ascorbic-acid-2-o-glucoside and castanospermine

It has been shown that ascorbate (AsA) and its stable derivative, ascorbic acid 2-O-alpha-glucoside (AA-2G), do not elicit neurite outgrowth in PC12 cells. However, these ascorbates are synergistically enhanced by both dibutyryl cyclic AMP (Bt(2)cAMP)- and nerve growth factor (NGF)-induced neurite outgrowth in this model. In the present study, the effects of a series of novel lipophilic ascorbate derivatives, 6-acylated ascorbic acid 2-O-alpha-glucosides (6-Acyl-AA-2G), on neurite outgrowth induced by Bt(2)cAMP and NGF were examined in PC12 cells. We found that all the tested acylated ascorbate derivatives enhanced neurite formation induced by both agents in a dose-dependent manner. Of the 6-Acyl-AA-2G derivatives, 6-octanoyl ascorbic acid 2-O-alpha-glucoside (6-Octa-AA-2G) enhanced the Bt(2)cAMP-induced phosphorylated MAPK p44 and p42 expression. A alpha-glucosidase inhibitor, castanospermine, completely abrogated the promotion of neurite outgrowth and MAPK expression by 6-Octa-AA-2G. Addition of 6-Octa-AA-2G (0.5 mM) to PC12 cells caused a rapid and significant increase in intracellular AsA content, which reached a maximum and was maintained from 12 to 24 h after the culture. These findings suggest that 6-Acyl-AA-2G is rapidly hydrolyzed to AsA within the cell and enhances neurite differentiation through the interaction with the inducer-activated MAPK pathway.

Topics: Acylation; Animals; Ascorbic Acid; Blotting, Western; Cell Count; Cell Line; Cyclic AMP; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Inhibitors; Extracellular Space; Indolizines; Mitogen-Activated Protein Kinases; Nerve Growth Factor; Neurites; PC12 Cells; Rats

Hepatocyte growth factor (HGF), a cytokine which is generally produced by mesenchymal cells, has mitogenic, motogenic and morphogenic activities in epithelial cells and it also has tumor-suppressing activities. Induction of HGF production may be involved in organ regeneration, wound healing and embryogenesis. We examined the effects of ascorbic acid (AsA), which stimulates the proliferation of fibroblasts, and its stable derivative, 2-O-alpha-D-glucopyranosyl-L-ascorbic acid (AA-2G), on HGF production by human skin fibroblasts. Basal HGF secretion was significantly stimulated by more than 0.1 mM AsA or AA-2G. Both vitamins synergistically enhanced HGF secretion stimulated by growth factors such as epidermal growth factor (EGF), platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF), cholera toxin and other inducers. Induction by EGF or bFGF was most markedly potentiated by the vitamins. HGF production by the KG-1 human leukemia cell line was also augmented by AsA or AA-2G. Another stable AsA derivative, ascorbic acid 2-phosphate (AA-2P) effectively promoted basal and EGF-induced HGF secretion by the fibroblasts, but ascorbic acid 2-sulfate (AA-2S) was much less effective. Intracellular AsA levels increased after the addition of AA-2G and AA-2P as well as AsA, but not after AA-2S. The effect of AA-2G was completely abrogated by the simultaneous addition of castanospermine, an alpha-glucosidase inhibitor, suggesting that the active form of AA-2G is AsA. Constitutive and EGF-induced HGF gene expression was also up-regulated after adding AsA or AA-2G to the cells. These results indicated that AsA acts alone or in synergy with several inducers to stimulate the production and gene expression of HGF in human skin fibroblasts and that the stable AsA derivative AA-2G is as effective as AsA in promoting HGF production.

Topics: Acetylcysteine; Antioxidants; Ascorbic Acid; Cells, Cultured; Child; Child, Preschool; Drug Synergism; Epidermal Growth Factor; Female; Fibroblasts; Gene Expression Regulation; Glutathione; Hepatocyte Growth Factor; Humans; Indolizines; Skin

The intestinal absorption efficacy of 2-O-alpha-D-glucopyranosyl-L-ascorbic acid (AA-2G), which has been recently synthesized and characterized as a stable ascorbate (AsA), was determined in guinea pigs by the perfusion technique. Perfusion of AA-2G in isotonic phosphate buffer to the small intestine resulted in a decrease of AA-2G accompanied by an increase of AsA in the perfusate. The results showed that intact AA-2G was not detected in the plasma of the portal vein of guinea pigs at 2 h after perfusion. The disappearance of AA-2G from perfusate was completely inhibited by the addition of castanospermine, a specific alpha-glucosidase inhibitor, or by carbohydrates such as maltose. These results indicate that ascorbic acid released from AA-2G by alpha-glucosidase on the brush border membrane is effectively taken up across the intestinal ascorbate transport channels, into a serosal site, whereas AA-2G permeation was poor via the passive transport system.

Topics: Animals; Ascorbic Acid; Carbohydrates; Enzyme Inhibitors; Glycoside Hydrolase Inhibitors; Guinea Pigs; Hydrolysis; Indolizines; Intestinal Absorption; Intestine, Small; Male; Maltose

Using an antigen-specific plaque forming cell (PFC) assay, we have studied the effect of ascorbic acid 2-O-alpha-glucoside (AA-2G), a new stable derivative of ascorbic acid (AsA), on antibody production in cultured murine splenocytes in comparison with that of AsA. A single addition of AA-2G (0.0625-1.0 mM) remarkably stimulated both anti-SRBC (sheep red blood cell, TD antigen) and anti-TNP (trinitrophenyl, TI antigen) PFC responses in a dose-dependent manner, although AsA failed to stimulate their responses. However, repeated additions of AsA at 12-h intervals during the cultivation resulted in enhancement of the anti-SRBC PFC response, and the magnitude of stimulation was comparable to that obtained by a single addition of AA-2G. AA-2G's effect was abrogated in the presence of castanospermine (alpha-glucosidase inhibitor) in the medium, indicating that the immunostimulation brought about by AA-2G is attributed to AsA released from the glucoside by alpha-glucosidase of cultured cells. In fact, the cells maintained a certain amount of AsA for a relatively long time after exposure to AA-2G. In contrast, AsA levels in the cells treated with AsA quickly decreased. AA-2G markedly enhanced lipopolysaccharide(LPS)-induced proliferative response, and could affect the concanavalin A (Con A) response weakly. These results suggest that AsA effectively potentiates B-cell functions in the humoral immune system. Thus, we conclude that AA-2G is capable of enhancing antibody production in the cultured splenocytes via a continuous supplementation of AsA and that AA-2G is available for long-term cell cultures as a useful tool for analyzing biological function of AsA.

Topics: Animals; Antibody Formation; Ascorbic Acid; Cells, Cultured; Erythrocytes; Female; Indolizines; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Spleen

We evaluated the effect of 2-O-alpha-D-glucopyranosyl-L-ascorbic acid (AA-2G) on collagen synthesis in cultured human skin fibroblasts and on proliferation of fibroblasts. At concentrations of 0.1-0.5 mmol/L, AA-2G effectively stimulated collagen synthesis with an effectiveness comparable to that of L-ascorbic acid. On the other hand, 6-O-alpha-D-glucopyranosyl-L-ascorbic acid showed a weak effect. The stimulation of collagen synthesis by AA-2G was attenuated by the addition of a collagen synthesis inhibitor, L-azetidine 2-carboxylic acid, in a dose-dependent manner. In addition, AA-2G-induced stimulation of collagen synthesis could be completely inhibited by the addition of castanospermine, an inhibitor of neutral alpha-glucosidase. Relatively high alpha-glucosidase activity, which would contribute to release of ascorbic acid from AA-2G, could be detected in the lysate of cultured fibroblasts. The stimulatory activity of AA-2G on collagen synthesis was observed after 5 d in culture, whereas L-ascorbic acid tended to lose its stimulatory activity. Continuous supplementation of AA-2G (0.25 mmol/L) to culture medium for 24 d enhanced the cell growth four times that of the control. These results indicate that AA-2G is gradually cleaved by the cellular alpha-glucosidase to release L-ascorbic acid, which adequately stimulates collagen synthesis and proliferation of human skin fibroblasts.

Topics: Alkaline Phosphatase; Ascorbic Acid; Cells, Cultured; Child, Preschool; Collagen; DNA; Female; Fibroblasts; Glucosidases; Glycoside Hydrolase Inhibitors; Humans; Indolizines; Skin