Regulation of [15N]Urea Synthesis from [5-15N]Glutamine

Abstract

ammonia. The formation of [15N]citrulline (atom % ex- cess) was increased with higher pH. Flux through phos- phate-dependent glutaminase (PDG) and [15N]urea syn- thesis were stimulated (p < 0.05) at pH 7.6 or with GLU and decreased (p < 0.05) at pH 6.8. Insulin had no signif- icant effect on flux through PDG or on [15N]urea synthe- sis. Decreased [15N]urea production at pH 6.8 was asso- ciated with depleted aspartate and glutamate levels. Pyruvate attenuated this decrease in the aspartate and glutamate pools and stimulated [15N]urea synthesis. Production of Asp from pyruvate was increased with increasing medium pH. Approximately 80% of Asp was derived from [3-13C]pyruvate regardless of incubation pH or addition of hormone. Furthermore, approxi- mately 20, 40, and 50% of the mitochondrial N-acetylglu- tamate (NAG) pool was derived from [3-13C]pyruvate at pH 6.8, 7.4, and 7.6, respectively. Both the concentration and formation of [13C]NAG from [3-13C]pyruvate were increased (p < 0.05) with glucagon and decreased (p < 0.05) with insulin or at pH 6.8. The data suggest a corre- lation between changes in [15N]urea synthesis and alter- ations in the level and synthesis of [13C]NAG from pyru- vate. The current observations suggest that the stimulation of [15N]urea synthesis in acute alkalosis is mediated via increased flux through PDG and subse- quent increased utilization of [5-15N] of glutamine for [15N]citrulline synthesis and/or increased synthesis of NAG from glutamate and pyruvate. The opposite may have occurred in acute acidosis. Glucagon, but not insu- lin, stimulated [15N]urea synthesis via increased flux through PDG and synthesis of NAG. Pyruvate stimu- lated urea synthesis via increased availability of aspar- tate and/or increased synthesis ofNAG. The formation of NAG and aspartate from pyruvate are both pH-sensitive processes.