Increased survival of rat hepatocytes in serum-free medium by inhibition of a trypsin-like protease associated with their plasma membranes

Abstract

Bovine pancreatic trypsin-inhibitor (bPTI) is required for survival of adult rat hepatocytes for more than 2 days in primary cultures in serum-free medium. Of the various protease inhibitors tested, all trypsin inhibitors increased the survival of rat hepatocytes in serum-free medium, their potencies being in the order bPTI greater than alpha 2-plasmin inhibitor greater than leupeptin greater than soybean trypsin inhibitor greater than alpha 1-antitrypsin = alpha 2-macroglobulin. Elastatinal, a specific inhibitor of elastase, was also effective. bPTI did not inhibit the degradation of proteins with short or long lives, suggesting that it did not increase the survival of hepatocytes by inhibiting cellular protein degradation. alpha 2-Plasmin inhibitor immobilized on Sepharose 4B caused dose-dependent increase in survival. Plasma membranes purified from adult rat liver had significant protease activity, about 80% of which was sensitive to bPTI, alpha 2-plasmin inhibitor and leupeptin. From its specificity for substrates and sensitivity to inhibitors, the membrane-bound protease was characterized as a trypsin-like protease. The effects of various inhibitors on the membrane-bound protease correlated well with their abilities to increase survival of rat hepatocytes. Therefore, it seems that bPTI acts on the cell surface and increases hepatocyte survival in serum-free cultures by inhibiting a trypsin-like protease associated with the plasma membranes.