Effects of aging in vitro on intracellular proteolysis in cultured rabbit lens epithelial cells in the presence and absence of serum

Abstract

Alterations in proteolytic capabilities have been associated with abnormalities in the aged eye lens, but in vivo tests of this hypothesis have been difficult to pursue. To simulate aging, we cultured cells from an 8-yr-old rabbit to early (population-doubling level 20 to 30) and late (population-doubling level greater than 125) passage. Long-lived (t1/2 greater than 10 h) and short-lived (t1/2 less than 10 h) intracellular proteins were labeled with [3H]leucine, and the ability of the cells to mount a proteolytic response to the stress of serum withdrawal was determined. For early passage cells, the average t1/2 of long-lived proteins in the presence and absence of serum was 62 and 39 h, respectively. For late-passage cells, the average t1/2 of long-lived proteins in the presence and absence of serum was 58 and 43 h, respectively. The net increase in intracellular proteolysis in the absence of serum was 59 and 35% for early and late-passage cells, respectively. Thus, in vitro-aged rabbit lens epithelial cells amount only 60% the proteolytic response to serum removal shown in "younger" cells. The enhanced ability of early passage cells to respond to serum removal seems to involve lower homeostatic levels of proteolysis in the presence of serum and greater enhancement of proteolysis in the absence of serum. Less than 2% of the protein is in the pool of short-lived proteins. Rates of proteolysis of short-lived proteins in the presence and absence of serum were indistinguishable. With respect to basal proteolytic rates in the presence of serum and ability to mount a proteolytic response upon serum withdrawal, these rabbit lens epithelial cells are similar to bovine lens epithelial cells and fibroblasts.