Ammonia production and glutamine incorporation into glutathione in the functioning rat kidney

Abstract

Incorporation of glutamine's γ-glutamyl moiety into glutathione was studied in the isolated perfused rat kidney under two conditions: (1) a resting state and (2) during reabsorption of a filtered glycylglycine load (designed to stimulate glutathione breakdown). Glutamine uptake and ammonia production were monitored simultaneously with [14C]glutamine (γ-glutamyl moiety) incorporation into glutathione.Renal glutathione content was highly dependent upon the presence of glutamine; in its absence, glutathione levels fell 40% in 30 min indicating synthesis does not keep pace with degradation. Presenting the kidney with a glycylglycine load doubled the rate of glutathione degradation. However, perfusing kidneys with physiological L-glutamine concentration (1 mM) maintained glutathione levels significantly above those in glutamine's absence in either condition 1 or 2. Evidence that glutamine functions as the γ-glutamyl donor in glutathione synthesis was established by the significant incorporation of [14C]glutamine into glutathione during condition 1 and the accelerated incorporation rate during glycylglycine loading (condition 2). Stimulating glutathione breakdown (condition 2) resulted in a significant increase in renal glutamine uptake and ammonia production apparently via the γ-glutamyltransferase pathway since inhibiting this activity blocked the incremental rise in glutamine uptake during glycylglycine loading. These results are consistent with the interpretation that stimulating γ-glutamyl cycle activity with a γ-glutamyl acceptor (glycylglycine) accelerates γ-glutamyl donor uptake (glutamine) and ammonia production via the γ-glutamyltransferase pathway.