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>A role for Na+/H+ exchangers and intracellular pH in regulating vitamin C-driven electron transport across the plasma membrane
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A role for Na+/H+ exchangers and intracellular pH in regulating vitamin C-driven electron transport across the plasma membrane
pAscorbate (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 (Nasup+/sup/Hsup+/sup-exchanger) inhibitors amiloride and 5-(iN/i-ethyl-iN/i-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./p
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机译：p抗坏血酸（维生素C）是tPMET（质膜电子传输）系统的主要电子供体，该系统最初在人的红细胞中被鉴定出来。这种质膜氧化还原系统似乎将电子从细胞内抗坏血酸转移到细胞外氧化剂（例如，未结合转铁蛋白的铁）。尽管已经在有核细胞中观察到了这种现象，但是其机理和调控尚不清楚。在本研究中，我们研究了在K562细胞和原代星形胶质细胞培养物中这种现象的两个方面。使用铁氰化物作为分析氧化剂，我们证明通过促进葡萄糖转运蛋白吸收脱氢抗坏血酸盐并随后积累细胞内抗坏血酸盐可以增强tPMET。另外，我们证明了这种刺激不是由于从细胞中释放出的抗坏血酸，而是仅取决于维生素在细胞内的限制。底物饱和动力学表明，迄今尚未确定的还原酶依赖于细胞内抗坏血酸的广泛循环，通过质膜进行酶催化反应。 NHE（Na + / H + -exchangeer）抑制剂阿米洛利和5-（ N -乙基-<碳酸氢根减少了i> N -异丙基）阿米洛利，提示tPMET活性可能受细胞内pH值的调节。支持该假设的是，氯化铵脉冲诱导的细胞内酸化显着抑制了星形胶质细胞中的tPMET，而碳酸氢盐诱导的细胞内碱化显着地刺激了tPMET。这些结果表明，抗坏血酸依赖的tPMET是酶催化的，并受NHE活性和细胞内pH的调节。
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