ascorbic-acid and ethylisopropylamiloride

Ascorbate (vitamin C) is the major electron donor to a tPMET (transplasma membrane electron transport) system that was originally identified in human erythrocytes. This plasma membrane redox system appears to transfer electrons from intracellular ascorbate to extracellular oxidants (e.g. non-transferrin-bound iron). Although this phenomenon has been observed in nucleated cells, its mechanism and regulation are not well understood. In the present study we have examined both facets of this phenomenon in K562 cells and primary astrocyte cultures. Using ferricyanide as the analytical oxidant we demonstrate that tPMET is enhanced by dehydroascorbate uptake via facilitative glucose transporters, and subsequent accumulation of intracellular ascorbate. Additionally, we demonstrate that this stimulation is not due to ascorbate that is released from the cells, but is dependent only on a restricted intracellular pool of the vitamin. Substrate-saturation kinetics suggest an enzyme-catalysed reaction across the plasma membrane by an as-yet-unidentified reductase that relies on extensive recycling of intracellular ascorbate. Inhibition of ascorbate-stimulated tPMET by the NHE (Na(+)/H(+)-exchanger) inhibitors amiloride and 5-(N-ethyl-N-isopropyl)amiloride, which is diminished by bicarbonate, suggests that tPMET activity may be regulated by intracellular pH. In support of this hypothesis, tPMET in astrocytes was significantly inhibited by ammonium chloride-pulse-induced intracellular acidification, whereas it was significantly stimulated by bicarbonate-induced intracellular alkalinization. These results suggest that ascorbate-dependent tPMET is enzyme-catalysed and is modulated by NHE activity and intracellular pH.

Topics: Amiloride; Animals; Animals, Newborn; Ascorbic Acid; Astrocytes; Cell Line, Tumor; Cell Membrane; Cells, Cultured; Dehydroascorbic Acid; Electron Transport; Ferricyanides; Fluorometry; Humans; Hydrogen-Ion Concentration; Mice; Mice, Inbred C57BL; Oxidation-Reduction; Rats; Rats, Wistar; Sodium-Hydrogen Exchangers

The characteristics of vitamin C (ascorbic acid, ASC) transport were studied in polarized cultured monolayers of the chick (Gallus gallus) renal proximal tubule in Ussing chambers. Under voltage clamp conditions, monolayers responded to apical addition of ASC in a dose-dependent manner, with positive short circuit currents (I(SC)), ranging from 3 microA/cm(2) at 5 microM ASC to a maximal response of 27 microA/cm(2) at 200 microM, and a half-maximal response at 40 microM. There was no effect of basolateral addition of ASC, indicating a polarized transport process. The oxidized form of ASC, dehydroascorbic acid had negligible effects. The I(SC) response to ASC was completely eliminated with Na(+) ion replacement, and was also eliminated by bilateral reduction of bath Cl(-), from 137 to 2.6 mM. There was significant inhibition of the I(SC) responses to 30 microM ASC by the flavanoid quercetin (50 microM) and by 100 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and 5-ethylisopropylamiloride (EIPA), blockers of anion exchangers and sodium-proton exchangers, respectively. There was no inhibition, however, by the chloride channel blocker 5-nitro-2(3-phenylpropylamino)benzoic acid (NPPB). Phorbol 12-myristate 13 acetate (PMA), the phorbol ester activator of protein kinase C, caused a 37% decrease in the I(SC) response to ASC. Chicken-specific primers to an EST homolog of the human vitamin C transporter SVCT1 (SLC23A1) were designed and used to probe transporter expression in these cells. RT-PCR analysis demonstrated the presence of chicken SVCT1 in both cultured cells and in freshly isolated proximal tubule fragments. These data indicate the presence of an electrogenic, sodium-dependent vitamin C transporter (SVCT1) in the chick renal proximal tubule. Vitamin C transport and conservation by the kidney is likely to be especially critical in birds, due to high plasma glucose levels and resulting high levels of reactive oxygen species.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Amiloride; Animals; Ascorbic Acid; Cells, Cultured; Chickens; Electrophysiology; Gene Expression Regulation; Humans; Kidney Tubules, Proximal; Nitrobenzoates; Organic Anion Transporters, Sodium-Dependent; Quercetin; Sodium-Coupled Vitamin C Transporters; Symporters; Tetradecanoylphorbol Acetate

The Na(+)/H(+) exchanger is the main intracellular pH (pH(i)) regulator in hepatic stellate cells (HSCs) and plays a key role in regulating proliferation and gene expression. We evaluated the effect of specific inhibition of this exchanger on HSC proliferation and collagen synthesis in vivo and in vitro.. Rat HSCs were incubated in the presence of platelet-derived growth factor (PDGF), transforming growth factor (TGF)-beta1, iron ascorbate (FeAsc), and ferric nitrilotriacetate solution (FeNTA) with or without the Na(+)/H(+) exchanger inhibitor 5-N-ethyl-N-isopropyl-amiloride (EIPA). pH(i) and Na(+)/H(+) exchanger activity, cell proliferation, and type I collagen accumulation were measured by using the fluorescent dye 2',7'-bis-(carboxyethyl)-5(6)-carboxyfluorescein, by immunohistochemistry for bromodeoxyuridine, and by enzyme-linked immunosorbent assay, respectively. In vivo liver fibrosis was induced by dimethylnitrosamine administration and bile duct ligation (BDL) in rats treated or not treated with amiloride.. PDGF, FeAsc, and FeNTA increased Na(+)/H(+) exchange activity and induced HSC proliferation. TGF-beta1 had no effect on the Na(+)/H(+) exchanger and was able, as for FeAsc and FeNTA, to induce type I collagen accumulation. EIPA inhibited all the effects determined by PDGF, FeAsc, and FeNTA and had no effect on TGF-beta1-induced collagen accumulation. In vivo, amiloride reduced HSC proliferation, activation, collagen deposition, and collagen synthesis.. The Na(+)/H(+) exchanger can play a key role in the development of liver fibrosis and in HSC activation in vivo.

Topics: Amiloride; Animals; Anti-Arrhythmia Agents; Antineoplastic Agents; Ascorbic Acid; Carcinogens; Cell Division; Collagen; Diuretics; Ferric Compounds; Ferrous Compounds; Gene Expression; Hydrogen-Ion Concentration; In Situ Nick-End Labeling; Liver; Liver Cirrhosis; Male; Nitrilotriacetic Acid; Procollagen; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; RNA, Messenger; Sodium-Hydrogen Exchangers; Thymidine Phosphorylase; Transforming Growth Factor beta

beta-Carotene absorbed 2 equimolar amounts of NO2 accompanying the complete destruction of beta-carotene. Electron spin resonance study using 2-phenyl-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl revealed that no significant amounts of NO were released by the interaction. Nitrogen atoms derived from NO2 were tightly bound to the beta-carotene molecules. Destruction of beta-carotene was inhibited little by alpha-tocopherol and polyunsaturated fatty esters, and slightly by ascorbyl palmitate, indicating that beta-carotene was a more effective scavenger of NO2. ONOOH/ONOO- and 3-morpholinosydononimine similarly destroyed beta-carotene. The results suggest that beta-carotene contributes to the prevention of cytotoxicity and genotoxicity of NO2 and ONOOH/ONOO- derived from NO.

Topics: Amiloride; Ascorbic Acid; beta Carotene; Docosahexaenoic Acids; Electron Spin Resonance Spectroscopy; Free Radical Scavengers; Nitrates; Nitrogen Dioxide; Vitamin E