REGULATION OF [N-15]UREA SYNTHESIS FROM [5-N-15]GLUTAMINE - ROLE OF PH, HORMONES, AND PYRUVATE

We have utilized both [5-N-15]glutamine and [3-C-13] pyruvate as metab olic tracers in order to: (i) examine the effect of pH, glucagon (GLU) , or insulin on the precursor-product relationship between (NH3)-N-15, [N-15]citrulline, and, thereby, [N-15]urea synthesis and (ii) elucida te the mechanism(s) by which pyruvate stimulates [N-15] urea synthesis . Hepatocytes isolated from rat were incubated at pH 6.8, 7.4, or 7.6 with 1 mM [5-N-15]glutamine and 0.1 mM (NH4Cl)-N-14 in the presence or the absence of [3-C-13] pyruvate (2 mM). A separate series of experim ents was performed at pH 7.4 in the presence of insulin or GLU. (NH3)- N-15 enrichment exceeded or was equal to that of [N-15]citrulline unde r all conditions except for pH 7.6, when the N-15 enrichment in citrul line exceeded that in ammonia. The formation of [N-15]citrulline (atom % excess) was increased with higher pH. Flux through phosphate-depend ent glutaminase (PDG) and [N-15]urea synthesis were stimulated (p < 0. 05) at pH 7.6 or with GLU and decreased (p < 0.05) at pH 6.8. Insulin had no significant effect on flux through PDG or on [N-15]urea synthes is, Decreased [N-15]urea production at pH 6.8 was associated with depl eted aspartate and glutamate levels, Pyruvate attenuated this decrease in the aspartate and glutamate pools and stimulated [N-15]urea synthe sis. Production of Asp from pyruvate was increased with increasing med ium pH. Approximately 80% of Asp was derived from [3-C-13]pyruvate reg ardless of incubation pH or addition of hormone. Furthermore, approxia tely 20, 40, and 50% of the mitochondrial N-acetylglutamate (NAG) pool was derived from [3-C-13]pyruvate at pH 6.8, 7.4, and 7.6, respective ly. Both the concentration and formation of [C-13]NAG from [3-C-13]pyr uvate were increased (p < 0.05) with glucagon and decreased (p < 0.05) with insulin or at pH 6.8. The data suggest a correlation between cha nges in [N-15]urea synthesis and alterations in the level and synthesi s of [C-13]NAG from pyruvate, The current observations suggest that th e stimulation of [N-15]urea synthesis in acute alkalosis is mediated v ia increased flux through PDG and subsequent increased utilization of [5-N-15] Of glutamine for [N-15]citrulline synthesis and/or increased synthesis of NAG from glutamate and pyruvate. The opposite may have oc curred in acute acidosis. Glucagon, but not insulin, stimulated [N-15] urea synthesis via increased flux through PDG and synthesis of NAG. Py ruvate stimulated urea synthesis via increased availability of asparta te and/or increased synthesis of NAG, The formation of NAG and asparta te from pyruvate are both pH-sensitive processes.