Cholinergic-induced production of reactive oxygen species in human neuroblastoma cells.
Stimulation of human SH-SY5Y neuroblastoma cells by a muscarinic receptor agonist, carbachol (CCh; 1 mM), elevated levels of free intracellular calcium and subsequently increased the production of reactive oxygen species (ROS). Quinuclidinylbenzilate (QNB) binding increased at 1 h after CCh, but returned back to the control level at 3 h. Production of ROS increased, however, during the 3 h time period. CCh also increased the translocation of protein kinase C (PKC) to the membrane. ROS production was completely blocked by atropine and a PKC inhibitor, Ro 31-8220. These results show that increased ROS production was a result of muscarinic receptor stimulation, and that PKC had an active role in this cellular stimulation. ROS production upon cellular stimulation by CCh was completely inhibited also by superoxide dismutase, and partially by catalase, indicating that the formation of superoxide anion dominated in cholinergic-induced generation of ROS in human neuroblastoma cells. These results also show that muscarinic stimulation causes sustained ROS production in human neuroblastoma cells. The slow increase in ROS production by CCh suggest a stepwise cascade of events leading to oxidative stress with a triggering role of cholinergic muscarinic receptors in this process.
Naarala
J
J
National Public Health Institute, Laboratory of Toxicology, Kuopio, Finland. jonne.naarala@ktl.fi
Tervo
P
P
Loikkanen
J
J
Savolainen
K
K
D001692:Biological Transport; D002118:Calcium; D002217:Carbachol / Q000494:pharmacology*; D006801:Humans; D018721:Muscarinic Agonists / Q000494:pharmacology*; D009447:Neuroblastoma / Q000378:metabolism / Q000473:pathology; D015240:Phorbol 12,13-Dibutyrate / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D011813:Quinuclidinyl Benzilate / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D018721:Muscarinic Agonists; D017382:Reactive Oxygen Species; D015240:Phorbol 12,13-Dibutyrate; D011813:Quinuclidinyl Benzilate; D002217:Carbachol; D011493:Protein Kinase C; D002118:Calcium