANG III to the low-affinity 125I-ANG III-binding site could not be inhibited with ANG II. These data imply that previously measured differences in the biological potency of cerebroventricularly applied ANG III and ANG II probably do not result from differential binding of these peptides to central angiotensin receptors.     

Specific High-Affinity Binding of L-[3H]Aspartate to Rat Brain Membranes

Abstract: The binding of L-[³H]aspartate was investigated in washed membranes prepared from whole rat brain. We were able to differentiate two separate binding sites differing in their Na dependence. The Na-independent binding was saturable, reversible, and optimal at 20°C and at pHs in the neutral range. The dissociation constant (K_d) at 20°C was about 200 n M . This binding site seemed to be modulated by magnesium and calcium at physiological concentrations. None of the amino acids tested was a potent competitor for Na-independent L-[³H]aspartate binding. This binding site was unevenly distributed in the rat central nervous system: cerebellum = cerebral cortex > ponsmedulla > spinal cord. Destruction of the intrinsic neurons of the cerebellum by injecting kainic acid 30 days before sacrifice resulted in a 53% reduction in Na-independent binding in this region. The Na-dependent binding of L-[³H]-aspartate (K_d= 484 n M ) was strongly inhibited by D-aspartate, L-glutamate, D,L-aspartate β-hydroxamate; was unaffected by calcium and magnesium; and showed a different pattern of distribution: cerebral cortex > cerebellum = pons-medulla = spinal cord. This binding in cerebellum was unaffected by injections of kainic acid.     

NMDA Receptors with Different Sensitivities to Magnesium and Ifenprodil Control the Release of [14C]Acetylcholine and [3H]Spermidine from Rat Striatal Slices in Vitro

Abstract: KCI (20–100 mM) and W-methyl-D-aspartate (NMDA, 100–1,000 μM) produce concomitant concentration-dependent increases in the release of previously captured [¹⁴C]acetylcholine and [³H]spermidine from rat striatal slices in vitro. The effects of NMDA (300μM) on striatal [¹⁴C]acetylcholine and [³H]spermidine release were blocked with equal potencies by the competitive NMDA antagonist CGP 37849, the glycine site antagonist L-689,560, and the NMDA channel blocker dizocilpine. In contrast, although NMDA-evoked [¹⁴C]acetylcholine release was antagonized by ifenprodil (IC₅₀= 5.3 μM) and MgCl₂, (IC₅₀= 200 μM), neither compound antagonized the NMDA-evoked release of [³H]spermidine at concentrations up to 100 μM (ifenprodil) or 1 mM (MgCl₂). Distinct NMDA receptor subtypes with different sensitivities to magnesium and ifenprodil therefore exist in the rat striaturn.     

[3H]Homoquinolinate Binds to a Subpopulation of NMDA Receptors and to a Novel Binding Site

Abstract: NMDA receptors mediate several important functions in the CNS; however, little is known about the pharmacology, biochemistry, and function of distinct NMDA receptor subtypes in brain tissue. To facilitate the study of native NMDA receptor subpopulations, we have determined the radioligand binding properties of [³H]homoquinolinate, a potential subtype-selective ligand. Using quantitative receptor autoradiography, NMDA-specific [³H]homoquinolinate binding selectively labeled brain regions expressing NR2B mRNA (layers I–III of cerebral cortex, striatum, hippocampus, and septum). NMDA-specific [³H]homoquinolinate binding was low in brain regions that express NR2C and NR2D mRNA (cerebellar granular cell layer, NR2C; glomerular layer of olfactory bulb, NR2C/NR2D; and midline thalamic nuclei, NR2D). In forebrain, the pattern of NMDA-specific [³H]homoquinolinate binding paralleled NR2B and not NR2A distribution. In addition to NMDA-displaceable binding, there was a subpopulation of [³H]homoquinolinate binding sites in the forebrain, cerebellum, and choroid plexus that was not displaced by NMDA or l -glutamate. In contrast, we found that the derivative of homoquinolinate, 2-carboxy-3-carboxymethylquinoline, markedly inhibited the NMDA-insensitive binding of [³H]homoquinolinate without inhibiting the NMDA-sensitive population. [³H]Homoquinolinate may be useful for selectively characterizing NR2B-containing NMDA receptors in a preparation containing multiple receptor subtypes and for characterizing a novel binding site of unknown function.     

Secretin Receptors on Neuroblastoma Cell Membranes: Characterization of 125I-Labeled Secretin Binding and Association with Adenylate Cyclase

Secretin, a gut-brain peptide, elicited cyclic AMP production in a clone of neuroblastoma cells derived from the C1300 mouse tumor. Adenylate cyclase (EC 4.6.1.1) in plasma membranes from these cells was stimulated by secretin greater than vasoactive intestinal peptide greater than peptide histidine isoleucine amide, but not by the related peptides glucagon, gastric inhibitory polypeptide, or human growth hormone releasing factor. Hill coefficients for stimulation approximated one and the response to submaximal peptide concentrations was additive, as expected for hormones competing for a single receptor associated with the enzyme. Binding of 125I-labeled secretin to the neuroblastoma plasma membranes was saturable, time-dependent, and reversible. The KD determined from kinetic and equilibrium binding studies approximated 1 nM. The binding site displayed marked ligand specificity that paralleled that for stimulation of adenylate cyclase. The secretin receptor was regulated by guanine nucleotides, with guanosine 5'-(beta, gamma-imino)-triphosphate being the most potent to accelerate the rate of dissociation of bound secretin. These findings demonstrate the functional association of the secretin receptor with adenylate cyclase in neuronally derived cells.     

Multiple [35S]t-Butylbicyclophosphorothionate Binding Sites in Rat and Chicken Cerebral Hemispheres

Specific binding of [35S]t-butylbicyclophosphorothionate (TBPS) to membranes from cerebral hemispheres of adult rat and chicken was determined over a range of radioligand concentrations from 0.25 to 500 nM. Scatchard plots of these data were curvilinear and nonlinear regression analysis indicated binding to two sites that differ in affinity. For rat cerebrum, KD(1) = 1.15 nM, Bmax(1) = 0.085 pmol/mg; KD(2) = 232 nM, Bmax(2) = 16.9 pmol/mg. For chicken cerebrum, KD(1) = 1.39 nM, Bmax(1) = 0.111 pmol/mg; KD(2) = 166 nM, Bmax(2) = 17.6 pmol/mg. This multiplicity of [35S]TBPS binding was further confirmed when unlabeled TBPS or picrotoxinin displaced radioligand. The displacement curves were biphasic and yielded Hill coefficients from 0.65 to 0.70. These displacement curves were also resolved into two components with distinct IC50 values for unlabeled TBPS (rat, 1.55 and 271 nM; chicken, 2.40 and 224 nM). The IC50 values were similar to the dissociation constants obtained from equilibrium binding measurements.     

Comparison of the Kinetics of [3H]Diazepam and [3H]Flunitrazepam Binding to Cortical Synaptosomal Membranes

Abstract: [³H]Diazepam and [³H]flunitrazepam ([³H]FNP) binding to washed and frozen synaptosomal membranes from rat cerebral cortex were compared. In Tris-citrate buffer, γ -aminobutyric acid (GABA) and NaCl both increased [³H]diazepam binding more than [³H]FNP binding. GABA and pentobarbital both enhanced this effect of NaCl. Because of the extremely rapid dissociation of [³H]diazepam in the absence of NaCl and GABA, the B_max (maximal binding capacity) was smaller by the filtration assay than by the centrifugation assay. [³H]FNP, which dissociates more slowly, had the same B_max in both assays. [³H]Diazepam association had two components, and was faster than [³H]FNP association. [³H]Diazepam dissociation, which also had two components, was faster than that of [³H]FNP, and also had a greater fraction of rapidly dissociating species. [³H]FNP dissociation was similar when initiated by diazepam, flunitrazepam, clonazepam, or Ro15-1788, which is a benzodiazepine antagonist. [³H]Diazepam dissociation with Ro15-1788, flunitrazepam, or clonazepam was slower than with diazepam. GABA and NaCl, but not pentobarbital, increased the percentage of slowly dissociating species. This effect of NaCl was potentiated by GABA and pentobarbital. The results support the cyclic model of benzodiazepine receptors existing in two interconvertible conformations, and suggest that, distinct from their binding affinity, some ligands (like flunitrazepam) are better than others (like diazepam) in inducing the conversion of the receptor to the higher-affinity state.     

Characterization of [3H]Hemicholinium-3 Binding Sites in Human Brain Membranes: A Marker for Presynaptic Cholinergic Nerve Terminals

We report here on the binding properties of [3H]hemicholinium-3, a selective inhibitor of the high-affinity choline uptake process, to human brain membranes. Under the assay conditions described, the binding of [3H]hemicholinium-3 exhibited a dependency of physiological conditions on pH, temperature, and NaCl concentrations. Striatal binding proved to be specific, to a single site, saturable, and reversible, with an apparent KD of 10 nM and a Bmax of 82 fmol/mg of protein. [3H]Hemicholinium-3 specific binding exhibited a pharmacological profile and an ionic dependency suggestive of physiologically relevant interactions and comparable with those reported for the high-affinity choline uptake. Moreover, specific [3H]hemicholinium-3 binding exhibited an uneven regional distribution: striatum much greater than nucleus basalis greater than spinal cord much greater than midbrain = cerebellum greater than or equal to hippocampus greater than neocortex = anterior thalamus greater than posterior thalamus much much greater than white matter. This distribution closely corresponds to the reported activity of both enzymatic cholinergic presynaptic markers and high-affinity choline uptake in mammalian brain. There are no significant differences between these results and those previously found in the rat brain using this radioligand. Our results demonstrate, for the first time, the presence of [3H]hemicholinium-3 binding sites in human brain and strongly support the proposal that this radioligand binds to the carrier site mediating the high-affinity choline uptake process on cholinergic neurons. Thus, [3H]hemicholinium-3 binding may be used in postmortem human brain as a selective and quantifiable marker of the presynaptic cholinergic terminals.     

Compared Binding Properties of 125I-Labeled Analogues of Neurotensin and Neuromedin N in Rat and Mouse Brain

Abstract: Neurotensin and neuromedin N are two structurally related peptides that are synthesized by a common precursor. The purpose of the present work was to characterize neuromedin N receptors in rat and mouse brain and to compare these receptors with those of neurotensin. A radiolabeled analogue of neuromedin N has been prepared by acylation of the N-terminal amino group of the peptide with the ¹²⁵I-labeled Bolton-Hunter reagent. This ¹²⁵I-labeled derivative of neuromedin N bound to newborn mouse brain homogenate with high affinity (K _d = 0.5 n M ). Cross-competition experiments between radiolabeled and unlabeled neurotensin and neuromedin N indicated that each peptide was able to displace completely and specifically the other peptide from its interaction with its receptor. Independently of the radioligand used, the affinity of neurotensin was always better than that of neuromedin N. Quantitative radioautographic studies demonstrated that the ratio of labeling intensities obtained with ¹²⁵I-labeled analogues of neurotensin and neuromedin N remained constant in all the brain areas. Our results do not support the existence of a specific neuromedin N receptor in rat and mouse brain and can be explained by the presence of a common receptor for both peptides.     

Ethanol-Induced Inhibition of [3H]Thienylcyclohexylpiperidine (TCP) Binding to NMDA Receptors in Brain Synaptic Membranes and to a Purified Protein Complex

Abstract: N -Methyl- d -asparate receptors (NMDARs) are a major target of ethanol effects in the nervous system. Haloperidol-insensitive, but dizocilpine (MK-801)-sensitive, binding of N -[1-(2-[³H]thienyl)cyclohexyl]piperidine ([³H]TCP) to synaptic membranes has the characteristics of ligand interaction with the ion channel of NMDARs. In the present studies, ethanol produced a concentration-dependent decrease in the maximal activation of [³H]TCP binding to synaptic membranes by NMDA and Gly, but a moderate change in the activation by l -Glu when l -Glu was present at concentrations < 100 µ M . However, ethanol (100 m M ) inhibited completely the activation of [³H]TCP binding produced by high concentrations of l -Glu (200–400 µ M ). It also inhibited strongly the activation of [³H]TCP binding by spermidine or spermidine plus Gly. In a purified complex of proteins that has l -Glu-, Gly-, and [³H]TCP-binding sites, ethanol (100 m M ) decreased significantly the maximal activation of [³H]TCP binding produced by either l -Glu or Gly. Activation constants ( K _act) for l -Glu and Gly acting on the purified complex were 12 and 28 µ M, respectively. Ethanol had no significant effect on the K _act of l -Glu but caused an increase in the K _act of Gly. These studies have identified at least one protein complex in neuronal membranes whose response to both l -Glu and Gly is inhibited by ethanol. These findings may explain some of the effects of acute and chronic ethanol treatment on the function and expression of the subunits of this complex in brain neurons.     

Evidence for Distinct 5-Hydroxytryptamine2 Binding Site Subtypes in Cortical Membrane Preparations

5-Hydroxytryptamine (5-HT) displays a sixfold higher affinity for 5-HT2 binding sites labeled by [3H]ketanserin in rat (IC50 = 200 +/- 40 nM) and human (IC50 = 190 +/- 50 nM) cortex than for 5-HT2 sites in bovine cortex (IC50 = 1,200 +/- 130 nM). The Hill slopes of the 5-HT competition curves are 0.67 +/- 0.04 in rat, 0.69 +/- 0.08 in human, and 0.96 +/- 0.02 in bovine cortex. Scatchard analysis of (+/-)-[3H]4-bromo-2,5-dimethoxyamphetamine ([3H]DOB) binding in the rat indicates a population of binding sites with a KD of 0.38 +/- 0.04 nM and a Bmax of 1.5 +/- 0.05 pmol/g tissue. In contrast, specific [3H]DOB binding cannot be detected in bovine cortical membranes. These data indicate that species variations exist in 5-HT2 binding site subtypes and that [3H]ketanserin appears to label a homogeneous population of 5-HT2 binding site subtypes in bovine cortex.     

Heterogeneity of Benzodiazepine Receptors: Experimental Differences Between [3H]Flunitrazepam and [3H]Ethyl-β-carboline-3-carboxylate Binding Sites in Rat Brain Membranes

Abstract: This study was designed to analyze possible differences in the binding of [³H]flunitrazepam ([³H]FNZP) and [³H]ethyl - β - carboline - 3 - carboxylate ([³H]β-CCE), to rat brain membranes, in various experimental conditions. In cerebral cortex, hippocampus, cerebellum, and orain stem the number of binding sites for [³H]β-CCE was higher than for [³H]FNZP; both were displaced by clonazepam. Until the 7th day of postnatal brain development the numbers of [³H]FNZP and [³H]β-CCE sites were equivalent; but later on, the β-carboline sites increased to a higher level. Noradrenergic denervation by 6-hydroxydopamine was followed in the hippocampal formation. Already after 2 days, there was a decrease in [³H]FNZP sites, which reached 70% of control after 14 days. Similar results were obtained with DSP-4 denervation. This change was only in B_max and not in K_D, In contrast, the [³H]β-CCE sites did not change with denervation. Neonatal injection of l - 2,4,5 - trihydroxyphenylalamine or DSP-4 produced in the adult a decrease in [³H]FNZP sites in the cerebral cortex, in parallel with the noradrenergic denervation. On the other hand, there was an increase in the cerebellum and brain stem, in correspondence with the hyperinnervation by sprouting. In these rats, the number of sites for [³H]β-CCE did not change in the different brain regions. With 0.1% Triton X-100, applied to synaptosomal membranes, [³H]FNZP binding was reduced by 35%, while that of [³H]β-CCE was not significantly changed. These results suggest that there is heterogeneity of binding sites for benzodiazepine receptors in rat brain. A tentative interpretation of the experiments involving noradrenergic denervation and hyperinnervation, as well as those with Triton X-100, is that [³H]FNZP binds to pre- and postsynaptic receptors, while [³H]β-CCE binds mainly to postsynaptic benzodiazepine receptors.     

Characterization of 125I-Lysergic Acid Diethylamide Binding to Serotonin Receptors in Rat Frontal Cortex

Acetylcholinesterase (AChE) is found both in motor end-plate (MEP)-free and MEP-rich regions of rat or mouse muscle. We studied the developmental aspects of the localization of asymmetric 16S AChE in both regions of the sternocleidomastoid muscle, which has a well-defined zone of motor innervation. In the rat, the proportion of 16S AChE to total AChE increases in the MEP-rich region, and becomes significantly higher than in the MEP-free regions between the first and the second weeks after birth. In the mouse, at birth, the MEP-rich region already has a higher relative content in 16S AChE than the MEP-free regions. Total 16S AChE amounts increase during postnatal development, not only in the MEP-rich region but also in the MEP-free regions. Thus, 16S AChE is not eliminated from MEP-free regions during muscle maturation and growth. Two distinct pools of 16S AChE are distinguished in the muscles, both of which increase during postnatal development: junctional and background 16S AChE.     

Species Differences in the Binding of [3H]Nitrobenzylthioinosine to the Nucleoside Transport System in Mammalian Central Nervous System Membranes: Evidence for Interconvertible Conformations of the Binding Site/Transporter Complex

The binding of [3H]nitrobenzylthioinosine (NBMPR) to specific sites in CNS membranes was investigated using cortical tissue from a variety of mammalian species. Mass law analysis of the site-specific binding of NBMPR data revealed that rat, mouse, guinea pig, and dog cortical membranes each contained an apparent single class of high-affinity (KD 0.11-4.9 nM) binding sites for NBMPR; rabbit cortical membranes, however, exhibited two distinct classes of NBMPR binding sites with KD values of 0.4 nM and 13.8 nM. Dipyridamole, a potent inhibitor of nucleoside transport, produced a biphasic profile of inhibition of the binding of NBMPR to guinea pig, rabbit, and dog membranes (IC50 less than 20 nM and IC50 greater than 6 microM for NBMPR binding sites displaying high and low affinity for dipyridamole, respectively). These results are indicative of heterogeneity of NBMPR binding sites in mammalian cortical membranes. Rat and mouse cortical membranes appear to possess only one type of NBMPR binding site, which has low affinity for dipyridamole. Detailed analysis of inhibitor-induced dissociation of NBMPR from its sites in each species led to the conclusion that these multiple forms of NBMPR binding sites are different conformations of a single site associated with the CNS nucleoside transport system, rather than two distinct sites. It is also suggested that the affinity of dipyridamole for each conformation of NBMPR site indicates the susceptibility of that conformation of the nucleoside transport system to inhibition by dipyridamole.     

Modulation of [3H]Diazepam Binding in Rat Cortical Membranes by GABAA Agonists

GABAA receptor agonists modulate [3H]diazepam binding in rat cortical membranes with different efficacies. At 23 degrees C, the relative potencies for enhancement of [3H]diazepam binding by agonists parallel their potencies in inhibiting [3H]gamma-aminobutyric acid [( 3H]GABA) binding. The agonist concentrations needed for enhancement of [3H]diazepam binding are up to 35 times higher than for [3H]GABA binding and correspond closely to the concentrations required for displacement of [3H]bicuculline methochloride (BMC) binding. The maximum enhancement of [3H]diazepam varied among agonists: muscimol = GABA greater than isoguvacine greater than 3-aminopropane sulphonic acid (3APS) = imidazoleacetic acid (IAA) greater than 4,5,6,7-tetrahydroisoxazolo (4,5,6)-pyridin-3-ol (THIP) = taurine greater than piperidine 4-sulphonic acid (P4S). At 37 degrees C, the potencies of agonists remained unchanged, but isoguvacine, 3 APS, and THIP acquired efficacies similar to GABA, whereas IAA, taurine, and P4S maintained their partial agonist profiles. At both temperatures the agonist-induced enhancement of [3H]diazepam binding was reversible by bicuculline methobromide and by the steroid GABA antagonist RU 5135. These results stress the importance of studying receptor-receptor interaction under near-physiological conditions and offer an in vitro assay that may predict the agonist status of putative GABA receptor ligands.     

[3H]MK-801 Labels a Site on the N-Methyl-D-Aspartate Receptor Channel Complex in Rat Brain Membranes

The potent noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist [3H]MK-801 bound with nanomolar affinity to rat brain membranes in a reversible, saturable, and stereospecific manner. The affinity of [3H]MK-801 was considerably higher in 5 mM Tris-HCl (pH 7.4) than in previous studies using Krebs-Henseleit buffer. [3H]MK-801 labels a homogeneous population of sites in rat cerebral cortical membranes with KD of 6.3 nM and Bmax of 2.37 pmol/mg of protein. This binding was unevenly distributed among brain regions, with hippocampus greater than cortex greater than olfactory bulb = striatum greater than medulla-pons, and the cerebellum failing to show significant binding. Detailed pharmacological characterization indicated [3H]MK-801 binding to a site which was competitively and potently inhibited by known noncompetitive NMDA receptor antagonists, such as phencyclidine, thienylcyclohexylpiperidine (TCP), ketamine, N-allylnormetazocine (SKF 10,047), cyclazocine, and etoxadrol, a specificity similar to sites labelled by [3H]TCP. These sites were distinct from the high-affinity sites labelled by the sigma receptor ligand (+)-[3H]SKF 10,047. [3H]MK-801 binding was allosterically modulated by the endogenous NMDA receptor antagonist Mg2+ and by other active divalent cations. These data suggest that [3H]MK-801 labels a high-affinity site on the NMDA receptor channel complex, distinct from the NMDA recognition site, which is responsible for the blocking action of MK-801 and other noncompetitive NMDA receptor antagonists.     

Characterization of [3H]Guanine Nucleotide Binding Sites in Brain Membranes

[3H]GTP [guanosine triphosphate] and [3H]GMP-PNP [guanosine 5'-(beta, 8-imino)triphosphate, a nonmetabolized analog of GTP] have been utilized as ligands to characterize binding sites of guanine nucleotides to rat brain membranes. Binding of both [3H]GTP and [3H]GMP-PNP is saturable, with respective KD values of 0.76 and 0.42 microM. The number of binding sites for GMP-PNP (4 nmol/g) is three times greater than for GTP (1.5 nmol/g). This discrepancy is caused by rapid degradation of GTP to guanosine by brain membranes, which can be partially prevented by addition of 100 microM-ATP. The binding of [3H]guanine nucleotides is selective, with approximately equipotent inhibition by GTP, GDP, and GMP-PNP (at 0.2--1.0 microM), but no inhibition by other nucleotides at 100 microM concentrations. The bindings sites for guanine nucleotides in brain membranes appear not to be associated with microtubules, since treatments that reduce [3H]colchicine binding by 65% have no effect on [3H]GTP binding. [3H]Guanine nucleotide binding is widely distributed in various organs, with highest levels in liver and brain and lowest levels in skeletal muscle. The characteristics of these binding sites in brain show specificity properties of sites that regulate neurotransmitter receptors and adenylate cyclase.