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.     

Solubilization, Characterization, and Partial Purification of α-Amino-3-Hydroxy-5-Methyl-4-Isoxazolepropionic Acid-, Quisqualate-, Kainate-Sensitive l-Glutamate Binding Sites from Porcine Brain Synaptic Junctions

L-[3H]Glutamate binding sites were solubilized from porcine brain synaptic junctions by Triton X-114 in the presence of KCl. The solubilized binding sites bound L-[3H]glutamate reversibly with KD and Bmax values of 1.48 +/- 0.18 microM and 178.2 +/- 15.9 pmol/mg of protein, respectively. These binding sites appeared to be integral membrane glycoproteins, with sugar moieties recognized by wheat germ agglutinin. A 49.3-fold purification of these binding sites was achieved by Triton X-114 solubilization, anion-exchange chromatography, and affinity chromatography using wheat germ agglutinin-Sepharose. The apparent molecular mass of the partially purified binding sites was 620 +/- 50 kDa. L-[3H]Glutamate bound to the solubilized preparation could be effectively displaced by agonists of non-N-methyl-D-aspartate (NMDA) L-glutamate receptors but not by NMDA or alpha-amino-4-phosphonobutyrate. The rank order for the competitive ligands in displacing L-[3H]glutamate was: quisqualate greater than alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid greater than L-glutamate greater than kainate.     

Autoradiographic Characterization and Localization of Quisqualate Binding Sites in Rat Brain Using the Antagonist [3H]6-Cyano-7-Nitroquinoxaline-2,3-Dione: Comparison with (R,S)-[3H]α-Amino-3-Hydroxy-5-Methyl-4-Isoxazolepropionic Acid Binding Sites

Using quantitative autoradiography, we have investigated the binding sites for the potent competitive non-N-methyl-D-aspartate (non-NMDA) glutamate receptor antagonist [3H]6-cyano-7-nitro-quinoxaline-2,3-dione ([3H]-CNQX) in rat brain sections. [3H]CNQX binding was regionally distributed, with the highest levels of binding present in hippocampus in the stratum radiatum of CA1, stratum lucidum of CA3, and molecular layer of dentate gyrus. Scatchard analysis of [3H]CNQX binding in the cerebellar molecular layer revealed an apparent single binding site with a KD = 67 +/- 9.0 nM and Bmax = 3.56 +/- 0.34 pmol/mg protein. In displacement studies, quisqualate, L-glutamate, and kainate also appeared to bind to a single class of sites. However, (R,S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) displacement of [3H]CNQX binding revealed two binding sites in the cerebellar molecular layer. Binding of [3H]AMPA to quisqualate receptors in the presence of potassium thiocyanate produced curvilinear Scatchard plots. The curves could be resolved into two binding sites with KD1 = 9.0 +/- 3.5 nM, Bmax = 0.15 +/- 0.05 pmol/mg protein, KD2 = 278 +/- 50 nM, and Bmax = 1.54 +/- 0.20 pmol/mg protein. The heterogeneous anatomical distribution of [3H]CNQX binding sites correlated to the binding of L-[3H]glutamate to quisqualate receptors and to sites labeled with [3H]AMPA. These results suggest that the non-NMDA glutamate receptor antagonist [3H]CNQX binds with equal affinity to two states of quisqualate receptors which have different affinities for the agonist [3H]AMPA.     

Displaceable binding of [3H]l-glutamic acid to non-receptor materials

[3H]L-glutamic acid binding to microfuge tubes and glass was investigated in four buffers. Background binding to these materials was negligible, but was increased by centrifugation or suction in Tris-HCl and Tris-citrate buffer. This binding was much less or eliminated when HEPES-KOH, or Tris-acetate buffer was used instead. [3H]L-glutamate binding to microfuge tubes was inhibited by L- but not D-isomers of glutamate and aspartate. DL-2-amino-7-phosphonoheptanoic acid also did not inhibit the binding. Other compounds which showed low to moderate inhibition were: N-methyl-D-aspartate, quisqualate, L-glutamic acid diethyl ester, N-methyl-L-aspartate, kainate, and 2-amino-4-phosphonobutyrate. Binding was inhibited by denatured rat brain membranes. A protein-dependent [3H]glutamate binding was obtained with a repeatedly frozen-thawed membrane preparation when binding was done in Tris-acetate buffer. It is recommended that Tris-acetate or HEPES-KOH buffer should be used in the glutamate binding assay. If Tris-HCl or Tris-citrate buffer is used, appropriate control experiment should be done to correct for binding to microfuge tubes or glass fiber filters.     

Solubilization of quisqualate-sensitive L-[3H]glutamate binding sites from guinea pig brain membranes using a zwitterionic detergent

L-[3H]Glutamate binding sites were solubilized with a zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane-sulfonate (CHAPS) plus ammonium thiocyanate from guinea pig synaptosomal membranes. The binding of L-[3H]glutamate to the solubilized binding sites was saturable and reversible. Scatchard analysis suggested the existence of two different classes of binding sites with KDs of 63.8 and 644 nM. The L-[3H]glutamate binding was displaced by excitatory amino acids with such an order of potency that L-glutamate much greater than D-glutamate congruent to L-aspartate greater than D-aspartate. Quisqualate effectively displaced the glutamate binding in biphasic manner. L-Glutamic acid diethyl ester, the quisqualate receptor antagonist, also showed a moderate displacing ability. Other neuroactive amino acid analogues displaced the glutamate binding only weakly, except for L- and D-homocysteic acids which had moderate potency. It is very likely from these results that the glutamate binding sites solubilized in this study are relevant to the physiological glutamate receptors especially of quisqualate-type.     

Specific inhibition of the binding of the taste stimulus, L-alanine, by sulphydryl reagents, in Ictalurus punctatus

1. Taste receptors for L-alanine and L-arginine in the channel catfish, Ictalurus punctatus, are differentially reactive to N-ethylmaleimide (NEM) and p-chloromercuribenzenesulphonic acid (pCMBS). 2. The binding of L-[3H]alanine by a sedimentable membrane fraction (Fraction P2) isolated from taste epithelium was inhibited by both NEM and pCMBS while the binding of L[3H]arginine was unaffected. 3. Inhibition of the binding of L-[3H]alanine by pCMBS was reversible with dithiothreitol (DTT). 4. NEM (10(-3) M) inhibited multi-unit neural responses to both 10(-4) M L-alanine and 10(-4) M L-arginine, while pCMBS had little effect on neural responses. 5. Pretreatment of intact taste epithelium before the preparation of Fraction P2 with NEM caused strong inhibition of L-[3H]alanine binding, while pretreatment with pCMBS caused weak inhibition. 6. The presence of L-alanine during the reaction of pCMBS or NEM with taste plasma membranes did not substantially protect against the inhibition of L-[3H]alanine binding.     

L-(3H) glutamate binding to a membrane preparation from the optic lobe of the giant freshwater prawn Macrobrachium rosenbergii de Man.

Membrane preparation from the optic lobe of the giant freshwater prawn, Macrobrachium rosenbergii de Man, was examined for the presence of specific L-(3H) glutamate binding. The optic lobes were isolated from live animals. The tissue was homogenized and the membrane fraction isolated by differential centrifugation. The membrane suspension was incubated with 10-1,000 nM of L-(3H) glutamate at 37 degrees C for 60 min. Nonspecific binding was determined by incubating the mixture with 100 microM L-glutamate. L-(3H) glutamate specifically bound to the membrane fraction with a dissociation equilibrium constant (Kd) of 205 nM and maximum number of binding sites (Bmax) of 2.04 n mol/mg protein. By using LIGAND computerized program, the saturation isotherm binding pattern indicates a single type of binding. To determine the type of glutamate receptors, competitive inhibition and IC50 of several glutamate agonists and antagonists were determined. The study reveals a metabotropic type of binding site.     

Differences in L-[3H]Glutamate Accumulation and Endogenous L-Glutamate Content in Synaptic Vesicles from Mice Selectively Bred for Differences in Ethanol Sensitivity

The ATP-stimulated accumulation of L-[3H]glutamate by whole brain synaptic vesicle preparations from long-sleep and short-sleep mice, lines selectively bred for difference in sleep time response to acute ethanol administration, was examined. L-[3H]Glutamate accumulation in vesicles from short-sleep mice was approximately twice that observed in vesicles from long-sleep mice at three glutamate loading concentrations. The vesicular content of endogenous L-glutamate in crude and enriched vesicle preparations from short-sleep mice was approximately 1.5-fold higher than in vesicles from long-sleep mice. The data suggest that L-glutamate associated with synaptic vesicles may serve a role in glutamate neurosecretion.     

Solubilization of Quisqualate-Sensitive [3H]Glutamate Binding Activity from Rat Retina

Binding activity of a putative central neurotransmitter, L-glutamic acid, was examined in the supernatant preparations solubilized from rat retinal membranes by Nonidet P-40. [3H]Glutamate binding activity increased linearly with increasing concentrations of the solubilized proteins up to 15 micrograms. The binding activity reached an equilibrium within 10 min at 2 degrees C, while increasing with incubation time up to 60 min at 30 degrees C. Addition of an excess of nonradioactive glutamate rapidly decreased the activity at 30 degrees C. Scatchard analysis revealed that the solubilized retinal binding activity consisted of a single component with a KD of 0.25 microM and a Bmax of 57.4 pmol/mg protein. The solubilized binding activity exhibited a stereospecificity and a structure selectivity to L-glutamate, and was abolished by quisqualate, L-glutamate diethyl ester, and DL-2-amino-3-phosphonopropionate. None of the other agonists and antagonists for the central excitatory amino acid receptors affected the binding activity. Reduction of incubation temperature from 30 degrees C to 2 degrees C resulted in a drastic attenuation of the binding activity due to decrement of the number of the apparent binding sites. Cation-exchange column chromatography revealed that unidentified radioactive material was in fact formed during the incubation of [3H]glutamate with the retinal preparations at 30 degrees C. These results suggest that retinal [3H]glutamate binding activity may be derived at least in part from the quisqualate-sensitive membranous enzyme with a stereospecific and structure-selective high affinity for the central neurotransmitter.     

Biochemical characterization of an autoradiographic method for studying excitatory amino acid receptors using L-[3H]glutamate

A method was developed for radiolabeling excitatory amino acid receptors of rat brain with L-[3H]glutamate. Effective labeling of glutamate receptors in slide-mounted 10-microns sections was obtained using a low incubation volume (0.15 ml) and rapid washing: a procedure where high ligand concentrations were achieved with minimal waste. Saturation experiments using [3H]glutamate revealed a single binding site of micromolar affinity. The Bmax was trebled in the presence of Ca2+ (2.5 mM) and Cl- (20 mM) with no change in the Kd. Binding was rapid, saturable, stereospecific, and sensitive to glutamate receptor agonists. The proportions of [3H]glutamate binding sensitive to N-methyl-D-aspartate (NMDA), kainate, and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) were 34, 54, and 51%, respectively. NMDA inhibited binding at a distinct subset of L-[3H]glutamate sites, whereas AMPA and kainate competed for some common sites. Labeling of sections with L-[3H]glutamate in the presence of the selective agonists allowed autoradiographic visualization of glutamate receptor subtypes in brain tissue.     

Sodium gradient-dependent L-glutamate transport is localized to the canalicular domain of liver plasma membranes. Studies in rat liver sinusoidal and canalicular membrane vesicles

The driving forces for L-glutamate transport were determined in purified canalicular (cLPM) and basolateral (i.e. sinusoidal and lateral; blLPM) rat liver plasma membrane vesicles. Initial rates of L-glutamate uptake in cLPM vesicles were stimulated by a Na+ gradient (Na+o greater than Na+i), but not by a K+ gradient. Stimulation of L-glutamate uptake was specific for Na+, temperature sensitive, and independent of nonspecific binding. Sodium-dependent L-glutamate uptake into cLPM vesicles exhibited saturation kinetics with an apparent Km of 24 microM, and a Vmax of 21 pmol/mg X min at an extravesicular sodium concentration of 100 mM. Specific anionic amino acids inhibited L-[3H]glutamate uptake and accelerated the exchange diffusion of L-[3H]glutamate. An outwardly directed K+ gradient (K+i greater than K+o) further increased the Na+ gradient (Na+o greater than Na+i)-dependent uptake of L-glutamate in cLPM vesicles, resulting in a transient accumulation of L-glutamate above equilibrium values (overshoot). The K+ effect had an absolute requirement for Na+. In contrast, in blLPM the initial rates of L-glutamate uptake were only minimally stimulated by a Na+ gradient, an effect that could be accounted for by contamination of the blLPM vesicles with cLPM vesicles. These results indicate that hepatic Na+ gradient-dependent transport of L-glutamate occurs at the canalicular domain of the plasma membrane, whereas transport of L-glutamate across sinusoidal membranes results mainly from passive diffusion. These findings provide an explanation for the apparent discrepancy between the ability of various in vitro liver preparations to transport glutamate and suggest that a canalicular glutamate transport system may serve to reabsorb this amino acid from bile.     

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)     

Enhancement of γ-Aminobutyric Acid Binding by Quazepam, a Benzodiazepine Derivative with Preferential Affinity for Type I Benzodiazepine Receptors

We evaluated the effect of the two N-trifluoroethyl benzodiazepines, quazepam and its 2-oxo metabolite SCH 15725, which possess preferential affinity for type I benzodiazepine recognition sites, on the binding of [3H] gamma-aminobutyric acid ([3H]GABA) to rat brain membrane preparations. The study also included compounds such as diazepam and N-desalkyl-2-oxoquazepam (SCH 17514), which have equal affinity for the type I and type II receptor subtypes. Binding of [3H]GABA was studied in frozen-thawed and repeatedly washed cortical membranes incubated in 20 mM KH2PO4 plus 50 mM KCl, pH 7.4, at 4 degrees C in the absence and presence of quazepam or its metabolites. Addition of 10(-6) M quazepam increased by 30% specific [3H]GABA binding; as revealed by Scatchard plot analysis, the effect was due to an increase in the total number of GABA receptors. The effect of quazepam was concentration dependent, and it was shared by its active metabolite SCH 15725. The potency of quazepam and SCH 15725 in enhancing [3H]GABA binding was similar to that of diazepam, whereas CL 218872 and SCH 17514 were less active. Moreover, the [3H]GABA binding-enhancing effect of quazepam was mediated by an occupancy of benzodiazepine receptors, because it was specifically antagonized by 5 X 10(-6) M Ro15-1788.     

Transformation and nuclear translocation of brain type L corticosteroid receptors complexed with the mineralocorticoid antagonist ZK 91587, aldosterone or dexamethasone.

Type I corticosteroid receptors were determined in cytosol from hippocampus (HIPPO) and amygdala (AMYG), using [3H]aldosterone (ALDO), [3H]dexamethasone (DEX) or the mineralocorticoid antagonist [3H]ZK 91587 as ligands. Incubations with the first two compounds also contained the pure glucocorticoid RU 28362 to block type II receptors. Binding of the three ligands was comparable in cytosol from HIPPO and it was slightly higher for [3H]DEX in AMYG. However, after heat-induced receptor transformation, binding to DNA-cellulose was observed for [3H]ALDO-receptor complex obtained from HIPPO or AMYG, whereas it was negligible for [3H]ZK 91587. Receptors charged with [3H]DEX or [3H]ALDO showed similar retention on DNA-cellulose columns in the case of the AMYG, while binding to the polynucleotide was higher for [3H]ALDO in the HIPPO. Finally, only [3H]ALDO was taken up to a significant extent in purified cell nuclei prepared from slices of HIPPO and AMYG previously incubated with the three ligands. It is concluded that binding of a natural agonist steroid may be a prerequisite for type I receptor transformation and translocation from the cytoplasm into the nuclear fraction. DEX binding to type I receptors resembles a partial agonist with antagonist properties, whereas antagonists such as ZK 91587 are bound and retained in cytoplasm, without further translocation.     

Problems Associated with the Binding of L-Glutamic Acid to Synaptic Membranes: Methodological Aspects

Abstract: Specific L-[³H]glutamate binding was investigated in extensively washed synaptic membrane preparations from rat brain. Mild conditions of ultrasonication effected a significant enhancement of binding, attributable to the marked reduction in membrane vesicle size and the removal of endogenous interfering substances such as glutamate. Preincubation of freshly prepared membranes at 37°C for 30 min followed by further washing resulted in enhanced binding. Addition of supernatant from preincubated membranes effectively inhibited [³H]glutamate binding to control membranes; the possibility of the presence of an endogenous glutamate receptor inhibitor is discussed. Treatment of membranes with low concentrations of Triton X-100, in contrast with the findings for GABA, did not produce any significant enhancement of specific glutamate binding. While binding of [³H]glutamate is almost abolished in frozen or cold-stored membranes, lyophilisation had a remarkable effect, not only affording protection, but actually enhancing the binding properties of the synaptic membrane preparation.     

Pharmacology of putative glutamate receptors from insect skeletal muscles, insect central nervous system and rat brain

Binding of [3H]glutamate to housefly brain and honeybee brain and thoracic muscle membranes as well as to the American cockroach nerve cord was measured in Na+-free Tris-citrate buffer, 2.5 mM CaCl2, pH 7.4. The dissociation constants (KDS) ranged from 0.16 to 1.36 microM, and thoracic muscles had 2-4-fold higher density of receptors than brain tissue. The potent inhibitors of housefly brain binding were in decreasing order of effectiveness: L-glutamate greater than L-aspartate = L-cysteate = ibotenate greater than quisqualate greater than L-homocysteate greater than L-APB greater than L-APV greater than NMDA greater than D-APB greater than D-glutamate, with no inhibition by 100 microM of GDEE, dihydrokainate, D-APV, D-homocysteate or D-aspartate. The drug specificity of [3H]glutamate binding sites in housefly brain was generally similar to that of binding sites in housefly muscle, except that the former had a slightly higher affinity for L-APB, L-homocysteate and NMDA. [3H]Glutamate binding to insect tissues differed in its drug sensitivity from binding to rat brain. Binding to insect membranes was much less sensitive to L-APB, D-APB, APV, homocysteate, L-cysteate, quisqualate and ibotenate. However, the insect binding site was much more stereoselective for the L than D isomers of glutamate and aspartate, while the rat brain site was more stereoselective for APB. It is suggested that the observed [3H]glutamate binding to insect tissue is not to NMDA or kainate receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solubilisation of a Glutamate Binding Protein from Rat Brain

Rat brain synaptic plasma membranes were solubilised in either 1% Triton X-100 or potassium cholate and subjected to batch affinity adsorption on L-glutamate/bovine serum albumin reticulated glass fibre. The fibre was extensively washed, and bound proteins eluted with 0.1 mM L-glutamate in 0.1% detergent, followed by repeated dialysis to remove the glutamate from the eluted proteins. Aliquots of the dialysed extracts were assayed for L-[3H]glutamate binding activity in the presence or absence of 0.1 mM unlabelled L-glutamate (to define displaceable binding). Incubations were conducted at room temperature and terminated by rapid filtration through nitrocellulose membranes. Binding to solubilised fractions could be detected only following affinity chromatography. Binding was saturable and of relatively low affinity: KD = 1.0 and 1.8 microM for Triton X-100 and cholate extracts, respectively. The density of binding sites was remarkably high: approximately 18 nmol/mg protein for Triton X-100-solubilised preparations, and usually double this when cholate was employed. Analysis of structural requirements for inhibition of binding revealed that only a very restricted number of compounds were effective, i.e., L-glutamate, L-aspartate, and sulphur-containing amino acids. Binding was not inhibited significantly by any of the selective excitatory amino acid receptor agonists--quisqualate, N-methyl-D-aspartate, or kainate. The implication from this study is that the glutamate binding protein is similar if not identical to one previously isolated and probably is not related to the pharmacologically defined postsynaptic receptor subtypes, unless solubilisation of synaptic membranes resulted in major alterations to binding site characteristics. Since solubilisation with Triton X-100 is known to preserve synaptic junctional complexes, it seems likely that the origin of the glutamate binding protein may be extrajunctional, although its functional role is unknown.     

Cortisol 17 beta acid, transcortin, and the heterogeneity of rat brain glucocorticoid receptors

Binding of tracer or competing steroids to transcortin can compromise specificity studies on receptors for adrenal steroids. Recently Alexis et al. have used cortisol 17 beta acid at high concentrations to prevent steroid binding to any transcortin possibly contaminating rat brain cytosol preparations. On the basis of limited specificity studies of [3H]dexamethasone and [3H]corticosterone binding under such conditions, it was claimed that binding sites for the two steroids are indistinguishable, and it is thus unnecessary to invoke distinct binding sites for each glucocorticoid. We have extended these competition studies in the presence of cortisol 17 beta acid, and shown that in rat hippocampus Type I, corticosterone-preferring glucocorticoid receptors can be clearly distinguished both from transcortin and from Type II, dexamethasone-binding glucocorticoid receptors.     

Characterization of High-Affinity Dopamine D2 Receptors and Modulation of Affinity States by Guanine Nucleotides in Cholate-Solubilized Bovine Striatal Preparations

3,4-Dihydroxyphenylethylamine (dopamine) D2 receptors, solubilized from bovine striatal membranes using a cholic acid-NaCl combination, exhibited the typical pharmacological characteristics of both agonist and antagonist binding. The rank order potency of the agonists and antagonists to displace [3H]spiroperidol binding was the same as that observed with membrane-bound receptors. Computer-assisted analysis of the [3H]spiroperidol/agonist competition curves revealed the retention of high- and low-affinity states of the D2 receptor in the solubilized preparations and the proportions of receptor subpopulations in the two affinity states were similar to those reported in membrane. Guanine nucleotide almost completely converted the high-affinity sites to low-affinity sites for the agonists. The binding of the high-affinity agonist [3H]N-n-propylnorapomorphine ([3H]NPA) was clearly demonstrated in the solubilized preparations for the first time. Addition of guanylyl-imidodiphosphate completely abolished the [3H]NPA binding. When the solubilized receptors were subjected to diethylaminoethyl-Sephacel chromatography, the dopaminergic binding sites eluted in two distinct peaks, showing six- to sevenfold purification of the receptors in the major peak. Binding studies performed on both peaks indicated that the receptor subpopulation present in the first peak may have a larger proportion of high-affinity binding sites than the second peak. The solubilized preparation also showed high-affinity binding of [35S]guanosine-5'-(gamma-thio)triphosphate, a result suggesting the presence of guanine nucleotide binding sites, which may interact with the solubilized D2 receptors. These data are consistent with the retention of the D2 receptor-guanine nucleotide regulatory protein complex in the solubilized preparations and should provide a suitable model system to study the receptor-effector interactions.     

Glutamate Binding to Brain Membranes Is Increased in Pentylenetetrazole-Kindled Rats

Abstract: The specific binding of L-[³H]glutamate to its receptors was investigated on crude membrane preparations from different brain regions of pentylenetetrazole-kindled rats using a binding assay technique. Pentylenetetrazole kindling induced by 10 intraperitoneal applications of 45 mg/kg over a period of 20 days resulted in a significant increase of both the convulsive susceptibility of animals to the convulsant and the specific L-[³H]glutamate binding in hippocampus and in motor, frontal, and inferiotemporal (acoustic) cortex tested with a L-[³H]glutamate concentration of 50 n M . No differences were observed in the other brain structures studied. Kinetic studies indicated that the enhanced L-[³H]glutamate binding to hippocampal membranes from kindled rats reflects changes in the density of the glutamate binding sites rather than an increase in receptor affinity. To study the effect of acute generalized convulsions on L-[³H]glutamate binding to synaptosomal membranes of hippocampus and visual cortex, rats were treated 24 h before the experiment with 60 mg/kg of pentylenetetrazole, i.p. Under these conditions, no differences between treated and control rats were observed. From these findings, it is concluded that the increase in glutamate receptor density demonstrated in hippocampus and several neocortical brain structures of pentylenetetrazole-kindled rats may be the expression of a specific enhancement of susceptibility of glutamatergic systems to this excitatory amino acid developing in the course of formation of pentylenetetrazole-induced kindling.