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.     

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.     

Reduced Glycine Stimulation of [3H]MK-801 Binding in Alzheimer''s Disease

The novel N-methyl-D-aspartate receptor channel ligand (+)-[3H]5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5, 10-imine maleate ([3H]MK-801) has been utilized to label this receptor in human brain tissue. Characteristics of [3H]MK-801 binding to well-washed membranes from 17 control subjects and 16 patients with Alzheimer's disease were determined in frontal, parietal, and temporal cerebral cortex and cerebellar cortex. In control tissue the pharmacological specificity of the binding of this substance is entirely consistent with the profile previously reported for rat brain. Binding could be stimulated by the addition of glutamic acid to the incubation medium; addition of glycine produced further enhancement which was not prevented by strychnine. The specificity of the effects of these and other amino acids on the binding was the same as in the rat. In Alzheimer's disease significantly less binding was observed in the frontal cortex under glutamate- and glycine-stimulated conditions. This appears to be associated with a reduced affinity of the site whereas the pharmacological specificity of the site remained unchanged. The effect did not appear to be due to differences in mode of death between Alzheimer's disease and control subjects and is unlikely to be related to factors for which the groups were matched. In contrast, binding was not altered in the absence of added amino acids and presence of glutamate alone. These results imply that in the cerebral cortex the agonist site and a site in the cation channel of the receptor are not selectively altered, but that their coupling to a strychnine-insensitive glycine recognition site is impaired.     

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.     

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.     

Kinetic characterization of the phencyclidine-N-methyl-D-aspartate receptor interaction: evidence for a steric blockade of the channel

The nature of the interactions between the N-methyl-D-aspartate (NMDA) and the phencyclidine (PCP) receptors was studied in membranes obtained from rat cerebral cortex and washed repeatedly to remove endogenous excitatory amino acids. Binding of [3H]-N-[1-(2-thienyl)cyclohexyl]piperidine ([3H]TCP) to its receptor sites in these membranes proceeded slowly and did not reach equilibrium even after incubation for 4 h at 25 degrees C. The dissociation rate of [3H]TCP-receptor complexes was also slow (t1/2 = 128-165 min). Both association and dissociation followed first-order reaction kinetics, with similar time constants (0.0054 min-1). Addition of glutamate and glycine to the washed membranes was immediately followed by a marked increase in the rates of both association of [3H]TCP with the receptors and its dissociation from them (t1/2 = 8 min). Association now followed second-order reaction kinetics. Accelerated association of [3H]TCP with its binding sites could also be induced by NMDA or by glutamate alone, and glycine enhanced the effect. All effects of glutamate and glycine on [3H]TCP binding kinetics were blocked by the competitive NMDA receptor antagonist AP-5 [D-(-)-2-amino-5-phosphovaleric acid]. [3H]TCP-receptor interactions at equilibrium were not altered by AP-5 or by glutamate and glycine. The binding data were fitted to a model in which interactions of [3H]TCP with the receptor involve a two-step process: the outside ligand must cross a barrier (presumably a closed NMDA receptor channel in the absence of agonists). Once agonists are added, this limitation is removed (presumably because the channel is open).(ABSTRACT TRUNCATED AT 250 WORDS)     

Amino acids in ram testicular fluid and semen and their metabolism by spermatozoa

1. The testis of the ram secretes considerable amounts of amino acids (200μmoles/day) into the fluid collected from the efferent ducts. The principal amino acid in this testicular fluid is glutamate, which is present in concentrations about eight times those in testicular lymph or in blood from the internal spermatic vein. 2. The concentration of glutamate in seminal plasma from the tail of the epididymis is about ten times that in testicular fluid, and, though glutamate is the major amino acid in ejaculated seminal plasma, its concentration is less than in epididymal plasma. 3. After the intravenous infusion of [U-¹⁴C]glucose, labelled glutamate was found in the testicular fluid. Radioactivity was also detected in alanine, glycine, serine plus glutamine and aspartate. Alanine had the highest specific activity, about 50% of the specific activity of blood glucose. 4. When [U-¹⁴C]glutamate was infused, the specific activity of glutamate in testicular fluid was only about 2% that in the blood plasma. 5. Testicular and ejaculated ram spermatozoa oxidized both [U-¹⁴C]glutamate and [U-¹⁴C]leucine to a small extent, but neither substrate altered the respiration from endogenous levels. 6. No radioactivity was detected in testicular spermatozoal protein after incubation with [U-¹⁴C]glutamate or [U-¹⁴C]leucine. Small amounts of radioactivity were detected in protein from ejaculated ram spermatozoa after incubation with [U-¹⁴C]glutamate. 7. The carbon of [U-¹⁴C]glucose was incorporated into amino acids by testicular spermatozoa; most of the radioactivity occurred in glutamate.     

Prostaglandin D2 receptor of mastocytoma P-815 cells--possible regulation by phosphorylation and dephosphorylation

The 3H-labeled prostaglandin D2 [( 3H]PGD2) binding protein in the membrane fraction of mastocytoma P-815 cells was characterized. The specific binding of [3H]PGD2 to the cells or the membranes reached a maximum at pH 5.6, and was saturable, displaceable and of high affinity when incubated at 0 or 37 degrees C. The Bmax values for [3H]PGD2 binding in the two preparations at pH 5.6 were much higher at 0 degrees C than at 37 degrees C, whereas the Kd values were almost equal (85.3 nM for the cells and 80.5 nM for the membranes, respectively). High specific [3H]PGD2 binding activity in the mildly acid-treated cells was still observed when the external pH was raised from 5.6 to 7.2. Furthermore, specific [3H]PGD2 binding to the membranes (at 0 degrees C, pH 5.6) increased on addition of phosphatase inhibitors (NaF and molybdate) in the presence of 10 microM ATP, but practically disappeared on pretreatment of the membranes with phosphatase. On incubation of the membrane with [gamma-32P]ATP and molybdate, the stimulated incorporation of the [32P]phosphate into several peptides, including ones having an Mr of around 100,000-120,000, was observed. These results suggest that [3H]PGD2 binding in the mastocytoma P-815 cell membrane is controlled through phosphorylation-dephosphorylation of the receptor itself.     

Sodium ions regulate a specific population of acidic amino acid receptors in synaptic membranes

The regulatory effects of Na+ on C1-/Ca2+-dependent and C1-/Ca2+-independent L-glutamate binding sites were examined. In Tris-C1-/Ca2+ buffer, the binding of L-[3H]-glutamate to rat brain synaptic membranes was 5-fold higher than in Tris-acetate buffer. Low concentrations of Na+ (less than 5 mM) markedly depressed L-glutamate binding when assayed in Tris-C1/Ca2+ buffer, and this effect was attenuated by the selective blocker of C1-/Ca2+-dependent binding sites, DL-2-amino-4-phosphonobutyrate (APB). Scatchard analyses indicated that the effect of Na+ was due to a decrease in the number of C1-/Ca2+-dependent binding sites with no change in affinity. In Tris-acetate buffer, low concentrations of Na+ had little effect on L-glutamate binding. Dose-response curves for the inhibition of L-glutamate binding by DL-APB indicated a predominant high-affinity (Ki 5-10 microM) inhibitory component in Tris-C1-/Ca2+ buffer, but mainly a low-affinity component (Ki 1-2 mM) in Tris-acetate buffer and in Tris-C1-/Ca2+ buffer containing Na+. These data indicate that low concentrations of Na+ regulate specifically the C1-/Ca2+-dependent, APB-sensitive class of L-glutamate binding sites.     

LABELLING OF BRAIN PROTEINS AT EARLY PERIODS AFTER SUBCUTANEOUS INJECTION OF A MIXTURE OF [U-14C]GLUCOSE AND [3H]GLUTAMATE

To obtain evidence of the site of conversion of [U-¹⁴C]glucose into glutamate and related amino acids of the brain, a mixture of [U-¹⁴C]glucose and [³H]glutamate was injected subcutaneously into rats. [³H]Glutamate gave rise to several ³H-labelled amino acids in rat liver and blood; only ³H-labelled glutamate, glutamine or γ-aminobutyrate were found in the brain. The specific radioactivity of [³H]glutamine in the brain was higher than that of [³H]glutamate indicating the entry of [³H]glutamate mainly in the ‘small glutamate compartment’. The ¹⁴C-labelling pattern of amino acids in the brain and liver after injection of [U-¹⁴C]glucose was similar to that previously reported (Gaitonde et al., 1965). The specific radioactivity of [¹⁴C]glutamine in the blood and liver after injection of both precursors was greater than that of glutamate between 10 and 60 min after the injection of the precursors. The extent of labelling of alanine and aspartate was greater than that of other amino acids in the blood after injection of [U-¹⁴C]glucose. There was no labelling of brain protein with [³H]glutamate during the 10 min period, but significant label was found at 30 and 60 min. The highest relative incorporation of [¹⁴C]glutamate and [¹⁴C]aspartate in rat brain protein was observed at 5 min after the injection of [U-¹⁴C]glucose. The results have been discussed in the context of transport of glutamine synthesized in the brain and the site of metabolism of [U-¹⁴C]glucose in the brain.     

Evoked release from guinea pig cerebral cortex slices of endogenous 14C-labelled amino acids, labelled via D-[U-14C]glucose.

Release of endogenous amino acids labelled via D-[U-14C]glucose was compared with that of several exogenous labelled amino acids using slices of guinea pig cerebral cortex. Electrical field stimulation evoked a selective release of endogenous [14C]glutamate, [14C]aspartate, and gamma-amino[14C]butyrate (14C-labelled GABA). The selectivity of release correlated well with 14C incorporation into endogenous amino acids. Calculations of the fraction of the tissue radioactivity released indicated that the selectivity was not an artifact due to differential incorporation. Because glucose in mammalian brain is metabolized almost entirely by the so-called 'large compartment', it is tentatively concluded that the releasable 'transmitter pool' of glutamate, aspartate, and GABA is located in this 'large compartment'.     

Modulation of glutamate-induced uncompetitive blocker binding to the NMDA receptor by temperature and by glycine

The effect of temperature on the binding of [3H]-N-[1-(2-thienyl)cyclohexyl]piperidine [( 3H]TCP) to the ion channel of the N-methyl-D-aspartate (NMDA) receptors was studied in washed rat brain-cortex membranes. Raising the temperature from 5 to 33 degrees C resulted in a significant increase in the association rates of [3H]TCP binding measured in the presence of 1 microM glutamate and 1 microM glycine, but was less effective in the absence of the added agonists. No such effects of temperature on the dissociation rates of [3H]TCP-receptor complexes were observed. In the absence of agonists, neither the association nor the dissociation binding components varied with temperature, suggesting a diffusion-controlled limitation of access of the ligand to its site within the nonactivated NMDA receptor. No evidence was found for a temperature-dependent change in the density of [3H]TCP binding sites or for heterogeneity of [3H]TCP binding sites associated with the NMDA receptor, even though when approaching equilibrium the binding kinetics in the presence of glutamate and glycine deviated from an ordinary bimolecular reaction scheme. The data were fitted instead to a two-exponent binding function, comprising the sum of a fast and a slow binding component. Their corresponding time constants exhibited an increase with temperature, and the increase of each one was correlated significantly with the corresponding decrease in the equilibrium binding constant; however, there was no temperature-related change in the relative proportions of the two components, with the fast binding component (alpha) accounting for 50-70% of the site population.(ABSTRACT TRUNCATED AT 250 WORDS)     

Insulin biosynthesis in the bullhead, Ictalurus nebulosus, and the effect of temperature

Insulin biosynthesis in the brown bullhead, Ictalurus nebulosus (Le Sueur), was studied by measuring the incorporation in vitro of [(3)H]leucine into proteins of the principal islet. The tissue was incubated for 6-15h in Krebs-Ringer bicarbonate buffer with [(3)H]leucine, supplemented with amino acids and glucose. Proteins, precipitated with trichloroacetic acid and extracted with acid ethanol, were separated by gel-filtration on Biogel P-30 in 3m-acetic acid. Three major components were found after incubation of the islets at 22 degrees C. On the basis of the results of sulphitolysis, biological activity and the demonstrated precursor-product relationship, components I and II were identified as proinsulin and insulin respectively. The third component was not identified. At 12 degrees C, [(3)H]leucine was incorporated only into proinsulin. No radioactivity was found in insulin or the unidentified component III at 12 degrees C as was found after incubation at 22 degrees C. When the temperature was lowered from 22 degrees to 12 degrees C after 3h of a 15h incubation, decreased conversion of proinsulin into insulin resulted at the lower temperature compared with the control tissue maintained at 22 degrees C. When the temperature was raised from 12 degrees to 22 degrees C at 3h of a 15h incubation, conversion of proinsulin into insulin occurred. No conversion occurred in the control tissue with the temperature maintained at 12 degrees C. No qualitative difference in the incorporation of [(3)H]leucine into proinsulin and its conversion into insulin at 12 degrees and 22 degrees C could be demonstrated between islet tissue from fish acclimated to less than 12 degrees C or to 22 degrees C. The results suggest that the enzyme(s) responsible for converting proinsulin into insulin in the bullhead may be temperature sensitive with low activity at 12 degrees C.     

Complex binding of L-[3H]glutamate to hippocampal synaptic membranes in the absence of sodium

Specific binding of L-[3H]glutamate was investigated with a thoroughly washed synaptic membrane preparation from rat hippocampal formation, a region of brain densely innervated by putatively glutamatergic fibers. L-[3H]Glutamate bound rapidly, saturably, and reversibly to these membranes in the absence of Na+. Specific binding was greatest around 38 degrees C and at a slightly acidic pH. Saturation isotherms fit a model of two independent binding sites with dissociation constants of 11 and 570 nM and corresponding densities of 2.5 and 47 pmol/mg protein. All potent amino acid excitants, except N-methyl-D-aspartate and kainate, and several excitatory amino acid antagonists inhibited specific radioligand binding with IC50 values between 10(-7) M and 10(-4) M. In contrast, weak amino acid excitants and an inhibitor of glutamate uptake were nearly inactive. Displacement curves were analyzed with a computer program that assumed the simultaneous contributions of two independent sites at which each compound competitively inhibited the binding of L-[3H]glutamate. According to this analysis, ibotenate and the L- and D-isomers of glutamate and aspartate bind preferentially to the high-affinity site, whereas quisqualate, L-alpha-aminoadipate, and the L- and D-isomers of homocysteate bind preferentially to the low-affinity site. With the notable exception of gamma-D-glutamylglycine, all of the more potent antagonists appear to bind preferentially to the low-affinity site. Both sites exhibit marked stereoselectivity for L-glutamate. D- and L-Homocysteate and most excitatory amino acid antagonists increased specific binding at concentrations below those required to demonstrate inhibition. Some properties of the low-affinity binding site resemble those of junctional glutamate receptors on insect muscle, but neither site appears to correspond to the "N-methyl-D-aspartate receptor" or the "quisqualate receptor."     

AMINO ACID METABOLISM IN GLIAL CELLS: HOMEOSTATIC REGULATION OF INTRA- and EXTRACELLULAR MILIEU BY C-6 GLIOMA CELLS

Abstract— The amino acid and carbohydrate metabolism of confluent cultures of C-6 glioma cells has been investigated. It was observed that the presence of glutamine in the incubation fluid was essential to maintain high glutamine levels in the cells during a 2 h incubation. When cells were incubated in a cerebrospinal fluid-like medium glutamate, glutamine, aspartate and γ-aminobutyrate (GABA) levels were comparable to those occurring in whole forebrain of adult rat in vivo. Glucose uptake was high, approx 1 μmol/mg protein/2 h, 50% of which was accounted for by lactate production. Of the remaining glucose uptake a substantial proportion was unaccounted for by known oxygen-coupled citric acid cycle flux, or glycogen or amino acid synthesis. Interestingly, the cells released into the medium significant amounts of the neuroinhibitory amino acids, GABA and glycine, and rapidly cleared the medium of the neuroexcitatory amino acids glutamate and aspartate. Metabolism of [2-¹⁴C]glucose and [³H]acetate by the cells indicated rapid labelling of the glutamate and aspartate pools of the cells by glucose in 1 h, but the relative specific activities of glutamine and GABA were much lower. The metabolism of tracer concentrations of [³H]acetate to glutamate by the cells indicated greater dilution of this isotope compared to that of labelled glucose. However, the ratio of ³H to ¹⁴C radioactivity in glutamate and other amino acids was similar to that in the mixture of glucose and acetate added to the medium. Therefore, some active route of acetate metabolism which communicates metabolically with the route of glucose metabolism to glutamate appears to exist in the cells. Significant acetate activation and fatty acid turnover would explain the present results. Some of the amino acid labelling patterns observed in these studies are not consistent with these glial-like cells behaving as models for the small compartment of amino acid metabolism in brain. Enzyme measurements corroborated the metabolic studies. Glutamate decarboxylase activity was 3–10% of the level found in whole brain. GABA transaminase was also low compared to brain as was glutamine synthetase. Glutamate dehydrogenase was present at levels equal to or higher than those of whole brain.     

Characterization of water-soluble products of palmitic acid beta-oxidation by a rat brain preparation

Abstract: (1) [1-¹⁴C]Palmitic acid was oxidized to CO₂ and a water-soluble material by a rat brain preparation. The radioactive CO₂ and water-soluble material were produced in a ratio of 1.0:1.3 when the mitochondrial fraction was used, and 1.0:10 or more with the postnuclear fraction. There was a lag period of 10 min for CO₂ production. These conversions were stimulated by carnitine and inhibited by cyanide. (2) Of the total radioactivity in the water-soluble material obtained with the mitochondrial fraction, 65% after 10 min of incubation and 80% thereafter were associated with amino acids, mostly with aspartate and glutamate. The remaining radioactivity, 35 and 20%, respectively, was associated with organic acids, 60–65% in citrate. The water-soluble material obtained with the postnuclear fraction contained an equal amount of radioactivity in organic and amino acids during the course of the experiment. In the organic acids, succinate was the highest labeled product during 10–40 min of incubation, whereas citrate was the highest labeled at the end of 60 min of incubation. After 60 min, the radioactivity in the amino acids was markedly associated with glutamate, and its radioactivity was 10 times greater with the postnuclear fraction than with the mitochondrial one. (3) An experiment with rat liver preparations was also camed out. The liver mitochondrial fraction showed an accumulation of radioactive organic acids within 10 min of incubation, which was followed by a linear production of ¹⁴CO₂. With the liver postnuclear fraction, the radioactivity was found mostly in the organic acids during the course of the experiment. In the liver system, the radioactive amino acids accounted for only 25% or less of the total radioactivity in the water-soluble material.     

Synthesis and characterization of a radiolabelled derivative of the phencyclidine/N-methyl-D-aspartate receptor ligand (+) MK-801 with high specific radioactivity

A [3H]-labelled derivative of the drug (+)MK-801 with a high specific radioactivity was synthesized by first preparing a tribromo derivative of (+)MK-801 followed by catalytic reduction in the presence of [3H]-gas and subsequent purification of the radioactive product by reversed-phase high performance liquid chromatography (RP-HPLC). This resulted in pure (+) [3H]MK-801 with a specific radioactivity of 97 Ci/mmol. The (+) [3H]MK-801 was shown to interact with high affinity and selectivity with the phencyclidine (PCP) receptor in guinea pig brain membrane suspensions. The PCP receptor is associated with a cation channel that is chemically gated by glutamate and N-methyl-D-aspartate (NMDA). Drugs that interact with the PCP receptor block this channel. The (+) [3H]MK-801 described here will be useful to investigate the biochemistry of PCP/NMDA receptors in experiments where a high specific radioactivity is essential.     

A comparison of methods for removal of endogenous GABA from brain membranes prepared for binding assays

Two commonly used procedures for removing endogenous GABA from brain homogenates were evaluated by measuring residual GABA using high performance liquid chromatography (HPLC). The effect of these treatments on [³H]muscimol binding to the GABA receptor was also determined. Membranes subjected to osmotic lysing and eight washes with Tris-citrate buffer contained significant quantities of residual GABA whereas lysing and incubation with Triton X-100 followed by three buffer washes resulted in GABA levels below the limits of detection. The apparent affinity for [³H]muscimol was significantly higher in the Triton X-100 treated membranes and this was probably a result of the lower amount of GABA present in these membranes. The effect of Triton treatment or buffer washing on residual levels of glutamate, glutamine, aspartate, and taurine were also determined.     

Inhibitory Modulation by Sodium Ions of the N-Methyl-D-Aspartate Recognition Site in Brain Synaptic Membranes

Specific binding of radiolabeled L-glutamic acid (Glu) was examined using rat brain synaptic membranes treated with a low concentration of Triton X-100. The binding drastically increased in proportion to increasing concentrations of the detergent used up to 0.1%. Addition of 100 mM sodium acetate significantly potentiated the binding in membranes not treated with Triton X-100, whereas it markedly inhibited the binding in Triton-treated membranes. The binding in Triton-treated membranes was inversely dependent on incubation temperature and reached a plateau within 10 min after the initiation of incubation at 2 degrees C, whereas the time required to attain equilibrium at 30 degrees C was less than 1 min. Sodium acetate invariably inhibited the binding detected at both temperatures independently of the incubation time via decreasing the affinity for the ligand. The binding was significantly displaced by agonists and antagonists for an N-methyl-D-aspartate (NMDA)-sensitive subclass of brain excitatory amino acid receptors, but not by those for the other subclasses. Inclusion of sodium acetate reduced the potencies of NMDA agonists to displace the binding without virtually affecting those of NMDA antagonists. Moreover, sodium ions inhibited the ability of Glu to potentiate the binding of N-[3H] [1-(2-thienyl)cyclohexyl]piperidine to open NMDA channels in Triton-treated membranes. These results suggest that sodium ions may play an additional modulatory role in the termination process of neurotransmission mediated by excitatory amino acids via facilitating a transformation of the NMDA recognition site from a state with high affinity for agonists to a state with low affinity.     

Biological activity, binding, and metabolic fate of Ac-[Nle4, D-Phe7]alpha-MSH4-11NH2 with the F1 variant of B16 melanoma cells

The alpha-MSH (alpha-melanocyte-stimulating hormone) agonist, Ac-[Nle4, D-Phe7]alpha-MSH4-11NH2 (hereafter called ND4-11 alpha-MSH), is at least 10-fold more potent than alpha-MSH as a stimulus of tyrosinase activity in F1 variant cells of B16 melanoma. The binding to these cells during an incubation with 5 nM (3H)ND4-11 alpha-MSH at 37 degrees C is maximal at 0-30 min, 22 fmol/10(6) cells, but declines to 40% of this value at 4 hr. in the presence of 5 nM (3H)ND4-11 alpha-MSH at 37 degrees C, the acid soluble (cell surface) radioactivity decreased rapidly from 11.4 fmol/10(6) cells at 5 min to 4.6 fmol/10(6) cells at 4 hr. Chromatographic analysis of media and cellular samples revealed that there was no evidence of degradation of (3H)ND4-11 alpha-MSH in the medium but there was evidence of intracellular degradation of (3H)ND4-11 alpha-MSH. Ammonium chloride (10mM) resulted in an increase in acid resistant radioactivity (internalized hormone) at 4 hr. The binding to F1 variant cells during an incubation with 0.155 nM or 5 nM (3H)ND4-11 alpha-MSH at 4 degrees C was constant from 4 hr to 24 hr. Under these conditions, there was no time-dependent change in the acid soluble radioactivity from 4 to 24 hr. Scatchard analysis of (3H)ND4-11 alpha-MSH binding to F1 variant cells at 4 degrees C demonstrated that there were approximately 4500 receptors per cell and an association constant of 17.1 nM-1. These results are consistent with a process of (3H)ND4-11 alpha-MSH binding to its receptor followed by internalization of the receptor-hormone complex and then intracellular degradation of the hormone.