烟酰胺腺嘌呤二核苷酸磷酸特异性异柠檬酸脱氢酶对大鼠肝细胞尿素合成的调节作用

NADP-specific isocitrate dehydrogenase in regulation of urea synthesis in rat hepatocytes.

作者信息

Petcu L G, Plaut G W

出版信息

Biochem J. 1980 Sep 15;190(3):581-92. doi: 10.1042/bj1900581.

DOI:10.1042/bj1900581

PMID:7470071

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC1162135/

Abstract

The effect of inhibition of NADP-specific isocitrate dehydrogenase (EC 1.1.1.42) by DL-threo-alpha-methylisocitrate (3-hydroxy-1,2,3-butanetricarboxylase) on urea synthesis was studied in isolated rat hepatocytes. alpha-Methylisocitrate substantially inhibited the rate of urea synthesis (35--84%) with substrates requiring net reductive amination of 2-oxoglutarate to glutamate for aspartate synthesis (i.e., L-serine, D-alanine, or NH4Cl + L-lactate). alpha-Methylisocitrate did not inhibit synthesis of urea from substrates not requiring reductive formation of glutamate (i.e. L-alanine, L-glutamine, L-asparagine, or NH4Cl + L-ornithine). The rate-limiting role of NADPH in urea synthesis was correlated with the decrease in NADPH content that occurred upon addition of NH4Cl or of alpha-methylisocitrate to hepatocytes incubated with lactate and pyruvate, indicating utilization of NADPH for reductive amination of 2-oxoglutarate and inhibition of NADPH generation via NADP-isocitrate dehydrogenase, respectively. Similar results were obtained with D-alanine and L-serine; however, alpha-methylisocitrate or NH4Cl did not substantially decrease NADPH content when L-alanine was the substrate. Inhibitors or ornithine--2-oxo acid transaminase (L-canaline or gabaculine) decreased the uptake of ornithine by hepatocytes and inhibited the alpha-methylisocitrate insensitive urea synthesis from ornithine and NH4Cl. Canaline did not inhibit urea synthesis from lactate, ornithine, and NH4Cl but the inhibition by alpha-methylisocitrate of urea formation from this combination was appreciably larger with canaline (approx. 82%) than without canaline (approx. 48%). Inhibition of urea synthesis from NH4Cl + lactate by alpha-methylisocitrate was partially prevented by oleate, octanoate, or 3-hydroxybutyrate. When the NADH content of hepatocytes was increased by 3-hydroxybutyrate, the addition of NH4Cl and/or alpha-methylisocitrate caused a decline in NADH (and NADPH) content, suggesting that reducing equivalents from NADH as well as from NADPH can support net reductive amination of 2-oxoglutarate when required for urea synthesis.

摘要

在分离的大鼠肝细胞中研究了 DL-苏式-α-甲基异柠檬酸（3-羟基-1,2,3-丁三羧酸）对 NADP 特异性异柠檬酸脱氢酶（EC 1.1.1.42）的抑制作用对尿素合成的影响。α-甲基异柠檬酸显著抑制了尿素合成速率（35%-84%），这些底物需要将 2-氧代戊二酸净还原胺化为谷氨酸以合成天冬氨酸（即 L-丝氨酸、D-丙氨酸或 NH₄Cl + L-乳酸）。α-甲基异柠檬酸不抑制由不需要还原形成谷氨酸的底物合成尿素（即 L-丙氨酸、L-谷氨酰胺、L-天冬酰胺或 NH₄Cl + L-鸟氨酸）。NADPH 在尿素合成中的限速作用与向用乳酸和丙酮酸孵育的肝细胞中添加 NH₄Cl 或α-甲基异柠檬酸后 NADPH 含量的降低相关，分别表明 NADPH 用于 2-氧代戊二酸的还原胺化以及通过 NADP-异柠檬酸脱氢酶抑制 NADPH 的生成。用 D-丙氨酸和 L-丝氨酸也得到了类似的结果；然而，当以 L-丙氨酸为底物时，α-甲基异柠檬酸或 NH₄Cl 并未显著降低 NADPH 含量。鸟氨酸-2-氧代酸转氨酶抑制剂（L-刀豆氨酸或加巴喷丁）降低了肝细胞对鸟氨酸的摄取，并抑制了由鸟氨酸和 NH₄Cl 合成的对α-甲基异柠檬酸不敏感的尿素。刀豆氨酸不抑制由乳酸、鸟氨酸和 NH₄Cl 合成尿素，但与不加刀豆氨酸（约 48%）相比，刀豆氨酸存在时α-甲基异柠檬酸对由该组合形成尿素的抑制作用明显更大（约 82%）。油酸、辛酸或 3-羟基丁酸部分阻止了α-甲基异柠檬酸对由 NH₄Cl + 乳酸合成尿素的抑制作用。当 3-羟基丁酸增加肝细胞的 NADH 含量时，添加 NH₄Cl 和/或α-甲基异柠檬酸会导致 NADH（和 NADPH）含量下降，这表明当尿素合成需要时，来自 NADH 以及 NADPH 的还原当量都可以支持 2-氧代戊二酸的净还原胺化。