Differential alteration of catecholamine release during chemical hypoxia is correlated with cell toxicity and is blocked by protein kinase C inhibitors in PC12 cells.
Release of neurotransmitters, including dopamine and glutamate, has been implicated in hypoxia/ischemia-induced alterations in neuronal function and in subsequent tissue damage. Although extensive studies have been done on the mechanism underlying the changes in glutamate release, few have examined the mechanism that is responsible for the changes in catecholamines. Rat pheochromocytoma-12 (PC12) cells synthesize, store, and release catecholamines including DA and NE. Therefore, we used HPLC and ED to evaluate extracellular DA and NE concentrations in a medium during chemical hypoxia in PC12 cells. Chemical hypoxia produced by KCN induced differential release of DA and NE. Under normal glucose conditions, KCN induced release of NE, but not DA. Under glucose-free conditions, KCN-induced release of DA was elevated transiently, whereas the release of NE increased progressively. Under parallel conditions, KCN biphasically elevated the level of cytosolic free calcium ([CA(2+)](i)) in glucose-free DMEM, peaking at 95 +/- 18 nM at 1,107 +/- 151 s, followed by a new plateau level at 249 +/- 24 nM sustained from 4,243 +/- 466 to 5,263 +/- 440 s. Cell toxicity, as measured by LDH release, was increased significantly by KCN in glucose-free DMEM but was diminished in the presence of glucose, and was correlated with DA release by chemical hypoxia. The protein kinase C (PKC) inhibitor GO6976 or staurosporine inhibited KCN-induced LDH release as well as the release of NE and DA. Taken together, selective activation of DA but not NE was correlated with the LDH release by chemical hypoxia, and was diminished with GO6976 or staurosporine. These results suggest that selective activation of PKC isoforms is involved in the chemical hypoxia-induced DA release, which may lead to neuronal cell toxicity.
Kuo
J S
JS
Department of Education and Medical Research, Taichung Veterans General Hospital, Taichung, Taiwan, Republic of China.
Cheng
F C
FC
Shen
C C
CC
Ou
H C
HC
Wu
T F
TF
Huang
H M
HM
D000818:Animals; D002118:Calcium / Q000378:metabolism; D015687:Cell Hypoxia; D002851:Chromatography, High Pressure Liquid; D004298:Dopamine / Q000378:metabolism*; D004563:Electrochemistry; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005947:Glucose / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D009638:Norepinephrine / Q000378:metabolism*; D016716:PC12 Cells; D011493:Protein Kinase C / Q000037:antagonists & inhibitors*; D051381:Rats
D004791:Enzyme Inhibitors; D007770:L-Lactate Dehydrogenase; D011493:Protein Kinase C; D005947:Glucose; D002118:Calcium; D004298:Dopamine; D009638:Norepinephrine