D-Aspartic acid is a novel endogenous neurotransmitter.

D-aspartic acid (D-Asp) is present in invertebrate and vertebrate neuroendocrine tissues, where it carries out important physiological functions and is implicated in nervous system development. We show here that D-Asp is a novel endogenous neurotransmitter in two distantly related animals, a mammal (Rattus norvegicus) and a mollusk (Loligo vulgaris). Our main findings demonstrate that D-Asp is present in high concentrations in the synaptic vesicles of axon terminals; synthesis for this amino acid occurs in neurons by conversion of L-Asp to D-Asp via D-aspartate racemase; depolarization of nerve endings with K(+) ions evokes an immediate release of D-Asp in a Ca(2+) dependent manner; specific receptors for D-Asp occur at the postsynaptic membrane, as demonstrated by binding assays and by the expansion of squid skin chromatophores; D-aspartate oxidase, the specific enzyme that oxidizes D-Asp, is present in the postsynaptic membranes; and stimulation of nerve endings with D-Asp triggers signal transduction by increasing the second messenger cAMP. Taken together, these data demonstrate that D-Asp fulfills all criteria necessary to be considered a novel endogenous neurotransmitter. Given its known role in neurogenesis, learning, and neuropathologies, our results have important implications for biomedical and clinical research.

Thyroid hormones and D-aspartic acid, D-aspartate oxidase, D-aspartate racemase, H2O2, and ROS in rats and mice.

Total concentrations of thyroid hormones T(3) and T(4), and of their free forms, FT(3) and FT(4), D-aspartic acid (D-Asp), D-aspartate oxidase (D-AspO), D-aspartate racemase, H(2)O(2), and ROS (reactive oxygen species) were determined in rats and mice. T(3) and T(4) were 1 and 50 ng/ml, respectively, in serum, and 750 and 40000 ng/g, respectively, in thyroid. Concentrations of the free forms FT(3) and FT(4) were ca. 250 times lower than their respective total concentrations. The endogenous content of D-Asp in thyroid gland was ca. 100 nmol/g tissue, whereas the activity of D-AspO was ca. 80 units/mg thyroid, and that of D-aspartate racemase was ca. 15 units/mg thyroid. H(2)O(2) Concentration in rat and mouse thyroid gland was ca. 290 pmol/g thyroid, and the concentration of ROS was ca. 10 pmol/DCF/min/mg protein. H(2)O(2) is essential for the iodination of the tyrosyl residues to produce mono- and diiodotyrosine that are the precursors for the synthesis of T(3) and T(4). Production of H(2)O(2) in thyroid glands occurs by oxidation of endogenous D-Asp by D-AspO (D-Asp+O(2)+H(2)O-->alpha-oxaloacetate+NH(3)+H(2)O(2)). D-Aspartate racemase catalyzes the in vivo production of D-Asp from L-Asp. Thus, interaction of endogenous D-Asp, D-AspO, and D-aspartate racemase in thyroid gland constitutes an additional biochemical pathway for the production of H(2)O(2) and consequently for the synthesis of thyroid hormones.

Distribution of free D-aspartic acid and D-aspartate oxidase in frog Rana esculenta tissues.

In this paper, we examined the distribution pattern of D-aspartic acid (D-Asp), as well as D-aspartate oxidase (D-AspO), D-amino acid oxidase (D-AAO), and L-amino acid oxidase (L-AAO) activities in different tissues of frog, Rana esculenta. High concentrations of free D-Asp were found in the testes (0.21+/-0.02 micromol/g b.w), in the liver (0.20+/-0.03 micromol/g b.w), and in the Harderian gland (HG) (0.19+/-0.03 micromol/g b.w). A higher activity of both D-AspO and D-AAO with respect to L-AAO was endogenously present in all examined frog tissues, particularly within the kidney, liver, and brain. Our in vivo experiments, consisting of i.p. injections of 2.0 micromol/g b.w. D-Asp in frogs, revealed that all examined tissues can take up and accumulate D-Asp and that this amino acid specifically triggers D-AspO activity. Indeed, no increase in both D-AAO and L-AAO was found in all frog tissues after D-Asp treatment. The optimum pH for D-AspO activity was around 8.2 and the optimum temperature was about 37 degrees C. Furthermore, its activity linearly increased with increasing D-Asp incubation times. In vitro experiments assaying the substrate specificity of D-AspO indicated that the enzyme had greater affinity for N-methyl-D-aspartate than for D-Asp and D-glutamate. This study provides evidence of the presence of free D-Asp in frog R. esculenta tissues, along with its role in triggering D-AspO activity. These findings suggest that D-AspO could play an essential role in decreasing excessive amounts of D-Asp in frog tissues, a phenomenon that, if left unchecked, could have detrimental physiological effects on the animal.

Evidence for the involvement of D-aspartic acid in learning and memory of rat.

D-Aspartic acid (D-Asp) is an endogenous amino acid present in neuroendocrine systems. Here, we report evidence that D-Asp in the rat is involved in learning and memory processes. Oral administration of sodium D-aspartate (40 mM) for 12-16 days improved the rats' cognitive capability to find a hidden platform in the Morris water maze system. Two sessions per day for three consecutive days were performed in two groups of 12 rats. One group was treated with Na-D-aspartate and the other with control. A significant increase in the cognitive effect was observed in the treated group compared to controls (two-way ANOVA with repeated measurements: F ((2, 105)) = 57.29; P value < 0.001). Five further sessions of repeated training, involving a change in platform location, also displayed a significant treatment effect [F ((2, 84)) = 27.62; P value < 0.001]. In the hippocampus of treated rats, D-Asp increased by about 2.7-fold compared to controls (82.5 +/- 10.0 vs. the 30.6 +/- 5.4 ng/g tissue; P < 0.0001). Moreover, 20 randomly selected rats possessing relatively high endogenous concentrations of D-Asp in the hippocampus were much faster in reaching the hidden platform, an event suggesting that their enhanced cognitive capability was functionally related to the high levels of D-Asp. The correlation coefficient calculated in the 20 rats was R = -0.916 with a df of 18; P < 0.001. In conclusion, this study provides corroborating evidence that D-aspartic acid plays an important role in the modulation of learning and memory.

D-Aspartate binding sites in rat Harderian gland.

Radioligand binding of D-[(3)H]aspartic and L-[(3)H]glutamic acids to plasma membranes from rat Harderian gland was evaluated. Binding was optimal under physiological conditions of pH and temperature, and equilibrium was reached within 50 min. Specific binding for D-Asp and L-Glu was saturable, and Eadie-Hofstee analysis revealed interaction with a single population of binding sites (for D-Asp K(d) = 860 +/- 28 nM, B(max) = 27.2 +/- 0.5 pmol/mg protein; for L-Glu, K(d) = 580 +/- 15 nM and B(max) = 51.3 +/- 0.8 pmol/mg protein). L-[(3)H]glutamate had higher affinity and a greater percentage of specific binding than did D-[(3)H]aspartate. The pharmacological binding specificity of L-[(3)H]glutamate indicated an interaction with NMDA-type receptors. Specifically, the order of potency of the displacing compound tested was L-Glu > D-Asp > NMDA > MK801 > D-AP5 > glycine. For D-[(3)H]aspartate, the data revealed an interaction of D: -Asp with either NMDA-type receptors or putative specific binding sites.

The role and molecular mechanism of D-aspartic acid in the release and synthesis of LH and testosterone in humans and rats.

BACKGROUND: D-aspartic acid is an amino acid present in neuroendocrine tissues of invertebrates and vertebrates, including rats and humans. Here we investigated the effect of this amino acid on the release of LH and testosterone in the serum of humans and rats. Furthermore, we investigated the role of D-aspartate in the synthesis of LH and testosterone in the pituitary and testes of rats, and the molecular mechanisms by which this amino acid triggers its action. METHODS: For humans: A group of 23 men were given a daily dose of D-aspartate (DADAVIT) for 12 days, whereas another group of 20 men were given a placebo. For rats: A group of 10 rats drank a solution of either 20 mM D-aspartate or a placebo for 12 days. Then LH and testosterone accumulation was determined in the serum and D-aspartate accumulation in tissues. The effects of D-aspartate on the synthesis of LH and testosterone were gauged on isolated rat pituitary and Leydig cells. Tissues were incubated with D-aspartate, and then the concentration (synthesis) of LH and cGMP in the pituitary and of testosterone and cAMP in the Leydig cells was determined. RESULTS: In humans and rats, sodium D-aspartate induces an enhancement of LH and testosterone release. In the rat pituitary, sodium D-aspartate increases the release and synthesis of LH through the involvement of cGMP as a second messenger, whereas in rat testis Leydig cells, it increases the synthesis and release of testosterone and cAMP is implicated as second messenger. In the pituitary and in testes D-Asp is synthesized by a D-aspartate racemase which convert L-Asp into D-Asp. The pituitary and testes possesses a high capacity to trapping circulating D-Asp from hexogen or endogen sources. CONCLUSION: D-aspartic acid is a physiological amino acid occurring principally in the pituitary gland and testes and has a role in the regulation of the release and synthesis of LH and testosterone in humans and rats.

Characterization and putative role of a type I gonadotropin-releasing hormone in the cephalochordate amphioxus.

GnRH, originally isolated from mammalian hypothalamus, is a key player in the control of vertebrate reproduction. Employing reverse-phase chromatography, we purified a peptide of relative molecular mass of 1182.60 Da from the cephalochordate amphioxus Branchiostoma lanceolatum. We found that its amino acid sequence (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH(2)) was identical to that of mammalian GnRH. The highest concentrations (4.04 +/- 0.3 microg/g tissue), localized in the anterior part of the body, occurred in November, a time when amphioxus gonads prepare for the seasonal spawning. Furthermore, the biological activity of amphioxus GnRH was investigated by examining its capability to elicit LH release from the rodent pituitary gland. The origins of GnRH can be traced back to the origins of chordates. The seasonal variations of amphioxus GnRH also suggest an ancient role of this peptide in the control of reproduction in chordates, even before the evolution of a proper pituitary gland.

D-aspartate prevents corticostriatal long-term depression and attenuates schizophrenia-like symptoms induced by amphetamine and MK-801.

Since their discovery in the mammalian CNS, D-aspartate and D-serine have aroused a strong interest with regard to their role as putative neuromodulatory molecules. Whereas the functional role of D-serine as an endogenous coagonist of NMDA receptors (NMDARs) has been elucidated, the biological significance of D-aspartate in the brain is still mostly unclear. In the present study, we demonstrated that nonphysiological high levels of D-aspartate (1) increased in vivo NMDAR activity, (2) attenuated prepulse inhibition deficits induced by amphetamine and MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine hydrogen maleate], (3) produced striatal adaptations of glutamate synapses resembling those observed after chronic haloperidol treatment, and (4) enhanced hippocampal NMDAR-dependent memory. This evidence was obtained using two different experimental strategies that produced an abnormal increase of endogenous D-aspartate levels in the mouse: a genetic approach based on the targeted deletion of the D-aspartate oxidase gene and a pharmacological approach based on oral administration of D-aspartate. This work provides in vivo evidence of a neuromodulatory role exerted by D-aspartate on NMDAR signaling and raises the intriguing hypothesis that also this D-amino acid, like D-serine, could be used as a therapeutic agent in the treatment of schizophrenia-related symptoms.

Increased levels of d-aspartate in the hippocampus enhance LTP but do not facilitate cognitive flexibility.

In the present study, we demonstrate a direct role for d-aspartate in regulating hippocampal synaptic plasticity. These evidences were obtained using two different experimental strategies which enabled a non-physiological increase of endogenous d-aspartate levels in the mouse hippocampus: a genetic approach based on the targeted deletion of d-aspartate oxidase gene and another based on the oral administration of d-aspartate. Overall, our results indicate that increased d-aspartate content does not affect basal properties of synaptic transmission but enhances long-term potentiation in hippocampal slices from both genetic and pharmacological animal models. Besides electrophysiological data, behavioral analysis suggests that altered levels of d-aspartate in the hippocampus do not perturb basal spatial learning and memory abilities, but may selectively interfere with the dynamic NMDAR-dependent processes underlying cognitive flexibility.

N-methyl-D-aspartic acid (NMDA) in the nervous system of the amphioxus Branchiostoma lanceolatum.

BACKGROUND: NMDA (N-methyl-D-aspartic acid) is a widely known agonist for a class of glutamate receptors, the NMDA type. Synthetic NMDA elicits very strong activity for the induction of hypothalamic factors and hypophyseal hormones in mammals. Moreover, endogenous NMDA has been found in rat, where it has a role in the induction of GnRH (Gonadotropin Releasing Hormone) in the hypothalamus, and of LH (Luteinizing Hormone) and PRL (Prolactin) in the pituitary gland. RESULTS: In this study we show evidence for the occurrence of endogenous NMDA in the amphioxus Branchiostoma lanceolatum. A relatively high concentration of NMDA occurs in the nervous system of this species (3.08 +/- 0.37 nmol/g tissue in the nerve cord and 10.52 +/- 1.41 nmol/g tissue in the cephalic vesicle). As in rat, in amphioxus NMDA is also biosynthesized from D-aspartic acid (D-Asp) by a NMDA synthase (also called D-aspartate methyl transferase). CONCLUSION: Given the simplicity of the amphioxus nervous and endocrine systems compared to mammalian, the discovery of NMDA in this protochordate is important to gain insights into the role of endogenous NMDA in the nervous and endocrine systems of metazoans and particularly in the chordate lineage.