Induction of mitochondrial metabolism and pH-modulated ammoniagenesis by rocking LLC-PK1 cells.

Abstract

In an effort to find a model system in which the regulation of renal ammoniagenesis could be delineated, the LLC-PK1 line of cultured pig kidney epithelial cells was examined. Ammonia production by normally cultured LLC-PK1 cells (monolayers on 75-cm2 flasks, under 6 ml still serum-containing medium) was found to be neither modulated by direct (3 h) nor adapted by chronic (3 day) manipulation of medium pH (production at pH 7.05, 7.40, or 7.60 not significantly different). Considering that the mitochondrial glutamine to alpha-ketoglutarate pathway is the major regulatory site of ammoniagenesis, and that mitochondrial metabolism might be restricted under the normal lactate-generating or hypoxic culture conditions, we examined ammonia production by rocked flasks of LLC-PK1 cells. Flask were rocked continually at a rate of 2.5 oscillations/min, thereby exposing the cells to the atmosphere 40-50% of the time and also maximizing medium O2 tension and nutrient-waste exchange. Mitochondrial enzyme activities in rocked LLC-PK1 cells were shown to be consistently 1.5-fold greater than those in normally cultured cells. Ammonia production by rocked cells was not only directly modulated by short incubations with low and high pH media (production at pH 7.05 greater than 7.40 greater than 7.60) but was adapted by chronic (3 day) manipulations of medium pH (16 h after return to pH 7.40 medium production by pH 7.05- greater than 7.40- greater than 7.60-adapted cells). Thus rocker culture of the LLC-PK1 cell line both induces mitochondrial metabolism and converts cellular ammoniagenesis to a pH-sensitive phenomenon.(ABSTRACT TRUNCATED AT 250 WORDS)