Intralysosomal Generation of Ammonia from Urea by Endocytosed Urease Results in Secretion of Free Lysosomal Arylsulfatase-A and Increased Activity of Membrane-Bound β-Glucosidase in Cultured Brain Cells

Abstract

Hyperammonemia interferes with normal brain function. The effect of ammonia on free and membrane-bound lysosomal enzymes and on mucopolysaccharide metabolism was studied in cultured rat brain cells (ROC-1, hybridoma between C6-astrocytoma and oligodendrocytes). Intralysosomal ammoniagenesis was achieved from urea by endocytosed Jackbean urease followed by incubation of the cultures with urea. The intralysosomal location of urease was evidenced by the protective effects of leupeptin and urea on the stability of intracellular urease. Ammonia formed from urea resulted in an increased secretion of lysosomal arylsulfatase-A (AS-A), but not of the membrane-bound lysosomal beta-glucosidase into the culture medium, thus intralysosomal AS-A activity decreased. Lysosomal, membrane-bound beta-glucosidase activity increased, presumably due to intralysosomal proteolytic protection following an increased lysosomal pH. Intralysosomal ammoniagenesis temporarily impaired 35SO4-glycosaminoglycan degradation of prelabeled cells. The results support the hypothesis that hyperammonemic states may interfere with lysosomal functions in vivo as well in cultured cells.