Induction of a 55-kDa PKN cleavage product by ischemia/reperfusion model in the rat retina.
OBJECTIVE
To investigate the physiological role of a protein kinase, PKN, and its relation to apoptosis in vivo.


METHODS
An ischemia/reperfusion model of the rat retina was created by elevating the intraocular pressure. Retinal samples were obtained after ischemic insult (15-45 minutes) followed by reperfusion (1-7 days). The effect of ischemia on the fragmentation of PKN was examined by immunoblotting and immunocytochemical procedures using the antibody against PKN. N-methyl-D-aspartate (NMDA) or a caspase-3 inhibitor (DEVD-CHO) was administered intravitreally to investigate its effect on the induction of PKN fragmentation. The retinal cell loss in each sample was evaluated by toluidine blue staining.


RESULTS
Ischemia induced a 55-kDa PKN cleavage fragment corresponding to the molecular size of the constitutively active fragment of PKN. The appearance of the cleavage fragment depended on the duration of reperfusion and correlated with the occurrence of retinal cell loss. Immunocytochemical analysis revealed that ischemia increased PKN immunoreactivity in the inner layers of the retina. DEVD-CHO significantly inhibited the appearance of the 55-kDa fragment and protected against retinal cell loss. The administration of NMDA also induced cleavage of PKN.


CONCLUSIONS
PKN is specifically cleaved by caspase-3 or a related protease during apoptosis in vivo, and PKN cleavage is at least partially initiated by activation of the NMDA receptor.
Sumioka
K
K
Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, Japan.
Shirai
Y
Y
Sakai
N
N
Hashimoto
T
T
Tanaka
C
C
Yamamoto
M
M
Takahashi
M
M
Ono
Y
Y
Saito
N
N
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D053148:Caspase 3; D061945:Caspase Inhibitors; D004195:Disease Models, Animal; D019084:Fluorescent Antibody Technique, Indirect; D015151:Immunoblotting; D008297:Male; D008970:Molecular Weight; D016202:N-Methylaspartate / Q000494:pharmacology; D009842:Oligopeptides / Q000494:pharmacology; D011493:Protein Kinase C; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D015427:Reperfusion Injury / Q000201:enzymology* / Q000473:pathology / Q000517:prevention & control; D012164:Retinal Diseases / Q000201:enzymology* / Q000473:pathology / Q000517:prevention & control
D061945:Caspase Inhibitors; D009842:Oligopeptides; C108637:aspartyl-glutamyl-valyl-aspartal; D016202:N-Methylaspartate; C061570:protein kinase N; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D011493:Protein Kinase C; C505407:CASP3 protein, human; C505410:Casp3 protein, rat; D053148:Caspase 3