Ascorbic acid accumulation and transport in human fibroblasts.
Ontology highlight
ABSTRACT:
SUBMITTER: Welch RW
PROVIDER: S-EPMC1134483 | biostudies-other | 1993 Sep
REPOSITORIES: biostudies-other
Similar Datasets
Project description:Intracellular vitamin C, or ascorbic acid, has been shown to prevent the apoptosis of cultured vascular pericytes under simulated diabetic conditions. We sought to determine the mechanism by which ascorbate is transported into pericytes prior to exerting this protective effect. Measuring intracellular ascorbate, we found that pericytes display a linear uptake over 30 min and an apparent transport Km of 21 ?M, both of which are consistent with activity of the Sodium-dependent Vitamin C Transporter 2 (SVCT2). Uptake of both radiolabeled and unlabeled ascorbate was prevented by inhibiting SVCT2 activity, but not by inhibiting the activity of GLUT-type glucose transporters, which import dehydroascorbate to also generate intracellular ascorbate. Likewise, uptake of dehydroascorbate was prevented with the inhibition of GLUTs, but not by inhibiting the SVCT2, indicating substrate specificity of both transporters. Finally, presence of the SVCT2 in pericytes was confirmed by western blot analysis, and immunocytochemistry was used to localize it to the plasma membrane and intracellular sites. Together, these data clarify previous inconsistencies in the literature, implicate SVCT2 as the pericyte ascorbate transporter, and show that pericytes are capable of concentrating intracellular ascorbate against a gradient in an energy- and sodium-dependent fashion.
| S-EPMC4351875 | biostudies-literature
Project description:Effect of Ascorbic acid on human genome
2009-10-15 | E-MEXP-1861 | biostudies-arrayexpress
Project description:The purpose of this study is to present a novel strategy to enhance collagen production in cells. To identify amino acid analogs with excellent collagen production-enhancing effects, human dermal fibroblasts (HDFs) were treated with 20 kinds of amidated amino acids and 20 kinds of free amino acids, individually at 1 mM. The results showed that glycinamide enhanced collagen production (secreted collagen level) most effectively. Glycine also enhanced collagen production to a lesser degree. However, other glycine derivatives, such as N-acetyl glycine, N-acetyl glycinamide, glycine methyl ester, glycine ethyl ester, and glycyl glycine, did not show such effects. Glycinamide increased type I and III collagen protein levels without affecting COL1A1 and COL3A1 mRNA levels, whereas transforming growth factor-β1 (TGF-β1, 10 ng mL-1) increased both mRNA and protein levels of collagens. Ascorbic acid (AA, 1 mM) increased COL1A1 and COL3A1 mRNA and collagen I protein levels. Unlike TGF-β1, AA and glycinamide did not increase the protein level of α-smooth muscle actin, a marker of differentiation of fibroblasts into myofibroblasts. The combination of AA and glycinamide synergistically enhanced collagen production and wound closure in HDFs to a level similar to that in cells treated with TGF-β1. AA derivatives, such as magnesium ascorbyl 3-phosphate (MAP), 3-O-ethyl ascorbic acid, ascorbyl 2-O-glucoside, and ascorbyl tetraisopalmitate, enhanced collagen production, and the mRNA and protein levels of collagens at 1 mM, and their effects were further enhanced when co-treated with glycinamide. Among AA derivatives, MAP had a similar effect to AA in enhancing wound closure, and its effect was further enhanced by glycinamide. Other AA derivatives had different effects on wound closure. This study provides a new strategy to enhance cell collagen production and wound healing using glycinamide in combination with AA.
| S-EPMC9138459 | biostudies-literature
Project description:The accumulation of senescent cells is implicated in the pathology of several age-related diseases. While the clearance of senescent cells has been suggested as a therapeutic target for patients with osteoarthritis (OA), cellular senescence of bone-resident osteoblasts (OB) remains poorly explored. Since oxidative stress is a well-known inducer of cellular senescence, we here investigated the effect of antioxidant supplementation on the isolation efficiency, expansion, differentiation potential, and transcriptomic profile of OB from osteoarthritic subchondral bone. Bone chips were harvested from sclerotic and non-sclerotic regions of the subchondral bone of human OA joints. The application of 0.1 mM ascorbic acid-2-phosphate (AA) significantly increased the number of outgrowing cells and their proliferation capacity. This enhanced proliferative capacity showed a negative correlation with the amount of senescent cells and was accompanied by decreased expression of reactive oxygen species (ROS) in cultured OB. Expanded cells continued to express differentiated OB markers independently of AA supplementation and demonstrated no changes in their capacity to osteogenically differentiate. Transcriptomic analyses revealed that apoptotic, cell cycle-proliferation, and catabolic pathways were the main pathways affected in the presence of AA during OB expansion. Supplementation with AA can thus help to expand subchondral bone OB in vitro while maintaining their special cellular characteristics. The clearance of such senescent OB could be envisioned as a potential therapeutic target for the treatment of OA.
| S-EPMC5751120 | biostudies-literature
Project description:Defective airway mucus clearance is a defining characteristic of cystic fibrosis lung disease, and improvements to current mucolytic strategies are needed. Novel approaches targeting a range of contributing mechanisms are in various stages of preclinical and clinical development. ARINA-1 is a new nebulized product comprised of ascorbic acid, glutathione, and bicarbonate. Using microoptical coherence tomography, we tested the effect of ARINA-1 on central features of mucociliary clearance in F508del/F508del primary human bronchial epithelial cells to assess its potential as a mucoactive therapy in cystic fibrosis. We found that ARINA-1 significantly augmented mucociliary transport rates, both alone and with CFTR (cystic fibrosis transmembrane conductance regulator) modulator therapy, whereas airway hydration and ciliary beating were largely unchanged compared with PBS vehicle control. Analysis of mucus reflectivity and particle-tracking microrheology indicated that ARINA-1 restores mucus clearance by principally reducing mucus layer viscosity. The combination of bicarbonate and glutathione elicited increases in mucociliary transport rate comparable to those seen with ARINA-1, indicating the importance of this interaction to the impact of ARINA-1 on mucus transport; this effect was not recapitulated with bicarbonate alone or bicarbonate combined with ascorbic acid. Assessment of CFTR chloride transport revealed an increase in CFTR-mediated chloride secretion in response to ARINA-1 in CFBE41o- cells expressing wild-type CFTR, driven by CFTR activity stimulation by ascorbate. This response was absent in CFBE41o- F508del cells treated with VX-809 and primary human bronchial epithelial cells, implicating CFTR-independent mechanisms for the effect of ARINA-1 on cystic fibrosis mucus. Together, these studies indicate that ARINA-1 is a novel potential therapy for the treatment of impaired mucus clearance in cystic fibrosis.
| S-EPMC7462336 | biostudies-literature
Project description:Postharvest deterioration is among the major challenges for the fruit industry. Regulation of the fruit softening rate is an effective strategy for extending shelf-life and reducing the economic losses due postharvest deterioration. The tomato myoinositol monophosphatase 3 gene SlIMP3, which showed highest expression level in fruit, was expressed and purified. SlIMP3 demonstrated high affinity with the L-Gal 1-P and D-Ins 3-P, and acted as a bifunctional enzyme in the biosynthesis of AsA and myoinositol. Overexpression of SlIMP3 not only improved AsA and myoinositol content, but also increased cell wall thickness, improved fruit firmness, delayed fruit softening, decreased water loss, and extended shelf-life. Overexpression of SlIMP3 also increased uronic acid, rhamnose, xylose, mannose, and galactose content in cell wall of fruit. Treating fruit with myoinositol obtained similar fruit phenotypes of SlIMP3-overexpressed fruit, with increased cell wall thickness and delayed fruit softening. Meanwhile, overexpression of SlIMP3 conferred tomato fruit tolerance to Botrytis cinerea. The function of SlIMP3 in cell wall biogenesis and fruit softening were also verified using another tomato species, Ailsa Craig (AC). Overexpression of SlDHAR in fruit increased AsA content, but did not affect the cell wall thickness or fruit firmness and softening. The results support a critical role for SlIMP3 in AsA biosynthesis and cell wall biogenesis, and provide a new method of delaying tomato fruit softening, and insight into the link between AsA and cell wall metabolism.
| S-EPMC9129080 | biostudies-literature
Project description:A human lung-cancer PC-9 subline with acquired resistance to vincristine (VCR), a chemotherapeutic agent, was established with incremental increases of the drug. The resistant PC-9 subline (PC-9/VCR) shows a 12-fold increase in resistance to VCR and a unique cross-resistance pattern: high cross-resistance to the potent VCR analogue colchicine (6.9-fold) and vinblastine (2.5-fold); lower cross-resistance to actinomycin D (1.8-fold), cisplatin (1.2-fold) and adriamycin (1.3-fold) and a sensitivity to melphalan and VP-16 which is similar to that of the parental cell line. A reduced accumulation of VCR in the resistant cells was demonstrated. Interestingly, the VCR resistance of the PC-9/VCR cell line was partially reversed by ascorbic acid, and the drug uptake was enhanced. In contrast, ascorbic acid had no effect on drug tolerance and drug accumulation was not observed in either PC-9 parental cells or known multidrug-resistant (MDR) cells, suggesting that VCR resistance in PC-9/VCR cells results essentially from reduced drug accumulation. It is worth noting that, whereas reduced drug accumulation in the PC-9/VCR cells was susceptible to modulation by ascorbic acid, the increased efflux rate characteristic of the resistant cells was not. Further, there was a higher efflux rate in resistant cells than in parental cells. DNA Southern- and RNA Northern-blot hybridization analyses indicate that PC-9/VCR cells do not contain amplified mdr genes or overexpress P-glycoprotein. In addition, the calcium-channel blocker verapamil, which acts as a competitive inhibitor of drug binding and efflux, did not affect the resistant phenotype of PC-9/VCR cells. These findings suggest an ascorbic acid-sensitive drug uptake mechanism which is important in mediating VCR resistance per se in human lung-cancer cells; this differs from the P-glycoprotein-mediated MDR mechanism.
| S-EPMC1137052 | biostudies-other
Project description:Several amidated biologically active peptides such as pancreastatin, thyrotropin-releasing hormone, pancreatic polypeptide and amylin are produced in endocrine pancreatic tissue which contains the enzyme necessary for their final processing, i.e. peptidylglycine alpha-amidating mono-oxygenase (EC 1.14.17.3). The enzyme needs ascorbic acid for activity as well as copper and molecular oxygen. The present work shows that pancreatic islet cells prepared from overnight cultures of isolated islets from 5-7-day-old rats accumulate 14C-labelled ascorbic acid by a Na(+)-dependent active transport mechanism which involves a saturable process (estimated Km 17.6 microM). Transport was inhibited by ouabain, phloridzin, cytochalasin B, amiloride and probenecid. Glucose inhibited or stimulated uptake, depending on the length of incubation time of the cells. The uptake of dehydroascorbic acid was linearly dependent on concentration. Dehydroascorbic acid was converted to ascorbic acid by an unknown mechanism after uptake. The uptake of both ascorbic acid and dehydroascorbic acid was inhibited by tri-iodothyronine, and uptake of ascorbic acid, but not of dehydroascorbic acid, was inhibited by glucocorticoids. Isolated secretory granules contained a fairly low concentration of iron but a high concentration of copper.
| S-EPMC1149973 | biostudies-other
Project description:The soybean sprout is a nutritious and delicious vegetable that is rich in ascorbic acid (AsA). Hydrogen gas (H2) may have potential applications in the vegetable processing industry. To investigate whether H2 is involved in the regulation of soybean sprouts AsA biosynthesis under UV irradiation, we set 4 different treatments: white light(W), W+HRW, UV-A and UV-A+HRW. The results showed that H2 significantly blocked the UV-A-induced accumulation of ROS, decreased TBARS content and enhanced SOD and APX activity in soybean sprouts. We also observed that the UV-A induced accumulation of AsA was enhanced more intensely when co-treated with HRW. Molecular analyses showed that UV-A+HRW significantly up-regulated AsA biosynthesis and recycling genes compared to UV-A in soybean sprouts. These data demonstrate that the H2 positively regulates soybean sprouts AsA accumulation under UV-A and that this effect is mediated via the up-regulation of AsA biosynthesis and recycling genes.
| S-EPMC5703957 | biostudies-literature
Project description:Salt stress has long been a prominent obstacle that restricts crop growth, and increasing the L-ascorbic acid (ASA) content of crops is an effective means of alleviating this stress. 2-Keto-L-gulonic acid (2KGA) is a precursor used in industrial ASA production as well as an ASA degradation product in plants. However, to date, no study has investigated the effects of 2KGA on ASA metabolism and salt stress. Here, we evaluated the potential of using 2KGA to improve crop resistance to salt stress (100mM NaCl) through a cultivation experiment of non-heading Chinese cabbage (Brassica campestris ssp. chinensis). The results showed that the leaf and root biomass were significantly improved by 2KGA application. The levels of metabolites and enzymes related to stress resistance were increased, whereas the hydrogen peroxide (H2O2) and malondialdehyde (MDA) contents were decreased. Lipid peroxidation and cell membrane damage were alleviated following 2KGA treatment. Positive correlations were found between photosynthetic pigments and organic solutes, ASA and photosynthetic pigments, and ASA and antioxidant enzymes. In contrast, negative correlations were observed between antioxidant enzymes and H2O2/MDA. Moreover, the expression levels of L-gulono-1,4-lactone oxidase, GDP-mannose pyrophosphorylase, dehydroascorbate reductase-3, and ascorbate peroxidase were increased by 2KGA treatment. These results suggested that exogenous 2KGA application can relieve the inhibitory effect of salt stress on plant growth, and the promotion of ASA synthesis may represent a critical underlying mechanism. Our findings have significant implications for the future application of 2KGA or its fermentation residue in agriculture.
| S-EPMC8599927 | biostudies-literature