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 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 mu-calpain, but there has been no direct evidence for PKM formation in vasospasm. The present immunoblot study with anti-PKCalpha antibody shows a significant decrease in cytosolic 80-kD PKCalpha and a concomitantly significant increase in membrane PKCalpha 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 PKCalpha 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 PKCalpha in spastic canine BA is translocated to the cell membrane, where PKCalpha is rapidly cleaved into PKM as a result of proteolysis of the isozyme by mu-calpain but not by m-calpain. The authors hypothesize that mu-calpain is continuously activated in spastic canine BA and produces PKM by limited proteolysis of PKCalpha.
Sato
M
M
Department of Neurosurgery, Hyogo College of Medicine, Japan.
Tani
E
E
Matsumoto
T
T
Fujikawa
H
H
Imajoh-Ohmi
S
S
D000818:Animals; D001488:Basilar Artery / Q000201:enzymology* / Q000503:physiopathology; D004285:Dogs; D004789:Enzyme Activation; D015151:Immunoblotting; D007527:Isoenzymes / Q000096:biosynthesis* / Q000276:immunology; D011493:Protein Kinase C / Q000096:biosynthesis* / Q000276:immunology; D051571:Protein Kinase C-alpha; D014661:Vasoconstriction
D007527:Isoenzymes; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha