Pilocarpine protects cobalt chloride-induced apoptosis of RGC-5 cells: involvement of muscarinic receptors and HIF-1 alpha pathway.
The retina is the most metabolically active tissue in the human body and hypoxia-induced retinal ganglion cell (RGC) death has been implicated in glaucomatous optic neuropathy. The aim of this study is to determine whether muscarinic receptor agonist pilocarpine, a classic antiglaucoma drug, possesses neuroprotection against cobalt chloride (CoCl(2))-mimetic hypoxia-induced apoptosis of rat retinal ganglion cells (RGC-5 cells) and its underlying mechanisms. Cell viability was determined by Cell Counting Kit-8 assay and apoptosis was examined by annexin V and mitochondrial membrane potential (MMP) assays. Expressions of hypoxia-induced factor-1 alpha (HIF-1 alpha), p53, and BNIP3 were investigated by quantitative real-time PCR and western blot analysis. After treatment of 200 microM CoCl(2) for 24 h, RGC-5 cells showed a marked decrease of cell viability by approximately 30%, increased apoptosis rate and obvious decline in MMP, which could largely be reversed by the pretreatment of 1 microM pilocarpine mainly via the activation of muscarinic receptors. Meanwhile, pretreatment of 1 microM pilocarpine could significantly prevent CoCl(2)-induced HIF-1 alpha translocation from cytoplasm to nucleus and down-regulate the expression of HIF-1 alpha, p53, and BNIP3. These studies demonstrated that pilocarpine had effective protection against hypoxia-induced apoptosis in RGCs via muscarinic receptors and HIF-1 alpha pathway. The findings suggest that HIF-1 alpha pathway as a "master switch" may be used as a therapeutic target in the cholinergic treatment of glaucoma.
Zhu
Xu
X
Department of Pharmacology, Institute of Medical Sciences, Shanghai Jiao Tong University School of Medicine, 280 South Chongqing Road, Shanghai, 200025, China.
Zhou
Wei
W
Cui
Yongyao
Y
Zhu
Liang
L
Li
Juan
J
Feng
Xuemei
X
Shao
Biyun
B
Qi
Hong
H
Zheng
Jun
J
Wang
Hao
H
Chen
Hongzhuan
H
D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D003035:Cobalt / Q000037:antagonists & inhibitors / Q000633:toxicity; D015536:Down-Regulation / Q000187:drug effects / Q000502:physiology; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D002534:Hypoxia, Brain / Q000188:drug therapy* / Q000378:metabolism / Q000503:physiopathology; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000187:drug effects* / Q000378:metabolism; D008565:Membrane Proteins / Q000187:drug effects / Q000378:metabolism; D024101:Mitochondrial Proteins; D018721:Muscarinic Agonists / Q000494:pharmacology* / Q000627:therapeutic use; D009498:Neurotoxins / Q000037:antagonists & inhibitors / Q000633:toxicity; D010862:Pilocarpine / Q000494:pharmacology / Q000627:therapeutic use; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D011518:Proto-Oncogene Proteins / Q000187:drug effects / Q000378:metabolism; D051381:Rats; D011976:Receptors, Muscarinic / Q000187:drug effects / Q000378:metabolism; D012162:Retinal Degeneration / Q000188:drug therapy* / Q000378:metabolism / Q000503:physiopathology; D012165:Retinal Ganglion Cells / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D016159:Tumor Suppressor Protein p53 / Q000187:drug effects / Q000378:metabolism
C505650:BNIP3 protein, rat; C497444:Hif1a protein, rat; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D018721:Muscarinic Agonists; D009498:Neurotoxins; D011518:Proto-Oncogene Proteins; D011976:Receptors, Muscarinic; D016159:Tumor Suppressor Protein p53; D010862:Pilocarpine; D003035:Cobalt; C018021:cobaltous chloride