Characteristics of choline acetyltransferase and cholinesterases in two types of cultured cells from embryonic chick brain

Cells that were mechanically dissociated from the brains of 7-day-old chick embryos were grown in culture for 7–8 days. Two major cell populations were observed: (1) cells that aggregated and sent out processes, (2) flat cells that proliferated rapidly and formed a confluent layer by day 4 of culture. Many of the cells of the first type had the morphological, histochemical and biochemical attributes of neurons. They possessed choline acetyltransferase (ChAc) and acetylcholinesterase (AChEs) activities. The flat cells possessed neither of the activities, but did have butyrylcholinesterase (BuChEs) activity and a choline independent acetylase activity (CIA) that may be carnitine acetyltransferase.The activities of ChAc and AChEs in the cultured neurons increased approximately 9-fold and 5-fold, respectively, over an 8-day period. The patterns of change of these enzymes were not unlike those seen in vivo in intact developing chick brain.The addition of thyroxine (10^?6M) to these cultures increased the activities of neuronal AChEs and flat cell BuChEs by 30–70%.     

Different properties of two brain extracts separated in sephadex G-50 that modify synaptosomal ATPase activities

We have previously reported that Na⁺,K⁺-ATPase of nerve ending membranes is stimulated by catecholamines only in the presence of a brain soluble fraction. The filtration of this soluble fraction through Sephadex G-50 permitted the separation of two extracts of maximal UV absorbance (peaks I and II) which showed different effects on ATPases. Peak I stimulated both Na⁺,K⁺-ATPase and Mg²⁺-ATPase activities and peak II inhibited Na⁺,K⁺-ATPase activity. We have now studied the activity of ATPases in the presence of the whole eluate obtained from the Sephadex G-50 column. It was observed that maximal effects on ATPases were obtained with peaks I and II. Peak I and peak II fractions were unable to modify the activity of acetylcholinesterase or 5-nucleotidase present in the synaptosomal membranes. The stimulatory effect of peak I on ATPases was concentration dependent (up to 1100), it was stable at different pHs and it was reverted by catecholamines. The inhibitory effect of peak II on Na⁺,K⁺-ATPase was concentration dependent (up to 150,000), it was stable only at acid pH, and it was partially reverted by catecholamines. These findings indicate that the factors responsible for the effects of peaks I and II have different properties and that their actions on ATPases show enzyme specifity.     

Metabolic routes of GABA formation in chick embryo brain

Sobue and Nakajima (1978) reported that GABA formation from putrescine is significant in chick embryo brain between days 6 and 8 of incubation. They attributed an important functional role to the putrescine-derived GABA. We found that depletion of putrescine and spermidine in chick embryos by inhibition of ornithine decarboxylase activity did not decrease the in vivo rate of GABA formation, showing that putrescine is, from a quantitative point of view, a negligible source for GABA in chick embryo brain. The changes of brain GABA levels obtained after administration of glutamate decarboxylase inhibitors and in vitro determinations of glutamate decarboxylase activity were compatible with the assumption that GABA is mainly formed by decarboxylation of l-glutamate, even during early brain development. Participation of the NAD⁺-dependent, aerobic transformation of glutamate into GABA (Seiler and Wagner, 1976) in the overall GABA production of chick embryo brain could, however, not be excluded.     

Ethanol exerts differential effects on high affinity choline uptake in neuron-enriched cultures from 8-day-old chick embryo cerebral hemispheres

Neuronal-enriched cultures were prepared from 8-day-old chick embryo cerebral hemispheres and exposed to ethanol (50 mM) from day 4 to 8 in culture. At day 8, both control and ethanol-treated cultures were processed for [³H]choline uptake in situ. Uptake was performed on cultures containing either Na⁺-plus or Na⁺-free (Li⁺) HEPES buffer. Total choline uptake as well as Na⁺-dependent and Na⁺-independent choline uptake were calculated. The K_m and V_max were calculated using the Lineweaver-Burke analysis. Our analysis of the data revealed that ethanol-treated cultures exhibited two values for V_max, one similar to that found in control cultures and one significantly lower than controls. No differences were observed in K_m values between control and ethanol-treated cultures. We interpret the low V_max to represent a population of cholinergic neurons which have been arrested at an immature stage as a result of ethanol insult.     

Hydrolysis of exogenous [3H]phosphatidylcholine by brain membranes and cytosol

Phosphatidylcholine, in addition to the widely studied inositol phospholipids, is cleaved to produce second messengers in neuronal signal transduction processes. Because of the difficulty in labelling and measuring the metabolism of endogenous phosphatidylcholine in brain tissue, we investigated the utility of measuring the hydrolysis of exogenous labelled substrate incubated with rat cerebral cortical cytosol and membrane fractions as has been successful in studies of phosphoinositide hydrolysis. In the cytosol [³H]phosphatidylcholine was hydrolyzed at a linear rate for 60 min of incubation and GTPS stimulated hydrolysis by 63%. The products of phospholipase C and phospholipase D, phosphorylcholine and choline, contributed only 44% of the [³H]phosphatidylcholine hydrolytic products in the cytosol, with phospholipase D activity slightly predominating. GTPS stimulated cytosolic phospholipase C and reduced phospholipase D activity. [³H]Phosphatidylcholine was hydrolyzed much more slowly by membranes than by cytosol. In membranes the production of [³H]phosphorylcholine and [³H]choline were approximately equal, contributing 27% of the total [³H]phosphatidylcholine hydrolysis, and GTPS only caused a slight stimulation of phospholipase C activity. Chronic lithium treatment (4 weeks) appeared to slightly reduce [³H]phosphatidylcholine metabolism in the cytosol and in membranes, but no statistically significant reductions were achieved. Cytosol and membrane fractions from postmortem human brain metabolized [³H]phosphatidylcholine slowly, and GTPS had no effects. In summary, exogenous [³H]phosphatidylcholine was hydrolyzed by brain cytosol and membranes, and this was stimulated by GTPS, but the complex contributions of multiple metabolic pathways complicates the application of this method for studying individual pathways, such as phospholipase D which contributes only a fraction of the total processes hydrolyzing exogenous [³H]phosphatidylcholine.     

Adenosine transport by cultured glial cells from chick embryo brain

The transport of adenosine was studied in pure cultures of glial cells from chick embryo brain. In order to avoid complications in uptake measurements due to adenosine metabolism, cultures were depleted of ATP by incubation with cyanide and iodoacetate prior to addition of [³H]adenosine. Under the 5- to 25-s periods used for the transport assay, no adenosine metabolism could be detected. Initial rates of adenosine transport under these conditions obeyed the Michaelis-Menten relationship with K_m = 370 μM and V_max = 10.3 nmol/min/mg cell protein. ATP depletion or elimination of Na⁺ from the assay medium had no significant effect on initial rates of adenosine uptake. However, when assays were carried out under conditions of significant adenosine metabolism (10-min uptake in the absence of metabolic inhibitors), a high-affinity incorporation process could be demonstrated in the glial cells (K_m = 12 μM; V_max = 0.34 nmol/ min/mg protein). The transport activity expressed in ATP-depleted glial cells was most sensitive to inhibition by nitrobenzylthioinosine, dipyridamole, and N⁶-benzyladenosine. In decreasing order of potency, N⁶-methyladenosine, 2-chloroadenosine, inosine, and thymidine also blocked adenosine translocation in glial cultures. Thus, adenosine transport by cultured glial cells occurs by means of a low-affinity, facilitated diffusion system which is similar to the nucleoside transporter in cells of nonneural origin.     

Control of aerobic glycolysis in the brain in vitro

Protoveratrine-(5 M) stimulated aerobic glycolysis of incubated rat brain cortex slices that accompanies the enhanced neuronal influx of Na⁺ is blocked by tetrodotoxin (3 M) and the local anesthetics, cocaine (0.1 mM) and lidocaine (0.5 mM). On the other hand, high [K⁺]-stimulated aerobic glycolysis that accompanies the acetylcholine-sensitive enhanced glial uptakes of Na⁺ and water is unaffected by acetylcholine (2 mM). Experiments done under a variety of metabolic conditions show that there exists a better correlation between diminished ATP content of the tissue and enhanced aerobic glycolysis than between tissue ATP and the ATP-dependent synthesis of glutamine. Whereas malonate (2 mM) and amino oxyacetate (5 mM) suppress ATP content and O₂ uptake, stimulate lactate formation, but have little effect on glutamine levels, fluoroacetate (3 mM) suppresses glutamine synthesis in glia, presumably by suppressing the operation of the citric acid cycle, with little effect on ATP content, O₂ uptake, and lactate formation. Exogenous citrate (5 mM), which may be transported and metabolized in glia but not in neurons, inhibits lactate formation by cell free acetone-dried powder extracts of brain cortex but not by brain cortex slices. These results suggest that the neuron is the major site of stimulated aerobic glycolysis in the brain, and that under our experimental conditions glycolysis in glia is under lesser stringent metabolic control than that in the neuron. Stimulation of aerobic glycolysis by protoveratrine occurs due to diminution of the energy charge of the neuron as a result of stimulation of the sodium pump following tetrodotoxin-sensitive influx of Na⁺; stimulation by high [K⁺, NH₄ ⁺, or Ca²⁺ deprivation occurs partly by direct stimulation of key enzymes of glycolysis and partly by a fall in the tissue ATP concentration.     

A CD80-transfected human breast cancer cell variant induces HER-2/neu–specific T cells in HLA-A*02–matched situations in vitro as well as in vivo

Adjuvant treatment is still only working in a small percentage of breast cancer patients. Therefore, new strategies need to be developed. Immunotherapies are a very promising approach because they could successfully attack tumor cells in the stage of dormancy. To assess the feasibility of using an allogeneic approach for vaccination of breast cancer patients, we selected a CD80-transfected breast cancer cell line based on its immunogenic properties. Using CD80⁺ KS breast cancer cells and human leukocyte antigen (HLA)-A*02–matched peripheral blood mononuclear cells (PBMCs) of breast cancer patients in allogeneic mixed lymphocyte–tumor cell cultures (MLTCs), it was possible to isolate HLA-A*02–restricted cytotoxic T cells (CTLs). Furthermore, a genetically modified KS variant expressing influenza A matrix protein serving as a surrogate tumor-associated antigen (TAA) was able to stimulate flu peptide-specific T cells alongside the induction of alloresponses in MLTCs. KS breast cancer cells were demonstrated to express already known TAAs such as CEA, MUC-1, MAGE-1, MAGE-2, and MAGE-3. To further improve antigenicity, HER-2/neu was added to this panel as a marker antigen known to elicit HLA-A*02–restricted CTLs in patients with breast cancer. Thus, the antigen-processing and antigen-presentation capacity of KS cells was further demonstrated by the stimulation of HER-2/neu–specific CD8⁺ T cells in PBMCs of breast cancer patients in vitro. These results gave a good rationale for a phase I/II trial, where the CD80⁺ HER-2/neu–overexpressing KS variant is actually used as a cellular vaccine in patients with metastatic breast cancer. As a proof of principle, we present data from two patients where a significant increase of interferon- (IFN-) release was detected when postvaccination PBMCs were stimulated by allogeneic vaccine cells as well as by HLA-A*02–restricted HER-2/neu epitopes. In whole cell vaccine trials, monitoring is particularly challenging because of strong alloresponses and limited knowledge of TAAs. In this study, a panel of HER-2/neu epitopes, together with the quantitative real time (qRT)-PCR method to analyze vaccine-induced cytokines secreted by T cells, proved to be highly sensitive and feasible to perform an immunological staging following vaccination.     

Three-dimensional structure of α-conotoxin EI determined by 1H NMR spectroscopy H resonance assignments of α-conotoxin EI

-Conotoxin EI is an 18-residue peptide (RDOCCYHPTCNMSNPQIC; 4–10, 5–18) isolated from the venom of Conus ermineus, the only fish-hunting cone snail of the Atlantic Ocean. This peptide targets specifically the nicotinic acetylcholine receptor (nAChR) found in mammalian skeletal muscle and the electric organ Torpedo, showing a novel selectivity profile when compared to other -conotoxins. The 3D structure of EI has been determined by 2D-NMR methods in combination with dynamical simulated annealing protocols. A total of 133 NOE-derived distances were used to produce 13 structures with minimum energy that complied with the NOE restraints. The structure of EI is characterized by a helical loop between Thr⁹ and Met¹² that is stabilized by the Cys⁴-Cys¹⁰ disulfide bond and turns involving Cys⁴-Cys⁵ and Asn¹⁴-Pro¹⁵. Other regions of the peptide appear to be flexible. The overall fold of EI is similar to that of other 4/7-conotoxins (PnIA/B, MII, EpI). However, unlike these other 4/7-conotoxins, EI targets the muscular type nAChR. The differences in selectivity can be attributed to differences in the surface charge distribution among these 4/7-conotoxins. The implications for binding of EI to the muscular nAChR are discussed with respect to the current NMR structure of EI.     

Biochemical characterization of [3H]norepinephrine uptake in dissociated brain cell cultures from chick embryos

The uptake of [³H]norepinephrine ([³H]NE) was studied in dissociated brain cell cultures prepared from 8-day-old chick embryos using the whole brain (minus optic lobes). Uptake of [³H]NE, 5×10^–9 M, 10 min incubation, in freshly dissociated noncultured embryonic chick brain cells, was detected in 6-day-old embryos; it was temperature and drug (cocaine, metanephrine) sensitive and increased with brain development. In cultured cells, which were assayed at various days in culture, the increase in [³H]NE accumulation per culture was less than that seen in freshly dissociated noncultured embryonic cells. When [³H]NE uptake was expressed per mg protein, a decrease with days in culture was observed, reflecting perhaps a dilution of growth or proliferation of cells not accumulating NE. Metanephrine, 5×10^–6 M, an inhibitor of extraneuronal uptake, inhibited [³H]NE in 5-day-old cultures whereas desmethylimipramine, an inhibitor of neuronal uptake, inhibited [³H]NE uptake in 15- and 20-day-old cultures. Cocaine, another neuronal inhibitor, inhibited [³H]NE at 10 and 15 days only. We interpret these findings to suggest that during early growth in culture most neuroblasts accumulate NE nonspecifically and, as neuronal maturation proceeds, NE accumulation becomes specific.     

Culture of chick embryo neural retina in serum-free medium : Expression of cholinergic proteins

Dissociated retinal cells from 8-day-old chick embryos were cultivated in serum-containing and in defined serum-free media. Under the latter conditions, and using polylysine as a substrate, the proliferation of glial cells was almost completely prevented, and pure (>90%) neuronal cultures could be maintained for up to 7 days in vitro. The specific but not the total activities of choline acetyltransferase and of the nicotinic and the muscarinic acetylcholine receptor were increased under serum-free culture conditions. Autoradiographic studies with [¹²⁵I]α-bungarotoxin, a selective ligand for nicotinic cholinergic receptors, showed that serum-free culture conditions may constitute a useful tool for identifying biochemically different types of retinal neurons in tissue culture.     

Regulation of enzymes responsible for neurotransmitter synthesis and degradation in cultured rat sympathetic neurons. II. Regulation of 16 S acetylcholinesterase by conditioned medium

The molecular forms of acetylcholinesterase (AcChE) have been studied in primary cultures of newborn rat sympathetic neurons grown either in the absence (CM^? cultures) or in the presence (CM⁺ cultures) of muscle conditioned medium. The cultures were treated with a mitotic poison to eliminate non-neuronal cells. CAT activity increased with time in culture 4- to 20-fold faster in CM⁺ than in CM^? cultures. In agreement with previous experiments (J. P. Swerts, A. Le Van Thaï, A. Vigny, and M. J. Weber, 1983, Develop. Biol.100, 1–11), AcChE activity developed at a 3-fold lower rate in CM⁺ than in CM^? cultures. This deficit in AcChE activity in CM⁺ cultures resulted from a deficit in the number of enzyme molecules immunoprecipitable with an antiserum raised against rat brain AcChE. In both types of cultures, AcChE forms were separated by sucrose gradient sedimentation into three main peaks corresponding to the 16 S and 10 S forms and a mixture of the 6.5 and 4 S forms. In 3-day-old CM⁺ and CM^? cultures, the 16 S form represented 2% of the total activity. After 12–26 days, the percentage of 16 S form raised to 15–30% in CM^? cultures, but remained lower than 5% in CM⁺ cultures. This difference was also observed when AcChE molecular forms were analyzed in the presence of protease inhibitors. A similar result was obtained by comparing cultures grown with and without a macromolecular factor partially purified from conditioned medium. These results suggest that an inverse relationship exists between the presence of 16 S AcChE and the presence of cholinergic synapses in these cultures.     

Effects of phenylalanine and its deaminated metabolites on Na+, K+-ATPase activity in synaptosomes from rat brain

The effects of phenylalanine (PHE) and its deaminated metabolites phenylpyruvate (PHP), phenyllactate (PHL) and phenylacetate (PHA) on sodium and potassium activated adenosinetriphosphatase (Na⁺, K⁺-ATPase) in synaptosomes from rat brain were investigated. At very low concentrations (5–10 M), PHE, PHL and PHA inhibited the activity, while PHP stimulated the activity. At intermediate concentrations (50–100 M), all compounds had no effect, but at higher (0.5–1.0 mM) concentrations they inhibited the enzyme activity. Thus all the compounds tested showed a biphasic effect on the enzyme activity. Hydroxylamine inhibited the Na⁺, K⁺-ATPase activity when present alone; simultaneous addition of hydroxylamine and PHE, however, eliminated the inhibitory effects of each other. Reversal of mutual inhibition also occurred in the presence of hydroxylamine and very low (5–10 M) concentrations of PHL or PHA. The inhibitory effects of PHE at all concentrations, and of PHL or PHA at low concentrations, were also eliminated in the presence of EGTA. The data indicate that inhibition of brain membrane Na⁺, K⁺-ATPase by PHE and by low concentrations of PHL and PHA may involve metal ions, but that the inhibition by high concentrations of these metabolites must occur by a different mechanism. Since Na⁺, K⁺-ATPase plays a central role in neuronal function, and the presence of excess PHE and its deaminated metabolites occurs in brain tissue under conditions of experimentally induced hyperphenylalaninemia and genetic phenylketonuria, the neurologic impairment in experimental and genetic PKU may in part be related to the deleterious effects of these compounds on brain ATPase.     

The assembly of the DNA complex present in chick embryo cell cytosol

• 1.1. Chromatography of chick embryo fibrobast cytosol labelled with [³H]thymidine or [³H]uridine precursors has shown the presence of early labelled DNA and RNA eluting at a position corresponding to a relative molecular mass of approximately 1.5–10⁵.
• 2.2. The early DNA-RNA (heteroduplex?) then moves progressively to a higher molecular weight peak, relative molecular mass approximately 10⁶.
• 3.3. The process appears similar in cytosol from cultured cells and from whole aminiotically labelled chick embryo: consequently the cytosolic DNA complex is not an artefact of cell culturing.
• 4.4. The relative contribution of artefactual and specific cytosol-associated DNA material is discussed: it is concluded that while both are present in cytosol as prepared, it is possible to discriminate between specific and artefactual DNA material.

     

Activity of NAD-dependent glucose-6-phosphate dehydrogenase in yeast-like organisms

Summary From tested yeast-like organisms, onlyGeotrichum candidum showed the same activity of glucose-6-phosphate dehydrogenase with both NAD⁺ and NADP⁺. i. e. 0.017–0.019 mol NADH/min. mg dry weight of cell free extracts. Omission of Mg⁺⁺ in the reaction mixture did not influence the activity of the enzyme in the presence of NAD⁺. Cell free extracts ofEndomyces magnusii showed only low activity of this enzyme and the ratio of its activity in the presence of NAD⁺ and NADP⁺, respectively, varied in individual cultures.Rhodotorula glutinis showed only an NADP⁺-dependent activity.     

Localization of ornithine decarboxylase in the chick embryo during organogenesis

Summary The localization of ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis and thus in cell growth, was determined in the 4.5-day-old chick embryo, using two independent methods of analysis. ODC protein was identified by indirect immunofluorescence with a monospecific ODC antibody, and catalytically active ODC was identified by autoradiography with -(5-³H) difluoromethylornithine. Both methods revealed a basically similar distribution of ODC within the embryo. Among the organs, the brain exhibited the highest ODC levels. ODC levels were also high in spinal cord, mesonephric tubules and heart. Similar levels, but confined to limited areas, were found in liver tissue, head mesenchyme, and the oral and pharyngeal regions. Organs that exhibited high ODC levels are all engaged in rapid growth, as well as in extensive tissue remodeling and differentiation.     

The activity of erythrocyte and brain Na+/K+ and Mg2+-ATPases in rats subjected to acute homocysteine and homocysteine thiolactone administration

Hyperhomocysteinemia is associated with various pathologies including cardiovascular disease, stroke, and cognitive dysfunctions. Systemic administration of homocysteine can trigger seizures in animals, and patients with homocystinuria suffer from epileptic seizures. Available data suggest that homocysteine can be harmful to human cells because of its metabolic conversion to homocysteine thiolactone, a reactive thioester. A number of reports have demonstrated a reduction of Na⁺/K⁺-ATPase activity in cerebral ischemia, epilepsy and neurodegeneration possibly associated with excitotoxic mechanisms. The aim of this study was to examine the in vivo effects of d,l-homocysteine and d,l-homocysteine thiolactone on Na⁺/K⁺- and Mg²⁺-ATPase activities in erythrocyte (RBC), brain cortex, hippocampus, and brain stem of adult male rats. Our results demonstrate a moderate inhibition of rat hippocampal Na⁺/K⁺-ATPase activity by d,l-homocysteine, which however expressed no effect on the activity of this enzyme in the cortex and brain stem. In contrast,d,l-homocysteine thiolactone strongly inhibited Na⁺/K⁺-ATPase activity in cortex, hippocampus and brain stem of rats. RBC Na⁺/K⁺-ATPase and Mg²⁺-ATPase activities were not affected by d,l-homocysteine, while d,l-homocysteine thiolactone inhibited only Na⁺/K⁺-ATPase activity. This study results show that homocysteine thiolactone significantly inhibits Na⁺/K⁺-ATPase activity in the cortex, hippocampus, and brain stem, which may contribute at least in part to the understanding of excitotoxic and convulsive properties of this substance.     

Stimulation of DNA synthesis and mitotic activity of chick embryo hepatocytes in primary culture

Summary Monolayer cultures were prepared from hepatocytes of 15 d chick embryos and maintained at high cell density in a chemically defined medium. In the absence of growth stimulatory conditions DNA synthesis was observed only during the first 10 to 16 h of culture. Thus, after a 12 h exposure to [³H]thymidine ([³H]dThd, 4 to 16 h) 9.1±1% ( ,n=4) of the hepatocyte nuclei were labeled. Labeled mitotic nuclei, up to late telophase, were regularly observed in these cultures. Beyond 16 h less than 2% labeled nuclei were found (12 h of [³H]dThd), which indicates that the hepatocytes entered proliferative quiescence. DNA synthesis of “resting” hepatocytes was stimulated by insulin and, only slightly, by hydrocortisone, glucagon, or fetal bovine serum. Triiodothyronine (T₃), or the nucleoside inosine (i) did not stimulate. Combination of insulin (I) with hydrocortisone (H), T₃ (T), or glucagon (G) resulted in a more than additive effect. Nearly maximal stimulation occurred with the combinations IHT and ITG. Labeling increased at 10 ng/ml of each component and was maximal at 1 to 10 μg/ml. A lag period of 8 to 10 h after hormone administration (IHiTG, 10 μg/ml) was observed before nuclear labeling increased. Within the subsequent 10 h a considerable proportion of the hepatocytes (up to 30% or more) entered DNA synthesis. Mitotic activity (with nuclei in prophase up to late telophase) also was stimulated. An increase of both total DNA and protein content was measured in several experiments. Hormonal stimulation of hepatocyte DNA synthesis and mitotic activity was associated with decreased β-naphthoflavone-mediated induction of cytochrome P450. A causal relationship between these two phenomena remains to be established. It is suggested that chick embryo hepatocyte cultures are a useful tool for studies on hepatocyte proliferation and differentiation. The present study is based on original observations by Dr. F. R. Althaus (presently at the Institute of Pharmacology and Biochemistry, University of Zürich, Switzerland). This contribution of his and his incisive criticism are acknowledged. The study was supported by Grant 3.893.81 from the Swiss National Research Foundation.     

Glutamate as a precursor of GABA in rat brain and peripheral tissues

Summary The formation of GABA from L-glutamate was investigated in homogenates of rat brain, liver, and kidney, using highly purified [¹⁴C]-L-glutamic acid as substrate and a thin-layer chromatographic separation of products. In agreement with other workers, liberation of [¹⁴C]-CO₂ was found to be stoichiometric with GABA formation in brain homogenates, but not in liver or kidney extracts. Subcellular fractionation and dialysis experiments suggested that most of the GABA synthesis in these peripheral tissues, unlike brain, does not occur via a direct decarboxylation of glutamate and requires one or more cofactors other than pyridoxal phosphate. NAD stimulated GABA formation in dialyzed extracts, and inhibition of GABA-transaminase, bothin vitro andin vivo, caused marked inhibition of GABA formation from glutamate in peripheral extracts. Although a very low GAD activity in liver and kidney cannot be excluded, these experiments suggest a major pathway from glutamate to GABA in these homogenates which includes (1) conversion of glutamate to -ketoglutarate by glutamate dehydrogenase or transaminases, (2) conversion of -ketoglutarate to succinic semialdehyde, and (3) formation of GABA from succinic semialdehyde and glutamate by GABA-transaminase.