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      Ascorbate Transport in Pig Coronary Artery Smooth Muscle: Na + Removal and Oxidative Stress Increase Loss of Accumulated Cellular Ascorbate


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          Pig deendothelialized coronary artery rings and smooth muscle cells cultured from them accumulated ascorbate from medium containing Na<sup>+</sup>. The accumulated material was determined to be ascorbate using high-performance liquid chromatography. We further characterized ascorbate uptake in the cultured cells. The data fitted best with a Hill coefficient of 1 for ascorbate (K<sub>asc</sub> = 22 ± 2 μ M) and 2 for Na<sup>+</sup> (K<sub>Na</sub> = 84 ± 10 m M). The anion transport inhibitors sulfinpyrazone and 4,4′-diisothiocyanatostilbene-2,2′-disulfonate (DIDS) inhibited the uptake. Transferring cultured cells loaded with <sup>14</sup>C-ascorbate into an ascorbate-free solution resulted in a biphasic loss of radioactivity – an initial sulfinpyrazone-insensitive faster phase and a late sulfinpyrazone-sensitive slower phase. Transferring loaded cells into a Na<sup>+</sup>-free medium increased the loss in the initial phase in a sulfinpyrazone-sensitive manner, suggesting that the ascorbate transporter is bidirectional. Including peroxide or superoxide in the solution increased the loss of radioactivity. Thus, ascorbate accumulated in coronary artery smooth muscle cells by a Na<sup>+</sup>-dependent transporter was lost in an ascorbate-free solution, and the loss was increased by removing Na<sup>+</sup> from the medium or by oxidative stress.

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          Free radicals in toxicology.

          Free radicals are recognized more and more frequently as being involved in the mechanism of toxicity of chemicals. In some cases, the organic radicals are involved, but often oxygen radicals result from redox cycling chemicals. Free radicals are usually very reactive, which, in addition to causing toxicities, can make them difficult to detect. Electron spin resonance (ESR) techniques are frequently used, but generally the radicals must be trapped to form a more stable radical for detection. Quantitation is therefore often very difficult. Free radicals of many xenobiotics are formed during their metabolism by enzymes such as cytochrome P450 or peroxidases. In some cases, chemicals can redox cycle using reductases, such as cytochrome P450 reductase, which can catalyze one-electron reductions. Some redox cycling xenobiotics reduce molecular oxygen by one electron to generate superoxide. Superoxide can cause toxicities against which superoxide dismutase is protective. However, in the presence of transition metals such as iron, superoxide can generate the very reactive hydroxyl radical by the iron-catalyzed Haber-Weiss reaction. Iron is therefore normally tightly controlled by transport and storage proteins. Chemicals that can release iron from these proteins can be very toxic, causing lipid, protein, and nucleic acid oxidation. The oxidation of these species, such as a low-density lipoprotein, is generally protected by a complex antioxidant system involving glutathione and glutathione peroxidase, vitamin E, ascorbic acid, etc.
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            Ascorbic acid transport and distribution in human B lymphocytes.

            Ascorbic acid (vitamin C) transport was investigated in human B lymphocytes. The vitamin was transported by two components. The first was a high-affinity activity with an apparent Km of 7-10 microM and Vmax of 0.14 mM/h (3.11 x 10(-4) mumol x h-1 x mg protein-1). The activity was concentration and temperature dependent, saturable, and inhibited by carbonylcyanide-p-trifluoromethoxyphenylhydrazone and ouabain and generated ascorbic acid accumulation against a concentration gradient. Kinetics for the second component were indeterminate because ascorbate was not accumulated against a concentration gradient. Subcellular fractionation revealed that intracellular ascorbic acid in human B lymphocytes was > 90% localized to the cytosol and not protein bound. Kinetic parameters of high-affinity ascorbic acid transport could operate effectively with plasma concentrations normally found in humans.
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              Ischemia/Reperfusion-Induced Injury of Forebrain Mitochondria and Protection by Ascorbate


                Author and article information

                J Vasc Res
                Journal of Vascular Research
                S. Karger AG
                October 2000
                02 October 2000
                : 37
                : 5
                : 390-398
                aDepartment of Medicine, McMaster University, Hamilton, bDepartment of Physiology, University of Western Ontario, London, Ontario, Canada
                25755 J Vasc Res 2000;37:390–398
                © 2000 S. Karger AG, Basel

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                Page count
                Figures: 7, References: 38, Pages: 9
                Research Paper

                General medicine,Neurology,Cardiovascular Medicine,Internal medicine,Nephrology
                Vitamin C,Membrane transport,Oxidative stress,Ischemia-reperfusion


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