Fate of glutamate carbon and nitrogen in isolated guinea-pig kidney-cortex tubules. Evidence for involvement of glutamate dehydrogenase in glutamine sythesis from glutamate

Abstract

1. The pathways and the fate of glutamate carbon and nitrogen were investigated in isolated guinea-pig kidney-cortex tubules. 2. At low glutamate concentration (1 mM), the glutamate carbon skeleton was either completely oxidized or converted into glutamine. At high glutamate concentration (5 mM), glucose, lactate and alanine were additional products of glutamate metabolism. 3. At neither concentration of glutamate was there accumulation of ammonia. 4. Nitrogen-balance calculations and the release of 14CO2 from L-[ 1-_4C]glutamate (which gives an estimation of the flux of glutamate carbon skeleton through a-oxoglutarate dehydrogenase) clearly indicated that, despite the absence of ammonia accumulation, glutamate metabolism was initiated by the action of glutamate dehydrogenase and not by transamination reactions as suggested by Klahr, Schoolwerth & Bourgoignie [(1972) Am. J. Physiol. 222,[813][814][815][816][817][818][819][820] and Preuss [(1972) Am. J. Physiol. 222, 1395-13971. Additional evidence for this was obtained by the use of (i) amino-oxyacetate, an inhibitor of transaminases, which did not decrease glutamate removal, or (ii) L-methionine DL-sulphoximine, an inhibitor of glutamine synthetase, which caused an accumulation of ammonia from glutamate. 5. Addition of NH4Cl plus glutamate caused an increase in both glutamate removal and glutamine synthesis, demonstrating that the supply of ammonia via glutamate dehydrogenase is the rate-limiting step in glutamine formation from glutamate. NH4Cl also inhibited the flux of glutamate through glutamate dehydrogenase and the formation of glucose, alanine and lactate. 6. The activities of enzymes possibly involved in the glutamate conversion into pyruvate were measured in guinea-pig renal cortex. 7. Renal arteriovenousdifference measurements revealed that in vivo the guinea-pig kidney adds glutamine and alanine to the circulating blood.Since the initial work of Krebs (1935), it is known that, in contrast with kidney-cortex slices of dog, cat, pig, rat, sheep and pigeon, slices of guinea pig and rabbit do not release ammonia when incubated with glutamate. Krebs concluded that this was so in both the latter species because the ammonia formed by the deamination of glutamate via glutamate dehydrogenase was used for glutamine synthesis (see Krebs, 1935). However, the role of glutamate dehydrogenase in the metabolism of glutamate by guinea-pig and rabbit kidney slices has been questioned by and Preuss (1972), because these authors observed that glucose was formed from glutamate without concomitant accumulation of Vol. 188 ammonia; they concluded that the initial step in glutamate metabolism was transamination rather than oxidative deamination. So the pathways of renal metabolism of glutamate in these species remain uncertain.In an attempt to clarify this subject, we studied glutamate metabolism in isolated guinea-pig kidney tubules. The data obtained support the view of Krebs (1935) that glutamate dehydrogenase plays a central role in the renal metabolism of glutamat...

Glutamine synthesis from aspartate in guinea-pig renal cortex

“…The addition of aspartate (5 mM) plus NH4CI (5 mM) did not increase glutamine synthesis, as has been observed when glutamate was the substrate [6], and no ammonia was removed (Table 2); on the contrary, both glutamine synthesis and aspartate removal were inhibited. This probably resulted from the decrease in the availability of 2-oxoglutarate for the aspartate aminotransferase reaction secondary to the end-product inhibition of glutamate dehydrogenase by high concentrations of ammonia, as previously demonstrated with glutamate as substrate [6]; calculations from the data of Table 2 show that flux through glutamate dehydrogenase, taken as the glutamine plus the ammonia found, decreased from a mean value of 412.4 ,mol/g dry wt.…”

“…This probably resulted from the decrease in the availability of 2-oxoglutarate for the aspartate aminotransferase reaction secondary to the end-product inhibition of glutamate dehydrogenase by high concentrations of ammonia, as previously demonstrated with glutamate as substrate [6]; calculations from the data of Table 2 show that flux through glutamate dehydrogenase, taken as the glutamine plus the ammonia found, decreased from a mean value of 412.4 ,mol/g dry wt. with aspartate alone to a mean value of 234.6 ,mol/g dry wt.…”

“…Of the two glutamate molecules formed by the transamination of two aspartate molecules with two 2-oxoglutarate molecules, one can be converted into glutamine by glutamine synthetase, which is very active in guineapig renal cortex [6,7]. Evidence is presented in Table 2 that glutamate dehydrogenase, which plays a major role in glutamate metabolism in guinea-pig tubules [6], provides from the second glutamate molecule available the ammonia needed for the formation of the glutamine amide nitrogen. The suppression of glutamine synthesis by methionine sulphoximine, a potent inhibitor of glutamine synthetase [17], was accompanied by a large accumulation of the ammonia released by glutamate dehydrogenase, whose role was masked by the very efficient operation of glutamine synthetase.…”

“…Addition of quinolinate, an inhibitor of phosphoenolpyruvate carboxykinase [18] whose inhibitory effect is established in guinea-pig renal cortex [6], caused a large inhibition of both aspartate removal and glutamine synthesis ( .r port across the mitochondrial membrane [19], caused a large accumulation of pyruvate in tubules metabolizing aspartate; this effect was accompanied by an inhibition of both aspartate removal and glutamine synthesis. This observation clearly demonstrates that pyruvate and the mitochondrial pyruvate transporter are involved in the conversion of aspartate into glutamine in guinea-pig kidney tubules.…”

“…In a previous paper [6], we have shown that, under appropriate experimental conditions, aspartate was synthesized from glutamate by isolated guinea-pig kidney-cortex tubules. Since aspartate aminotransferase (EC 2.6.1.1) is a near-equilibrium enzyme, we considered that aspartate could also be a substrate for the guinea-pig renal cortex.…”