Effects of Anion Channel Blockers on Hyposmotically Induced Amino Acid Release From the In Vivo Rat Cerebral Cortex

A cortical cup model with continuous perfusion of artificial cerebrospinal fluid (containing 134 mM NaCl) was used to investigate the effects of anion channel blockers on the hyposmotically-induced release of amino acids from the in vivo rat cerebral cortex. The hyposmotic stimulus (25 mM NaCl) evoked a release of taurine, glutamate, aspartate, glycine, phosphoethanolamine and GABA. Topically applied anion channel blockers 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (1 mM); 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid (2 mM); 5-nitro-2-(3-phenylpropylamino) benzoic acid (350 M); niflumic acid (500 M); tamoxifen (20 M) and arachidonic acid (0.5 M) all significantly reduced the hyposmotically-induced release of taurine. The releases of glutamate, aspartate, glycine, phosphoethanolamine and GABA were variably susceptible to inhibition by these compounds. These results demonstrate that osmoregulatory processes in cortical cells, in vivo, involve amino acids, with taurine playing a dominant role. The efflux of taurine and, to a lesser extent, the other amino acids may be mediated by anion channels.     

Role of aspartate aminotransferase and mitochondrial dicarboxylate transport for release of endogenously and exogenously supplied neurotransmitter in glutamatergic neurons

Evoked release of glutamate and aspartate from cultured cerebellar granule cells was studied after preincubation of the cells in tissue culture medium with glucose (6.5 mM), glutamine (1.0 mM),d[³H] aspartate and in some cases aminooxyacetate (5.0 mM) or phenylsuccinate (5.0 mM). The release of endogenous amino acids and ofd-[³H] aspartate was measured under physiological and depolarizing (56 mM KCl) conditions both in the presence and absence of calcium (1.0 mM), glutamine (1.0 mM), aminooxyacetate (5.0 mM) and phenylsuccinate (5.0 mM). The cellular content of glutamate and aspartate was also determined. Of the endogenous amino acids only glutamate was released in a transmitter fashion and newly synthesized glutamate was released preferentially to exogenously suppliedd-[³H] aspartate, a marker for exogenous glutamate. Evoked release of endogenous glutamate was reduced or completely abolished by respectively, aminooxyacetate and phenylsuccinate. In contrast, the release ofd-[³H] aspartate was increased reflecting an unaffected release of exogenous glutamate and an increased psuedospecific radioactivity of the glutamate transmitter pool. Since aminooxyacetate and phenylsuccinate inhibit respectively aspartate aminotransferase and mitochondrial keto-dicarboxylic acid transport it is concluded that replenishment of the glutamate transmitter pool from glutamine, formed in the mitochondrial compartment by the action of glutaminase requires the simultaneous operation of mitochondrial keto-dicarboxylic acid transport and aspartate aminotransferase which is localized both intra- and extra-mitochondrially. The purpose of the latter enzyme apparently is to catalyze both intra- and extra-mitochondrial transamination of -ketoglutarate which is formed intramitochondrially from the glutamate carbon skeleton and transferred across the mitochondrial membrane to the cytosol where transmitter glutamate is formed. This cytoplasmic origin of transmitter glutamate is in aggreement with the finding thatd-[³H] aspartate readily labels the transmitter pool even when synthesis of endogenous transmitter is impaired in the presence of AOAA or phenylsuccinate.Special issue dedicated to Dr Elling Kvamme     

Kappa opioid agonists inhibit transmitter release from guinea pig hippocampal mossy fiber synaptosomes

Opioid agonists specific for the , , and opioid receptor subtypes were tested for their ability to modulate potassium-evoked release of L-glutamate and dynorphin B-like immunoreactivity from guinea pig hippocampal mossy fiber synaptosomes. The opioid agonists U-62,066E and (–) ethylketocyclazocine, but not the agonist [D-Ala²,N-MePhe⁴,Gly⁵-ol]-enkephalin (DAGO) nor the agonist [D-Pen^2,5]enkephalin (DPDE), inhibited the potassium-evoked release of L-glutamate and dynorphin B-like immunoreactivity. U-62,066E, but not DAGO or DPDE, also inhibited the potassium-evoked rise in mossy fiber synaptosomal cytosolic Ca²⁺ levels, indicating a possible mechanism for agonist inhibition of transmitter release. DAGO and DPDE were found to be without any effect on cytosolic Ca²⁺ levels or transmitter release in this preparation. The U-62,066E inhibition of the potassium-evoked rise in synaptosomal cytosolic Ca²⁺ levels was partially attenuated by the opioid antagonist quadazocine and insensitive to the -opioid specific antagonist ICI 174,864 and the opioid-preferring antagonists naloxone and naltrexone. Quadazocine also reversed U-62,066E inhibition of the potassium-evoked release of L-glutamate, but not dynorphin B-like immunoreactivity. These results suggest that opioid agonists inhibit transmitter release from mossy fiber terminals through both opioid and non- opioid receptor mediated mechanisms.     

17beta-estradiol effect on the extracellular concentration of amino acids in the glutamate excitotoxicity model in the rat

Estrogen has demonstrated a neuroprotective role in a rat model of glutamate excitotoxicity and other neurodegenerative disorders. We studied the effect of 17-estradiol on glutamate-induced increases in amino acids levels (aspartate, histidine, taurine and GABA) in the rat cortex. Local perfusion of glutamate produced a transient increase of aspartate, histidine, taurine and GABA in the extracellular fluid. Pretreatment with 17-estradiol significantly reduced the increases of taurine and moderately attenuated that of histidine, whereas aspartate and GABA releases were not modified. The effect of 17-estradiol on histidine release was reversed by the antiestrogen tamoxifen, suggesting a receptor-dependent mechanism. Good correlations between the volumes of the glutamate-induced lesions and the extracellular concentrations of taurine and aspartate were observed. These findings suggest that the attenuation of the glutamate-induced release of taurine by 17-estradiol may participate in the neuroprotective effects of 17-estradiol and that increased levels of aspartate and taurine are markers for the severity of the glutamate-induced cortical lesions.     

Displacement of endogenous glutamate withd-aspartate: an effective strategy for reducing the calcium-independent component of glutamate release from synaptosomes

d-aspartate was used in the present study to partially deplete the cytosolic pool of glutamate, which is released independent of extracellular Ca²⁺, prior to measuring the K⁺-evoked release of this endogenous acidic amino acid from rat hippocampal mossy fiber synaptosomes. This pretreatment is known to be an effective method for substantially reducing the Ca²⁺-independent component of glutamate release. The rate of glutamate efflux is dependent on the concentration of sodium ions in the external medium and can be stimulated by exposure of hippocampal mossy fiber synaptosomes to externald-aspartate (50 M). Following the partial displacement of this cytosolic pool of glutamate withd-aspartate, the K⁺-evoked release of the residual, presumably vesicular, pool of endogenous glutamate has a strict requirement for external calcium and is highly dependent on the extent to which depolarization elevates the level of free cytosolic calcium. It is concluded that the protocol described in this study for the displacement of cytosolic glutamate withd-aspartate provides a useful alternative method of controlling for the Ca²⁺-independent component of glutamate release in synaptosomal preparations.Abbreviations used Ca calcium - Ca²⁺ free calcium - EGTA (ethylene-dioxy)diethylenedinitrilotetraacetic acid - KBM Krebs-bicarbonate medium The animals involved in this study were procured, maintained and used in accordance with the Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources, National Research Council.     

Interactions of γ-L-glutamyltaurine with excitatory aminoacidergic neurotransmission

The in vitro effects of -L-glutamyltaurine on different stages of excitatory aminoacidergic neurotransmission were tested with -D-glutamyltaurine as reference. -L-Glutamyltaurine enhanced the K⁺-stimulated release of [³H]glutamate from cerebral cortical slices (25% at 0.1 mM) and slightly inhibited the uptake by crude brain synaptosomal preparations (about 10% at 1 mM). -L-Glutamyltaurine was also a weak displacer of glutamate and its agonists from their binding sites in brain synaptic membrane preparations, being, however, less selective to quisqualate (QA) sites than -D-glutamyltaurine. The basal influx of Ca²⁺ into cultured cerebellar granular cells was not affected by 1 mM -L-glutamyltaurine, but the glutamate- and its agonist-activated influx was significantly inhibited in low-Mg²⁺ (0.1 mM) and Mg²⁺-free media. The glutamate-evoked increase in free intracellular Ca²⁺ and the kainate-activated formation of cGMP in cerebellar slices were both markedly inhibited by 0.1 mM -L-giutamyltaurine. We propose that -L-glutamyltaurine may act as endogenous modulator in excitatory aminoacidergic neurotransmission.     

Stereospecificity and electrogenicity of amino acid transport in Riccia fluitans

In the aquatic liverwort Riccia fluitans, membrane depolarization (_m), change in membrane conductance (g_m), and current-voltage (I-V) characteristics in the presence of different amino acids as well as the uptake of ¹⁴C-labeled amino acids were measured. L-isomers of the tested amino acids generate larger electrical effects (_m, g_m) than D-isomers, and the I-V characteristics show that the positive electrical inward-current of 20 mA m^-2 generated by 0.5 mM D-serine is only about 50% of the current generated by adding 0.5 mM L-serine. Whereas - and -amino acids rapidly depolarize the membrane to the same extend, with -aminobutyric acid (-AB) and dipeptides no significant electrical effects have been measured. The uptake kinetics of ¹⁴C-labeled amino acids display three components: (I) A saturable high-affinity component with K_s-values of 48 M D-alanine, 12 M -aminoisobutyric acid (AIB), 9 M L-alanine, 8 M L-proline, and 6 M L-serine, respectively; (2) an apparently linear low-affinity component, and (3) an also linear but unspecific component at concentrations >20 times the given K_s-value. Uptake of ¹⁴C-labeled AIB can be inhibited competitively by all tested neutral amino acids, the L-isomers being more effective than the D-isomers, as well as by ammonium or methylamine. Vice versa, AIB competitively inhibits uptake of L-serine and L-alanine. It is concluded that an uncharged stereospecific carrier for the investigated amino acids exists in the plasmalemma of Riccia fluitans. Accumulation ratios of about 50 suggest secondary active transport driven by a transmembrane electro-chemical gradient (mainly _m) which is generated by the electrogenic proton pump. It is suggested that this carrier binds to the amino group forming either a charged binary complex with positively charged amines (Felle 1980), or an uncharged complex with -AB or dipeptides, whereas electrogenic transport of - and -amino acids is mediated by a ternary carrier complex, probably charged by a proton.Symbols and Abbreviations _m membrane potential (mV) - E_co equilibrium potential (mV) of the transport system - g_m membrane (slope) conductance (Sm^-2) - g_m change in g_m - I-V curve current-voltage curve - AIB -aminoisobutytric acid - -AB -aminobutyric acid     

Autotrophic CO2 fixation in Chlorobium limicola. Evidence for the operation of a reductive tricarboxylic acid cycle in growing cells

Chlorobium limicola was grown on a mineral salts medium with CO₂ as the main carbon source supplemented with specifically labeled ¹⁴C propionate and the incorporation of ¹⁴C into alanine ( intracellular pyruvate), aspartate ( oxaloacetate), and glutamate ( -ketoglutarate) was studied in long term labeling experiments. During growth in presence of propionate 30% of the cell carbon were derived from propionate and 70% from CO₂. Propionate was not oxidized to CO₂.All three amino acids were found to be labeled. The labeling patterns indicate that propionate was assimilated via propionyl CoA, methylmalonyl CoA and succinyl CoA. When 1-¹⁴C propionate was the labeled precursor no radioactivity was found in the carboxyl group(s) of alanine, aspartate and glutamate, excluding the incorporation of propionate into the amino acids via succinate oxidation to fumarate. With 1-¹⁴C propionate preferentially aspartate (C-3) and glutamate (C-2) became labeled, with 2-¹⁴C propionate alanine (C-3) and glutamate (C-4). These findings indicate that propionate was incorporated into the amino acids via succinyl CoA, -ketoglutarate, isocitrate, and citrate, followed by a si-type cleavage of citrate to oxaloacetate and acetyl CoA (or acetate). Similar experiments with U-¹⁴C acetate confirm these conclusions. Thus, all reactions of the proposed reductive tricarboxylic acid cycle could be demonstrated in autotrophically growing cells.     

Receptor Interactions of β-N-Oxalyl-L-α,β-Diaminopropionic Acid,the Lathyrus sativus Putative Excitotoxin,with Synaptic Membranes

Direct evidence for the excitotoxicity of -N-oxalyl-L-,-diaminopropionic acid (ODAP), the Lathyrus sativus neurotoxin has been studied by examining the binding of chemically synthesized [2,3 ³H]ODAP ([³H]ODAP) to synaptic membranes. [³H]ODAP binding to membranes was mostly nonspecific, with only a very low specific binding (15–20% of the total binding) and was also not saturable. The low specific binding of [³H]ODAP remained unaltered under a variety of assay conditions. A low B_max of 3.2 ± 0.4 pmol/mg and K_d 0.2 ± 0.08 M could be discerned for the high affinity interactions under conditions wherein more than 80–90% of the binding was nonspecific. While ODAP could inhibit the binding of [³H]glutamate to chick synaptic membranes with a K_i of 10 ± 0.9 M, even L-DAP, a non neurotoxic amino acid was also equally effective in inhibiting the binding of [³H]glutamate. The very low specific binding of [³H]ODAP to synaptic membranes thus does not warrant considering its interactions at glutamate receptors as a significant event. The results thus suggest that the reported in vitro excitotoxic potential of ODAP may not reflect its true mechanism of neurotoxicity.     

Excitatory Sulfur-Containing Amino Acid-Induced Release of [3H]GABA from Rat Olfactory Bulb

The effect of L-cysteine sulfinic acid (CSA) and L-homocysteic acid (HCA) on the release of tritiated -amino butyric acid ([³H]GABA), from the external plexiform layer (EPL) of the rat olfactory bulb, was compared with that of glutamate. These amino acids induced release of GABA was strongly inhibited by the glutamate uptake blocker, pyrrolidine-2,4-dicarboxylate (2,4,PDC) (50 M), while it was not inhibited by the specific GABA uptake blockers nipecotic acid (0.5 mM) or NO-711 (5M). Only the HCA induced GABA release was 60% inhibited by -alanine (0.5 mM), a glial GABA uptake blocker and 78% by the NMDA receptor antagonist 2-amino-5-phosphonopentanoic acid (AP-5) (100 M). The non-NMDA receptor antagonists 6-cyano-2,3-dihydroxy-7-nitro-quinoxaline (CNQX) up to 500 M had no effect on HCA or CSA stimulated GABA release. These results bring evidence for an excitatory role of HCA and CSA together with glutamate on GABAergic neuronal or glial elements, in the olfactory bulb. This role could be mediated through the reversal of the glutamate or/and the glial GABA transporter and through the activation of a NMDA type receptor.     

β-Dl-Methylene-aspartate,an inhibitor of aspartate aminotransferase,potently inhibitsl-glutamate uptake into astrocytes

[³H]Glutamate uptake into astrocytes in primary culture was potently inhibited by the aspartate analoguesl- andd-aspartic acid,Dl-threo--hydroxy-aspartic acid,l-aspartic acid--hydroxymate (IC50's: 136, 259, 168, and 560 M, respectively) and by -Dl-methylene-aspartate, a suicide inhibitor of asparate aminotransferase (IC50: 524 M), and by the endogenous sulphur-containing amino acidl-cysteinesulfinic acid (IC50: 114 M). [³H]Glutamate uptake was not significantly affected by either N-methyl-d-aspartate orDl-homocysteine thiolactone. These results demonstrate that other excitatory amino acids including aspartate andl-cysteinesulfinic acid (but excludingl-homocysteic acid) interact with the glutamate transport system of astrocytes. Inhibition of glutamate uptake may significantly increase the level of neuronal excitability.     

Studies on the release of exogenous and endogenous GABA and glutamate from rat brain synaptosomes

The aim of the present study was to determine whether exogenous radioactive GABA and glutamate previously taken up by rat brain synaptosomes are released preferentially with respect to the endogenous unlabeled amino acids. Preferential release was monitored by comparing the specific radioactivity of the amino acids released to that present in synaptosomes at the beginning and at the end of the release period. The GABA released spontaneously or by depolarizing the synaptosomes with high K⁺ in the presence of Ca²⁺ had the same specific radio-activity as that present in synaptosomes before or after superfusion. Depolarization with veratridine or superfusion with OH-GABA caused a moderate increase (15–20%) in the specific radioactivity of the GABA released and a corresponding slight decrease in that of superfused synaptosomes. In conditions causing a supraadditive release of exogenous and endogenous GABA (see ref. 13), the specific radioactivity of the GABA released was increased 20–30%. The GABA with higher-than-average specific radioactivity is probably representative of the cytoplasmic pool of this amino acid. The glutamate released spontaneously had a specific radioactivity lower than that present in synaptosomes at the start of superfusion, and also the specific radioactivity in superfused synaptosomes was lower than at the start of superfusion. The glutamate released by aspartate (by heteroexchange), by veratridine, or by high K⁺ had a specific radioactivity higher than that of the amino acid released spontaneously, similar to that present in synaptosomes at the start of superfusion, and higher than that found in superfused synaptosomes. These findings suggest that exogenous radioactive glutamate is released preferentially with respect to the endogenous amino acid and to the glutamate synthesized from glucose during the superfusion period.     

Electrically induced release of amino acids formed from (U14C) glucose in rat brain cortex slices,studied by a simplified dansylation procedure

The nonessential amino acids glutamate, aspartate, glutamine, -minobutyrate (GABA), alanine, glycine, and proline present in rat thin brain cortex slices were labeled by in vitro incubation of these with [U-¹⁴C]glucose, and the efflux of such endogenous radioactive amino acids and of lactate was studied in a superfused system, under control conditions or when the slices were depolarized by various procedures. When electrical stimuli known to induce selective neurotransmitter release (1 or 1.5 volt, sine wave 60 Hz) were applied for 10 sec to the slices, no significant increase in amino acid efflux was found. When more intense stimuli (4 volt, 60 Hz) were applied for 60 sec, or extracellular potassium was raised to 56 mM, both conditions being known to induce nonselective substance release, the efflux of essentially all amino acids and of lactate was markedly increased. Increases in efflux were proportionately larger for glutamate, aspartate, and -aminobutyrate, and this could be accounted for by their greater intracellular chemical (or electrochemical) potentials, but not because of a selective release mechanism for them. Amino acids were analyzed as their 1-dimethylaminonaphthalene-5-sulfonyl (dansyl) derivatives, by a modification of existing procedures in which the dansyl (DNS) derivatives were efficiently extracted from acidified incubation fluid into an organic phase. This rapidly desalted the derivatives and allowed their concentration and chromatographic separation on thin-layer silica gel sheets with little loss.     

β-Alanine Release from the Adult and Developing Hippocampus Is Enhanced by Ionotropic Glutamate Receptor Agonists and Cell-Damaging Conditions

The release of the inhibitory amino acid -alanine was investigated in hippocampal slices from adult (3-month-old) and developing (7-day-old) mice, using a superfusion system. The release was enhanced by -alanine itself and the structural analogs taurine and -aminobutyrate. It was dependent on Na⁺, but independent of Ca²⁺ in both mature and immature hippocampus, being thus mostly mediated by uptake carriers operating in an outward direction. The release was potentiated in the developing mice, but not affected in the adults, by the ionotropic glutamate receptor agonists N-methyl-D-aspartate, kainate, 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate and tetrazolylglycine in a receptor-mediated manner. Cell-damaging conditions, including hypoxia, hypoglycemia, ischemia, oxidative stress and the presence of free radicals, greatly enhanced -alanine release at both ages, but more markedly in the adults. The great amounts of -alanine, together with the inhibitory amino acids taurine and -aminobutyrate, released simultaneously with the excitatory amino acids in the hippocampus may constitute an important protective mechanism against excitotoxicity, which leads to neuronal death.     

Salinity dependence,uptake kinetics,and specificity of amino-acid absorption across the body surface of the oligochaete annelidEnchytraeus albidus

Enchytraeus albidus is able to absorb dissolved¹⁴C-labeled neutral amino acids (glycine, L-alanine, L-valine,-aminoisobutyric acid) and an amino-acid mixture from ambient water across the body surface against considerable concentration gradients. Saturation kinetics and susceptibility of glycine uptake to competitive inhibition by alanine suggest mediated transport. Absorption of neutral amino acids is an active process. Exchange diffusion of preloaded-aminoisobutyric acid against external glycine or-aminoisobutyric acid could not be detected. Results on inhibition of glycine uptake by a variety of low-molecular-weight substances indicate that glycine absorption is highly specific for neutral amino acids and somewhat less for basic amino acids; it is unspecific for non--amino acids, acidic amino acids, carbohydrates, and organic acids. Rates of transintegumentary net influx of glycine are nearly identical to¹⁴C-glycine influx, suggesting that only small amounts of amino acids are released, as compared with the capacity for uptake. Thus,¹⁴C-amino-acid influx data are used for characterization of the uptake system. Glycine uptake is positively correlated to external salinity. In fresh water, absorption is nearly zero; between 10 and 20 S, uptake increases markedly reaching maximum values at 30 S; these remain almost constant at 40 S. Transport constants and maximum uptake rates increase with rising salinities. Since absorption of glycine and L-valine is susceptible to sodium depletion, similar mechanisms presumably underly salinity-dependent uptake of amino acids and sodium-dependent solute transport. Oxygen consumption is not significantly modified by different external salinities. Estimates of nutritional profit gained from absorption of amino acids vary between 4 and 15 % of metabolic rate for glycine absorption and between 10 and 39 % for uptake of an amino-acid mixture, according to external concentrations (10 and 50 µM) and salinities (20 and 30 S).     

Baclofen-induced,calcium-dependent stimulation of in vivo release ofd-[3H]aspartate from rat hippocampus monitored by intracerebral microdialysis

The release ofd-[³H]aspartate (used as a tracer for endogenous glutamate and aspartate) was studied at high K⁺ (100 mM) and under ischemia in rats implanted with 0.3 mm diameter dialysis tubing through the hippocampus. The effect on thed-[³H]aspartate release of the two -aminobutyric acid (GABA) agonists 4,5,6,7-tetrahydroisoxazolo[5,4-c]-pyridin-3-ol (THIP) and (±)--(p-chlorophenyl)GABA (baclofen), which specifically activate GABA_A and GABA_B receptors, respectively, was studied. Initial experiments employing HPLC analysis showed a coincident increase in the amounts of glutamate, aspartate and the amount of radioactivity following introduction of K⁺ (100 mM) or a period of ischemia suggesting that thed-[³H]aspartate labels the transmitter pools of the two amino acids under the present experimental conditions. The presence of 10 mM baclofen or 10 mM THIP in the perfusion medium did not inhibit ischemia inducedd-[³H]aspartate release. On the contrary, 10 mM baclofen alone (but not 0.1 or 1 mM) in the perfusion medium induced release ofd-[³H]aspartate in a calcium dependent manner, whereas 10 mM THIP had no significant releasing effect.Special issue dedicated to Dr. Elling Kvamme     

Energy Metabolism of Synaptosomal Subpopulations from Different Neuronal Systems of Rat Hippocampus: Effect of L-Acetylcarnitine Administration In Vivo

The maximum rate (V_max) of some enzyme activities related to glycolysis, Krebs' cycle, acetylcholine catabolism and amino acid metabolism were evaluated in different types of synaptosomes obtained from rat hippocampus. The enzyme characterization was performed on two synaptosomal populations defined as large and small synaptosomes, supposed to originate mainly from the granule cell glutamatergic mossy fiber endings and small cholinergic nerve endings mainly arising from septohippocampal fiber synapses, involved with cognitive processes. Thus, this is an unique model of pharmacological significance to study the selective action of drugs on energy metabolism of hippocampus and the sub-chronic i.p. treatement with L-acetylcarnitine at two different dose levels (30 and 60 mg · kg^–1, 5 day a week, for 4 weeks) was performed. In control animals, the results indicate that these two hippocampal synaptosomal populations differ for the potential catalytic activities of enzymes of the main metabolic pathways related to energy metabolism. This energetic micro-heterogeneity may cause their different behaviour during both physiopathological events and pharmacological treatment, because of different sensitivity of neurons. Therefore, the micro-heterogeneity of brain synaptosomes must be considered when the effect of a pharmacological treatment is to be evaluated. In fact, the in vivo administration of L-acetylcarnitine affects some specific enzyme activities, suggesting a specific molecular trigger mode of action on citrate synthase (Krebs' cycle) and glutamate-pyruvate-transaminase (glutamate metabolism), but mainly of small synaptosomal populations, suggesting a specific synaptic trigger site of action. These observations on various types of hippocampal synaptosomes confirm their different metabolic machinery and their different sensitivity to pharmacological treatment.     

Effect of chemical modifiers of amino acid residues on proton conduction by the H+-ATPase of mitochondria

The effect of chemical modifiers of amino acid residues on the proton conductivity of H⁺-ATPase in inside out submitochondrial particles has been studied. Treatment of submitochondrial particles prepared in the presence of EDTA (ESMP) with the arginine modifiers, phenylglyoxal or butanedione, or the tyrosine modifier, tetranitromethane, caused inhibition of the ATPase activity. Phenylglyoxal and tetranitromethane also caused inhibition of the anaerobic release of respiratory H⁺ in ESMP as well as in particles deprived of F₁ (USMP). Butanedione treatment caused, on the contrary, acceleration of anaerobic proton release in both particles. The inhibition of proton release caused by phenylglyoxal and tetranitromethane exhibited in USMP a sigmoidal titration curve. The same inhibitory pattern was observed with oligomycin and withN,N-dicyclohexylcarbodiimide. In ESMP, relaxation of H⁺ exhibited two first-order phases, both an expression of the H⁺ conductivity of the ATPase complex. The rapid phase results from transient enhancement of H⁺ conduction caused by respiratory H⁺ itself. Oligomycin,N,N-dicyclohexylcarbodiimide, and tetranitromethane inhibited both phases of H⁺ release, and butanedione accelerated both. Phenylglyoxal inhibited principally the slow phase of H⁺ conduction. In USMP, H⁺ release followed simple first-order kinetics. Oligomycin depressed H⁺ release, enhanced respiratory H⁺, and restored the biphasicity of H⁺ release. Phenylglyoxal and tetranitromethane inhibited H⁺ release in USMP without modifying its first-order kinetics. Butanedione treatment caused biphasicity of H⁺ release from USMP, introducing a very rapid phase of H⁺ release. Addition of soluble F₁ to USMP also restored biphasicity of H⁺ release. A mechanism of proton conduction by F _o is discussed based on involvement of tyrosine or other hydroxyl residues, in series with the DCCD-reactive acid residue. There are apparently two functionally different species of arginine or other basic residues: those modified by phenylglyoxal, which facilitate H⁺ conduction, and those modified by butanedione, which retard H⁺ diffusion.     

Study of amino acid formation during palmitate oxidation in rat brain mitochondria

The interrelation of palmitate oxidation with amino acid formation in rat brain mitochondria has been investigated in purified mitochondria of nonsynaptic origin by measuring the formation of aspartate, -ketoglutarate, and glutamate during palmitate oxidation, and also by assaying¹⁴C-products of [1-¹⁴C]palmitate oxidation. Oxidation of palmitate (or [1-¹⁴C]palmitate) resulted in the formation of aspartate (or¹⁴C-aspartate), and the oxidation was inhibited by aminooxyacetate (an inhibitor of transaminase), Palmitate oxidation also resulted in -ketoglutarate formation, which was sensitive to the effect of aminooxyacetate. Addition of NH₄Cl was found to increase¹⁴C-products and formation of -ketoglutarate, whereas glutamate formation was not increased unless the rate of palmitate oxidation was reduced by 50% by aminooxyacetate or -ketoglutarate was added exogenously. Exogenous -ketoglutarate was found to decrease¹⁴C-products, but not aspartate formation. These results indicated that palmitate oxidation was closely related to aspartate formation via aspartate aminotransferase. During palmitate oxidation without aminooxyacetate or added -ketoglutarate, however, -ketoglutarate was not available for glutamate formation via glutamate dehydrogenase. We discuss the possibility that this was because (a) oxidative decarboxylation of -ketoglutarate to form succinyl-CoA was favored over glutamate formation for the competition for -ketoglutarate in the same pool, and (b) the pool of -ketoglutarate produced in the aspartate aminotransferase reaction did not serve as substrate for glutamate formation.     

Mode of uptake of sterol by Arthrobacter simplex

The mechanism of uptake of water-insoluble -sitosterol by a newly isolated strain of Arthrobacter simplex SS-7 was studied. The production of an extracellular sterol-pseudosolubilizing protein during growth of A. simplex on -sitosterol was demonstrated by isolating the factor from the cell-free supernatant and its subsequent purification by Sephadex G-150 column chromatography. The M _r of the purified sterol-pseudosolubilizing protein determined by SDS–PAGE was 19kDa. The rate of sterol pseudosolubilization (5.2×10^–3g l^–1h^–1) could not adequately account for the rate of sterol uptake (72×10^–3g l^–1h^–1) and the specific growth rate (56×10^–3 h^–1). However in the unfavourable growth condition, when the cells were treated with sodium azide at the level of 30–60% of MIC, the sterol pseudosolubilization accounted for nearly 74% of the total growth containing 96% free cells. Cellular adherence to substrate particles was found to play an active role in the normal growth of the strain on -sitosterol. Unlike sodium acetate-grown cells, whose surface activity was negligible (60mNm^–1), the sterol-grown cells had strong surface activity (40mNm^–1). The high lipid content and long chain fatty acids in the cell-wall of -sitosterol-grown cells probably contribute to the high sterol adherence activity of the cells.