Relationship between intracellular pH and ammonia metabolism in LLC-PK1 cells

Abstract

Previous studies from our laboratory have confirmed that cultures of LLC-PK1 cells exhibit pH-responsive alterations in ammonia metabolism produced by changes in media bicarbonate concentration. To further elucidate the mechanism of ammonia regulation, studies were carried out using parallel cultures of still and rocked LLC-PK1 cells subjected to acute alterations in media pH by either metabolic or respiratory acid-base manipulations. When media pH was altered by modifying PCO2 levels, the response of ammonia and alanine production by rocked culture was identical to the changes observed with metabolic acid-base maneuvers. Furthermore, both metabolic and respiratory acute acidosis resulted in a fall of intracellular alpha-ketoglutarate concentrations in these cells. In contrast, standard still cultures subjected to acute acidosis/alkalosis by metabolic and respiratory manipulations did not exert any significant change in ammonia and alanine production or in intracellular alpha-ketoglutarate concentration. Measurements of intracellular pH (pHi) by the 5,5-[2-14C]dimethyloxazolidine-2,4-dione method in rocked cells demonstrated changes in pHi parallel to media pH changes induced by both metabolic and respiratory acid-base maneuvers. Despite the absence of pH-responsive ammonia-genesis in still cultured cells the pHi values were altered in a fashion similar to their rocked counterparts, indicating the lack of an effect of the pHi signal on ammonia metabolism.