Group of Molecular Toxicology, Department of Biochemistry and Molecular and Cell Biology, Veterinary Faculty, University of Zaragoza, Zaragoza, Spain.
Am J Physiol Cell Physiol. 2022 May 1;322(5):C803-C813. doi: 10.1152/ajpcell.00404.2021. Epub 2022 Mar 9.
Nicotinamide is an important regulator of Pi homeostasis after conversion into NAD/NADH. In this work, we have studied the classical inhibition of Pi transport by these compounds in the brush border membrane vesicles (BBMV) of rat kidney and rat intestine, and we examined the effects in opossum kidney (OK) cells and in phosphate transporter-expressing oocytes. In BBMV, NAD required preincubation at either room temperature or on ice to inhibit Pi uptake in BBMV. However, no effects were observed in the known Slc34 or Slc20 Pi transporters expressed in oocytes, in OK cells, or in isolated rat cortical nephron segments. In BBMV from jejunum or kidney cortex, the inhibition of Pi transport was specific, dose-related, and followed a competitive inhibition pattern, as shown by linear transformation and nonlinear regression analyses. A value of 538 µM NAD in kidney BBMV was obtained. Ribosylation inhibitors and ribosylation assays revealed no evidence that this reaction was responsible for inhibiting Pi transport. An analysis of the persistence of NAD/NADH revealed a half-life of just 2 min during preincubation. Out of several metabolites of NAD degradation, only ADP-ribose was able to inhibit Pi uptake. Pi concentration also increased during 30 min of preincubation, up to 0.67 mM, most likely as a metabolic end product. In conclusion, the classical inhibition of Pi transport by NAD/NADH in BBMV seems to be caused by the degradation metabolites of these compounds during the preincubation time.
烟酰胺在转化为 NAD/NADH 后是调节 Pi 稳态的重要调节剂。在这项工作中，我们研究了这些化合物在大鼠肾脏和肠道刷状缘膜囊泡（BBMV）中对 Pi 转运的经典抑制作用，并在负鼠肾脏（OK）细胞和表达磷酸盐转运体的卵母细胞中检查了这些作用。在 BBMV 中，NAD 需要在室温或冰上预孵育才能抑制 BBMV 中的 Pi 摄取。然而，在卵母细胞中表达的已知 Slc34 或 Slc20 Pi 转运体、OK 细胞或分离的大鼠皮质肾单位段中未观察到任何作用。在空肠或肾皮质的 BBMV 中，Pi 转运的抑制是特异性的、剂量相关的，并遵循竞争性抑制模式，如线性变换和非线性回归分析所示。在肾 BBMV 中获得 NAD 的 值为 538 µM。核糖基化抑制剂和核糖基化测定未显示出该反应负责抑制 Pi 转运的证据。对 NAD/NADH 持续时间的分析表明，在预孵育期间仅持续 2 分钟。在 NAD 降解的几种代谢物中，只有 ADP-核糖能够抑制 Pi 摄取。Pi 浓度在 30 分钟的预孵育期间也增加到 0.67 mM，很可能是作为代谢终产物。总之，在 BBMV 中，NAD/NADH 对 Pi 转运的经典抑制作用似乎是由这些化合物在预孵育期间的降解代谢物引起的。
