Monteil C, Marouillat S, Fillastre J P, Morin J P
INSERM U-295-Université de Rouen, Saint Etienne du Rouvray, France.
Epithelial Cell Biol. 1995;4(3):131-9.
In vivo, bicarbonate can affect proximal tubule intermediary metabolism, including gluconeogenesis, ammoniagenesis and maintenance of the mitochondrial substrate supply. In vitro, rabbit proximal tubule cells (RPTC) in primary culture revert from gluconeogenesis to glycolysis and their mitochondrial metabolism remains lower than in vivo. To determine whether the bicarbonate buffer system could have an effect on these deregulations, RPTC in primary culture grown in the absence of insulin and glucose in the culture medium were developed either with the standard sodium bicarbonate buffer with 5% CO2 or with a Hepes hydrogen ion buffer in the presence of 0.5% CO2. Duration of the bicarbonate-free cultures was increased until at least day 17 after seeding, compared with day 11 in bicarbonate-buffered cultures. As could be expected, succinate dehydrogenase activity remained stable as a function of time in bicarbonate-free cultures while an early marked decrease of this activity occurred from seeding in cultures developed in the presence of bicarbonate buffer. Compared to bicarbonate-buffered cells, higher phosphoenolpyruvate carboxykinase activity concomitant with lower intracellular lactate dehydrogenase activity was observed in cultures developed in the absence of bicarbonate, which is indicative of closer carbohydrate metabolism orientation to the in vivo situation for RPTC. Immunofluorescence staining of RPTC with monoclonal antibodies directed to neutral endopeptidase (NEP), and dipeptidyl-peptidase IV (DPP II) showed similar extensive labelling with DPP and NEP in both culture conditions. Confocal microscopy analysis of NEP subcellular distribution, showed exclusive targetting of NEP to the apical plasma membranes. In both models, cAMP production was stimulated by parathyroid hormone and unaffected by arginine vasopressin. In conclusion, bicarbonate withdrawal from the culture medium (without changing the pH of the medium) allows a marked improvement of mitochondrial capacity and carbohydrate metabolism pattern without any loss of differentiated properties.
在体内，碳酸氢盐可影响近端小管的中间代谢，包括糖异生、氨生成以及线粒体底物供应的维持。在体外，原代培养的兔近端小管细胞（RPTC）从糖异生转变为糖酵解，其线粒体代谢仍低于体内水平。为了确定碳酸氢盐缓冲系统是否会对这些失调产生影响，在无胰岛素和葡萄糖的培养基中培养原代RPTC，分别采用含5%二氧化碳的标准碳酸氢钠缓冲液或含0.5%二氧化碳的羟乙基哌嗪乙磺酸（Hepes）氢离子缓冲液。与碳酸氢盐缓冲培养至第11天相比，无碳酸氢盐培养的时间延长至至少接种后第17天。不出所料，在无碳酸氢盐培养中，琥珀酸脱氢酶活性随时间保持稳定，而在有碳酸氢盐缓冲的培养中，接种后该活性早期显著下降。与碳酸氢盐缓冲的细胞相比，在无碳酸氢盐培养中观察到磷酸烯醇式丙酮酸羧激酶活性较高，同时细胞内乳酸脱氢酶活性较低，这表明RPTC的碳水化合物代谢方向更接近体内情况。用针对中性内肽酶（NEP）和二肽基肽酶IV（DPP II）的单克隆抗体对RPTC进行免疫荧光染色，结果显示在两种培养条件下，DPP和NEP的标记情况相似。对NEP亚细胞分布的共聚焦显微镜分析表明，NEP仅靶向顶端质膜。在两种模型中，甲状旁腺激素刺激环磷酸腺苷（cAMP）的产生，而精氨酸加压素对此无影响。总之，从培养基中去除碳酸氢盐（不改变培养基的pH值）可显著改善线粒体功能和碳水化合物代谢模式，且不会丧失任何分化特性。
