Alessandra Santillo

Current knowledge of d-aspartate in glandular tissues

Free d-aspartate (d-Asp) occurs in substantial amounts in glandular tissues. This paper reviews the existing work on d-Asp in vertebrate exocrine and endocrine glands, with emphasis on functional roles. Endogenous d-Asp was detected in salivary glands. High d-Asp levels in the parotid gland during development suggest an involvement of the amino acid in the regulation of early developmental phases and/or differentiation processes. d-Asp has a prominent role in the Harderian gland, where it elicits exocrine secretion through activation of the ERK1/2 pathway. Interestingly, the increase in NOS activity associated with d-Asp administration in the Harderian gland suggests a potential capability of d-Asp to induce vasodilatation. In mammals, an increase in local concentrations of d-Asp facilitates the secretion of anterior pituitary hormones, i.e., PRL, LH and GH, whereas it inhibits the secretion of POMC/α-MSH from the intermediate pituitary and of oxytocin from the posterior pituitary. d-Asp also acts as a negative regulator for melatonin synthesis in the pineal gland. Further, d-Asp can stereo-specifically modulate the production of sex steroids, thus taking part in the endocrine control of reproductive activity. Although d-Asp receptors remain to be characterized, gene expression of NR1 and NR2 subunits of NMDAr responds to d-Asp in the testis.

Mast cells in nonmammalian vertebrates: an overview.

Mast cells are best known as multifunctional entities that may confer a benefit on immune system. This review presents the known facts on mast-cell system and breakthroughs in mast-cell biology in fish, amphibians, reptiles, and birds. As compared to mammals, there are relatively few data available on mast cells in many nonmammalian vertebrates. Nevertheless, like in mammals, mast cells in nonmammalian vertebrates contain a wide range of bioactive compounds including histamine, heparin, neuropeptides, and neutral proteases. In bony fishes, these cells secrete antimicrobial peptides as well. Mast cells have a widespread distribution in the brain, endocrine glands, intestine, liver, kidney, skin, tongue, and lungs, the highest concentration occurring in different tissues in the different taxa. Currently, researchers are grappling with the nature of scientific support to substantiate the functional importance of mast cells in nonmammalian vertebrates. Ultimately, the origin and evolution of vertebrate mast cell is of great interest to comparative immunologists seeking an underlying trend in the phylogenetic development of immunity.

D-aspartate modulates transcriptional activity in Harderian gland of frog, Rana esculenta: Morphological and molecular evidence

In the green frog, Rana esculenta, a substantial amount of D‐aspartate (D‐Asp) is found endogenously within the Harderian gland (HG) following its synthesis from L‐aspartate (L‐Asp) by an aspartate racemase. The frog HG is an orbital seromucoid gland that displays seasonal changes in secretory activity. Our in vivo experiments, consisting of i.p. injection of 2.0 μmol/g b.w. D‐Asp in frogs collected during two periods of differing glandular activity (high or medium‐low secretory activity), revealed that HG can to take up and accumulate D‐Asp and that this amino acid may modulate the exocrine secretion through a kinase pathway. At a time when the gland shows relatively low secretory activity, i.p. administration of D‐Asp rapidly induced activation of ERK1 and an increase in cells active in RNA synthesis. This increase in transcriptional activity was followed by a significant increase in mucous secretion. By contrast, administration of exogenous D‐Asp when HG was showing high activity rapidly induced inhibition of both ERK1 and transcriptional activity. Since D‐Asp is known to be recognized by receptors for N‐methyl‐D‐aspartic acid (NMDA), it is possible that in the HG, D‐Asp mediated NMDA activation may enhance the kinase pathway. The above activation of opposing stimulatory and inhibitory processes could reflect different levels of NMDA‐receptor activity, which could vary as a function of the level of gland activity. This study provides the first evidence of a role for this excitatory amino acid in exocrine secretion. The effects of D‐Asp in HG appear to be specific since they were not seen in frogs treated with other D‐ or L‐amino acids with known excitatory effects on neurosecretion.

d-aspartate affects NMDA receptor-extracellular signal–regulated kinase pathway and upregulates androgen receptor expression in the rat testis

Previous studies have demonstrated that D-aspartic acid (D-Asp) has a role in regulating the release and synthesis of testosterone in rats. In this study, we investigated the molecular pathway by which this amino acid triggers its action in the rat testis. We found expression of N-Methyl-D-Aspartic Acid (NMDA) receptor messenger RNAs for NR1, NR2A, and NR2D receptor subunits. After D-Asp administration, NR1 and NR2A messenger RNA levels were significantly higher than those of controls, whereas NR2D levels remained unchanged. Expression of extracellular signal-regulated kinase (ERK) 1 protein was higher than that of ERK2 protein in the testis of both D-Asp-treated rats and controls. D-Asp administration increased testis levels of both phosphorylated ERK (P-ERK) 1 and 2. Using immunohistochemical technique, NR1 and P-ERK 1 or 2 proteins were preferentially localized within the spermatogonia. Moreover, D-Asp administration increased both serum and testis testosterone levels but not estradiol levels. Finally, in D-Asp-treated rats, testicular androgen receptor protein levels were significantly increased, whereas both estrogen receptor α and P-450 aromatase levels were significantly decreased. Conclusively, our results, besides strengthening the evidence that D-Asp administration in rats induces testosterone synthesis, demonstrate for the first time that D-Asp (1) induces testicular NMDA receptor-ERK pathway, (2) upregulates androgen receptor expression, and (3) downregulates estrogen receptor expression.

D-Aspartate affects secretory activity in rat Harderian gland: molecular mechanism and functional significance.

In this paper, the role of d-aspartate in the rat Harderian gland (HG) was investigated by histochemical, ultrastructural, and biochemical analyses. In this gland, substantial amounts of endogenous d-Asp were detected, along with aspartate racemases that convert d-Asp to l-Asp and vice versa. We found that the gland was capable of uptaking and accumulating exogenously administered d-Asp. d-Asp acute treatment markedly increased lipid and porphyrin secretion and induced a powerful hyperaemia in inter-acinar interstitial tissue. Since d-Asp is known to be recognized by NMDA receptors, the expression of such receptors in rat HG led us to the hypothesis that d-Asp acute treatment induced the activation of the extracellular signal-regulated protein kinase (ERK) and nitric oxide synthase (NOS) pathways mediated by NMDA. Interestingly, as a result of enhanced oxidative stress due to increased porphyrin secretion, the revealed activation of the stress-activated protein kinase/c-jun N-terminal kinase (SAPK/JNK) pro-apoptotic pathway was probably triggered by the gland itself to preserve its cellular integrity.

Induced synthesis of P450 aromatase and 17β-estradiol by d-aspartate in frog brain

SUMMARY d-Aspartic acid is an endogenous amino acid occurring in the endocrine glands as well as in the nervous system of various animal phyla. Our previous studies have provided evidence that d-aspartate plays a role in the induction of estradiol synthesis in gonads. Recently, we have also demonstrated that d-aspartic acid induces P450 aromatase mRNA expression in the frog (Pelophylax esculentus) testis. P450 aromatase is the key enzyme in the estrogen synthetic pathway and irreversibly converts testosterone into 17β-estradiol. In this study, we firstly investigated the immunolocalisation of P450 aromatase in the brain of P. esculentus, which has never previously been described in amphibians. Therefore, to test the hypothesis that d-aspartate mediates a local synthesis of P450 aromatase in the frog brain, we administered d-aspartate in vivo to male frogs and then assessed brain aromatase expression, sex hormone levels and sex hormone receptor expression. We found that d-aspartate enhances brain aromatase expression (mRNA and protein) through the CREB pathway. Then, P450 aromatase induces 17β-estradiol production from testosterone, with a consequent increase of its receptor. Therefore, the regulation of d-aspartate-mediated P450 aromatase expression could be an important step in the control of neuroendocrine regulation of the reproductive axis. Accordingly, we found that the sites of P450 aromatase immunoreactivity in the frog brain correspond to the areas known to be involved in neurosteroid synthesis.

D-Aspartate Induces Proliferative Pathways in Spermatogonial GC-1 Cells

D‐aspartate (D‐Asp) is an endogenous amino acid present in vertebrate tissues, with particularly high levels in the testis. In vivo studies indicate that D‐Asp indirectly stimulates spermatogenesis through the hypothalamic‐pituitary‐gonadal axis. Moreover, in vitro studies have demonstrated that D‐Asp up‐regulates testosterone production in Leydig cells by enhancing expression of the steroidogenic acute regulatory protein. In this study, a cell line derived from immortalized type‐B mouse spermatogonia retaining markers of mitotic germ cells (GC‐1) was employed to explore more direct involvement of D‐Asp in spermatogenesis. Activity and protein expression of markers of cell proliferation were determined at intervals during incubation in D‐Asp‐containing medium. D‐Asp induced phosphorylation of ERK and Akt proteins, stimulated expression of PCNA and Aurora B, and enhanced mRNA synthesis and protein expression of P450 aromatase and protein expression of Estrogen Receptor β (ERβ). These results are the first demonstration of a direct effect of D‐Asp on spermatogonial mitotic activity. Considering that spermatogonia express the NR1 subunit of the N‐Methyl‐D‐Aspartic Acid receptor (NMDAR), we suggest that their response to D‐Asp depends on NMDAR‐mediated activation of the ERK and Akt pathways and is further enhanced by activation of the P450 aromatase/ERβ pathway. J. Cell. Physiol. 231: 490–495, 2016.

D-Aspartic acid implication in the modulation of frog brain sex steroid levels.

There is evidence that D-aspartate (D-Asp) modulates sex hormone levels in frog testis by regulating the activity of P450 aromatase (P450 aro), the key enzyme which converts Testosterone (T) in 17ß-Estradiol (E2). Here we report, for the first time, that there is a direct correlation among brain levels of D-Asp, P450 aro, E2 and Estradiol Receptor (ERα) in the male frogs during the reproductive as well as the post-reproductive phases of the breeding cycle, with highest levels being observed in the post-reproductive period. D-Asp i.p. administration to frogs ready for reproduction, induced an increase of brain P450 aro protein expression with concomitant enhancement of both E2 levels and ERα expression; at the same time, brain T levels and Androgen receptor expression decreased. In contrast, in the post-reproductive frogs, D-Asp treatment did not modify any of these parameters. Taken together, these results imply that the regulation of P450 aro expression by D-Asp could be an important step in the control of E2 levels in the frog brain.

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.

Effects of D‐Aspartate Treatment on D‐Aspartate Oxidase, Superoxide Dismutase, and Caspase 3 Activities in Frog (Rana esculenta) Tissues

Although D‐aspartate (D‐Asp) has been recognized to have a physiological role within different organs, high concentrations could elicit detrimental effects on those same organs. In this study, we examined the D‐aspartate oxidase (D‐AspO) activity and the expression of superoxide dismutase 1 (SOD1) and caspase 3 in different tissues of the frog Rana esculenta after chronic D‐Asp treatment. Our in vivo experiments, consisting of intraperitoneal (ip) injections of D‐Asp (2.0 μmol/g b.w.) in frogs for ten consecutive days, revealed that all examined tissues can take up and accumulate D‐Asp. Further, in D‐Asp treated frogs, i) the D‐AspO activity significantly increased in all tissues (kidney, heart, testis, liver, and brain), ii) the SOD1 expression (antioxidant enzyme) significantly increased in the kidney, and iii) the caspase 3 level (indicative of apoptosis) increased in both brain and heart. Particularly, after the D‐Asp treatment we found in both brain and heart (which showed the lowest SOD1 levels) a significant increase of the caspase 3 expression and, vice versa, in the kidney (which showed the highest SOD1 expression) a significant decrease of the caspase 3 expression. Therefore, we speculate that, in frog tissue, D‐AspO plays an essential role in modulating the D‐Asp concentration. In addition, exaggerated D‐Asp concentrations activated SOD1 as cytoprotective mechanism in the kidney, whereas, in the brain and in the heart, where the antioxidant action of SOD1 is limited, caspase 3 was activated.