Presence Of D-serine Research Articles

A raman spectroscopic investigation of conformation of flavin adenine dinucleotide, a coenzyme of d-amino acid oxidase

Based on silver nanoparticles application, surface-enhanced Raman scattering spectra of D-amino acid oxidase from pig kidney were recorded and analyzed. Spectral parameters characteristic of conformational changes in cofactor flavin adenine dinucleotide during activation of the enzyme by D-amino acids were revealed. It was found that enzyme substrate specificity determines the amount of time from the start of of the conformational changes of flavin adenine dinucleotide until they no longer occur: in the presence of D-alanine, registration of the said conformational changes takes up just a few seconds, while it takes 10 min in the presence of D-serine .

Oral administration of D-serine prevents the onset and progression of colitis in mice.

L-amino acids are the predominant forms of organic molecules on the planet, but recent studies have revealed that various foods contain D-amino acids, the enantiomers of L-amino acids. Though diet plays important roles in both the development and progression of inflammatory bowel disease (IBD), to our best knowledge, there has been no report on any potential interactions between D-amino acids and IBD. In this report, we aim to assess the effects of D-serine in a murine model of IBD. To induce chronic colitis, naïve CD4 T cells (CD4+ CD62+ CD44low) from wild-type mice were adoptively transferred into Rag2-/- mice, after or before the mice were orally administered with D-serine. In vitro proliferation assays were performed to assess naïve CD4 T cell activation under the Th-skewing conditions in the presence of D-serine. Mice treated with D-serine prior to the induction of colitis exhibited a reduction in T-cell infiltration into the lamina propria and colonic inflammation that were not seen in mice fed with water alone or L-serine. Moreover, D-serine suppressed the progression of chronic colitis when administered after the disease induction. Under in vitro conditions, D-serine suppressed the proliferation of activated CD4 T cells and limited their ability to differentiate to Th1 and Th17 cells. Our results suggest that D-serine not only can prevent, but also has efficacious effects as a treatment for IBD.

Agonist and antagonist NMDA receptor effect on cell fate during germ cell differentiation and regulate apoptotic process in 3D organ culture.

In this work, agonist and antagonist N-methyl-D-aspartate (NMDA) receptor activation effect on cell fate during germ cell differentiation and regulate apoptotic process in 3D organ culture were studied. Afterwards, the effect of D-serine, retinoic acid (RA) and MK801 on spermatogenesis development was investigated. The animals were injected a single dose (40mg/kg, intraperitoneal) of busulfan. After confirming the model, ten 5-day-old NMRI mice were used as spermatogonial stem cells (SSCs) transplantation donors. The SSCs were confirmed by detecting the promyelocytic leukaemia zinc finger (PLZF) protein. Then, tissue culture of the azoospermia model which had received SSCs was performed in various conditions (seven groups). The apoptosis markers levels of cells were significantly decreased in differentiation media containing RA and serine. In contrast, the expression of apoptotic markers including caspase 3, caspase 9 and Bax was increased in the presence of MK801. In conclusion, a new in vitro system capable of producing mature spermatozoa was developed that would be useful for investigating the medicinal effects of agents on the male reproductive system. Also, a comparison of spermatogenesis development in different media revealed that the presence of D-serine and RA (retinoic acid) in the culture medium has a positive effect on spermatogenesis.

Modulating NMDA Receptor Function with d-Amino Acid Oxidase Inhibitors: Understanding Functional Activity in PCP-Treated Mouse Model

Deficits in N-methyl-D-aspartate receptor (NMDAR) function are increasingly linked to persistent negative symptoms and cognitive deficits in schizophrenia. Accordingly, clinical studies have been targeting the modulatory site of the NMDA receptor, based on the decreased function of NMDA receptor, to see whether increasing NMDA function can potentially help treat the negative and cognitive deficits seen in the disease. Glycine and D-serine are endogenous ligands to the NMDA modulatory site, but since high doses are needed to affect brain levels, related compounds are being developed, for example glycine transport (GlyT) inhibitors to potentially elevate brain glycine or targeting enzymes, such as D-amino acid oxidase (DAAO) to slow the breakdown and increase the brain level of D-serine. In the present study we further evaluated the effect of DAAO inhibitors 5-chloro-benzo[d]isoxazol-3-ol (CBIO) and sodium benzoate (NaB) in a phencyclidine (PCP) rodent mouse model to see if the inhibitors affect PCP-induced locomotor activity, alter brain D-serine level, and thereby potentially enhance D-serine responses. D-Serine dose-dependently reduced the PCP-induced locomotor activity at doses above 1000 mg/kg. Acute CBIO (30 mg/kg) did not affect PCP-induced locomotor activity, but appeared to reduce locomotor activity when given with D-serine (600 mg/kg); a dose that by itself did not have an effect. However, the effect was also present when the vehicle (Trappsol(®)) was tested with D-serine, suggesting that the reduction in locomotor activity was not related to DAAO inhibition, but possibly reflected enhanced bioavailability of D-serine across the blood brain barrier related to the vehicle. With this acute dose of CBIO, D-serine level in brain and plasma were not increased. Another weaker DAAO inhibitor NaB (400 mg/kg), and NaB plus D-serine also significantly reduced PCP-induced locomotor activity, but without affecting plasma or brain D-serine level, arguing against a DAAO-mediated effect. However, NaB reduced plasma L-serine and based on reports that NaB also elevates various plasma metabolites, for example aminoisobutyric acid (AIB), a potential effect via the System A amino acid carrier may be involved in the regulation of synaptic glycine level to modulate NMDAR function needs to be investigated. Acute ascorbic acid (300 mg/kg) also inhibited PCP-induced locomotor activity, which was further attenuated in the presence of D-serine (600 mg/kg). Ascorbic acid may have an action at the dopamine membrane carrier and/or altering redox mechanisms that modulate NMDARs, but this needs to be further investigated. The findings support an effect of D-serine on PCP-induced hyperactivity. They also offer suggestions on an interaction of NaB via an unknown mechanism, other than DAAO inhibition, perhaps through metabolomic changes, and find unexpected synergy between D-serine and ascorbic acid that supports combined NMDA glycine- and redox-site intervention. Although mechanisms of these specific agents need to be determined, overall it supports continued glutamatergic drug development.

Dynamic regulation of D-serine release in the vertebrate retina.

Activation of NMDA receptors (NMDARs) is essential for encoding visual stimuli into signals for the brain, although their over-activation can cause cell death. The recruitment of NMDARs is important for encoding light intensity in retinal ganglion cells. D-serine binding is essential for proper activation of NMDARs, although its role in signal processing and the mechanisms that underlie its availability are not well understood. In these light-evoked experiments, the addition of exogenous D-serine had a large effect on low contrast and low intensity NMDAR responses that decreased as the intensity was increased. The degradation of endogenous D-serine decreased the responses more at higher intensities. The results provide compelling evidence favouring a new interpretation of NMDAR recruitment in which light-evoked D-serine release serves an important regulatory control over the recruitment of NMDARs. The present study aimed to investigate the functional properties of NMDA receptor coagonist release and to specifically evaluate whether light-evoked release mechanisms contribute to the availability of the coagonist D-serine. Two different methods were involved in our approach: (i) whole-cell recordings from identified retinal ganglion cells in the tiger salamander were used to study light adaptation with positive and negative contrast stimuli over a range of ± 1 log unit against a steady background illumination and (ii) the mechanisms for intensity encoding to a range of light intensities covering 6 log10 units were investigated. This latter study employed extracellular recordings of the proximal negative field potential, pharmacologically manipulated to generate a pure NMDA mediated response. For the adaptation study, we examined the light-evoked responses under control conditions, followed by light stimuli presented in the presence of D-serine, followed by light stimulation in the presence of dichlorokynurenic acid to block the coagonist site of NMDA receptors. For the brightness encoding studies, we examined the action of D-serine on each intensity used and then applied the enzyme D-serine deaminase to remove significant levels of D-serine. These studies provided new insights into the mechanisms that regulate coagonist availability in the vertebrate retina. Our results strongly support the idea that light-evoked coagonist release, a major component of which is D-serine, is needed to provide the full range of coagonist availability for optimal activation of NMDA receptors.

Significance of the d -Serine-Deaminase and d -Serine Metabolism of Staphylococcus saprophyticus for Virulence

Staphylococcus saprophyticus is the only species of Staphylococcus that is typically uropathogenic and possesses a gene coding for a D-serine-deaminase (DsdA). As D-serine is prevalent in urine and toxic or bacteriostatic to many bacteria, it is not surprising that the D-serine-deaminase gene is found in the genome of uropathogens. It has been suggested that D-serine-deaminase or the ability to respond to or to metabolize D-serine is important for virulence. For uropathogenic Escherichia coli (UPEC), a high intracellular D-serine concentration affects expression of virulence factors. S. saprophyticus is able to grow in the presence of high D-serine concentrations; however, its D-serine metabolism has not been described. The activity of the D-serine-deaminase was verified by analyzing the formation of pyruvate from D-serine in different strains with and without D-serine-deaminase. Cocultivation experiments were performed to show that D-serine-deaminase confers a growth advantage to S. saprophyticus in the presence of D-serine. Furthermore, in vivo coinfection experiments showed a disadvantage for the ΔdsdA mutant during urinary tract infection. Expression analysis of known virulence factors by reverse transcription-quantitative PCR (RT-qPCR) showed that the surface-associated lipase Ssp is upregulated in the presence of D-serine. In addition, we show that S. saprophyticus is able to use D-serine as the sole carbon source, but interestingly, D-serine had a negative effect on growth when glucose was also present. Taken together, D-serine metabolism is associated with virulence in S. saprophyticus, as at least one known virulence factor is upregulated in the presence of D-serine and a ΔdsdA mutant was attenuated in virulence murine model of urinary tract infection.

Antinociceptive Effect of Rat D-Serine Racemase Inhibitors, L-Serine-O-Sulfate, and L-Erythro-3-Hydroxyaspartate in an Arthritic Pain Model

N-methyl-D-aspartic acid receptor (NMDAr) activation requires the presence of D-serine, synthesized from L-serine by a pyridoxal 5′-phosphate-dependent serine racemase (SR). D-serine levels can be lowered by inhibiting the racemization of L-serine. L-serine-O-sulfate (LSOS) and L-erythro-3-hydroxyaspartate (LEHA), among others, have proven to be effective in reducing the D-serine levels in culture cells. It is tempting then to try these compounds in their effectiveness to decrease nociceptive levels in rat arthritic pain. We measured the C-reflex paradigm and wind-up potentiation in the presence of intrathecally injected LSOS (100 μg/10 μL) and LEHA (100 μg/10 μL) in normal and monoarthritic rats. Both compounds decreased the wind-up activity in normal and monoarthritic rats. Accordingly, all the antinociceptive effects were abolished when 300 μg/10 μL of D-serine were injected intrathecally. Since no in vivo results have been presented so far, this constitutes the first evidence that SR inhibitions lower the D-serine levels, thus decreasing the NMDAr activity and the consequent development and maintenance of chronic pain.

Coactivation of NMDA Receptors by Glutamate and d-Serine Induces Dilation of Isolated Middle Cerebral Arteries

N-methyl-D-aspartate (NMDA) receptors are glutamate-gated cation channels that mediate excitatory neurotransmission in the central nervous system. In addition to glutamate, NMDA receptors are also activated by coagonist binding of the gliotransmitter, D-serine. Neuronal NMDA receptors mediate activity-dependent blood flow regulation in the brain. Our objective was to determine whether NMDA receptors expressed by brain endothelial cells can induce vasodilation of isolated brain arteries. Adult mouse middle cerebral arteries (MCAs) were isolated, pressurized, and preconstricted with norepinephrine. N-methyl-D-aspartate receptor agonists, glutamate and NMDA, significantly dilated MCAs in a concentration-dependent manner in the presence of D-serine but not alone. Dilation was significantly inhibited by NMDA receptor antagonists, D-2-amino-5-phosphonopentanoate and 5,7-dichlorokynurenic acid, indicating a response dependent on NMDA receptor glutamate and D-serine binding sites, respectively. Vasodilation was inhibited by denuding the endothelium and by selective inhibition or genetic knockout of endothelial nitric oxide synthase (eNOS). We also found evidence for expression of the pan-NMDA receptor subunit, NR1, in mouse primary brain endothelial cells, and for the NMDA receptor subunit NR2C in cortical arteries in situ. Overall, we conclude that NMDA receptor coactivation by glutamate and D-serine increases lumen diameter in pressurized MCA in an endothelial and eNOS-dependent mechanism.

Turning pyridoxal-5′-phosphate-dependent enzymes into thermostable binding proteins: d-Serine dehydratase from baker’s yeast as a case study

d-serine dehydratase from Saccharomyces cerevisae is a recently discovered dimeric enzyme catalyzing the β-elimination of d-serine to pyruvate and ammonia. The reaction is highly enantioselective and depends on cofactor pyridoxal-5′-phosphate (PLP) and Zn2+. In our work, the aldimine linkage tethering PLP to recombinant, tagged d-serine dehydratase (Dsd) has been reduced by treatment with NaBH4 so as to yield an inactive form of the holoenzyme (DsdR), which was further treated with a protease in order to remove the amino-terminal purification tag. Fourier Transform infrared (FT-IR) spectroscopic analysis revealed that both the reduced form (DsdR) and the reduced/detagged form (DsdRD) maintain the overall secondary structure of Dsd, but featured a significant increased thermal stability. The observed Tm values for DsdR and for DsdRD shifted to 71.5 °C and 73.3 °C, respectively, resulting in nearly 11 °C and 13 °C higher than the one measured for Dsd. Furthermore, the analysis of the FT-IR spectra acquired in the presence of d-serine and l-serine indicates that, though catalytically inert, DsdRD retains the ability to enantioselectively bind its natural substrate. Sequence analysis of d-serine dehydratase and other PLP-dependent enzymes also highlighted critical residues involved in PLP binding. In virtue of its intrinsic properties, DsdRD represents an ideal candidate for the design of novel platforms based on stable, non-consuming binding proteins aimed at measuring d-serine levels in biological fluids.

Evaluation of the E. coli d-serine ammonia lyase gene (Ec. dsdA) for use as a selectable marker in maize transformation

In this study, the d-serine ammonia lyase (dsdA) gene from Escherichia coli was evaluated as a selectable marker for maize transformation. Plants are incapable of utilizing the D-form of most amino acids, and d-serine has recently been demonstrated to be phytoinhibitory to plant growth. d-Serine ammonia lyase detoxifies d-serine via a substrate-specific reaction to pyruvate, ammonia, and water. d-Serine inhibits germination of isolated maize immature embryos and growth of embryogenic callus from wild-type plants at concentrations about approx. 2–15 mM. Transgenic plants were recovered in the presence of d-serine in tissue culture media with dsdA as the selection marker at efficiencies comparable to using a mutated acetohydroxy acid synthase selection marker gene and selection in the presence of imidazolinone herbicides. Immature embryos infected with an Agrobacterium strain containing an acetohydroxy acid synthase gene construct without dsdA did not yield any transgenic events on the selection medium with 10 mM d-serine, indicating that d-serine provided selection tight enough to prevent escapes. Molecular analysis confirmed the integration of the dsdA gene into the genome of the transgenic plants. No adverse phenotypes were observed in the greenhouse, and expression of the dsdA marker had no affect on agronomic characteristics or grain yield in multi-location field trials. Seed compositional analysis demonstrated no significant differences in the contents of seed protein, starch, fatty acids, fiber, phytic acid, and free amino acids between transgenic and non-transgenic control plants. These data indicate that the dsdA gene is properly expressed in maize and the d-serine ammonia lyase (DSDA) enzyme functions appropriately to metabolize d-serine during in vitro selection. Preliminary safety assessments indicated that no adverse affects would be expected if humans were exposed to the DSDA protein in the diet from an allergenicity or toxicity perspective. The dsdA gene in combination with phytoinhibitory levels of d-serine represents a new and effective selectable marker system for maize transformation.

Possible mechanisms of A beta(1-40)- or A beta(1-42)-induced cell death and their rescue factors

Amyloidbetapeptide (A beta) is implicated in neuronal cell death in Alzheimer's disease, but the molecular mechanisms are still unclear. We analyzed its mechanism and found several potential rescue factors against A beta-mediated apoptosis. A beta(1-40) stimulated phosphorylation of tau and JNK and induced cell death in SH-SY5Y cells. The cell death was inhibited by insulin-like growth factor-1, suggesting that the JNK pathway may be involved in A beta(1-40)-induced cytotoxicity. Using the human fetus brain cDNA library-targeted differential display technique, a new gene BF5-1 (32aa) was found as a rescue factor against A beta(1-40). BF5-1 has partially the same amino acid sequences as those of the C-terminus of cytochrome c oxidase subunit VIIb (COX-VIIb). COX-VIIb mRNA is increased in AD brains and its overexpression in cells enhanced A beta(1-40)-toxicity. These data suggest that BF5-1 may act as a dominant negative mutant of COX-VIIb. A beta(1-42) also induced cell death in rat neuroblastoma B104 cells, which was abolished by addition of IL-11. By cDNA subtraction analysis in the cell death, the enhanced expression of L-phosphoserine phosphatase was found, but this was also abolished by IL-11. The glutamate neurotoxicity was stimulated in the presence of D-serine, suggesting that NMDA receptors may be involved in A beta(1-42)-induced cytotoxicity. A beta(1-42) also induced increase of a new gene p18A beta rP (p18-amyloid-beta-responsive protein; 166 aa) mRNA expression; overexpression of this gene in PC12 cells induced cell death. By the application of a death trap method, a new gene, p60TRP (p60-Transcription-Regulating-Protein; rat:539 aa, human:547aa), was found as a potential rescue factor against the cell death by p18A beta rP. Thus, our cell death systems and/or new rescue proteins may provide suitable tools for the establishment of drug screening systems leading to the identification of new low-molecular candidates applicable for the treatment of AD.

Control by GABA and tachykinins of the evoked release of acetylcholine in striatal compartments under different modalities of NMDA receptor stimulation

The contribution of endogenously released dopamine, GABA and its co-transmitters, substance P (SP) and neurokinin A (NKA), to the control of the evoked release of acetylcholine was investigated in vitro in the striosomes and the matrix of the rat striatum under various modalities of NMDA receptor stimulation (NMDA 50 μM or 1 mM without or with 10 μM d-serine). Sulpiride, bicuculline, SR140333 and SR48968, the antagonists of D 2, GABA A, NK 1 and NK 2 tachykinin receptors, respectively, were used for this purpose. (1) In both striatal compartments, the dopamine-mediated inhibitory regulation of the evoked release of acetylcholine only occurred when d-serine was co-applied with 50 μM or 1 mM NMDA. (2) In striosomes, the dopamine-dependent inhibitory effects of SP and NKA on the evoked release of acetylcholine only occurred when d-serine was co-applied with 50 μM or 1 mM NMDA. (3) A similar inhibitory regulation by NKA, but not SP, was found in the matrix when 1 mM NMDA was co-applied with d-serine. (4) In contrast, the dopamine-dependent facilitatory effect of GABA on the evoked release of acetylcholine did not require added d-serine and was more important with 1 mM than 50 μM NMDA. In the presence of d-serine, and depending on the NMDA concentration, the facilitatory regulation of GABA was reduced (matrix) or suppressed (striosomes). This latter effect was partially restored in the presence of SR48968. Therefore, the dopamine-dependent inhibitory effects of tachykinins on the evoked release of acetylcholine only occurred when NMDA receptors were stimulated in the presence of saturating concentrations of d-serine.

Substrate inhibition of D-amino acid transaminase and protection by salts and by reduced nicotinamide adenine dinucleotide: isolation and initial characterization of a pyridoxo intermediate related to inactivation.

D-Amino acid transaminase, a pyridoxal phosphate (PLP) enzyme, is inactivated by its natural substrate, D-alanine, concomitant with its alpha-decarboxylation [Martinez del Pozo, A., Yoshimura, T., Bhatia, M. B., Futaki, S., Manning, J. M., Ringe, D., and Soda, K. (1992) Biochemistry 31, 6018-6023; Bhatia, M. B., Martinez del Pozo, A., Ringe, D., Yoshimura, T., Soda, K., and Manning, J. M. (1993) J. Biol. Chem. 268, 17687-17694]. beta-Decarboxylation of d-aspartate to d-alanine leads also to this inactivation [Jones, W. M., van Ophem, P. W., Pospischil, M. A., Ringe, D., Petsko, G., Soda, K., and Manning, J. M. (1996) Protein Sci. 5, 2545-2551]. Using a high-performance liquid chromatography-based method for the determination of pyridoxo cofactors, we detected a new intermediate closely related to the inactivation by d-alanine; its formation occurred at the same rate as the inactivation and upon reactivation it reverted to PLP. Conditions were found under which it was characterized by ultraviolet-visible spectral analysis and mass spectroscopy; it is a pyridoxamine phosphate-like compound with a C2 fragment derived from the substrate attached to the C'-4 of the pyridinium ring and it has a molecular mass of 306 consistent with this structure. In the presence of d-serine, slow accumulation of a quinonoid intermediate is also related to inactivation. The inactivation can be prevented by salts, which possibly stabilize the protonated aldimine coenzyme complex. The reduced cofactor, nicotinamide adenine dinucleotide, prevents D-aspartate-induced inactivation. Both of these events also are related to formation of the novel intermediate.

Occurrence of peptidyl d-amino acids in soluble fractions of several eubacteria, archaea and eukaryotes

The occurrence of peptidyl d-amino acids in the aqueous soluble fractions was investigated in various eubacteria, some archaea and some eukaryotes. The contents of the d-enantiomers of serine, alanine, proline, glutamate (glutamine), aspartate (asparagine) and phenylalanine were determined with cell- and tissue-extracts, by means of acid hydrolysis and high-performance liquid chromatography. The rate of d-enantiomer (%, the ratio in molar concentration of a d-amino acid to the total of the d-amino acid and the corresponding l-amino acid) of alanine and glutamate were high in some Gram-positive eubacteria: 11.7% in Staphylococcus epidermidis and 10.3% in Streptococcus pyogenes for alanine, and 22.3% for glutamate in Bacillus YN-1. The d-glutamate content was also high (8.0%) in the Gram-negative eubacterium, Thiobacillus ferrooxidans. d-Aspartate was common, as was d-glutamate: the highest d-aspartate content was detected in an archaeum, Pyrobaculum islandicum (4.0%). However, the content of d-aspartate was low, 0.2–1.8% in most other bacteria. The presence of d-serine was shown in some organisms, but that of d-proline was scarce. The d-enantiomer of phenylalanine was not detected in any of the organisms examined. These results indicate that of the bacteria examined herein most Gram-negative and some Gram-positive eubacteria, as well as archaea contain only low levels of d-amino acids in the soluble peptidyl fraction, and the levels were comparable to those in eukaryotes examined. To our knowledge, the general presence of peptidyl d-amino acids in these organisms, especially archaea and eukaryotic cells including those from rat liver tissues, has been shown here for the first time.

In vitro modulation of hippocampal pyramidal cell response by quinolones: effects of HA 966 and gamma-hydroxybutyric acid.

The influence of quinolones on electrically evoked pyramidal cell activity in the rat hippocampus in vitro was studied by using the slice technique. We hoped to learn more about the possible mechanisms for the development of side effects of different quinolones and to find a possible treatment. As reported earlier (W. Dimpfel, M. Spüler, A. Dalhoff, W. Hofmann, and G. Schlüter, Antimicrob. Agents Chemother. 35:1142-1146, 1991), the amplitude of the population spike increased in the presence of ciprofloxacin, lomefloxacin, or ofloxacin about twofold in comparison with reference values. This increase could be prevented in a concentration-dependent manner by the concomitant presence of 3-amino-1-hydroxy-2-pyrrolidone (HA 966), a compound acting at the so-called glycine site of the N-methyl-D-aspartate (NMDA) receptor, but not in the presence of aminophosphonovaleric acid (APV), which acts at a different recognition site of the NMDA receptor. Another tool, 6,7-dinitroquinoxaline-2,3-dione, an antagonist of the so-called AMPA receptor (named after the binding of L-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid [AMPA] to this site), could not antagonize the effect induced by the quinolones. Activation of the glycine site of the NMDA receptor induced by the presence of D-serine in the superfusion medium also resulted in a concentration-dependent increase in the population spike amplitude. This response remained unchanged in the presence of ciprofloxacin, whereas lomefloxacin and ofloxacin led to further increases in the amplitude, especially in the presence of higher concentrations of D-serine. These results also point to an involvement of the glycine site of the central NMDA receptor in the development of side effects by different quinolones. A complete attenuation of the quinolone-induced effects was obtained in the presence of 2.5 microM gamma-hydroxybutyric acid (GHB), a physiological neuromodulator which is marketed in some countries of Europe as a sedative. It is therefore concluded that the excitatory adverse effects of quinolones might be treated by the administration of GHB.

Regulation of endogenous noradrenaline release from the bed nucleus of stria terminalis

The bed nucleus of stria terminalis (BNST) contains the highest concentration of noradrenaline (NA) in the brain. Minislices of the ventral portion of the bed nucleus of stria terminalis (vBNST) were used to study the release of endogenous NA. High K + induced a Ca 2+-dependent and reserpine-sensitive release of NA. Clonidine (1 μM), an α 2-noradrenergic receptor agonist, significantly decreased K +-induced release of NA, whereas yohimbine (1 μM), an α 2-noradrenergic antagonist, increased this release. N-Methyl-D-aspartate (NMDA), a specific agonist of NMDA-type glutamate receptors, evoked the release of NA from vBNST minislices. In the presence of D-serine (10 μM), an agonist at the glycine site associated with the NMDA receptor, the NMDA effect was significantly higher. Glycine (1 μM) also increased NA release evoked by NMDA. However, glycine exhibited a significant effect by itself, suggesting the existence of strychnine-sensitive glycine receptors in vBNST. Endogenous NA release induced by 40 mM K + and NMDA was not additive. Thus, vBNST minislices seem to be a good model to study the release of endogenous NA in the CNS. Such NA release in the vBNST is regulated by α 2-noradrenergic receptors and by glutamate through NMDA receptors.

Release of endogenous catecholamines from the striatum and bed nucleus of stria terminalis evoked by potassium and N-methyl-D-aspartate: in vitro microdialysis studies.

Induced release of endogenous dopamine and noradrenaline from coronal slices containing the striatum and the bed nucleus of the stria terminalis, respectively, was studied by means of in vitro microdialysis. A Ca(+2)-dependent and reserpine-sensitive K(+)-induced release of catecholamines was detected in both nuclei. We confirmed that N-methyl-D-aspartate (2.5 and 5.0 mM in the dialysis perfusion solution) induces the release of dopamine from the striatum, and this effect was blocked by prior dialysis perfusion with 500 microM MK-801, a noncompetitive N-methyl-D-aspartate receptor antagonist. Infusion of N-methyl-D-aspartate (1-10 mM) or glutamate through the dialysis probe did not produce any detectable modification in the extracellular levels of noradrenaline in the bed nucleus of the stria terminalis. In addition, perfusion with D-serine (100 microM) alone or in the presence of desipramine (10 microM), resulted in a slight increase in extracellular noradrenaline in the bed nucleus of the stria terminalis. However, N-methyl-D-aspartate in the presence of D-serine and desipramine produced a marked increase in extracellular noradrenaline from the bed nucleus of the stria terminalis. These results indicate that N-methyl-D-aspartate receptors might regulate the release of noradrenaline from the bed nucleus of the stria terminalis as is the case of dopamine release in the striatum. The in vitro microdialysis seems to be a suitable complement to the in vivo microdialysis for the study of catecholamine release in discrete regions of the central nervous system and its local regulation by excitatory amino acid receptors.

The NMDA receptor antagonist eliprodil (SL 82.0715) blocks voltage-operated Ca 2+ channels in rat cultured cortical neurons

The effect of the non-competitive NMDA receptor antagonist eliprodil on NMDA receptor- and voltage-operated Ca 2+ currents was investigated in rat cultured cortical neurons by using the whole-cell patch clamp technique. With neurons voltage-clamped at −40 mV, eliprodil reduced in concentration-dependent manner the inward current induced by N- methyl- D-aspartate (NMDA) (10 μM) in the presence of D-serine with an IC 50 of 0.67 μM ( I max=83%). Eliprodil also blocked the total inward Ba 2+ current carried in part by L- and N-type Ca 2+ channels with an IC 50 of 1.48 μM ( I max=87%). These results suggest that the neuroprotective properties of eliprodil could be due to its combined ability to antagonize the NMDA receptor- and voltage-operated Ca 2+ channels.

D-Aminoaciduria in mutant mice lackingd-amino-acid oxidase activity

Urine of mutant ddY/DAO(-) mice lackingD-amino-acid oxidase activity contained more serine and proline than that of normal ddY/DAO(+) mice.D-Amino-acid oxidase treatment of urinary amino acids decreased the serine and proline, suggesting that they containedD-isomers. An HPLC analysis confirmed the presence ofD-serine. Urinary serine and proline contents were not decreased when the ddY/DAO(-) mice were fed a diet which did not contain supplementaryD-methionine or when they were given water containing antibiotics. These results suggest that theD-serine andD-proline do not derive from theD-methionine supplemented in the diet or from intestinal bacteria. In urine of the ddY/DAO(-) mice, a substance which seemed to beD-methionine sulfoxide and/orD-methionine sulfone was present. It is probably a metabolite of theD-methionine supplemented in the diet. TheD-aminoaciduria in the mutant mice lackingD-amino-acid oxidase activity indicates that this enzyme is involved in the metabolism of theD-amino acids in normal mice.

Conversion of phosphatidylcholine to phosphatidylserine by various phospholipases D in the presence of l- or d-serine

The conversion of phosphatidylcholine (PC) to phosphatidylserine (PS) by four phospholipase D preparations was studied in the presence of l-serine or d-serine. The biphasic reaction was carried out at 30 ± 0.5°C using aqueous phase containing phospholipase D with serine and ethyl acetate containing PC. As a solvent candidate for PS formation, ethyl acetate and diethyl ether demonstrated efficient transphosphatidylation. Among the phospholipase D preparations examined, three from Streptomyces and one from cabbage origin, a Streptomyces phospholipase D displayed the highest catalytic activity for PS formation and the apparent rate constant ratio of transphosphatidylation and hydrolysis, (k′3/k3)app of the phospholipase D was the highest. At a concentration of 3.4 M serine, which is the solubility of serine in buffer of pH 5.6 at 30 ± 0.5°C, the phospholipase D yielded 98.5 ± 0.5 and 100% of PS using 17.8 mM PC with l-serine and d-serine, respectively. The increase in serine concentration exhibited no inhibition. PC was almost completely converted to PS (98 ± 0.8% yield) below 53.4 mM PC, keeping other reaction conditions constant. At all the concentrations of both the stereoisomers of serine, the initial rate of transphosphatidylation was higher with d-serine than with l-serine using all the Streptomyces phospholipase D preparations. On the other hand, cabbage phospholipase D barely showed any transphosphatidylation in the presence of d-serine and displayed only hydrolytic activity, while partial transphosphatidylation was observed with l-serine, yielding a maximum 41.3 ± 1.9% of PS. The PS products formed with l- and d-isomers of serine by the phospholipase D retained their original stereoisomeric forms of serine. Repeated batch operation conducted with the phospholipase D immobilized in amphiphilic gels showed some encouraging results and 96 ± 1.45% of PC to PS conversions were achieved in the first four batch cycles.