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Kiyoshi Sumioka, Yasuhito Shirai, Norio Sakai, Takeshi Hashimoto, Chikako Tanaka, Misao Yamamoto, Mikiko Takahashi, Yoshitaka Ono, Naoaki Saito; Induction of a 55-kDa PKN Cleavage Product by Ischemia/Reperfusion Model in the Rat Retina. Invest. Ophthalmol. Vis. Sci. 2000;41(1):29-35.
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© ARVO (1962-2015); The Authors (2016-present)
purpose. 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
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.
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