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.
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 micromol/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.
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.
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.
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