Generation of the catalytic fragment of protein kinase C alpha in vasospastic canine basilar artery

Abstract

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