Influence of ammonia and pH on protein and amino acid metabolism in LLC-PK1 cells.

Abstract

Metabolic acidosis inhibits protein synthesis (PS) and stimulates protein degradation (PD) in muscle and cultured myocytes but causes hypertrophy of the proximal tubule. The reason for this tissue-specific difference in response to acidosis is unknown, but it might be related to stimulation of renal ammonia production since ammonia reportedly increases PS and inhibits PD in cultured kidney cells. We examined how ammonia and pH could interact to change protein turnover in confluent LLC-PK1 cells. Varying extracellular pH from 6.95 to 7.60 did not alter PS or PD even though intracellular pH changed predictably. Six millimolar NH4Cl did not change PS while 20 mM inhibited PS; there was no interaction with pH. This unexpected difference from the reported stimulation of PS by NH4Cl could be explained by our use of L-[U-14C]phenylalanine rather than radiolabelled leucine to measure PS. NH4Cl was found to inhibit leucine degradation which would increase radiolabelled leucine available for incorporation into protein. Either 6 mM or 20 mM NH4Cl inhibited PD measured as the release of L-[14C]phenylalanine from prelabelled protein. Experiments with an inhibitor of lysosomal function, chloroquine, suggest that NH4Cl inhibits lysosomal proteolysis. There was no interaction of cell pH and ammonia-induced changes in PD. Thus, the response of renal cells to acidification differs markedly from myocytes and ammonia changes protein turnover primarily by suppressing PD.