IDENTIFICATION OF MU-CALPAINS, M-CALPAINS AND CALPASTATIN AND CAPTUREOF MU-CALPAIN ACTIVATION IN ENDOTHELIAL-CELLS

The presence of the calpain-calpastatin system in human umbilical vein endothelial cells (HUVEC) was investigated by means of ion exchange c hromatography, Western blot analysis, and Northern blot analysis. On D EAE anion exchange chromatography, calpain and calpastatin activities were eluted at approximately 0.30 M and 0.15-0.25 M NaCl, respectively . For half-maximal activity, the protease required 800 mu M Ca2+, comp arable to the Ca2+ requirement of m-calpain. By Western blot analysis, the large subunit of mu-calpain (80 kDa) was found to be eluted with calpastatin (110 kDa). Both the large subunit of m-calpain (80 kDa) an d calpastatin were detected in the respective active fractions. By Nor thern blot analysis, mRNAs for large subunits of mu- and m-calpains we re detected in single bands, each corresponding to approximately 3.5 K b. Calpastatin mRNA was observed in two bands corresponding to approxi mately 3.8 and 2.6 Kb. Furthermore, the activation of mu-calpain in HU VEC by a calcium ionophore was examined, using an antibody specificall y recognizing an autolytic intermediate form of mu-calpain large subun it (78 kDa). Both talin and filamin of HUVEC were proteolyzed in a cal cium-dependent manner, and the reactions were inhibited by calpeptin, a cell-permeable calpain specific inhibitor. Proteolysis of the cytosk eleton was preceded by the appearance of the autolytic intermediate fo rm of mu-calpain, while the fully autolyzed postautolysis form of mu-c alpain (76 kDa) remained below detectable levels at all time points ex amined. These results indicate that the calpain-calpastatin system is present in human endothelial cells and that mu-calpain may be involved in endothelial cell function mediated by Ca2+ via the limited proteol ysis of various proteins. (C) 1997 Wiley-Liss, Inc.