Schoolwerth A C, Smith B C, Drewnowska K
Department of Internal Medicine, Medical College of Virginia, Richmond 23298.
Am J Physiol. 1992 Jun;262(6 Pt 2):F1007-14. doi: 10.1152/ajprenal.1992.262.6.F1007.
To examine the interrelationships of proton compartmentation and ammoniagenesis, experiments were performed in tubules and mitochondria isolated from dog kidney cortex. Tubules were incubated in Krebs-Henseleit buffer at different pH (pHe), and cytosolic pH (pHi) was estimated with the fluorescent probe 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Mitochondrial pH (pHm) was determined simultaneously in intact tubules by use of dimethyloxazolidine-2,4-dione. Over the pHe range 6.9-7.7, pHi was similar in control and acidotic dogs and linearly related to pHe. At pHe 7.4 in control tubules. pHm was 7.78 +/- 0.07, and varied little over the pHe range of 7.0-7.7. The pH gradient across the mitochondrial membrane rose at acid pHe. pHm was more alkaline when estimated in tubules from acidotic dogs compared with controls. Ammonium and glucose productions from glutamine were inversely related to pHe and pHi in tubules from both control and acidotic animals and were higher in acidosis. In contrast, ammonium production by isolated mitochondria did not vary as pHe was altered. Enzyme fluxes, calculated from metabolite changes, demonstrated that glutamate dehydrogenase (GDH) flux was altered. Enzyme fluxes, calculated from metabolite changes, demonstrated that glutamate dehydrogenase (GDH) flux was inversely and glutaminase (PDG) flux was linearly related to pHe. Ammonium production was significantly greater in mitochondria from acidotic dogs because of accelerated flux through PDG but not GDH. The present study demonstrates significant difference between proton compartmentation and regulation of ammoniagenesis in kidneys from acidotic dog compared with rat.
为了研究质子区室化与氨生成之间的相互关系，我们在从犬肾皮质分离出的肾小管和线粒体中进行了实验。将肾小管置于不同pH值（细胞外pH，pHe）的 Krebs-Henseleit 缓冲液中孵育，并用荧光探针 2',7'-双（2-羧乙基）-5（6）-羧基荧光素估算胞质pH值（细胞内pH，pHi）。通过使用二甲基恶唑烷-2,4-二酮在完整的肾小管中同时测定线粒体pH值（pHm）。在pHe范围为6.9 - 7.7时，对照犬和酸中毒犬的pHi相似，且与pHe呈线性相关。在对照肾小管中，pHe为7.4时，pHm为7.78±0.07，在7.0 - 7.7的pHe范围内变化不大。线粒体膜两侧的pH梯度在酸性pHe时升高。与对照相比，在酸中毒犬的肾小管中估算的pHm更偏碱性。对照动物和酸中毒动物的肾小管中，谷氨酰胺产生铵和葡萄糖的量与pHe和pHi呈负相关，且在酸中毒时更高。相比之下，分离的线粒体产生铵的量不会随pHe的改变而变化。根据代谢物变化计算的酶通量表明，谷氨酸脱氢酶（GDH）通量发生了改变。根据代谢物变化计算的酶通量表明，谷氨酸脱氢酶（GDH）通量与pHe呈负相关，而谷氨酰胺酶（PDG）通量与pHe呈线性相关。由于通过PDG而非GDH的通量加速，酸中毒犬的线粒体中铵的产生显著增加。本研究表明，与大鼠相比，酸中毒犬肾脏中质子区室化和氨生成调节存在显著差异。
