GENERATION OF THE CATALYTIC FRAGMENT OF PROTEIN-KINASE-C-ALPHA IN SPASTIC CANINE BASILAR ARTERY

In previous studies of topical application of calphostin C, a specific inhibitor of the regulatory domain of protein kinase C (PKC), and cal peptin, a selective inhibitor of calpain, to spastic canine basilar ar tery (BA) researchers have suggested that the catalytic fragment of PK C (known as PKM) is probably formed by a limited proteolysis of contin uously activated mu-calpain, but there has been no direct evidence for PKM formation in vasospasm. The present immunoblot study with anti-PK C alpha antibody shows a significant decrease in cytosolic 80-kD PKC a lpha and a concomitantly significant increase in membrane PKC alpha in the spastic canine BA. In addition, an immunoblot study in which clea vage site-directed antibodies were used demonstrated a significant inc rease in immunoreactive 45-kD PKM. The changes in membrane PKC alpha a nd PKM were enhanced with the lapse of time after subarachnoid hemorrh age. The cleavage site-directed antibodies distinguish the proteolyzed from the unproteolyzed forms of PKC for in situ analyses of enzyme re gulation mediated by proteolysis. The data indicate that PKC alpha in spastic canine BA is translocated to the cell membrane, where PKC alph a is rapidly cleaved into PKM as a result of proteolysis of the isozym e by mu-calpain but not by m-calpain. The authors hypothesize that mu- calpain is continuously activated in spastic canine BA and produces PK M by limited proteolysis of PKC alpha.