INCREASED ACTIVITIES OF CATHEPSIN-B AND OTHER LYSOSOMAL HYDROLASES INFIBROBLASTS AND BONE TISSUE-CULTURED IN THE PRESENCE OF CYSTEINE PROTEINASES INHIBITORS

Leupeptin is an established, reversible inhibitor of cathepsin B, a ly sosomal cysteine proteinase. Yet, in rat fibroblasts as well as in foe tal mouse calvaria, we observed an increase of the activity of catheps in B in homogenates of cells and tissue harvested after culture in the presence of leupeptin. This effect was also seen for other lysosomal hydrolases, namely sphingomyelinase, N-acetyl-beta-glucosaminidase, ar ylsulphatase A and phospholipase A1 in fibroblasts, and beta-glucuroni dase in mouse calvaria. In calvaria, antipain, another reversible cyst eine proteinase inhibitor, caused a similar effect, whereas E-64, an i rreversible inhibitor, was consistently inhibitory of the cathepsin B activity; yet it also caused an increase of beta-glucuronidase activit y. The effect of leupeptin in fibroblasts was dose and time-dependent, required the continuous presence of the inhibitor, and was not depend ent from protein synthesis. Actually, addition of cycloheximide caused a severe loss of activity of cathepsin B and of sphingomyelinase. In the presence of both cycloheximide and leupeptin, however, these two a ctivities were retained to a value corresponding to that found in exce ss in cells cultivated with leupeptin alone. The data therefore sugges ts that leupeptin exerts the effects described in this paper by preven ting the degradation of cathepsin B, sphingomyelinase and probably sev eral other lysosomal hydrolases by cysteine proteinases. We therefore propose that cysteine proteinases play a key role in the control of th e steady-state levels of these enzymes in normal conditions.