Pro-protein convertase-2/carboxypeptidase-E mediated neuropeptide processing of RGC-5 cell after in vitro ischemia.
OBJECTIVE
To observe the change of the neuropeptide pro-protein processing system in the ischemic retina ganglion cell-5 (RGC-5) cells, pro-protein convertase-2 (PC2), carboxypeptidase-E (CPE) and preproneuropeptide Y (preproNPY) protein levels in the ischemic RGC-5 cells and conditioned medium were analyzed.


METHODS
The RGC-5 cell was differentiated in 0.1 mumol/L staurosporine for 24 h and then stressed by different doses of oxygen and glucose deprivation (OGD). The acute or chronic OGD-induced cell death rates were obtained by using PI or TUNEL staining. The protein expression levels were determined by using the Western blot method and PC2 activity analysis.


RESULTS
The ischemia caused substantial cell death in an OGD dose-dependent manner. In the cells, proPC2 and preproNPY protein levels gradually increased whereas proCPE gradually decreased. After OGD, PC2 activity was decreased. In the conditioned medium, proPC2 and PC2 proteins gradually decreased whereas proCPE, CPE, and preproNPY proteins gradually increased.


CONCLUSIONS
These results demonstrated that OGD inhibited the neuropeptide pro-protein processing system by reducing PC2 activity and the maturation of proPC2. The aggregation of the pro-proteins and the increase of the active CPE excision adversely exacerbated the cell injury. The pro-protein processing system might play a critical role in the ischemic stress of RGC-5 cells.
Tang
Song-Shan
SS
Department of Biochemistry, School of Basic Courses, Guangdong Pharmaceutical University, Guangzhou 510006, China. songstang@gmail.com
Zhang
Juan-Hui
JH
Liu
Huan-Xin
HX
Zhou
Dong
D
Qi
Rong
R
D000818:Animals; D043423:Carboxypeptidase H / Q000378:metabolism*; D016923:Cell Death / Q000187:drug effects / Q000502:physiology; D002454:Cell Differentiation / Q000187:drug effects; D015687:Cell Hypoxia / Q000187:drug effects / Q000502:physiology; D002461:Cell Line, Transformed; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000502:physiology*; D005947:Glucose / Q000172:deficiency; D020287:In Situ Nick-End Labeling / Q000379:methods; D007211:Indoles; D009478:Neuropeptide Y / Q000378:metabolism*; D045706:Proprotein Convertase 2 / Q000378:metabolism*; D011498:Protein Precursors / Q000378:metabolism*; D051381:Rats; D012165:Retinal Ganglion Cells / Q000187:drug effects / Q000378:metabolism*; D019311:Staurosporine / Q000494:pharmacology; D013997:Time Factors
D004791:Enzyme Inhibitors; D007211:Indoles; D009478:Neuropeptide Y; D011498:Protein Precursors; C007293:DAPI; C042084:preproneuropeptide Y; D043423:Carboxypeptidase H; D045706:Proprotein Convertase 2; D019311:Staurosporine; D005947:Glucose