Effect of ferric nitrilotriacetate on predominantly cortical neuronal cell cultures

Predominately neuronal cell cultures were produced as described in previous communications. Neuronal cells were exposed to ferric nitrilotriacetate (Fe-NTA) at varying concentrations. Studies of the neuronal cells were performed at 13 and 20 days in culture. In addition to morphologic studies, biochemical assays including choline acetyltransferase (ChAT) activity, specific [³H]flunitrazepam (FLU) binding, clonazepam (CLO)-displaceable [³H]FLU binding, Ro5-4864-displaceable [³H]FLU binding, high-affinity [³H]GABA uptake, and protein determinations were performed. The data demonstrate that chelated ferric iron has an adverse effect on predominately neuronal cultures after 7 days of exposure as measured by choline acetyltransferase activity, while other measures remained unaffected; however, after 14 days of exposure all measures were significantly decreased. The effects of Fe-NTA exposure appear to be both concentration and duration-of-exposure related.     

Effect of ferric nitrilotriacetate on predominately cortical glial cell cultures

Cultured glial cells were exposed to ferric nitrilotriacetate (Fe-NTA) at varying concentrations. Studies of the exposed glial cells were performed at days 29 and 36 post-conceptional age (culture days 8 and 15). In addition to morphologic studies, biochemical assays including [³H]-flunitrazepam (FLU) specific binding, Ro5-4864-displaceable³H-FLU binding, and protein determinations were performed. At day 29 post-conceptional age, significant decreases in³H-FLU specific binding, Ro5-4864-displaceable³H-FLU binding, and protein determinations were discernible only in the presence of 100 M Fe-NTA. At day 36 post-conceptional age³H-FLU specific binding was significantly decreased at 20, 60, and 100 M Fe-NTA concentrations, while Ro5-4864-displaceable³H-FLU binding and protein determinations were significantly reduced at 60 and 100 M Fe-NTA concentrations. The effects of Fe-NTA exposure appear to be both concentration and duration-of-exposure related. When compared to previously reported neuronal cell culture, studies utilizing³H-FLU specific binding, Ro5-4864-displaceable³H-FLU binding, and protein determinations, glial cells appear to be significantly more resistant to chelated iron exposure.     

Simultaneous Isolation of Glial and Neuronal Fractions from Rat Brain Homogenates: Comparison of High-Affinity L-Glutamate Transport Properties

An improved three-step Percoll density gradient centrifugation technique is described for simultaneous isolation of glial plasmalemmal vesicles (GPV) and synaptosomal vesicles (SYN) from a rat brain homogenate. While electron microscopy revealed that fractions contained intact vesicles with markedly distinct morphological features, measures of high-affinity [³H]choline uptake, glutamine synthetase and carbonic anhydrase activities, as well as Western blot analyses for glial fibrillary acidic protein and neuron specific enolase, served to confirm the low level of neuronal contamination in GPV fractions as well as the low level of glial contamination in SYN fractions. In addition, GPV and SYN fractions were used to characterize the kinetic and pharmacological properties of sodium-dependent [³H]L-glutamate transport. In conclusion, these results demonstrate the usefulness of this method for obtaining highly-enriched, functionally viable populations of glial and neuronal elements which are suitable for studies of their respective cell functions in vitro.     

Neurochemical correlates of cyanide-induced hypoxic neuronal damage in vitro

Neuronal cortical cell cultures obtained from fetal mice were subjected to an hypoxic insult produced by sodium cyanide (1 mM) for 24 h. Neurochemical assays were performed 13–14 days after plating on intact cells in situ to determine if there was a specific pattern of cellular dysfunction in addition to morphologic change. Ro5-4864-displaceable benzodiazepine (BDZ) binding and high-affinity [³H]-alanine uptake were not reduced when compared to control values. However, specific and clonazepam-displaceable BDZ binding (81±4% and 50±9% of control values, respectively), high-affinity [³H]GABA uptake (75±2%), and choline acetyltransferase activity (82±2%) were significantly lower. When the data were expressed in terms of protein content, high-affinity [³H]-alanine uptake was significantlyincreased in cyanide-exposed and magnesium-treated cultures (123±5% and 117±3%, respectively) as was R05-4864-displaceable BDZ binding (152±14%), consistent with stimulation of nonneuronal BDZ binding and increased glial neurotransmitter uptake. Moreover, pretreatment of the cultures with magnesium effectively prevented both the morphologic and neurochemical evidence of hypoxic injury. These data lend further support to the notion that the release of excitatory neurotransmitters may mediate neurotoxicity in developing brain.     

GLUCOSE AND AMINO ACID METABOLISM IN ISOLATED NEURONAL AND GLIAL CELL FRACTIONS IN VITRO

Abstract—

• 1 The metabolism of three substrates, [U-¹⁴C]glucose, [U-¹⁴C]pyruvate and [U-¹⁴C]glutamate has been studied in vitro in neuronal and glial cell fractions obtained from rat cerebral cortex by a density gradient technique.
• 2 The mixed cell suspension, after washing, metabolized glucose and glutamate in a manner essentially similar to the tissue slice. Exceptions were a reduced ability to generate lactate from glucose and alanine from glutamate, and a lowered effect of added glucose in suppressing the production of aspartate from glutamate.
• 3 After 2 hr incubation with [U-¹⁴C]glucose, the concentration of the amino acids glutamate, glutamine, GABA, aspartate and alanine were raised in the neuronal, compared to the glial fraction to 234 per cent, 176 per cent, 202 per cent, 167 per cent and 230 per cent respectively although both were lower than in the tissue slice. Incorporation of radio-activity was absolutely lower in the neuronal fraction, however, and the specific activities of the amino acids were: glutamate 12 per cent, GABA 18 per cent, aspartate 34 per cent, and alanine 33 per cent of those in the glial fraction.
• 4 After the incubation with [U-¹⁴C]pyruvate, the pool size of the amino acids were higher than after incubation with glucose, except for GABA, which was reduced to one-third. The concentrations of the amino acids glutamate, glutamine, GABA, aspartate, and alanine in the neuronal fraction were respectively 46 per cent, 143 per cent, 105 per cent, 97 per cent, and 57 per cent of those in the glial. Thus, with the exception of alanine, the specific activity of the neuronal amino acids compared to the glial was little increased when pyruvate replaced glucose as substrate.
• 5 After 2 hr incubation with [U-¹⁴C]glutamate in the presence of non-radioactive glucose, the pool sizes of all the amino acids were increased in both neuronal and glial fractions, with the exception of neuronal alanine and glial glutamine. The concentrations of the amino acids glutamine, GABA, aspartate and alanine were raised in the neuronal fraction, compared to the glial, to 425 per cent, 187 per cent, 222 per cent, and 133 per cent respectively. The specific activities of all the amino acids were higher than with glucose alone with the exception of alanine, and neuronal GABA. Neuronal glutamine and aspartate had specific activities respectively 102 per cent and 84 per cent of glial.
• 6 An unidentified amino acid, with R_F comparable to that of alanine and specific activity close to that of glutamate, was also present after incubation. It was relatively concentrated in the neuronal fraction.
• 7 The distribution of the enzymes glutamate dehydrogenase, aspartate aminotransferase, glutamate decarboxylase and glutamine synthetase between the cell fractions was studied. With the exception of glutamine synthetase, none of the enzymes was lost from the cell fractions during their preparation. Only 14 per cent of the glutamine synthetase, compared with 75 per cent of total protein, was recovered in the fractions. Of the enzymes, glutamate dehydrogenase activity was 406 per cent, and glutamate synthetase activity 177 per cent in the neuronal fraction compared to the glial in the absence of detergent. In the presence of detergent, glutamate dehydrogenase control was 261 per cent, aspartate aminotransferase activity 237 per cent is the neuronal as compared to the glial fraction.
• 8 Incorporation of radioactivity into acid-insoluble material from either glutamate or pyruvate was twice as high into the neuronal as the glial fraction.
• 9 The extent to which these differences may be extrapolated back to the intact tissue is considered, and certain correction factors calculated. The significance of the observations for an understanding of the compartmentation of amino acid pools and metabolism in the brain, and the possible identification of such compartments, is discussed.

     

Effects of phorbol ester on immunoreactive protein kinase C,insulin binding,and glucose uptake in astrocytic glial and neuronal cells from the brain

Phorbol esters, potent stimulators of protein kinase C (PKC), stimulate [³H]2-deoxy-d-glucose (dGlc) uptake and [¹²⁵I] insulin binding in cultured glial cells but not neuronal cells from neonatal rat brains. Using an antibody to the and forms of PKC we have demonstrated that both neuronal and glial cells contain an immunoactive PKC of Mr 80 kD, although the PKC level in neurons is greater than 4-fold that in glia. The majority of immunoactive PKC (63%) is cytosolic in glial cells although the reverse is true in neuronal cells, in which 88% of the PKC is membrane-bound in the basal state. The most potent phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), stimulates a redistribution of this enzyme in neuronal and glial cells. The TPA-stimulated translocation of PKC from cytosol to membrane precedes TPA's effecs of [³H]dGlc uptake and insulin binding in glial cells.     

Neuronal and Glial Release of [3H]GABA from the Rat Olfactory Bulb

Abstract: GABA uptake and release mechanisms have been shown for neuronal as well as glial cells. To explore further neuronal versus glial components of the [³H]-γ-aminobutyric acid ([³H]GABA) release studies were performed with two different microdissected layers of the olfactory bulb of the rat: the olfactory nerve layer (ONL), consisting mainly of glial cells, and the external plexiform layer (EPL) with a high density of GABAergic dendritic terminals. In some experiments substantia nigra was used as a GABAergic axonal system and the trigeminal ganglia as a peripheral glial model. Spontaneous release of [³H]GABA was always lower in neuronal elements as compared with glial cells. A veratridine-evoked release was observed from the ONL but not from the trigeminal ganglia. Tetrodotoxin (TTX) abolished the veratridine-evoked release from the ONL, which also showed a partial inhibition when high magnesium concentrations were used in a Ca²⁺-free solution. β-Alanine was strongly exchanged with [³H]GABA from the ONL of animals with the olfactory nerve lesioned and from animals with no lesion; but only a small heteroexchange was found from the external plexiform layer. The β-alanine heteroexchange was able to deplete the releasable GABA store from the ONL of lesioned animals. In nonlesioned animals and the external plexiform layer, the veratridine-stimulated release of [³H]GABA was not significantly reduced after the β-alanine heteroexchange. Stimulation of the [³H]GABA release by high concentrations of potassium elicited a higher release rate from axonal terminals than from dendrites or glia. Neurones and glia showed a similar inhibition of [³H]GABA release when a high magnesium concentration was added to a calcium-free solution. When D-600 was used as a calcium-flux blocker no inhibition of the release was observed in glial cells, whereas an almost complete blockage was found in both neuronal preparations (substantia nigra and EPL). These results provide further evidence for differential release mechanisms of GABA from CNS neurones and glial cells.     

Substrate Specificity of [3H] Muscimol Binding to a Particulate Fraction of a Neuron-enriched Culture of Embryonic Rat Brain

Abstract: The effects of some GABA analogues and some drugs on the binding of [³H]muscimol (3.08 nM) to thoroughly washed subcellular particles prepared from a neuron-enriched culture of embryonic rat brain were examined using Na+-free Tris-citrate medium and a centrifugation method. Competition for [³H]muscimol binding sites by excess(10^?5 M) unlabelled GABA provided estimates of “specific” binding. In accord with in vivo neuropharmacological studies on GABA receptors and with in vitro studies on cerebral membrane preparations, [³H]muscimol binding was potently inhibited by muscimol itself (IC₅₀, 2.5 nM), GABA (1C₅₀, 43 nM), isoguvacine (IC₅₀, 61 nM), and 3-aminopropanesulphonic acid (IC₅₀, 160 nM), and less potently inhibited by the GABA antagonist bicuculline methobromide (IC₅₀, 800 nM). δ- Aminovaleric acid (IC₅₀, 2.6 μM), the glycinelp-alanine antagonist strychnine (IC₅₀, 6.6 μM), and the predominantly glial GABA uptake inhibitors β-alanine (IC₅₀, 23 μM) and p-proline (IC₅₀, 66 μM) also inhibited [³H]muscimol binding. Other inhibitors of Na+-dependent GABA uptake, (±)-nipecotic acid, L- 2,4-diaminobutyric acid, and guvacine, as well as picrotoxinin, were relatively inactive as inhibitors of [³H]muscimol binding (IC₅₀≥ 1 mM). In addition to revealing that GABA receptors are present on neuronal membranes before the formation of most synapses, this binding of [³H]muscimol that occurs to neuronal, but not to glial, membranes might be useful as a “neuronal marker” and for the further characterization and isolation of GABA receptors.     

The effect of iron on mammalian cortical neurons in culture

We added iron in the ferric form to predominantly neuronal, cortical cell cultures, and determined clonazepam-displaceable [³H]diazepam binding, choline acetyltransferase activity, high-affinity [³H]GABA uptake, and glutamic acid decarboxylase activity. Chronic exposure (14 days) to low concentrations (0.01, 0.04, and 0.1 g/ml) of added ferric iron resulted in a significant decrease in each of the measures studied.     

Subclasses of Adenosine Receptors in Brain Membranes from Adult Tissue and from Primary Cultures of Chick Embryo

Abstract: Membranes from adult chicken brain have high-affinity binding sites for N⁶-cyclohexyl[³H]adenosine (CHA) (K_D= 4 nM, Bmax = 0.6 pmol/mg protein). This CHA binding could be attributed to adenosine receptors of the A₁ type, since substituted adenosine analogs, e.g. N⁶-(l -2-phenylisopropyl)adeno sine (IC50 = 60 nM), were very potent displacers. Binding sites for 1,3-diethyl- 8-[³H]phenylxanthine (DPX) in adult brain membranes have a moderate affinity (K_D= 50 nM, Bmax = 1.5 pmol/mg). The association of DPX with these sites could be completely displaced by 8-phenyltheophylline (IC₅₀= 300 nM) and other xanthines, but only 45% of specific DPX binding could be displaced by phenylisopropyladenosine. This suggests that about half of DPX sites are putative A₁ receptors and the other half are of the A₂ type. Primary cultures of pure glial and neuronal cells from chick embryo brain were also examined for adenosine receptors. Specific binding of CHA could not be detected in these preparations, but both glial and neuronal membranes have specific sites for DPX. At a [³H]DPX concentration of 20 nM, specific binding was 50% higher (per mg protein) in glial than in neuronal membranes. The maximum binding of DPX to glial membranes (B_max= 1.6 pmol/mg) was comparable to values for adult brain, but the glial affinity (K_D= 90 nM) was somewhat less. Phenylisopropyladenosine was able to displace less than 20% of the total glial sites for DPX. This finding was in accord with the lack of CHA sites and demonstrates that A₁ receptors make little contribution to DPX binding in glial membranes. In decreasing order of potency, 8-phenyltheophylline, CHA, theophylline, caffeine, and 3-isobutyl-I-methylxanthine completely displace DPX association with glia. DPX binding to glial membranes thus appears due to a single class of receptors, which may prove to be of the A₂ type.     

The effect of fluorocitrate on transmitter amino acid release from rat striatal slices

In order to study the role of glutamine from glial cells for the synthesis of transmitter amino acids, the effect of the gliotoxic substance fluorocitrate on amino acid release from slices was investigated. In vivo treatment with 1 nmol fluorocitrate reduced the Ca²⁺ dependent K⁺ evoked release of endogenous glutamate and GABA from the slices, whereas the glutamine efflux decreased and alanine efflux increased. The K⁺ evoked release of [³H]d-aspartate increased during fluorocitrate treatment. The latter is consistent with an inhibited uptake ofd-aspartate into glial cells. Incubation of striatal slices with fluorocitrate (0.1 mM) decreased the glutamine efflux and increased the alanine efflux. Similar to the in vivo condition, fluorocitrate increased the K⁺ evoked [³H]d-asparate release, but the K⁺ evoked release of endogenous glutamate and GABA increased rather than decreased. The ratio between the K⁺ evoked release of exogenousd-aspartate to endogenous glutamate increased in both cases. The results suggest an important role of glial cells in the synthesis and inactivation of transmitter amino acids.Special Issue dedicated to Prof. Holger Hydén.     

NEURONAL-GLIAL CONTRIBUTIONS TO TRANSMITTER AMINO ACID METABOLISM: STUDIES WITH KAINIC ACID-INDUCED LESIONS OF RAT STRIATUM

Abstract– Various aspects of amino acid metabolism were studied in striatum of rats with unilateral, kainic acid-induced lesions. Tissue slices were prepared from the lesioned and the contralateral, unlesioned, striatum. The preparations were incubated with a mixture of d -[2-¹⁴C]glucose and [³H]acetate in a Krebs-Ringer bicarbonate medium to evaluate oxidative metabolism. Glutamate and aspartate levels were decreased in the slices prepared from the lesioned striata by 35-40% and that of GABA by 75% compared to the levels found in the slices from the contralateral striata; glutamine levels were not different in the two preparations. Glucose utilization was decreased 60% in the slices from the lesioned striatum; this was caused not only by decreased levels of glutamate, aspartate and GABA but also by a decreased rate of labelling of glutamate and aspartate. On the other hand, the metabolism of [³H]acetate was greatly increased. The specific activities of glutamate and aspartate were 4-5-fold higher in the slices from kainic acid-lesioned striata; those of glutamine and GABA were unchanged. Thus, there was a 6-7-fold increase in the ratio of ³H to ¹⁴C in the specific activities of glutamate, aspartate and GABA with no change in this ratio in glutamine. The labelling of glutamine relative to that of glutamate, especially from [³H]acetate, suggested that the compartmentation of the glutamate-glutamine system was greatly altered in the kainate-lesioned striatum which now more closely resembled a single compartment system. The activities of lactate dehydrogenase, glutamate dehydrogenase, GABA transaminase and ‘cytoplasmic’ aspartate aminotransferase were decreased in homogenates of lesioned striatum. Succinate dehydrogenase, glutaminase (phosphate-activated) and ‘mitochondrial’ aspartate aminotransferase activities were unchanged whilst that of glutamine synthetase was increased. The results are consistent with hypotheses concerning the assignment of labelled acetate metabolism to glial cells as well as the distribution of the above enzymes between glia, neurones and nerve endings.     

INCORPORATION IN VIVO OF RADIOACTIVE LEUCINE INTO NEURONAL AND GOAL NUCLEAR PROTEINS OF RAT BRAIN

Purified neuronal and glial nuclei were separated from rat brain cells. The fraction rich in neuronal nuclei contained 68 ± 9 per cent neuronal nuclei and the fraction rich in glial nuclei contained 89 ± 6 per cent glial nuclei. The fraction rich in neuronal nuclei isolated from cells of adult rat brain incorporated l -[4,5-³H]leucine into TCA-insoluble material at a rate comparable to those of the microsomal and the soluble fractions of the brain, and at a much higher rate than the fraction rich in glial nuclei. The proteins soluble in buffered-saline, the acid-soluble deoxyribonucleoproteins, and the residual proteins of the neuronal nuclei are apparently the proteins which account for the higher specific activity of neuronal proteins compared with glial nuclear proteins. In liver and kidney, the incorporation of [³H]leucine into nuclear proteins was lower than into other subcellular fractions from the same organs.     

Propionate increases neuronal histone acetylation, but is metabolized oxidatively by glia. Relevance for propionic acidemia

In propionic acidemia, propionate acts as a metabolic toxin in liver cells by accumulating in mitochondria as propionyl-CoA and its derivative, methylcitrate, two tricarboxylic acid cycle inhibitors. Little is known about the cerebral metabolism of propionate, although clinical effects of propionic acidemia are largely neurological. We found that propionate was metabolized oxidatively by glia: [3-(14)C]propionate injected into mouse striatum or cortex, gave a specific activity of glutamine that was 5-6 times that of glutamate, indicating metabolism in cells that express glutamine synthetase, i.e., glia. Further, cultured cerebellar astrocytes metabolized [3-(14)C]propionate; cultured neurons did not. However, both cultured cerebellar neurons and astrocytes took up [3H]propionate, and propionate exposure increased histone acetylation in cultured neurons and astrocytes as well as in hippocampal CA3 pyramidal neurons of wake mice. The inability of neurons to metabolize propionate may be due to lack of mitochondrial propionyl-CoA synthetase activity or transport of propionyl residues into mitochondria, as cultured neurons expressed propionyl-CoA carboxylase, a mitochondrial matrix enzyme, and oxidized isoleucine, which becomes converted into propionyl-CoA intramitochondrially. The glial metabolism of propionate suggests astrocytic vulnerability in propionic acidemia when intramitochondrial propionyl-CoA may accumulate. Propionic acidemia may alter both neuronal and glial gene expression by affecting histone acetylation.     

Receptors for phorbol esters are primarily localized in neurons: Comparison of neuronal and glial cultures

Binding of [³H]PDB has been measured in the present study to determine the levels of protein kinase C in the neuronal and astrocytic glial cells in culture from rat brain. Binding of [³H]PDB to homogenates of cultured neuronal cells from the brains of normotensive and hypertensive rats was time-dependent and specific. The relative potency for competition by various phorbol esters to [³H]PDB binding was TPA > -PDD > POE > -PDD 4phorbol. Scatchard analysis showed that neuronal cultures from normotensive rat brains contained 2–3 fold more phorbol ester receptors compared with the glial cultures from the same brains. No differences in theK _d andB _max were observed between neuronal cultures from normotensive and spontaneously hypertensive rat brains. These studies suggest that the phorbol ester receptors are primarily localized in neuronal cells.     

Rat mesencephalic neuronal cells cultured for different periods as a model of dopamine transporter ontogenesis

Ventral mesencephalic neurons contained only low-affinity and sodium-independent binding sites of [³H]WIN 35,428 (marker of dopamine transporter) during the first 10d in primary cultures. These sites were present in cytosol, and they are not very probably related to dopamine transporter. After 12 d in culture, membrane-bound, high-affinity, and sodium-dependent [³H]WIN 35,428 binding sites were detected. In membranes prepared from cells 14 d in culture, cocaine displaced [³H]WIN 35,428 binding with similar potency to that in striatal membranes of adult rat brain. The high-affinity [³H]WIN 35,428 binding sites in mesencephalic neuronal cell cultures are very probably related to dopamine transporter. The development of high-affinity [³H]WIN 35,428 binding sites in neurons cultured for different time periods could be a useful model of dopamine transporter ontogenesis.     

Ca2+-Guanine Nucleotide Interactions in Brain Membranes. II. Characteristics of [3H]Guanosine Triphosphate and [3H]β, γ-Imidoguanosine 5'-Triphosphate Binding and Catabolism in the Rat Hippocampus and Striatum

Abstract: With [³H]guanosine triphosphate ([³H]GTP) and [³H]β, γ -imidoguanosine 5′-triphosphate ([³H]GppNHp) as the labelled substrates, both the binding and the catabolism of guanine nucleotides have been studied in various brain membrane preparations. Both labelled nucleotides bound to a single class of noninteracting sites (K_D= 0.1-0.5 μm ) in membranes from various brain regions (hippocampus, striatum, cerebral cortex). Unlabelled GTP, GppNHp, and guanosine diphosphate (GDP) but not guanosine monophosphate (GMP) and guanosine competitively inhibited the specific binding of [³H]guanine nucleotides. Calcium (0.1–5 mm ) partially prevented the binding of [³H]GTP and [³H]GppNHp to hippocampal and striatal membranes. This resulted from both an increased catabolism of [³H]GTP (into [³H]guanosine) and the likely formation of Ca-guanine nucleotide^2- complexes. The blockade of guanine nucleotide catabolism was responsible for the enhanced binding of [³H]GTP to hippocampal membranes in the presence of 0.1 mm -ATP or 0.1 mm -GMP. Striatal lesions with kainic acid produced both a 50% reduction of the number of specific guanine nucleotide binding sites and an acceleration of [³H]GTP and [³H]GppNHp catabolism (into [³H]guanosine) in membranes from the lesioned striatum. This suggests that guanine nucleotide binding sites were associated (at least in part) with intrinsic neurones whereas the catabolising enzyme(s) would be (mainly) located to glial cells (which proliferate after kainic acid lesion). The characteristics of the [³H]guanine nucleotide binding sites strongly suggest that they may correspond to the GTP subunits regulating neurotransmitter receptors including those labelled with [³H]5-hydroxytryptamine ([³H]5-HT) in the rat brain.     

Inhibition of Astrocyte Glutamine Production by α-Ketoisocaproic Acid

Abstract: We have evaluated the effect of α-ketoisocaproic acid (KIC), the ketoacid of leucine, on the production of glutamine by cultured astrocytes. We used ¹⁵NH₄Cl as a metabolic tracer to measure the production of both [5-¹⁵N]glutamine, reflecting amidation of glutamate via glutamine synthetase, and [2-¹⁵N]glutamine, representing the reductive amination of 2-oxoglutarate via glutamate dehydrogenase and subsequent conversion of [¹⁵N]-glutamate to [2-¹⁵N]glutamine. Addition of KIC (1 mM) to the medium diminished the production of [5-¹⁵N]glutamine and stimulated the formation of [2-¹⁵N]glutamine with the overall result being a significant inhibition of net glutamine synthesis. An external KIC concentration as low as 0.06 mM inhibited synthesis of [5-¹⁵N]glutamine and a level as low as 0.13 mM enhanced labeling (atom% excess) of [2-¹⁵N]glutamine. Higher concentrations of KIC in the medium had correspondingly larger effects. The presence of KIC in the medium did not affect flux through glutaminase, which was measured using [2-¹⁵N]glutamine as a tracer. Nor did KIC inhibit the activity of glutamine synthetase that was purified from sheep brain. Addition of KIC to the medium caused no increased release of lactate dehydrogenase from the astrocytes, suggesting that the ketoacid was not toxic to the cells. KIC treatment was associated with an approximately twofold increase in the formation of ¹⁴CO₂ from [U-¹⁴C]glutamate, indicating that transamination of glutamate with KIC increases intraastrocytic α-ketoglutarate, which is oxidized in the tricarboxylic acid cycle. KIC inhibited glutamine synthesis more than any other ketoacid tested, with the exception of hydroxypyruvate. The data indicate that KIC diminishes flux through glutamine synthetase by lowering the intraastrocytic glutamate concentration below the K_m of glutamine synthetase for glutamate, which we determined to be ～7 mM.     

Substance P-Induced Reduction in the Initial Accumulation of Cytosolic myo-[3H]Inositol in Rat Parotid Acinar Cells Mediated by the NK1 Tachykinin Receptor

Abstract: Stimulation of rat parotid acinar cells by the tachykinin neurokinin (NK) 1 receptor agonist substance P (SP) resulted in a significant reduction in the initial accumulation of cytosolic myo-[³H]inositol. This effect was rapid, because a reduction of ～15% could be seen already at 30 s, with the maximal effect (～45%) being observed at 15 min. The response to SP stimulation Was temperature dependent, because at 4°C no reduction was found, jln addition, at 4°C, cytosolic myo-[³H]inositol represented only 10% of the labeled inositol accumulated at 37°C. The SP-induced reduct on in cytosolic ravo[³H]inositol accumulation was concentration dependent; the EC₅₀ obtained for SP was 5.8 ± 2.5 nM. Spantide [N Arg¹, D-Trp⁷⁹, Leu]SP), a SP antagonist, used at a concentration oif 10⁵ A/, gave a competitive shift of the dose-response curve to SP. Various tachykinins and their analogs were evaluated for their ability to reduce cytosolic mvo-[³H]inositol. [L-Pro⁹]SP and SP methyl ester, two highly selective agonists of NK1 receptors, reduced the initial accumulation of myo-H]inositol with EQo values of 2.3 and 67.0 nM, respectively. Long SP C-terminal fragments were more potent than shorter ones. SP N-terminal fragments and SP free acid were -without effect. [Pro⁷]NKB, a selective NKB analog, had no effect. The rank order of potency of mammalian tachykinins was SP > NKA > NKB. These findings and the close correlation between EC50 values and IC₅₀ values obtained in binding studies implicate the NK 1 receptor. In addition, stimulation of muscarinic receptors by carbachol alscp resulted in a reduction in level of cytosolic mjw-[³H]inositol, with this effect being reversed by atropine. Moreover, atropine was unable tjo alter the SP-induced reduction in cytosolic myo-[³H]inositol accumulation. Other neurotransmitters, such as glutamic acid, serotonin, chplecystokinin, neurotensin, bradykinin, and neuropeptide Y, were without effect on initial cytosolic myo-[³H]inositol accumulation. In conclusion, NK1 and muscarinic receptors seem to regulate the membrane transport of inositol in acinar cells of the rat parotid gland. Measurement of the initial accumulation of cytosolic myo-[³H]inositol in this tissue could profitably be adopted as a very simple, rapid, [sensitive, and specific biochemical procedure for screening the activity of potential agonists and antagonists at NK1 receptors.     

Regulation of glutamine synthetase, aspartokinase, and total protein turnover in Klebsiella aerogenes

When suspensions of Klebsiella aerogenes are incubated in a nitrogen-free medium there is a gradual decrease in the levels of acid-precipitable protein and of aspartokinase III (lysine-sensitive) and aspartokinase I (threonine-sensitive) activities. In contrast, the level of glutamine synthetase increases slightly and then remains constant. Under these conditions, the glutamine synthetase and other proteins continue to be synthesized as judged (a) by the incorporation of [¹⁴C]leucine into the acid-precipitable protein fraction and into protein precipitated by anti-glutamine synthetase antibodies, (b) by the fact that growth-inhibiting concentrations of chloramphenicol also inhibit the incroporation of [¹⁴C]leucine into protein and into protein precipitated by anti-glutamine synthetase antibody, and (c) by the fact that chloramphenicol leads to acceleration in the loss of aspartokinases I and III and promotes a net decrease in the level of glutamine synthetase and its cross-reactive protein. The loss of aspartokinases I and III in cell suspensions is stimulated by glucose and is inhibited by 2,4-dinitrophenol. Glucose also stimulates the loss of aspartokinases and glutamine synthetase in the presence of chloramphenicol. Cell-free extracts of K. aerogenes catalyze rapid inactivation of endogenous glutamine synthetase as well as exogeneously added pure glutamine synthetase. This loss of glutamine synthetase is not associated with a loss of protein that cross-reacts with anti-glutamine synthetase antibodies. The inactivation of glutamine synthetase in extracts is not due to adenylylation. It is partially prevented by sulfhydryl reagents, Mn²⁺, antimycin A, 2,4-dinitrophenol, EDTA, anaerobiosis and by dialysis. Following 18 h dialysis, the capacity of extracts to catalyze inactivation of glutamine synthetase is lost but can be restored by the addition of Fe²⁺ (or Ni²⁺ together with ATP (or other nucleoside di- and triphosphates. After 40–60 h dialysis Fe³⁺ together with NADH (but not ATP) are required for glutamine synthetase inactivation. The results suggest that accelerated protein degradation in cells exposed to nitrogen-limited conditions reflects the differential destruction of some proteins, including aspartokinases I and III, in order to sustain the biosynthesis of others such as glutamine synthetase. The loss of glutamine synthetase activity in cell-free extracts is likely mediated in part by mixed-function oxidation systems and could represent a ‘marking’ step in protein turnover.