THE BINDING OF 5-HYDROXYTRYPTAMINE TO BUTANOL EXTRACTS OF RAT BRAIN STEM

Abstract— When butanol-water extracts of rat brain stem were incubated with [³H]5-HT, (5 × 10⁻⁷ m ), and the components resolved by chromatography on LH₂₀ Sephadex, a peak representing approximately 70% of the eluted radioactivity was found in chloroform-methanol 4:1. The peak was not found in identically prepared extracts from rat diaphragm, neither was a similar peak found when brain extracts were incubated with [¹⁴C]ACh (10⁻⁶ m ), suggesting a degree of selectivity. Binding was not saturated at concentrations of 5 × 10⁻⁵ m -5-HT. The binding was highly sensitive to the presence of water, requiring about 15% (v/v) for optimum binding. The implications of these findings are discussed in terms of a possible '5-HT receptor'.     

[1251]2α-BUNGAROTOXIN AND [3H]QUINUCLIDINYLBENZILATE BINDING IN CENTRAL NERVOUS SYSTEMS OF DIFFERENT SPECIES

Abstract— [¹²⁵I]Diiodo α-bungarotoxin ([¹²⁵I]₂BuTx) and [³H]quinuclidinylbenzilate ([³H]QNB) binding sites were measured in post-nuclear membrane fractions prepared from whole brains or brain regions of several species. Species studied included Drosophila melanogaster (fruit fly), Torpedo californiea (electric ray), Carassius auratus (goldfish), Ram pipiens (grass frog), Kana cutesheiana (bullfrog), Rattus norvegicus (rat, Sprague-Dawley), Mus muscalus (mouse, Swiss random, C58/J, LG/J), Oryctolagus cuniculus (rabbit, New Zealand Whitc), and Bos (cow). Acetyl-CoA: choline O -acetyltransferase (EC 2.3.1.6) levels were also determined in the post nuclear supernatants and correlated with the number of binding sites.
All species and regions except Drosophila had 16–150 fold more [³H]QNB binding sites than [¹²⁵I]₂BuTx binding sites. Brain regions with the highest levels of [¹²⁵I]₂BuTx binding were Drosophila heads (300 fmol/mg), goldfish optic tectum (80fmol/mg), and rat and mouse hippocampus (3040 fmol/mg). The highest levels of [³H]QNB binding were seen in rat and mouse caudate (1.3–1.6 pmol/mg). Lowest levels of [³H]QNB and [¹²⁵I]₂BuTx binding were seen in cerebellum. The utility of [¹²⁵I]₂BuTx and [³H]QNB binding as quantitative measures of nicotinic and muscarinic acetylcholine receptors in CNS is discussed.     

Ascorbic Acid Inhibits 125I-SCH 23982 Binding but Increases the Affinity of Dopamine for D1 Dopamine Receptors

Abstract: Effects of ascorbic acid (AA) on ¹²⁵I-SCH 23982 binding to D₁ dopaminergic receptors in membrane preparations from rat striatum were investigated. AA in the range of 0.03 µ M –0.33 m M inhibited 75% of specific binding of ¹²⁵I-SCH 23982 in a dose-dependent manner. At higher concentrations, this inhibition of binding activity by AA was less potent, and 3.3 m M AA inhibited only 30% of specific binding. Reduced glutathione did not alter the inhibition of binding by 0.33 m M AA, but reduced the inhibition by 3.3 m M AA to 8% of specific binding. The loss of specific binding by AA was rescued by 1 m M EDTA, an inhibitor of lipid peroxidation. In the absence of AA, competition experiments with the agonist, dopamine, revealed the presence of high-affinity ( K _h = 224.9 ± 48.9 n M ) and low-affinity ( K _l = 21,100 ± 2,400 n M ) binding sites. Although the maximum binding of ¹²⁵I-SCH 23982 decreased to 40% without affecting the K _D value in the presence of 1.67 m M AA, the value of the high-affinity site for dopamine was increased ( K _h = 23.3 ± 9.4 n M ) and that of the low-affinity site was decreased ( K _l = 136,800 ± 40,900 n M ). These results suggest that AA may affect D₁ dopamine receptor function by lipid peroxidation, competition with dopamine for low-affinity sites, and reduced oxidation of dopamine.     

INCORPORATION OF PHENYLALANINE AS A SINGLE UNIT INTO RAT BRAIN PROTEIN: RECIPROCAL INHIBITION BY PHENYLALANINE AND TYROSINE OF THEIR RESPECTIVE INCORPORATIONS

Abstract— Of seven amino acids studied, glutamic acid and phenylalanine were incorporated in highest amounts into the hot-TCA-insoluble material of the 100,000 g supernatant fraction of rat brain homogenate. The system for incorporation of phenylalanine was RNase-insensitive and required ATP (apparent K_m = 0.64 m m ), KC1 (apparent K_m = 14 m m ) and MgCl₂ (optimal concentration range 4-15 m m ). The apparent K_m for phenylalanine was 2.9 m m . [¹⁴C]Phenylalanine did not undergo modification before incorporation. Tyrosine and phenylalanine inhibited the incorporation, respectively, of [¹⁴C]phenylalanine and [¹⁴C]tyrosine when incubated simultaneously or successively. The K_m and K_t (3.3 m m ) values for phenylalanine in the incorporation reaction and as inhibitor of the incorporation of [¹⁴C]tyrosine were similar. We suggest that both the enzyme and the acceptor for the incorporation of these two amino acids are the same. [¹⁴C]Phenylalanine and [¹⁴C]tyrosine entered into COOH-terminal positions in the reactions described. Brain exhibited a 25- to 100-fold higher capacity to incorporate phenylalanine than that of liver, kidney or thyroid. The acceptor capacity in rat brain rapidly decreased from day 5 to day 15 of postnatal age and then slowly until age 150 days.     

CHARACTERIZATION OF BENZODIAZEPINE BINDING SITES IN CULTURED CELLS OF NEURAL ORIGIN

Abstract— The binding of [³H]diazepam to benzodiazepine receptors was investigated in cultured cell lines of neural origin. Two cell lines, the rat C6 glioma and mouse NB41A3 neuroblastoma possess large numbers of benzodiazepine binding sites, while the other neural cell lines examined had significantly fewer benzodiazepine binding sites. [³H]diazepam binding to membranes prepared from C6 or NB41A3 cells was saturable and of a relatively high affinity ( K _D± 12 and 20 n m , respectively) when compared with rat cerebral cortex ( K _D± 4.6 n m ). A single class of binding sites in both cell lines was demonstrated by Scatchard analysis. The maximum binding capacities ( B _max) in the C6 and NB41A3 cell lines were found to be 10 and 3.5 fold higher than in rat cerebral cortex, respectively. In contrast to the rat cerebral cortex, binding of [³H]diazepam in cultured cells was not displaced by the clinically active benzodiazepines clonazepam and oxazepam while the clinically inactive benzodiazepine Ro 5-4864 potently inhibited the binding of [³H]diazepam in both neural cell lines. In toto , this data suggests a change in the benzodiazepine binding sites in cultured cells of neural origin to that found in peripheral (kidney) tissue. The observation that cell lines derived from both neuronal and glial elements contain large numbers of benzodiazepine binding sites also suggests benzodiazepine receptors in the central nervous system may not be confined to a single cell type.     

[3H]SCH 58261, a Selective Adenosine A2A Receptor Antagonist, Is a Useful Ligand in Autoradiographic Studies

Abstract: We have characterized the new potent and selective nonxanthine adenosine A_2A receptor antagonist SCH 58261 as a new radioligand for receptor autoradiography. In autoradiographic studies using agonist radioligands for A_2A receptors ([³H]CGS 21680) or A₁ receptors ( N ⁶-[³H]cyclohexyladenosine), it was found that SCH 58261 is close to 800-fold selective for rat brain A_2A versus A₁ receptors ( K _i values of 1.2 n M versus 0.8 µ M ). Moreover, receptor autoradiography showed that [³H]SCH 58261, in concentrations below 2 n M , binds only to the dopamine-rich regions of the rat brain, with a K _D value of 1.4 (0.8–1.8) n M . The maximal number of binding sites was 310 fmol/mg of protein in the striatum. Below concentrations of 3 n M , the nonspecific binding was <15%. Three adenosine analogues displaced all specific binding of [³H]SCH 58261 with the following estimated K _i values (n M ): 2-hex-1-ynyl-5'- N -ethylcarboxamidoadenosine, 3.9 (1.8–8.4); CGS 21680, 130 (42–405); N ⁶-cyclohexyladenosine, 9,985 (3,169–31,462). The binding of low concentrations of SCH 58261 was not influenced by either GTP (100 µ M ) or Mg²⁺ (10 m M ). The present results show that in its tritium-labeled form, SCH 58261 appears to be a good radioligand for autoradiographic studies, because it does not suffer from some of the problems encountered with the currently used agonist radioligand [³H]CGS 21680.     

Studies of Inositol Phosphate Export from Neuronal Tissue In Vitro

Abstract: Recent in vivo microdialysis studies have demonstrated the presence of extracellular levels of inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃] that can be increased in a concentration-dependent manner by muscarinic receptor activation. The aim of the present study was to determine whether extracellular levels of Ins(1,4,5)P₃ could be measured in vitro. Despite rapid increases in internal Ins(1,4,5)P₃ levels after stimulation with 1 m M carbachol, there was no change in external levels in both rat brain cortical slices and human neuroblastoma SH-SY5Y cells. Suprafusion of myo -[³H]inositol-prelabelled hippocampal slices with 1 m M carbachol caused an increase in ³H-inositol phosphates over basal levels in the perfusate after 10 min, reaching a peak (223 ± 56% of basal) 20 min after suprafusion with carbachol was started. This response to carbachol was potentiated in the presence of 30 m M K⁺. Analysis of the individual ³H-inositol phosphates in the perfusate revealed that levels of [³H]inositol monophosphate, [³H]inositol bisphosphate, [³H]inositol trisphosphate, and [³H]inositol tetrakisphosphate were all significantly increased. A similar increase in extracellular ³H-inositol phosphates was demonstrated in SH-SY5Y cells incubated with 1 m M carbachol for 30 min. This response was again enhanced by 30 m M K⁺, although the intracellular response was not potentiated. Possible roles for extracellular inositol phosphates are discussed.     

45Ca2+ Uptake into Rat Whole Brain Synaptosomes Unaltered by Dihydropyridine Calcium Antagonists

Abstract: Voltage-dependent ⁴⁵Ca²⁺ uptake into rat whole brain synaptosomes was measured after 3-s KCl-induced depolarization to investigate possible inhibitory effects of calcium antagonists, nitrendipine, nimodipine, and nisoldipine. At a Ca²⁺ concentration of 1.2 m M , nitrendipine, in concentrations ranging from 0.1 n M to 10 μ M , had no effect on ⁴⁵Ca²⁺ uptake. When the Ca²⁺ concentration was lowered to 0.06 and 0.12 m M , nitrendipine, 10 μ M , inhibited ⁴⁵Ca²⁺ uptake in response to 109 m M KCl depolarization. However, in a separate concentration response study, nitrendipine, nimodipine, and nisoldipine, 0.1 n M to 10 μ M , failed to alter the uptake of ⁴⁵Ca²⁺ (0.06 m M Ca²⁺) into 30 m M KCl-depolarized synaptosomes. The high concentrations of these agents required to depress ⁴⁵Ca²⁺ uptake indicate that the dihydropyridine calcium antagonists are considerably less potent in brain tissue than in peripheral tissue.     

Properties of [3H]Prazosin-Labeled α1Adrenergic Receptors in Rat Brain and Porcine Neurointermediate Lobe Tissue

Abstract: [³H]Prazosin binding to α₁ receptors in homogenates of rat prefrontal cortical tissue and porcine pituitary neurointermediate lobe tissue was investigated. Competition curves produced by coincubating adrenergic agonists and antagonists with 0.5 n M [³H]prazosin and tissue revealed some anomalous binding properties. In the brain and pituitary tissue, agonist competition curves produced "shallow" slopes, with Hill coefficients significantly lower than unity. The IC₅₀ of the agonists epinephrine, norepinephrine, and clonidine for inhibition of 0.5 n M [³H]prazosin binding were significantly lower in the porcine pituitary than in the rat brain. Most antagonists, such as prazosin, chlorpromazine, and piperoxan, produced "steep" competition curves with Hill coefficients close to unity, with two notable exceptions. WB-4101 and phentolamine produced competition curves with Hill coefficients significantly less than unity in the rat brain preparation. Ketanserin, an antagonist, displayed a sevenfold higher affinity for the a, sites in the pituitary tissue than in the brain tissue. These anomalies in the binding results may indicate the presence of an endogenous modulatory factor affecting agonist and antagonist affinities for the a, receptor.     

Distribution and Ontogeny of 1S,3R-1-Aminocyclopentane-1,3-Dicarboxylic Acid-Sensitive and Quisqualate-Insensitive [3H]Glutamate Binding Sites in the Rat Brain

Abstract: Displacement of [³H]glutamate by 1 S ,3 R -1-aminocyclopentane-1,3-dicarboxylic acid and quisqualate (in the presence of saturating concentrations of ionotropic glutamate receptor agonists) was used to characterize optimal ionic conditions, distribution, and the ontogeny of glutamate receptor binding sites in rat brain. Using rat forebrain membranes or receptor autoradiography, optimal 1 S ,3 R -1-aminocyclopentane-1,3-dicarboxylic acid-sensitive [³H]glutamate binding was found in the presence of 100 m M bromide ions and in the absence of calcium ions. Under these conditions, [³H]glutamate binding was relatively quisqualate insensitive. In regions of the neonatal (11-day-old) and adult rat brain, this [³H]glutamate binding was highest in forebrain (striatum, cerebral cortex, and hippocampus) and hypothalamus/midbrain but was lower in the cerebellum, olfactory bulb, and pons/medulla regions. 1 S ,3 R -1-aminocyclopentane-1,3-dicarboxylic acid-sensitive and quisqualate-insensitive [³H]glutamate binding was present in the rat forebrain at 1 day of age and gradually increased more than twofold by day 50 (adult). Thus, in the presence of bromide ions and in the absence of calcium ions, [³H]glutamate labels a subpopulation of metabotropic glutamate receptors that are sensitive to 1 S ,3 R -1-aminocyclopentane-1,3-dicarboxylic acid but insensitive to quisqualate. Expression of [³H]glutamate binding under these conditions was both regionally and developmentally regulated in rat brain, suggesting that [³H]glutamate is labeling a distinct population of metabotropic glutamate receptors.     

Characterization of 5,7-Dichlorokynurenate-Insensitive d-[3H]Serine Binding to Synaptosomal Fraction Isolated from Rat Brain Tissues

Abstract: To explore target sites for endogenous d -serine that are different from the glycine site of the N -methyl- d -aspartate (NMDA) type glutamate receptor, we have studied the binding of d -[³H]serine to the synaptosomal P2 fraction prepared from the rat brain and peripheral tissues in the presence of an excess concentration (100 µ M ) of the glycine site antagonist 5,7-dichlorokynurenate (DCK). Nonspecific binding was defined in the presence of 1 m M unlabeled d -serine. Association, dissociation, and saturation experiments indicated that d -[³H]serine bound rapidly and reversibly to a single population of recognition sites in the cerebellar P2 fraction in the presence of DCK, with a K _D of 614 n M and a B _max of 2.07 pmol/mg of protein. d -Serine, l -serine, and glycine produced a total inhibition of the specific DCK-insensitive d -[³H]serine binding to the cerebellum with similar K _i values. Strychnine and 7-chlorokynurenate failed to inhibit the binding at 10 µ M . The profiles of displacement of the DCK-insensitive d -[³H]serine binding by various amino acids and glutamate and glycine receptor-related compounds differ from those of any other defined recognition sites. DCK-insensitive d -[³H]serine binding was at high levels in the cerebral cortex and cerebellum but very low in the kidney and liver. The present findings indicate that the DCK-insensitive d -[³H]serine binding site could be a novel candidate for a target for endogenous d -serine in mammalian brains.     

KINETICS OF [3H]ACETYLCHOLINE SYNTHESIS AND RELEASE IN PRIMARY CELL CULTURES FROM MAMMALIAN CNS

Abstract— The present study was undertaken to characterize the cholinergic system of primary cell cultures of mouse and rat CNS.
In confirmation of previous reports, primary cultures were found to contain choline acetyltransferase (ChAc). Furthermore they contain acetylcholine (ACh) as measured by two different bioassays. They also synthesize [³H]ACh from [³H]Choline offered to the cultures.
The formation of [³H]ACh is inhibited in the presence of hemicholinium-3 (10⁻⁶ m ) to 50% or ouabain (10⁻³ m ) to 20% of the values found in untreated cultures. Omission of Na ⁺ from the incubation solution also diminishes the [³H]ACh formation of the cells.
[³H]ACh is released upon depolarisation by K⁺ ions in a concentration dependent manner. The release can be prevented by lack of Ca²⁺ ions in the incubation solution.     

RELEASE OF [3H]GAMMA-AMINOBUTYRIC ACID FROM GLIAL CELLS IN RAT DORSAL ROOT GANGLIA.

Abstract— Desheathed rat dorsal root ganglia were incubated in a medium containing amino-oxyacetic acid and [³H]GABA. Under these conditions, [³H]GABA is taken up exclusively by the satellite glial cells in the ganglia. Efflux of [³H]GABA from the tissue was measured after passing the ganglia through a series of wash solutions. The spontaneous efflux of radioactivity, mostly [³H]GABA, was more rapid in the absence of amino-oxyacetic acid in the incubation and wash media.
Raising the potassium concentration in the wash media caused an increase in the efflux of [³H]GABA. This increase was sigmoidally related to the potassium concentration in the wash media, reaching a maximum at 64 m m -K⁺. The releasing effect of K⁺ was inhibited by removing calcium from the media. Reducing the calcium and raising the magnesium concentration in the wash solutions inhibited the increased efflux of [³H]GABA due to 64 m m -K⁺ by 48 per cent, while 5 mM-La³⁺ and diphenylhydantoin (0·005 and 0·5 m m ) had no effect on this increase.
Only a small increase in the efflux of [¹⁴C]glutamate was produced by 64 m m -K⁺ and it had no effect upon the effluxes of [³H]glycine, [³H]alanine or [³H]leucine. The efflux of lactate dehydrogenase was similarly unaffected by 64 mM-K⁺. The results suggest that glial cells in spinal ganglia can respond to depolarizing concentrations of potassium by releasing GABA in a calcium-dependent process.     

THE METABOLISM OF TRITIATED DOPAMINE IN REGIONS OF THE RAT BRAIN IN VIVO—II

Abstract— The levels of tritiated catecholamines and metabolites were measured in regions of the rat brain at intervals after the intraventricular injection of [³H]dopamine, [³H]nor-adrenaline and [³H]normetanephrine. The disappearance of catecholamines and appearance of metabolites with time and the regional turnover rates of these amines indicate that the major pathway of the metabolism of noradrenaline and dopamine actively released from physiological storage sites is to the neutral alcoholic metabolites. The acid metabolites, homovanillic acid and 3,4-dihydroxyphenylacetic acid appear to be only minor products of normal dopamine metabolism in rat brain regions including the striate, but are the main end products of the metabolism of excess exogenous dopamine.
The active metabolism of stored noradrenaline to alcohol metabolites is also indicated by the increase in neutral alcohol metabolites accompanying the increased noradrenaline turnover when rats were subjected to electroshock stress. Therefore in the rat brain, neutral alcohol metabolites of dopamine and noradrenaline have great significance in the study of physiological catecholamine turnover in any region.     

Glycine Receptors in the Human Substantia Nigra as Defined by [3H]Strychnine Binding

Abstract: Specific [³H]strychnine binding was used to identify the glycine receptor macromolecular complex in human spinal cord, substantia nigra, inferior olivary nucleus, and cerebral cortex. In material from control patients a high-affinity K d (3–8 n m ) was observed in the spinal cord and the substantia nigra, both the pars compacta and the pars reticulata. This is very similar to the values observed in the rat and bovine spinal cord (8 and 3 n m , respectively) and rat substantia nigra (12 n m ). In the human brain the distribution of [³H]strychnine binding (at 10 n m ) was: spinal cord – substantia nigra, pars compacta > substantia nigra, pars reticulata = inferior olivary nucleus > cerebral cortex. The binding capacity ( B _max) of the rat brain (substantia nigra or spinal cord) was approximately 10-fold that of the human brain. [ ³ H]Strychnine binding was significantly decreased in the substantia nigra from Parkinson's disease patients, both in the pars compacta (67% of control) and the pars reticulata (50% of control), but not in the inferior olivary nucleus. The results were reproduced in a preliminary experiment in rats with unilateral 6-hydroxydopamine lesions of the medial forebrain bundle. In the substantia nigra from patients who died with Huntington's disease, [³H]strychnine binding tended to be high (150% of control, NS) in both the pars compacta and the reticulata. [³H]Strychnine binding was unaltered in the substantia nigra of patients with senile dementia. Together with previous neurophysiological and neuropharmacological findings, those results support the hypothesis of glycine receptors occurring on dopamine cell bodies and/or dendrites in the substantia nigra.     

ETA and ETB Specific Ligands Synergistically Antagonize Endothelin-1 Binding to an Atypical Endothelin Receptor in Primary Rat Astrocytes

Abstract: Using a whole-cell binding procedure with long incubations at low temperature and subsequent acid stripping, we have characterized an atypical endothelin (ET) receptor in primary rat cortical astrocyte cultures. We found the following: (a) no competition for ¹²⁵I-ET-1 binding by the ET_A antagonists BQ-123 and LU 135252 or the ET_B agonist IRL 1620; (b) weak competition by the ET_B antagonist BQ-788 and by the predominant ET_B ligand ET-3; (c) potent synergistic competition of ET_A and ET_B ligands in combination for ¹²⁵I-ET-1 binding; (d) potent competition of ET-1 with any of the radioligands used, ¹²⁵I-ET-1, ¹²⁵I-IRL 1620, and [³H]BQ-123; (e) lack of competition of IRL 1620 and BQ-123 with the respective other radioligand; (f) shifting of the amount of acid-strippable ¹²⁵I-ET-1 binding from 20 to 80% by ET_B ligands and to 4% by ET_A ligands; and (g) as a control, typical ET_A and ET_B binding characteristics of the RAT-1 fibroblast and the U373MG astrocytoma cell line, respectively, under our assay conditions. The unusual binding properties of astrocytic ET receptors described in this study appear to be the result of several binding sites in the receptor for different ET ligands or ligand epitopes.     

CHARACTERIZATION OF THE BINDING OF [3H]MUSCIMOL, A POTENT γ-AMINOBUTYRIC ACID AGONIST, TO RAT BRAIN SYNAPTOSOMAL MEMBRANES USING A FILTRATION ASSAY

Abstract— The binding of [³H]muscimol, a potent GABA agonist, to crude synaptic membranes prepared from rat brain was studied using a filtration method to isolate membrane-bound ligand. Specific binding was found to be saturable and occurred to two binding sites of K _d5 5 and 30 n m . Binding was Na⁺-independent and enhanced by both freezing and Triton treatment. Regional and subcellular distribution studies and pharmacological characterization of specific [³H]muscimol binding are consistent with binding to the synaptic GABA receptor.     

Modulation of Neuropeptide FF Receptors by Guanine Nucleotides and Cations in Membranes of Rat Brain and Spinal Cord

Abstract: Using a radioligand binding assay, we examined ionic modulation and G protein coupling of neuropeptide FF(NPFF) receptors in membranes of rat brain and spinal cord. We found that NaCl (but not KCl or LiCl) and MgCl₂ increased specific ¹²⁵I-YLFQPQRFamide (¹²⁵I-Y⁸Fa) binding to NPFF receptors in both tissues in a dose-dependent manner, with optimal conditions being 60 m M NaCl and 1 m M MgCl₂. Guanine nucleotides dose-dependently inhibited specific ¹²⁵I-Y⁸Fa binding to rat brain and spinal cord membranes with maximal effects of 64 ± 6 and 71 ± 2%, respectively. The order of potency was nonhydrolyzable GTP analogues > GTP GDP > GMP, ATP. The guanine nucleotide inhibition was observed in the absence and presence of NaCl and MgCl₂. The mechanism of inhibition in spinal cord membranes appeared to be a reduction in the number of NPFF receptors; in one experiment, control K_D and B_max values were 0.068 n M and 7.2 fmol/mg of protein, respectively, and with 0.1 μ M guanylylimidodiphosphate the respective values were 0.081 n M and 4.9 fmol/mg, a 32% reduction in receptor number. Similar results were obtained with guanosine 5'-0-(3-thiotriphosphate). Our data suggest that ¹²⁵I-Y8Fa binding sites in rat CNS are G protein-coupled NPFF receptors regulated by GTP and cations.     

FAST AXOPLASMIC TRANSPORT OF A CALCIUM-BINDING PROTEIN IN MAMMALIAN NERVE

Abstract— Calcium is transported at a fast rate of 410 mm/day in cat sciatic nerve on injection of ⁴⁵Ca²⁺ into the L7 dorsal root ganglia. Nerve segments corresponding to the crest and the plateau regions of transported activity were analyzed by column chromatography on Sephadex G-100 and Biogel A 5m columns and the fast transported ⁴⁵Ca²⁺ found to be bound to a protein of 15,000 dalton. Using [³H]leucine as a precursor, a labeled calcium binding protein (CaBP) was found located at the same position in elution volumes from the columns as was the protein-bound ⁴⁵Ca^{2 +}. The level of [³H]-labeled CaBP in the crest and plateau regions were compared using column chromatography and polyacrylamide gel electrophoresis techniques and approx 3×4 times more [³H]-labeled activity was found in the crest as compared to the plateau. These findings indicate that Ca²⁺ is fast transported in association with the CaBP. The relation of CaBP to the transport filament model of axoplasmic transport and its possible role in nerve are discussed.     

AMMONIUM ION INHIBITION OF EVOKED RELEASE OF ENDOGENOUS GLUTAMATE FROM HIPPOCAMPAL SLICES

Abstract— The effect of pathophysiological levels (2-5 m m ) of ammonium chloride on the efflux of endogenous and exogenous [¹⁴C]glutamate from hippocampal slices was studied. The evoked release of glutamate which occurs dring tissue depolarization with 56 m m -KCl was greatly reduced when the tissue had been exposed to NH₄Cl for 40–80 min. This effect was seen whether or not glutamine (0.5 m m ) was present in the incubation medium. The effect was completely reversible. The spontaneous efflux and the evoked release of [¹⁴C]glutamate was, on the contrary, completely unaltered after exposure of the slice to ammonium ions. Nigher (20–36 m m ) amounts of NH₄Cl evoked a release of [¹⁴C]glutamate from the crude mitochondrial fraction, as did high concentrations of KCl. The results are discussed in relation to the compartmentation of glutamate metabolism and the pathogenesis of hepatic coma.