NADP-specific isocitrate dehydrogenase in regulation of urea synthesis in rat hepatocytes.
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.
D000596:Amino Acids / Q000378:metabolism; D000643:Ammonium Chloride / Q000378:metabolism; D000818:Animals; D066298:In Vitro Techniques; D007521:Isocitrate Dehydrogenase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007523:Isocitrates / Q000494:pharmacology; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D009249:NADP / Q000378:metabolism; D051381:Rats; D013379:Substrate Specificity; D014508:Urea / Q000096:biosynthesis*
D000596:Amino Acids; D007523:Isocitrates; D000643:Ammonium Chloride; D009249:NADP; C021058:threo-alpha-methylisocitrate; D014508:Urea; D007521:Isocitrate Dehydrogenase