Contribution of luminal ammoniagenesis to proximal tubule ammonia appearance in the rat

Abstract

The contribution of luminal ammoniagenesis in the late proximal convolute tubule (PCT) via phosphate-independent glutaminase [gamma-glutamyltransferase (gamma-GT)] remains controversial. If this pathway is important, it must rely on glutamine secretion, because filtered glutamine is reabsorbed in the early PCT. The contribution of gamma-GT to luminal ammoniagenesis was tested by use of in vivo microperfusion in conjunction with a new microfluorometric assay for glutamate. We first confirmed that aspartate completely blocked glutamate uptake in the PCT. Furthermore, the gamma-GT inhibitor acivicin completely eliminated glutamate entry, showing that passive glutamate entry was negligible. Thus the accumulation of glutamate can be used as an estimate of luminal glutamine deamidation. L-Phenylalanine was used to inhibit glutamine loss, and hippurate was used to stimulate gamma-GT activity; therefore luminal glutamine conversion to glutamate was promoted. Perfusing the tubule at 30 nl/min with a solution containing 10 mM each of hippurate, phenylalanine, and aspartate resulted in a glutamate delivery of 1.08 +/- 0.12 pmol.min-1.mm-1. Ammonia appearance was 10-fold higher, averaging 11.5 +/- 1.3 pmol.min-1.mm-1 under these same conditions. Thus the luminal conversion of glutamine to glutamate via gamma-GT is a small component of total ammoniagenesis in this segment.