L-Quisqualic acid transport into hippocampal neurons by a cystine-sensitive carrier is required for the induction of quisqualate sensitization.

A brief exposure of hippocampal slices to L-quisqualic acid sensitizes CA1 pyramidal neurons 30-250-fold to depolarization by two classes of excitatory amino acid analogues: (1) those whose depolarizing effects are rapidly terminated following washout, e.g. L-2-amino-4-phosphonobutanoic acid (L-AP4) and L-2-amino-6-phosphonohexanoic acid (L-AP6) and (2) those whose depolarizing effects persist following washout, e.g. L-aspartate-beta-hydroxamate (L-AbetaH). This process has been termed quisqualate sensitization. In this study we directly examine the role of amino acid transport systems in the induction of quisqualate sensitization. We report that L-quisqualate is a low-affinity substrate (K(M)=0.54 mM) for a high capacity (V(max)=0.9 nmol (mg protein)(-1) min(-1)) Na(+)-dependent transport system(s) and a high-affinity substrate (K(M)=0.033 mM) for a low-capacity (V(max)=0.051 nmol (mg protein)(-1) min(-1)) transporter with properties similar to the cystine/glutamate exchange carrier, System x(c-). We present evidence that suggests that System x(c-) participates in quisqualate sensitization. First, simultaneous application of L-quisqualate and inhibitors of System x(c-), but not inhibitors of Na(+)-dependent glutamate transporters, prevents the subsequent sensitization of hippocampal neurons to phosphonates or L-AbetaH. Second, L-quisqualic acid only sensitizes hippocampal neurons to other substrates of System x(c-), including cystine. Third, immunocytochemical analysis of L-quisqualate uptake demonstrates that only inhibitors of System x(c-) inhibit the highly concentrative uptake of L-quisqualate into a widely dispersed group of GABAergic hippocampal interneurons. We conclude that quisqualate sensitization is a direct consequence of the unique interaction of various excitatory amino acids, namely L-quisqualate, cystine, and phosphonates, with the exchange carrier, System x(c-). Therefore, the results of this study have important implications for the mechanism by which L-quisqualate, and other substrates of this transporter which are also excitatory amino acid agonists (such as glutamate and beta-N-oxalyl-L-alpha,beta-diaminopropionic acid, beta-L-ODAP) may trigger neurotoxicity.

Cyclobutane quisqualic acid analogues as selective mGluR5a metabotropic glutamic acid receptor ligands.

The conformationally constrained cyclobutane analogues of quisqualic acid (Z)- and (E)-1-amino-3-[2'-(3',5'-dioxo-1',2', 4'-oxadiazolidinyl)]cyclobutane-1-carboxylic acid, compounds 2 and 3, respectively, were synthesized. Both 2 and 3 stimulated phosphoinositide (PI) hydrolysis in the hippocampus with EC50 values of 18 +/- 6 and 53 +/- 19 microM, respectively. Neither analogue stimulated PI hydrolysis in the cerebellum. The effects of 2 and 3 were also examined in BHK cells which expressed either mGluR1a or mGluR5a receptors. Compounds 2 and 3 stimulated PI hydrolysis in cells expressing mGluR5a but not in those cells expressing mGluR1a. The EC50 value for 2 was 11 +/- 4 microM, while that for 3 was 49 +/- 25 microM. Both 2 and 3 did not show any significant effect on cells expressing the mGluR2 and mGluR4a receptors. In addition, neither compound blocked [3H]glutamic acid uptake into synaptosomal membranes, and neither compound was able to produce the QUIS effect as does quisqualic acid. This pharmacological profile indicates that 2 and 3 are selective ligands for the mGluR5a metabotropic glutamic acid receptor.

Persistent depression of synaptic responses occurs in quisqualate sensitized hippocampal slices after exposure to L-aspartate-beta-hydroxamate.

Exposure of slices of rat hippocampus to quisqualic acid produces an enhanced sensitivity of neurons to depolarization by other excitatory amino acid analogues, particularly amino acid phosphonates. The phosphonates may act at extracellular sites, since their depolarizing effects are rapidly reversed by washout with phosphonate-free incubation medium. We now wish to report a novel class of excitatory amino acid analogues that induce a persistent depolarization that is not reversed by washout. Exposure of quisqualate-sensitized slices of rat hippocampus to 400 microM L-aspartate-beta-hydroxamate for 8 min results in the complete depression of extracellular synaptic field potentials. This depression persists for at least 1 h after washout of the hydroxamate compound. Analogous compounds L-glutamate-gamma-hydroxamate, D-aspartate-beta-hydroxamate and the phosphonate derivative L-2-amino-3-phosphonopropanoic acid (L-AP3) induce a similar but weaker persistent depression of the field potentials. Previous studies also demonstrated that exposure of hippocampal slices to L-alpha-aminoadipate blocks or reverses quisqualate sensitization, making the neurons unresponsive to depolarization by phosphonate compounds. We now report that L-alpha-aminoadipate also blocks or reverses the persistent depolarization of quisqualate-sensitized neurons which is induced by exposure to the hydroxamates or L-AP3.

A 3-amino-4-hydroxy-3-cyclobutene-1,2-dione-containing glutamate analogue exhibiting high affinity to excitatory amino acid receptors.

The syntheses of several novel N-(hydroxydioxocyclobutenyl)-containing analogues of gamma-amino-butyric acid and L-glutamate were undertaken to test the hypothesis that derivatives of 3,4-dihydroxy-3-cyclobutene-1,2-dione (squaric acid), such as 3-amino-4-hydroxy-3-cyclobutene-1,2-dione, could serve as a replacement for the carboxylate moiety in neurochemically interesting molecules. The syntheses were successfully accomplished by preparation of a suitably protected diamine or diamino acid followed by reaction with diethyl squarate. Subsequent deprotection resulted in the isolation of the corresponding N-(hydroxydioxocyclobutenyl)-containing analogues 13, 14, and 18. These analogues were screened as displacers in various neurochemical binding site assays. The L-glutamate analogue 18, which showed high affinity as a displacer for kainate and AMPA binding, was also examined for agonist potency for CA1 pyramidal neurons of the rat hippocampal slice preparation. It rivaled AMPA as one of the most potent agonists for depolarizing pyramidal neurons in medium containing 2.4 mM Mg+2 ions in which kainate/AMPA receptors are active but NMDA receptors are inhibited (IC50 = 1.1 microM). It was 1 order of magnitude less potent for depolarizing pyramidal neurons under conditions in which kainate/AMPA receptors were inhibited by 10 microM CNQX but NMDA receptors were active in 0.1 mM Mg(+2)-containing medium (IC50 = 10 microM). Compound 18 did not induce sensitization of CA1 pyramidal cells to depolarization by phosphonate analogues of glutamate (the QUIS-effect).

Synthesis of oxadiazolidinedione derivatives as quisqualic acid analogues and their evaluation at a quisqualate-sensitized site in the rat hippocampus.

The ability of quisqualic acid (1) to sensitize neurons to depolarization by omega-phosphono alpha-amino acid analogues of excitatory amino acids is a highly specific phenomenon and is termed the QUIS effect. In an attempt to elucidate the structure-activity relationships for this sensitization, analogues 2-6 of quisqualic acid have been synthesized. Compounds 4, 5, and 6 showed no quisqualate sensitization with respect to L-2-amino-6-phosphonohexanoic acid (L-AP6), while compounds 2 and 3 were 1/10 and 1/1000, respectively, as active as quisqualic acid in sensitizing neurons toward L-AP6.

Utilization of the resolved L-isomer of 2-amino-6-phosphonohexanoic acid (L-AP6) as a selective agonist for a quisqualate-sensitized site in hippocampal CA1 pyramidal neurons.

Brief exposure of rat hippocampal slices to quisqualic acid (QUIS) sensitizes neurons to depolarization by the alpha-amino-omega-phosphonate excitatory amino acid (EAA) analogues AP4, AP5 and AP6. These phosphonates interact with a novel QUIS-sensitized site. Whereas L-AP4 and D-AP5 cross-react with other EAA receptors, DL-AP6 has been shown to be relatively selective for the QUIS-sensitized site. This specificity of DL-AP6, in conjunction with the apparent preference of this site for L-isomers, suggested that the hitherto unavailable L-isomer of AP6 would be a potent and specific agonist. We report the resolution of the D- and L-enantiomers of AP6 by fractional crystallization of the L-lysine salt of DL-AP6. We also report the pharmacological responses of kainate/AMPA, NMDA, lateral perforant path L-AP4 receptors and the CA1 QUIS-sensitized site to D- and L-AP6, and compare these responses to the D- and L-isomers of AP3, AP4, AP5 and AP7. The D-isomers of AP4, AP5 and AP6 were 5-, 3- and 14-fold less potent for the QUIS-sensitized site than their respective L-isomers. While L-AP4 and L-AP5 cross-reacted with NMDA and L-AP4 receptors, L-AP6 was found to be highly potent and specific for the QUIS-sensitized site (IC50 = 40 microM). Its IC50 values for kainate/AMPA, NMDA and L-AP4 receptors were > 10, 3 and 0.8 mM, respectively. As with AP4 and AP5, sensitization to L-AP6 was reversed by L-alpha-aminoadipate.

Quisqualic acid induced sensitization and the active uptake of L-quisqualic acid by hippocampal slices.

Hippocampal CA1 pyramidal cell neurons are sensitized to depolarization by L-2-amino-4-phosphonobutanoic acid (L-AP4) following exposure to L-quisqualic acid (QUIS). It has been proposed that induction of this 'QUIS-effect' involves uptake of L-QUIS by hippocampal cells. We have used o-phthaldialdehyde (OPA) derivatization and high performance liquid chromatographic (HPLC) separation of extracts from hippocampal slices which have been exposed to varied concentrations of L-QUIS to investigate L-QUIS uptake into hippocampal slices. We observe uptake rates such that the internal concentration of L-QUIS exceeds the bath concentration within 7 min. The fact that this uptake is concentrative indicates that it is mediated by an active transport system. In addition, uptake of L-QUIS may be linked to the induction of the QUIS-effect. At low concentrations of L-QUIS (< 4 microM), the QUIS-effect is only partially induced within the 4 min incubation time which maximally induces the effect when 16 microM L-QUIS is used. However, repeated 4 min exposure periods of slices to low L-QUIS concentrations will eventually induce the QUIS-effect even when each exposure is separated by extensive washout periods. Hence induction is dependent on both concentration and total exposure time. We also examined the effects of L-alpha-aminoadipic acid and L-serine-O-sulfate on the rate of L-QUIS uptake. Exposure of slices to these compounds prior to treatment with L-QUIS will block the physiological effects of L-QUIS. We found that these 'pre-blocking' compounds did not decrease the rate of L-QUIS uptake.(ABSTRACT TRUNCATED AT 250 WORDS)

Quisqualic acid analogues: synthesis of beta-heterocyclic 2-aminopropanoic acid derivatives and their activity at a novel quisqualate-sensitized site.

Hippocampal CA1 pyramidal cell neurons are sensitized over 30-fold to depolarization by L-2-amino-4-phosphonobutanoic acid (L-AP4) following exposure to L-quisqualic acid. This phenomenon has been termed the QUIS effect. In the present study several novel L-quisqualic acid analogues have been synthesized and tested for their interaction with the different components of the QUIS-effect system. Replacement of the oxadiazolidinedione ring of L-quisqualic acid with several other types of heterocyclic rings yielded the following quisqualic acid analogues: maleimide 2, N-methylmaleimide 3, N-(carboxymethyl)maleimide 4, succinimides 5A and 5B, and imidazolidinedione 6. None of these analogues were able to mimic the effects of L-quisqualic acid and sensitize hippocampal CA1 neurons to depolarization by L-AP4. Also, unlike L-serine O-sulfate, L-homocysteinesulfinic acid, or L-alpha-aminoadipic acid, none of the analogues were able to preblock or reverse the QUIS effect. However, when the IC50 values for inhibition of the CA1 synaptic field potential of analogues 2-6 were determined both before and after hippocampal slices were exposed to L-quisqualic acid, the IC50 values of analogues 3 and 4 were found to decrease more than 7-fold. Thus, these two compounds behave like L-AP4 rather than L-quisqualic acid in this system in that they exhibit increased potencies in slices that have been pretreated with L-quisqualic acid even though they cannot themselves induce this sensitization. Compounds 3 and 4, therefore, represent the first non-phosphorus-containing compounds to which hippocampal neurons become sensitized following exposure to L-quisqualic acid. No change in the IC50 values was observed for 5A or 5B. Analogues 2 and 6, on the other hand, displayed a high potency for inhibition of the evoked field potential even prior to treatment of the slices with L-quisqualic acid.

Synthesis of acyclic and dehydroaspartic acid analogues of Ac-Asp-Glu-OH and their inhibition of rat brain N-acetylated alpha-linked acidic dipeptidase (NAALA dipeptidase).

The following structural and conformationally constrained analogues of Ac-Asp-Glu-OH (1) were synthesized: Ac-Glu-Glu-OH (2), Ac-D-Asp-Glu-OH (3), Ac-Glu-Asp-OH (4), Ac-Asp-Asp-OH (5), Ac-Asp-3-aminohexanedioic acid (6), Ac-3-amino-3-(carboxymethyl)propanoyl-Glu-OH (7), N-succinyl-Glu-OH (8), N-maleyl-Glu-OH (9), N-fumaryl-Glu-OH (10), and Ac-delta ZAsp-Glu-OH (11). These analogues were evaluated for their ability to inhibit the hydrolysis of Ac-Asp-[3,4-3H]-Glu-OH by N-acetylated alpha-linked acidic dipeptidase (NAALA dipeptidase) in order to gain some insight into the structural requirements for the inhibition of this enzyme. Analogues 4-6 and 9 were very weak inhibitors of NAALA dipeptidase (Ki greater than 40 microM), while 2, 3, and 7 with Ki values ranging from 3.2-8.5 microM showed intermediate inhibitory activity. The most active inhibitors of NAALA dipeptidase were compounds 8, 10, and 11 with Ki values of 0.9, 0.4, and 1.4 microM, respectively. These results suggest that the relative spacing between the side chain carboxyl and the alpha-carboxyl group of the C-terminal residue may be important for binding to the active site of the enzyme. They also indicate that the chi 1 torsional angle for the aspartyl residue is in the vicinity of 0 degrees.

High-affinity transport of L-glutamine by a plasma membrane preparation from rat brain.

Plasma membrane vesicles prepared from rat brain contain a saturable, high-affinity transport system for L-glutamine that exhibits the following characteristics: (1) The rate of L-glutamine transport is linear up to 200 micrograms/mL membrane protein. (2) Transport of [3H]-L-glutamine is linear with time for at least 10 min, is significantly reduced by lowering the assay temperature to 4 degrees C, and is essentially abolished by the addition of excess unlabeled L-glutamine. (3) The transport rate is optimal in the range of pH 7.4-8.2. (4) The system exhibits a Km for L-glutamine of approximately 1.7 microM and a Vmax of approximately 46 pmol/(min.mg of protein). (5) The system is not highly dependent upon the addition of monovalent or divalent cations. (6) Inhibitor studies reveal that the amino acid amides exhibit the highest affinity for the system and that there is a high specificity for the L-isomers.

Asparaginase II of Saccharomyces cerevisiae. Characterization of the ASP3 gene.

Purified preparations of asparaginase II of Saccharomyces cerevisiae exhibit two protein bands upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cloning and sequencing of the ASP3 gene, and partial amino acid sequencing as asparaginase II, imply that both bands are encoded by ASP3 but have different N termini. Northern blot analysis using the cloned ASP3 gene as a probe indicates that nitrogen catabolite repression of asparaginase II is achieved by alteration in mRNA levels. Deletion of sequences greater than 600 base pairs upstream from the initiation AUG codon results in an altered response to certain nitrogen sources in strains containing the truncated gene.

Asparaginase II of Saccharomyces cerevisiae: selection of four mutations that cause derepressed enzyme synthesis.

A positive selection method was used to isolate four Saccharomyces cerevisiae mutations that cause derepressed synthesis of asparaginase II. The four mutations (and1, and2, and3, and4) were neither closely linked to each other nor linked to previously characterized mutations (asp3, asp6) which cause the complete loss of asparaginase II activity. One of the new mutations (and4) was shown to be allelic to gdh-CR, a pleiotropic mutation which causes derepressed synthesis of a number of enzymes of nitrogen catabolism.

Asparaginase II of Saccharomyces cerevisiae: positive selection of two mutations that prevent enzyme synthesis.

A positive selection method, D-aspartic acid beta-hydroxamate resistance, was used to isolate Saccharomyces cerevisiae strains lacking the ability to synthesize asparaginase II. Of 100 such mutant strains, 93 exhibited mutations which were allelic with asp3, a previously characterized mutation. The other seven strains carried a new mutation, asp6. The asp6 mutation segregated 2:2 in asp6 X wild-type crosses and assorted from the asp3 mutation in asp6 X asp3 crosses. All seven asp6 mutant isolates reverted at a relatively high frequency, whereas the asp3 mutant isolates did not revert under the same conditions. Various independent asp3 isolates were mated to give heteroallelic diploids, which when sporulated and spread on D-asparagine medium yielded no recombinant strains.

Asparaginase II of Saccharomyces cerevisiae: comparison of enzyme stability in vivo and in vitro.

Asparaginase II of Saccharomyces cerevisiae is a cell wall mannan containing glycoprotein. Recent studies have demonstrated that asparaginase II activity increases in exponentially growing cell cultures and then decreases as the cells enter the stationary phase. Enzyme inactivation has been attributed to a Zn2+-dependent protease which is synthesized de novo during the late exponential phase [Pauling, K.D., & Jones, G.E. (1980) J. Gen. Microbiol. 117, 423-430; Pauling, K.D., & Jones, G.E. (1980) Biochim. Biophys. Acta 616, 271-282]. We have investigated the mechanism of asparaginase II inactivation using both whole cell suspensions and highly purified enzyme. Our data indicate that the rate of asparaginase II inactivation in cell suspensions is primarily influenced by pH changes that occur as a consequence of cell growth and glucose fermentation and that enzyme inactivation is not dependent on Zn2+ or on de novo protein synthesis. Also, in vitro studies with purified enzyme show kinetics of inactivation that are similar to those observed in vivo. Consequently, involvement of a yeast protease in the inactivation process is relatively unlikely.

Transport and metabolic effects of alpha-aminoisobutyric acid in Saccharomyces cerevisiae.

alpha-Aminoisobutyric acid is actively transported into yeast cells by the general amino acid transport system. The system exhibits a Km for alpha-aminoisobutyric acid of 270 microM, a Vmax of 24 nmol/min per mg cells (dry weight), and a pH optimum of 4.1-4.3. alpha-Aminoisobutyric acid is also transported by a minor system(s) with a Vmax of 1.7 nmol/min per mg cells. Transport occurs against a concentration gradient with the concentration ratio reaching over 1000:1 (in/out). The alpha-aminoisobutyric acid is not significantly metabolized or incorporated into protein after an 18 h incubation. alpha-Aminoisobutyric acid inhibits cell growth when a poor nitrogen source such as proline is provided but not with good nitrogen sources such as NH+4. During nitrogen starvation alpha-aminoisobutyric acid strongly inhibits the synthesis of the nitrogen catabolite repression sensitive enzyme, asparaginase II. Studies with a mutant yeast strain (GDH-CR) suggest that alpha-aminoisobutyric acid inhibition of asparaginase II synthesis occurs because alpha-aminoisobutyric acid is an effective inhibitor of protein synthesis in nitrogen starved cells.

Nitrogen catabolite repression in a glutamate auxotroph of Saccharomyces cerevisiae.

The biosynthesis of asparaginase II in Saccharomyces cerevisiae is subject to nitrogen catabolite repression. In the present study we examined the physiological effects of glutamate auxotrophy on cellular metabolism and on the nitrogen catabolite repression of asparaginase II. Glutamate auxotrophic cells, incubated without a glutamate supplement, had a diminished internal pool of alpha-ketoglutarate and a concomitant inability to equilibrate ammonium ion with alpha-amino nitrogen. In the glutamate auxotroph, asparaginase II biosynthesis exhibited a decreased sensitivity to nitrogen catabolite repression by ammonium ion but normal sensitivity to nitrogen catabolite repression by all amino acids tested.

Temperature-sensitive glucosamine auxotroph of Saccharomyces cerevisiae.

Temperature-sensitive revertants were isolated from Saccharomyces cerevisiae D-glucosamine auxotrophs previously obtained in this laboratory (W. L. Whelan and C. E. Ballou, J. Bacteriol. 124:1545-1557, 1975). The auxotrophs lack the enzyme 2-amino-2-deoxy-D-glucose-6-phosphate ketol-isomerase (EC 5.3.1.19), and the revertants appear to be temperature sensitive in the formation of enzyme activity. The enzyme they produce under permissive conditions decays in activity at a rate comparable to that of the wild-type enzyme, and it has similar kinetic properties. The homozygous diploid mutant fails to sporulate at the nonpermissive temperature. Temperature shift experiments were carried out in an effort to determine what effect glucosamine deficiency had on mannoprotein secretion as reflected in the formation of external asparaginase. Although the results were complicated by the slow decay of the residual ketol-isomerase activity, they did show that mannoprotein synthesis or secretion was altered when the internal pool of D-glucosamine was depleted.

Nitrogen catabolite repression of asparaginase II in Saccharomyces cerevisiae.

The biosynthesis of asparaginase II in Saccharomyces cerevisiae is subject to strong catabolite repression by a variety of nitrogen compounds. In the present study, asparaginase II synthesis was examined in a wild-type yeast strain and in strains carrying gdhA, gdhCR, or gdhCS mutations. The following effects were observed: (i) In the wild-type strain, the biosynthesis of asparaginase II was strongly repressed when either 10 mM ammonium sulfate or various amino acids (10 mM) served as the source of nitrogen. (ii) In a yeast strain carrying the gdhA mutation, asparaginase II was synthesized at fully derepressed levels when 10 mM ammonium sulfate was the source of nitrogen. When amino acids (10 mM) served as the nitrogen source, asparaginase II synthesis was strongly repressed. (iii) In a strain carrying the gdhCR mutation, the synthesis of asparaginase II was partially (30 to 40%) derepressed when either 10 mM ammonium sulfate or amino acids were present in the medium. (iv) In a yeast strain containing both gdhA and gdhCR mutations, asparaginase II synthesis was fully derepressed when 10 mM ammonium sulfate was the nitrogen source and partially derepressed when 10 mM amino acids were present. (v) Yeast strains carrying the gdhCS mutation were indistinguishable from the wild-type strain with respect to asparaginase II synthesis.