Decreased proteolysis and increased amino acid efflux in aging human fibroblasts

The release of radioactivity was measured following variable times of dual labeling of normal human fibroblasts at early and late passage and late-passage cell strains derived from the progeria and Werner's syndrome of premature aging. In the rapid first phase to 20 min, all 3 late-passage (aged) cells released more acid-soluble radioactivity into the medium than early-passage normal cells in the order Werner's greater than progeria greater than late-passage normal greater than early-passage normal, virtually all of this radioactivity emanating from intracellular material that was acid-soluble after prelabeling (zero time). In the slower second phase from 20 min to 3 h, all 4 cell types showed approximately parallel profiles of release. Following preloading with the non-utilizable amino acid alpha-amino[14C] isobutyric acid, progeria and Werner's cells released radioactivity more rapidly than did early- and late-passage normal cells in that order. In contrast, direct measurements on the net loss of counts from acid-insoluble material, i.e. true proteolysis, revealed that all 3 aged cells degraded proteins of short half-life more slowly but proteins of long half-life were degraded at the same rate as those of young normal cells. The results indicate that aged cells have a reduced proteolytic capacity and increased amino acid efflux. The latter process is probably due to the higher proportion of labeled amino acids in the cell water of aged cells at zero time and possibly increased membrane leakiness. The reason for the decreased proteolysis is not clear but it may relate to the rising proportion of defective proteins, the increased protein content and the loss of replicative capacity in aging cells.