Regulatory Interactions Among Axon Terminals Affecting the Release of Different Transmitters from Rat Striatal Slices Under Hypoxic and Hypoglycemic Conditions

An in vitro model of ischemia was utilized to study the effects of both oxygen and glucose depletion on transmitter release from rat striatal slices. The spontaneous and stimulation-evoked releases of tritiated dopamine, gamma-aminobutyric acid, glutamate, and acetylcholine were measured. Hypoxia increased the evoked release of glutamate and dopamine without effect on the resting release. In contrast, hypoglycemia itself increased the resting release of dopamine. Hypoxia in combination with hypoglycemia provoked a massive release of glutamate, dopamine, and gamma-aminobutyric acid. The effect on acetylcholine release was less pronounced. Ca2+ withdrawal partly reduced the effect of hypoxia combined with hypoglycemia on dopamine release and application of tetrodotoxin (1 microM) abolished it. MK-801 (3 microM), an N-methyl-D-aspartate receptor antagonist, attenuated the effect of hypoxia and hypoglycemia on [3H]dopamine release. omega-Conotoxin (0.1 microM) had a similar effect on stimulation-evoked release under a hypoxic condition. The D2 receptor antagonist sulpiride (100 microM) failed to enhance the release of [3H]acetylcholine in hypoxia combined with hypoglycemia. It was suggested that in response to hypoxia combined with hypoglycemia there is a massive release of glutamate due to the increased firing rate which in turn releases dopamine from the axon terminals through stimulation of presynaptic N-methyl-D-aspartate receptors. Dopaminergic inhibitory control on ACh release seems not to be operative under conditions of hypoxia combined with hypoglycemia.     

Electrical Stimulation of the Stratum Radiatum Increases the Release and Neosynthesis of Aspartate, Glutamate, and γ-Aminobutyric Acid in Rat Hippocampal Slices

The release of endogenous aspartic, glutamic, and gamma-aminobutyric acids (Asp, Glu, GABA, respectively) was measured in the effluent from superfused hippocampal slices using a new and sensitive mass spectrometric method. The stimulation of the stratum radiatum of the rat dorsal hippocampus caused a Ca2+-dependent increase in the release of these amino acids. This release was accompanied by an increase in the incorporation of [13C2] from [13C]glucose into Asp, Glu, and GABA, suggesting an increase in their neosynthesis. The removal of Ca2+ from the superfusion fluid brought about a marked decrease in Asp and Glu release at rest, and prevented their stimulation-evoked release and the appearance of population spikes. The results support the hypothesis that Asp and Glu are excitatory neurotransmitters in intrinsic hippocampal circuits and are possibly released from the Schaffer collaterals and commissural fibres. The increase in GABA release and neosynthesis during stimulation of the stratum radiatum could be related to recurrent inhibition evoked by transsynaptic stimulation of the pyramidal cells.     

Regulation of Transmitter γ-Aminobutyric Acid (GABA) Synthesis and Metabolism Illustrated by the Effect of γ-Vinyl GABA and Hypoglycemia

The effect of different treatments on amino acid levels in neostriatum was studied to throw some light on the synthesis and metabolism of gamma-aminobutyric acid (GABA). Irreversible inhibition of GABA transaminase by microinjection of gamma-vinyl GABA (GVG) led to a decrease in aspartate, glutamate, and glutamine levels and an increase in the GABA level, such that the nitrogen pool remained constant. The results indicate that a large part of brain glutamine is derived from GABA. Hypoglycemia led to an increase in the aspartate level and a decrease in glutamate, glutamine, and GABA levels. The total amino acid pool was decreased compared with amino acid levels in normoglycemic rats. GVG treatment of hypoglycemic rats led to a decrease in the aspartate level and a further reduction in glutamate and glutamine levels. In this case, GABA accumulation continued, although the glutamine pool was almost depleted. The GABA level increased postmortem, but there were no detectable changes in levels of the other amino acids. Pretreatment of the rats with hypoglycemia reduced both glutamate and glutamine levels with a subsequent decreased postmortem GABA accumulation. The half-maximal GABA synthesis rate was obtained when the glutamate level was reduced by 50% and the glutamine level was reduced by 80%.     

The genetic code as a periodic table

Summary The contemporary genetic code is reflective of a significant correlation between the properties of amino acids and their anticodons in a periodic manner. Almost all properties of amino acids showed a greater correlation to anticodonic than to codonic dinucleoside monophosphate properties. The polarity and bulkiness of amino acid side chains can be used to predict the anticodon with considerable confidence. The results are most consistent with predictions of the direct interaction and ambiguity reduction hypotheses for the origin of the genetic code.     

Ethylenediamine as a Specific Releasing Agent of γ-Aminobutyric Acid in Rat Striatal Slices

Abstract: Following incubation with [¹⁴C]y-aminobutyric acid (GABA) or [³H]dopamine, slices of rat striatum were superfused with media containing 36 mM K⁺ or ethylenediamine (EDA), 1 or 5 mM. Both K⁺ and EDA induced a release of [¹⁴C]GABA, the K⁺-induced release being largely Ca²⁺-dependent, while the EDA-induced release was not. Whereas K⁺ also evoked a Ca²⁺-dependent release of [³H]dopamine, EDA evoked no release of dopamine. EDA may therefore have potential as a specific GABA releasing agent.     

Characterization of the Release of Cholecystokinin-8 from Isolated Nerve Terminals and Comparison with Exocytosis of Classical Transmitters

In the present study, the release of the neuropeptide cholecystokinin-8 (CCK) from purified nerve terminals (synaptosomes) of the rat hippocampus was characterized with respect to the subcellular distribution, the release upon addition of various agents, the release kinetics, the Ca2+ and ATP dependence of release, and the relationship between CCK release and elevations of intraterminal free Ca2+ concentration ([Ca]i). These characteristics were compared with those for the release of classical transmitters in similar preparations. CCK-like immunoreactivity (CCK-LI) is enriched in the purified synaptosomal fraction of hippocampus homogenates and released in a strictly Ca2(+)-dependent manner upon chemical depolarization, addition of 4-aminopyridine, or stimulation with the Ca2+ ionophore ionomycin. The presence of Ca2+ in the medium significantly stimulates the basal efflux of CCK-LI from synaptosomes. The release upon stimulation develops gradually in time with no significant release in the first 10 s and levels off after 3 min of depolarization. At this time, a large amount of CCK-LI is still present inside the synaptosomes. A correlation exists between the release of CCK-LI and the elevations of [Ca]i. The release of CCK-LI is decreased, but not blocked, upon ATP depletion. These characteristics markedly differ from those for classical transmitters, which show a fast component of Ca2(+)-dependent (exocytotic) release, an absolute dependence on cellular ATP, and no marked stimulation of basal efflux in the presence of Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Release of Endogenous Amino Acids from Superior Colliculus of the Rabbit: In Vitro Studies After Retinal Ablation

Tissue slices from the superior colliculi (SC) of the rabbit were superfused and investigated 1 week after unilateral eye removal. Amino acid levels were determined both in the tissue slices and in the medium after chemical depolarisation (56 mM K). The amino acid determinations were done fluorimetrically by precolumn derivation and HPLC separation. Colliculi contralateral to the enucleation exhibited a 16% reduction in glutamate compared with the ipsilateral colliculi. The Ca-dependent release of glutamate or other amino acids tested was not appreciably affected by enucleation. However, both the total and the Ca-independent release of glutamate was lower from contralateral SC slices compared with the ipsilateral slices. The results do not favour glutamate as the major optic nerve transmitter in the rabbit, but do not rule out glutamate as a transmitter in a minor population of retinal fibres.     

Homocysteic Acid as a Putative Excitatory Amino Acid Neurotransmitter: I. Postsynaptic Characteristics at N-Methyl-D-Aspartate-Type Receptors on Striatal Cholinergic Interneurons

The actions of the stereoisomers of homocysteic acid (HCA) were characterized at N-methyl-D-aspartate (NMDA)-type receptors which mediate excitatory amino acid-evoked [3H]acetylcholine ([3H]ACh) release from striatal cholinergic interneurons. Like NMDA, L-HCA and D-HCA evoked the release of [3H]ACh formed from [3H]choline in striatal slices. The concentration-response curve for L-HCA was virtually superimposable on that for NMDA, yielding an equal EC50 value (56.1 microM) and maximal response. However, D-HCA was weaker, with an EC50 value of 81.1 microM, and an apparently smaller maximal response. L-HCA-evoked [3H]ACh release was inhibited by the same categories of compounds which inhibit NMDA-evoked [3H]ACh release: the divalent ion Mg2+ (IC50 = 25.8 microM); competitive NMDA antagonists 2-amino-7-phosphonoheptanoate (IC50 = 51.2 microM) and 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (IC50 = 20.1 microM); and the dissociative anesthetics tiletamine (IC50 = 0.59 microM) and MK-801 (IC50 = 0.087 microM). Like NMDA, L-HCA produced a tachyphylaxis in this system. Tachyphylaxis to NMDA resulted in a decrease response to L-HCA, and conversely, tachyphylaxis to L-HCA resulted in a decrease response to NMDA. The results suggest that L-HCA is an agonist at the NMDA-type receptor and may represent an endogenous ligand for this excitatory amino acid receptor.     

Adenosine and Glutamate Modulate Each Other's Release from Rat Hippocampal Synaptosomes

Abstract: In rat hippocampal synaptosomes, adenosine decreased the K⁺ (15 mM) or the kainate (1 mM) evoked release of glutamate and aspartate. An even more pronounced effect was observed in the presence of the stable adenosine analogue, R-phenylisopropyladenosine. All these effects were reversed by the selective adenosine A₁ receptor antagonist 8-cyclo-pentyltheophylline. In the same synaptosomal preparation, K⁺ (30 mM) strongly stimulated the release of the preloaded [³H]adenosine in a partially Ca²⁺-dependent and tetrodotoxin (TTX)-sensitive manner. Moreover, in the same experimental conditions, both l -glutamate and l -aspartate enhanced the release of [³H]adenosine derivatives ([³H]ADD). The gluta-mate-evoked release was dose dependent and appeared to be Ca²⁺ independent and tetrodotoxin insensitive. This effect was not due to metabolism because even the nonmetabolizable isomers d -glutamate and d -aspartate were able to stimulate [³H]ADD release. In contrast, the specific glutamate agonists N-methyl-d -aspartate, kainate, and quisqualate failed to stimulate [³H]ADD release, suggesting that glutamate and aspartate effects were not mediated by known excitatory amino acid receptors. Moreover, NMDA was also ineffective in the absence of Mg²⁺ and l -glutamate-evoked release was not inhibited by adding the specific antagonists 2-amino-5-phosphonovaleric acid or 6–7-dinitroquinoxaline-2, 3-dione. The stimulatory effect did not appear specific for only excitatory amino acids, as γ-anunobutyric acid stimulated [³H]ADD release in a dose-related manner. These results suggest that, at least in synaptosomal preparations from rat hippocampus, adenosine and glutamate modulate each other's release. The exact mechanism of such interplay, although still, unknown, could help in the understanding of excitatory amino acid neurotoxicity.     

A New Classification Scheme of the Genetic Code

Since the early days of the discovery of the genetic code nonrandom patterns have been searched for in the code in the hope of providing information about its origin and early evolution. Here we present a new classification scheme of the genetic code that is based on a binary representation of the purines and pyrimidines. This scheme reveals known patterns more clearly than the common one, for instance, the classification of strong, mixed, and weak codons as well as the ordering of codon families. Furthermore, new patterns have been found that have not been described before: Nearly all quantitative amino acid properties, such as Woeses polarity and the specific volume, show a perfect correlation to Lagerkvists codon–anticodon binding strength. Our new scheme leads to new ideas about the evolution of the genetic code. It is hypothesized that it started with a binary doublet code and developed via a quaternary doublet code into the contemporary triplet code. Furthermore, arguments are presented against suggestions that a simpler code, where only the midbase was informational, was at the origin of the genetic code.     

Measurement of amino acid release from cultured cerebellar granule cells by an improved high performance liquid chromatography procedure

Endogenous amino acid release was examined in rat cerebellar primary cultures comprising more than 95% of glutamatergic granule cells. Eighteen amino acids were determined in the cell extracts and in the release fractions by high performance liquid chromatography, using precolumn derivatization witho-phthaldialdehyde and separation on a reverse-phase column using a multi-step gradient system of two solvents (0.1 M Na⁺acetate, pH 7.2/methanol: tetrahydrofuran, 97:3). The fluorimetric response was linear, at least in the range of 2–162 pmol, for all the amino acids analysed, with a detection limit of 1 pmole. We observed a good reproducibility in within-assay and between-assay coefficients of variation of the retention times and fluorescence yield. When cultured granule cells were exposed to the excitatory amino acid receptor agonist quisqualic acid (50 M), we observed a net increase in the release of glutamate (3 fold over the baseline) and a smaller increase in that of aspartate (2 fold) and taurine (1.6 fold). Other amino acids were not significantly affected. GABA levels were below detection limits, due to the minimal number of GABAergic neurons present in the cultures.     

The Release and Neosynthesis of Glutamic Acid Are Increased in Experimental Models of Hepatic Encephalopathy

The effects of ammonium ions on the release of glutamic acid from the rat cerebral cortex were measured in vivo using cortical cups and a multiple ion detection technique. The neosynthesis of this amino acid from glucose was also studied in two experimental models of hepatic encephalopathy: (1) rats receiving large amounts of ammonium acetate (i.p.) and (2) rats with a surgically constructed portocaval anastomosis. Intraperitoneal administration of 8 mmol/kg of ammonium acetate increased the cortical release of glutamic acid from 9.1 +/- 0.8 to 19 +/- 2 (nmol X cm-2 X min-1). Moreover, 20 min after ammonium acetate administration the rate of incorporation of 13C2, originating from [13C]glucose, into glutamic acid increased by 65%. In several brain areas of rats bearing a portocaval anastomosis and fed ad libitum for 4 weeks, the content of glutamic acid slightly increased and the rate of formation of [13C2]glutamate from [13C]glucose approximately doubled. These results indicate that ammonium ions increase the release and the formation of glutamic acid in the brain. The resulting increased concentration of this amino acid in the extracellular spaces may be one of the mechanisms of ammonia toxicity in vivo.     

Maitotoxin-Induced Release of γ-[3H]Aminobutyric Acid from Cultures of Striatal Neurons

The potent marine toxin, maitotoxin, induced the release of gamma-[3H]aminobutyric acid (GABA) from reaggregate cultures of striatal neurons in a dose-dependent manner. Maitotoxin-induced release occurred following a lag period of several minutes and was persistent. Release induced by 70 mM K+ on the other hand was immediate and transient in nature. Co2+ (3 mM) and Cd2+ (1 mM) inhibited maitotoxin-induced release of GABA as did removal of extracellular Ca2+. However, the organic calcium antagonists nisoldipine, nitrendipine, and D-600 at concentrations of 10(-6) M did not block maitotoxin-induced or 70 mM K+-induced release. High concentrations of D-600 (10(-4) M) partially blocked both maitotoxin- and 70 mM K+-induced release. The dihydropyridine calcium agonist BAY K8644 (10(-6) M) did not enhance maitotoxin-induced or 70 mM K+-induced release. Replacement of Na+ in the incubation medium with choline led to an increased basal output of GABA and an apparent inhibition of the effect of maitotoxin. These data are discussed with reference to the hypothesis that maitotoxin can directly activate voltage-sensitive calcium channels.     

Effect of Nitroprusside (Nitric Oxide) on Endogenous Dopamine Release from Rat Striatal Slices

It is becoming apparent that the synthesis of nitric oxide (NO) from L-arginine not only explains endothelium-dependent vascular relaxation, but is a widespread mechanism for the regulation of cell function and communication. We examined the role of NO on the endogenous dopamine (DA) release from rat striatum. Nitroprusside, in the concentration range of 3-100 microM, induced a dose-dependent increase in the endogenous DA release from rat striatal slices. The maximal response was 330% over the baseline release. A higher concentration of nitroprusside (300 microM) produced an inhibitory effect on the spontaneous release of DA. L-Arginine (10 and 100 microM), a substrate in the NO-forming enzyme system, also produced an elevation of DA release. L-Arginine-induced DA release was attenuated by NG-monomethyl-L-arginine, an inhibitor of NO synthase. NADPH (1 microM), a cofactor of NO synthase, enhanced L-arginine-induced DA release. These results suggest a possible involvement of NO in the DA release process in rat striatum.     

Amino Acids in Rat Striatal Dialysates: Methodological Aspects and Changes After Electroconvulsive Shock

Extracellular levels of amino acids were estimated in dialysates of the rat striatum that were collected 1, 2, and/or more than 5 days after surgery, before (resting release) and during exposure to high K concentrations (50 mM) or electroconvulsive shocks. The resting release of several amino acids (Glu, Asn, Thr, Tau, Tyr, Gly, and Ala) was higher 9 days as compared to 1 day after surgery. In the 1-day preparation the resting release correlated highly with that observed with push-pull cannulas. The correlation with the tissue content of the amino acids was high only when they were divided into two groups (putative transmitters and metabolic intermediates). High K exposure produced increased output of Ala, ethanolamine (Eam), Asp, Glu, Tau, and Gly and a decrease in the egress of Gln 1 or 2 days after surgery. The effects on Asp and Glu had disappeared, and that on Gln reversed after 4-9 days. Electrically induced convulsions produced increased output of Ala, Gln, and Eam 1 or 2 days and 2 weeks after implantation of the probe. Changes were seen not only during but also (and some cases even more prominent) after the seizure. This study shows the usefulness of dialysis to monitor extracellular transmitter amino acids in the striatum of conscious rats (also bilateral dialysis was possible) for only a limited time after implantation of the probe. The dialysis method is suitable for longer time, when metabolic changes in amino acids are to be followed. In addition to transmitter release, glycolysis can be monitored by the measurement of Ala in the dialysate.     

Codon Usage Bias and Mutation Constraints Reduce the Level of ErrorMinimization of the Genetic Code

Studies on the origin of the genetic code compare measures of the degree of error minimization of the standard code with measures produced by random variant codes but do not take into account codon usage, which was probably highly biased during the origin of the code. Codon usage bias could play an important role in the minimization of the chemical distances between amino acids because the importance of errors depends also on the frequency of the different codons. Here I show that when codon usage is taken into account, the degree of error minimization of the standard code may be dramatically reduced, and shifting to alternative codes often increases the degree of error minimization. This is especially true with a high CG content, which was probably the case during the origin of the code. I also show that the frequency of codes that perform better than the standard code, in terms of relative efficiency, is much higher in the neighborhood of the standard code itself, even when not considering codon usage bias; therefore alternative codes that differ only slightly from the standard code are more likely to evolve than some previous analyses suggested. My conclusions are that the standard genetic code is far from being an optimum with respect to error minimization and must have arisen for reasons other than error minimization.[Reviewing Editor: Martin Kreitman]     

Regional Acetylcholine Metabolism in Brain During Acute Hypoglycemia and Recovery

Insulin-induced hypoglycemia in normothermic rats caused progressive neurological depression and differentially altered regional cerebral acetylcholine metabolism. Reductions of plasma glucose from 7.7 mM (control) to 2.5-1.7 mM (moderate hypoglycemia associated with decreased motor activity) or 1.5 mM (severe hypoglycemia with lethargy progressing to stupor) decreased glucose concentrations in the cerebral cortex, striatum, and hippocampus to less than 10% of control. Moderate hypoglycemia diminished acetylcholine concentrations in cortex and striatum (21% and 45%, respectively) and reduced [1-2H2, 2-2H2]choline incorporation into acetylcholine (62% and 41%, respectively). Severe hypoglycemia did not reduce the acetylcholine concentration or synthesis in cortex and striatum further. The concentrations of choline rose in the cortex (+53%) and striatum (+130%) of animals that became stuporous but a similar rise in [1-2H2, 2-2H2]choline left the specific activities of choline in these structures unchanged. Even severe hypoglycemia did not alter the hippocampal cholinergic system. In rats that developed hypoglycemic stupor and were then treated with glucose, the animals recovered apparently normal behavior, and the concentrations of acetylcholine and the incorporation of [1-2H2, 2-2H2]-choline into acetylcholine returned to control values in the striatum but not in the cerebral cortex. Thus, impaired acetylcholine metabolism in selected regions of the brain may contribute to the early symptoms of neurological dysfunction in hypoglycemia.     

Diacylglycerol-Induced Stimulation of Neurotransmitter Release from Rat Brain Striatal Synaptosomes

These studies were undertaken to test the hypothesis that alterations in phosphatidylinositol metabolism can modulate neurotransmitter release in the central nervous system. The effects of 1,2-diacylglycerols (DAGs) on dopamine release in the rat central nervous system were determined by measuring dopamine release from rat striatal synaptosomes in response to two DAGs (sn-1,2-dioctanoylglycerol and 1-oleoyl-2-acetylglycerol) that can activate protein kinase C and one DAG (deoxydioctanoylglycerol) that does not activate this kinase. Dioctanoylglycerol and 1-oleoyl-2-acetylglycerol, at a concentration of 50 micrograms/ml, stimulated the release of labeled dopamine from striatal synaptosomes by 35-50 and 17%, respectively. Dioctanoylglycerol-induced release was also demonstrated for endogenous dopamine. In contrast, deoxydioctanoylglycerol (50 micrograms/ml) did not stimulate dopamine release. Dioctanoylglycerol-induced dopamine release was independent of external calcium concentration, indicating a utilization of internal calcium stores. Dioctanoylglycerol (50 micrograms/ml) also produced a 38% increase in labeled serotonin release from striatal synaptosomes. The addition of dioctanoylglycerol to the striatal supernatant fraction increased protein kinase C activity. These results are consistent with the concept that an increase in phosphatidylinositol metabolism can stimulate neurotransmitter release in the central nervous system via an increase in DAG concentration. The data suggest an involvement of protein kinase C in the DAG-induced release, but other sites for DAG action are also possible.     

Synthesis of polypeptides by microwave heating I. Formation of polypeptides during repeated hydration-dehydration cycles and their characterization

Summary Amino acid amides effectively reacted to produce polypeptides in response to microwave heating during repeated hydration-dehydration cycles. The polypeptides, formed from a mixture of glycinamide, alaninamide, valinamide, and aspartic acid -amide, had molecular weights ranging from 1000 to 4000 daltons. Amino acids were incorporated into the polypeptides in proportion to the starting concentrations, with the exception of glycine whose incorporation was 1.5 times higher than that of the other amino acids. The polypeptides had some definite secondary structure, such as -helix and -sheet, in aqueous solution. This reaction provides not only a convenient method for abiotic peptide formation but also a convenient method for the chemical synthesis of peptides.