Binding Sites for l-[3H]Glutamate on Hippocampal Synaptic Membranes: Three Populations Differentially Affected by Chloride and Calcium Ions

The effects of Cl- and Ca2+ were studied on the specific binding of L-[3H]glutamate to multiple sites on rat hippocampal synaptic membranes. Quisqualate (5 microM) or DL-2-amino-4-phosphonobutyrate (2-APB) (300 microM) was used to discriminate two previously identified classes of binding sites. Saturation isotherms and displacement curves constructed under different ionic conditions suggested that the effects of Cl- and Ca2+ could best be explained by postulating the existence of three major binding site populations in this preparation rather than two. The binding of L-glutamate to Glu A sites exhibits an absolute dependence on Cl-, and Ca2+ markedly increases the maximum density of these sites. Glu A sites bind quisqualate and 2-APB with relatively high affinity. Cl- (47 mM) more than doubles the maximum density of Glu B sites, but Ca2+ appears to have no effect. Glu B sites can be discriminated from the other classes by their relatively low affinity for quisqualate and 2-APB. There is reason to think that the Glu B population is heterogeneous. The novel Glu C population can be virtually selectively labeled by exposing 2-APB-sensitive binding sites to radioligand in Tris-HOAc buffer with Ca2+. Binding of L-[3H]glutamate to these sites is enhanced by both Cl- and Ca2+, but requires neither ion. Ca2+ appears to increase both the affinity of Glu C sites for L-glutamate and their maximum binding site density. In the presence of Ca2+ and Cl-, Glu C sites bind the radioligand with micromolar affinity (KD approximately 2 microM) and high capacity (Bmax approximately 160 pmol/mg protein).(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of Two [3H]Glutamate Binding Sites in Rat Hippocampal Membranes

The specific binding of L-[3H]glutamate was investigated in the presence and the absence of sodium ions in freshly prepared membranes from rat hippocampus. Sodium ions were found to have a biphasic effect; low concentrations induced a marked inhibition of the binding (in the range 0.5-5.0 mM), whereas higher concentrations resulted in a dose-dependent stimulation of binding (in the range 10-150 mM). These results permit the discrimination of two binding sites in hippocampal membranes. Both Na+-independent and Na+-dependent binding sites were saturable, exhibiting dissociation constants at 30 degrees C of 750 nM and 2.4 microM, respectively, with Hill coefficients not significantly different from unity, and maximal number of sites of 6.5 and 75 pmol/mg protein, respectively. [3H]Glutamate binding to both sites reached equilibrium between 5 and 10 min and was reversible. The relative potencies of a wide range of compounds, with known pharmacological activities, to inhibit [3H]glutamate binding were very different for the Na+-independent and Na+-dependent binding and suggested that the former sites were related to post-synaptic glutamate receptors, whereas the latter were related to high-affinity uptake sites. This conclusion was also supported by the considerable variation in the regional distribution of the Na+-dependent binding site, which paralleled that of the high-affinity glutamate uptake; the Na+-independent binding exhibited less regional variation.     

Characterisation of Na+-Independent L-[3H]Glutamate Binding Sites in Human Temporal Cortex

The binding of L-[3H]glutamate to membranes from human temporal cortex was studied in the absence of Na+, Ca2+, and Cl- ions. Pharmacological characterisation revealed that approximately 35% of specific binding at 50 nM L-[3H]glutamate was sensitive to a combination of kainate and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid. The remaining approximately 65% of specific binding was to a single population of sites with a KD of 844 nM and a Bmax of 0.92 pmol/mg protein. The pharmacological characteristics were consistent with an interaction at the N-methyl-D-aspartate subclass of excitatory amino acid receptor. The inclusion of Cl- ions revealed additional glutamate binding; this was sensitive to quisqualate and DL-2-amino-4-phosphonobutyrate, but not to kainate, DL-2-amino-7-phosphonoheptanoate, or alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid.     

A Novel Chloride-Dependent L-[3H]Glutamate Binding Site in Astrocyte Membranes

Membrane fractions prepared from astrocytes grown in culture exhibit a specific binding site for L-[3H]glutamate that is Cl--dependent and Na+-independent. The binding site is a single saturable site with a KD of about 0.5 microM, is inhibited by L-aspartate, L-cysteate, and quisqualate, and is insensitive to kainate, N-methyl-D-aspartate, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate, and 2-amino-4-phosphonobutyrate. The pharmacological characteristics of the binding site indicate that it is distinct from any site previously described in synaptic membrane preparations. Comparisons of ionic requirements, ligand specificity, and inhibitor sensitivities, however, suggest the described binding is the first step in a Cl--dependent high-affinity glutamate uptake system. Such binding studies provide a useful model system in which to investigate the close association between excitatory amino acids, astrocytes, the termination of glutamate's excitatory action by high-affinity uptake, and the excitotoxic action of acidic amino acids in membranes of a single cell type.     

L-[3H]Glutamate Binding to a Membrane Preparation from Crayfish Muscle

The binding of L-[3H]glutamate to an isolated membrane preparation from crayfish tail muscle has been studied. The muscle homogenate was osmotically shocked, frozen and thawed, and thoroughly washed before incubation with L-[3H]glutamate. The preparation showed high specific binding of L-glutamate with a KD of 0.12 microM and Bmax of 4.7 pmol/mg protein measured in Tris/HCl pH 7.3 and at 4 degrees C. Nonspecific binding was 5-10% of total binding. The glutamate binding was highly stereospecific [K0.5 (D-glutamate), 270 microM] and showed a high degree of discrimination between L-glutamate and L-aspartate [K0.5 (L-aspartate), 54 microM]. In mammalian CNS preparations potent agonists of L-glutamate such as kainate and N-methyl-D-aspartate had no effect at 1 mM, and quisqualate was a weak inhibitor of L-glutamate binding [K0.5 (quisqualate), 162 microM]. Ibotenate was the most potent inhibitor [K0.5 (ibotenate), 0.27 microM], and various esters of L-glutamate were of intermediate potency as displacers of L-[3H]glutamate binding (K0.5 values from 6 to 60 microM). The glutamate binding site from crayfish muscle is clearly different from any of the subclasses of glutamate receptors in mammalian CNS. A possible physiological function of the binding site is a postsynaptic receptor for glutamate, either an extra-junctional or a junctional receptor.     

Dementia of the Alzheimer's Type: Changes in Hippocampal L-[3H]Glutamate Binding

Abstract: Glutamate or a related excitatory amino acid is thought to be the major excitatory neurotransmitter of hippocampal afferents, intrinsic neurons, and efferents. We have used an autoradiographic technique to investigate the status of excitatory amino acid receptors in the hippocampal formation of patients dying with dementia of the Alzheimer type (DAT). We examined l-[³H]glutamate binding to sections from the hippocampal formation of six patients dying of DAT and six patients without DAT and found marked reductions in total [³H]glutamate binding in all regions of hippocampus and adjacent parahippocampal cortex in DAT brains as compared to controls. When subtypes of excitatory amino acid receptors were assayed, it was found that binding to the N -methyl-d-aspartate (NMDA)-sensitive receptor was reduced by 75–87%, with the greatest loss found in stratum moleculare and stratum pyramidale of CA1. Binding to quisqualate (QA)-sensitive receptors was reduced by 45–69%. There were smaller reductions (21–46%) in GABA_A receptors in DAT cases. Muscarinic cholinergic receptors assayed in adjacent sections of hippocampal formation were unchanged in DAT. Benzodiazepine receptors were reduced significantly only in parahippocampal cortex by 44%. These results suggest that glutamatergic neurotransmission within the hippocampal formation is likely to be severely impaired in Alzheimer's disease. Such impairment may account for some of the cognitive decline and memory deficits that characterize DAT.     

Binding Sites for [3H]Glutamate and [3H]Aspartate in Human Cerebellum

Abstract The binding of [³H]aspartate and [³H]glutamate to membranes prepared from frozen human cerebellar cortex was studied. The binding sites differed in their relative proportions, their inhibition by amino acids and analogues, and by the effects of cations. A proportion (about 30%) of [³H]glutamate binding was to sites similar to those labelled by [³H]aspartate. An additional component of [³H]gluta-mate binding (about 50%) was displaced by quisqualate and aL-amino-3-hydroxy-5-methylisoxazole-4-propionic acid, and may represent a “quisqualate-preferring” receptor. Neither N-methyl-d-aspartic acid-sensitive nor dl-2-amino-4-phosphonobutyric acid-sensitive [³H]glutamate binding was detected.     

Identification and Characterization of an N-Methyl-D-Aspartate-Specific L-[3H]Glutamate Recognition Site in Synaptic Plasma Membranes

Conditions have been developed for an L-[3H]glutamate binding assay in which 85-95% of the specific binding is to a site that corresponds to the N-methyl-D-aspartate subclass of acidic amino acid receptors. Incubation of synaptic plasma membranes with L-[3H]glutamate in 50 mM Tris/acetate, pH 7.4, for 2-20 min at 2 degrees C results in binding with pharmacological characteristics of the electrophysiologically defined N-methyl-D-aspartate receptor. The fraction of glutamate binding to this subclass of receptors, relative to the total, decreases with both increased time and temperature. This binding is reversible, is concentrated in the synaptic plasma membrane fraction, has a pH optimum of 7.0-7.4, and is linear with respect to tissue protein concentration. The binding is unaffected by 1 mM concentrations of the anions sulfate, chloride, bromide, thiocyanate, phosphate, acetate, nitrate, or carbonate and the monovalent cations potassium or ammonium. However sodium and the divalent cations copper, cobalt, zinc, cadmium, and manganese decrease binding to this N-methyl-D-aspartate site.     

Selective Loss of N-Methyl-d-Aspartate-Sensitive l-[3H]Glutamate Binding Sites in Rat Brain Following Portacaval Anastomosis

Excitatory amino acids have been implicated in the pathogenesis of hepatic encephalopathy. In the present study, kainate, quisqualate and N-methyl-D-aspartate (NMDA) subclasses of L-glutamate receptors were measured in adult rat brain by quantitative receptor autoradiography following surgical construction of an end-to-side portacaval anastomosis (PCA). PCA resulted in sustained hyperammonemia and decreased binding of L-glutamate to the NMDA receptor when compared to sham-operated controls. Decreases in binding ranged from 17 to 39% in several regions of cerebral cortex, hippocampus, striatum, and thalamus. Binding to quisqualate and kainate receptor subtypes was not altered. PCA leads to astrocytic changes in brain but does not result in any measurable loss of neuronal integrity. It is therefore proposed that decreased glutamate binding to the NMDA receptor following PCA results from increased extracellular glutamate caused by decreased reuptake into perineuronal astrocytes and a compensatory down-regulation of these receptors. Such changes could be of pathophysiological significance in hepatic encephalopathy.     

Properties of Quisqualate-Sensitive L-[3H]Glutamate Binding Sites in Rat Brain as Determined by Quantitative Autoradiography

Quisqualate, a glutamate analogue, displaced L-[3H]glutamate binding in a biphasic manner, corresponding to "high-affinity" and "low-affinity" binding sites. High-affinity quisqualate sites were termed "quisqualate-sensitive L-[3H]glutamate" binding sites. Quisqualate-sensitive L-[3H]glutamate binding was regionally distributed, with the highest levels present in the cerebellar molecular layer. This binding was stimulated by millimolar concentrations of chloride and calcium. The stimulatory effects of calcium required the presence of chloride ions, whereas chloride's stimulatory effects did not require calcium. All of the L-[3H]glutamate binding stimulated by chloride/calcium was quisqualate sensitive and only weakly displaced by N-methyl-D-aspartate, L-aspartate, or kainate. At high concentrations (1 mM), the anion blockers 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid both reduced, by 41 and 43%, respectively, the stimulatory effects of chloride. At concentrations of 100 microM, kynurenate, L-aspartate, (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and L-2-amino-4-phosphonobutyric acid (L-APB) failed to displace quisqualate-sensitive L-[3H]glutamate binding in the cerebellar molecular layer. In the presence of KSCN, however, 100 microM AMPA displaced 44% of binding. Quisqualate-sensitive L-[3H]glutamate binding was not sensitive to freezing, and, in contrast to other chloride- and calcium-dependent L-[3H]glutamate binding sites that have been reported, quisqualate-sensitive binding observed by autoradiography was enhanced at 4 degrees C compared with 37 degrees C. Quisqualate-sensitive L-[3H]glutamate binding likely represents binding to the subclass of postsynaptic neuronal glutamate receptors known as quisqualate receptors, rather than binding to previously described APB receptors, chloride-driven sequestration into vesicles, or binding to astrocytic membrane binding sites.     

Effects of Guanyl Nucleotides on [3H]Flunitrazepam Binding to Rat Hippocampal Synaptic Membranes: Equilibrium Binding and Dissociation Kinetics

The effects of guanyl nucleotides on the binding of [3H]flunitrazepam to rat hippocampal synaptic membranes were studied. In equilibrium binding studies, gamma-amino-n-butyric acid (GABA) increased and GTP decreased the binding affinity of [3H]flunitrazepam; GTP also caused a decrease in binding capacity. The effect, however, is variable. In studies of the dissociation kinetics of [3H]flunitrazepam using diazepam and the antagonist Ro 15-1788 as the displacers, there was evidence of two dissociation rate constants. GTP increased both the fast- and slow-dissociation rate constants and increased the ratio of the slow-dissociation binding state. The effect of GTP was mimicked by its nonhydrolyzable analogue 5'-guanylylimidodiphosphate but not by ATP and occurred when diazepam, but not when Ro 15-1788, was used as the displacer. GABA antagonized the effect of GTP on the dissociation of [3H]flunitrazepam. The nature of the benzodiazepine receptor, its actions, and the possible role of cyclic AMP as a second messenger are discussed.     

Enhanced [3H]DOPA and [3H]Dopamine Turnover in Striatum and Frontal Cortex In Vivo Linked to Glutamate Receptor Antagonism

Abstract: We tested the hypothesis that blockade of NMDA glutamate receptors in brain enhances dopamine turnover. We blocked this class of glutamate receptors in the rat brain in vivo with dizocilpine (MK-801) and measured the accumulation of radiolabeled DOPA and its metabolites as functions of time after intravenous bolus injection. Using the time courses of the accumulated metabolites, we calculated the turnover constants of enzymes mediating dopamine synthesis and catabolism. Dizocilpine treatment for 8 days enhanced the rates of DOPA decarboxylation and dopamine oxidation (monoamine oxidation) 4- and 16-fold, respectively, in neostriatum and 10- and 3-fold, respectively, in frontal cortex. The findings are not inconsistent with the hypothesis that the psychotomimetic properties of dizocilpine may be the manifestation of denervation hypersensitivity linked to activation of key enzymes of dopamine turnover in striatum.     

[3H]Thienylcyclohexylpiperidine Binding Activity in Brain Synaptic Membranes Treated with Triton X-100

Binding activity of [3H]thienylcyclohexylpiperidine was examined using rat brain synaptic membranes treated with Triton X-100. This compound is proposed to be a noncompetitive antagonist for the N-methyl-D-aspartate (NMDA)-sensitive subclass of brain excitatory amino acid receptors. The activity decreased in proportion to increasing concentrations of the detergent up to 0.08%. In vitro addition of L-glutamate (Glu) partially restored the decreased activity caused by this Triton treatment, whereas further addition of glycine (Gly) entirely reversed the loss of activity to the level found in membranes extensively washed but not treated with a detergent. These stimulatory effects were found to be due to the acceleration of the association of ligand. The rank order of potentiation of the activity coincided well with that of the affinity for the NMDA-sensitive subclass among numerous Glu analogs. The potentiation by Gly as well as Glu was invariably prevented by competitive NMDA antagonists, such as DL-2-amino-5-phosphonovalerate and (+/-)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonate, but not by strychnine. No significant difference was observed between pharmacological profiles of the activities in synaptic membranes treated and not treated with Triton X-100, except haloperidol. The potency of this sigma-ligand to inhibit the activity was greatly reduced by the Triton treatment in the presence of both Glu and Gly. These results suggest that the regulatory properties of Triton-treated synaptic membranes remain unchanged in terms of the interaction within the NMDA receptor complex.     

Optimal Conditions for [3H]Apomorphine Binding and Anomalous Equilibrium Binding of [3H]Apomorphine and [3H]Spiperone to Rat Striatal Membranes: Involvement of Surface Phenomena Versus Multiple Binding Sites

I. Binding of [³H]apomorphine to dopaminergic receptors in rat striatum was most reproducible and clearly detectable when incubations were run at 25°C in Tris-HCl buffer, pH 7.5, containing 1 mM-EDTA and 0.01% ascorbic acid, using a washed total-membrane fraction. The receptor binding was stereospecifically inhibited by (+)-butaclamol, and dopamine agonists and antagonists showed high binding affinity for these sites. Unlabelled apomorphine inhibited an additional nonstereospecific binding site, which was unrelated to dopamine receptors. EDTA in the incubation mixture considerably lowered nonstereospecific [³H]apomorphine binding, apparently by preventing the complexation of the catechol moiety with metal ions which were demonstrated in membrane preparations. Stereospecific [³H]apomorphine binding was not detectable in the frontal cortex, whereas in the absence of EDTA much saturable nonstereospecific binding occurred. II. Kinetic patterns of stereospecific [³H]spiperone and [³H] apomorphine binding to rat striatal membranes and the inhibition patterns of a dopamine antagonist and an agonist were evaluated at different temperatures in high-ionic-strength Tris buffer with salts added and low-ionic-strength Tris buffer with EDTA. Apparent K_D, values of spiperone decreased with decreasing tissue concentrations. K_D, values of both spiperone and apomorphine were little influenced by temperature changes. Scatchard plots of the stereospecific binding changed from linear to curved; the amount of nonstereospecific binding of the ³H ligands varied considerably, but in opposite directions for spiperone and apomorphine in the different buffers. In various assay conditions, interactions between agonists, and between antagonists, appeared fully competitive, but agonist-antagonist interactions were of mixed type. The anomalous binding patterns are interpreted in terms of surface phenomena occurring upon reactions of a ligand with complex physicochemical properties and nonsolubilized sites on membranes suspended in a buffered aqueous solution. It is concluded that anomalous binding patterns are not necessarily an indication of binding to multiple sites or involvement of distinct receptors for high-affinity agonist and antagonist binding.     

[3H]Aniracetam Binds to Specific Recognition Sites in Brain Membranes

Abstract: [³H]Aniracetam bound to specific and saturable recognition sites in membranes prepared from discrete regions of rat brain. In crude membrane preparation from rat cerebral cortex, specific binding was Na⁺ independent, was still largely detectable at low temperature (4°C), and underwent rapid dissociation. Scatchard analysis of [³H]aniracetam binding revealed a single population of sites with an apparent K_D value of ～70 nM and a maximal density of 3.5 pmol/mg of protein. Specifically bound [³H]aniracetam was not displaced by various metabolites of aniracetam, nor by other pyrrolidinone-containing nootropic drugs such as piracetam or oxiracetam. Subcellular distribution studies showed that a high percentage of specific [³H]aniracetam binding was present in purified synaptosomes or mitochondria, whereas specific binding was low in the myelin fraction. The possibility that at least some [³H]aniracetam binding sites are associated with glutamate receptors is supported by the evidence that specific binding was abolished when membranes were preincubated at 37°C under fast shaking (a procedure that substantially reduced the amount of glutamate trapped in the membranes) and could be restored after addition of either glutamate or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) but not kainate. The action of AMPA was antagonized by DNQX, which also reduced specific [³H]aniracetam binding in unwashed membranes. High levels of [³H]aniracetam binding were detected in hippocampal, cortical, or cerebellar membranes, which contain a high density of excitatory amino acid receptors. Although synaptosomal aniracetam binding sites may well be associated with AMPA-sensitive glutamate receptors, specifically bound [³H]aniracetam could not be displaced by cyclothiazide or GYKI 52466, which act as a positive and negative modulator of AMPA receptors, respectively.     

Ionic Regulation of the Binding of dl-[3H]2-Amino-4-Phosphonobutyrate to l-Glutamate-Sensitive Sites on Rat Brain Membranes

Abstract: The effects of ions on the binding of the excitatory amino acid analogue dl -[³H]2-amino-4-phosphon-obutyrate to l -glutamate-sensitive sites on rat brain synaptic membranes was investigated. The divalent cations manganese, magnesium, strontium, and particularly calcium, produced a marked enhancement in specific binding. However, this effect was manifest only in the presence of added chloride, or to a lesser extent, with bromide ions. Application of saturation analysis revealed that both chloride and calcium acted to increase the binding site density in a concentration-dependent manner, without affecting the dissociation constant. The only other ionic species found to have a significant effect on 2-amino-4-phosphonobutyrate binding was sodium, which produced an apparent reduction in site affinity, without modifying the binding site density. Although the significance of these striking ionic effects is as yet unknown, it seems feasible that chloride (and possibly also calcium) ions may serve a role in regulating the interaction of excitatory amino acids with their physiological receptors.     

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.     

Medium Isotope Effect in [3H]Diazepam Binding to Benzodiazepine Receptors of Synaptic Membranes

Replacement of H2O by D2O resulted in significantly higher amount of [3H]diazepam specifically bound to synaptic membranes. The isotope effect arises from increased number of binding sites in D2O and is associated with a stronger solvation of membrane receptors by heavy water.     

Characterization of [3H]Clonidine Binding to Two Sites in Calf Brain Membranes

Kinetic studies showed that under appropriate conditions, [3H]clonidine binds to two distinct receptor sites in calf cortex membranes. At 23 degrees C, binding was obtained at a low-affinity site (dissociation constant, KD = 5.4 nM) and a high-affinity site (KD = 1.1 nM). In contrast, at 0 degree C, selective binding occurred to the low-affinity site only. Consequently, at 0 degree C, it was possible to evaluate the interaction of drugs with the low-affinity receptor directly. On the other hand, competition with the high-affinity receptor could be ascertained by generating displacement curves representing the differential between specific binding values obtained at 23 and 0 degree C. Guanine nucleotides selectively decreased binding to the high-affinity site without apparent influence on the low-affinity [3H]clonidine binding. The activities of various pharmacological agents at the low- and high-affinity clonidine receptors are discussed and compared with WB-4101 binding data.     

Heterogeneity of [3H]Dipyridamole Binding to CNS Membranes: Correlation with [3H]Nitrobenzylthioinosine Binding and [3H]Uridine Influx Studies

The relationship between the nucleoside transport system and the nitrobenzylthioinosine-sensitive and -resistant [3H]dipyridamole binding sites was examined by comparing the characteristics of [3H]dipyridamole binding with those of [3H]nitrobenzylthioinosine binding and [3H]-uridine influx in rabbit and guinea pig cerebral cortical synaptosomes. Two distinct high-affinity synaptosomal membrane-associated [3H]dipyridamole binding sites, with different sensitivities to inhibition by nitrobenzylthioinosine, were characterized in the presence of 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS, 0.01%) to prevent [3H]dipyridamole binding to glass tubes and filters. The nitrobenzylthioinosine-resistant [3H]-dipyridamole binding sites represented a greater proportion of the total membrane sites in guinea pig than in rabbit (40 vs. 10% based on inhibition studies). In rabbit, nitrobenzylthioinosine-sensitive [3H]dipyridamole binding (KD = 1.4 +/- 0.2 nM) and [3H]nitrobenzylthioinosine binding (KD = 0.30 +/- 0.01 nM) appeared to involve the same membrane site associated with the nitrobenzylthioinosine-sensitive nucleoside transporter. By mass law analysis, [3H]-dipyridamole binding in guinea pig could be resolved into two components based on sensitivity to inhibition by 1 microM nitrobenzylthioinosine. The nitrobenzylthioinosine-resistant [3H]dipyridamole binding sites were relatively insensitive to inhibition by all of the nucleoside transport substrates and inhibitors tested, with the exception of dipyridamole itself. In guinea pig synaptosomes, 100 microM dilazep blocked nitrobenzylthioinosine-resistant [3H]uridine transport completely but inhibited the nitrobenzylthioinosine-resistant [3H]dipyridamole binding component by only 20%. Furthermore, a greater percentage of the [3H]dipyridamole binding was nitrobenzylthioinosine resistant in guinea pig compared with rabbit, yet both species had a similar percentage of nitrobenzylthioinosine-resistant [3H]uridine transport.(ABSTRACT TRUNCATED AT 250 WORDS)