Diphenyleneiodonium suppresses apoptosis in cerulein-stimulated pancreatic acinar cells.
NADPH oxidase has been considered a major source of reactive oxygen species in phagocytic and non-phagocytic cells. Apoptosis linked to oxidative stress has been implicated in pancreatitis. Recently, we demonstrated that NADPH oxidase subunits Nox1, p27phox, p47phox, and p67phox are constitutively expressed in pancreatic acinar cells, which are activated by cerulein, a cholecystokinin analogue. Cerulein induces an acute and edematous form of pancreatitis. We investigated whether inhibition of NADPH oxidase by diphenyleneiodonium suppresses the production of reactive oxygen species and apoptosis by determining viable cell numbers, DNA fragmentation, TUNEL staining, caspase-3 activity, and the expression of apoptosis-inducing factor in pancreatic acinar AR42J cells stimulated with cerulein. Inhibition on NADPH oxidase by diphenyleneiodonium was assessed by the alterations in NADPH oxidase activity and translocation of the cytosolic subunits p67phox and p47phox to the membrane. Intracellular Ca2+ level was monitored to investigate the relationship between NADPH oxidase and Ca2+ in cells stimulated with cerulein. As a result, cerulein induced the activation of NADPH, increased production of reactive oxygen species, and apoptotic indices determined by the expression of apoptosis-inducing factor, caspase-3 activation, TUNEL staining, DNA fragmentation, and cell viability. Treatment with DPI inhibited cerulein-induced activation of NADPH oxidase, the production of reactive oxygen species, and apoptosis, but not the increase of intracellular Ca2+ levels in pancreatic acinar cells. These results demonstrate that the cerulein-induced increase in intracellular Ca2+ level may be an upstream event of NADPH oxidase activation. Diphenyleneiodonium, an NADPH oxidase inhibitor, inhibits the expression of apoptosis-inducing factor and caspase-3 activation, and thus apoptosis in pancreatic acinar cells.
Yu
Ji Hoon
JH
Department of Pharmacology, Brain Korea 21 Project for Medical Science, College of Medicine, Yonsei University, Seoul, Republic of Korea.
Kim
Kyung Hwan
KH
Kim
Dong Goo
DG
Kim
Hyeyoung
H
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D051033:Apoptosis Inducing Factor / Q000235:genetics / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D053148:Caspase 3 / Q000378:metabolism; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D002108:Ceruletide / Q000494:pharmacology*; D053938:DNA Fragmentation / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D005786:Gene Expression Regulation / Q000187:drug effects; D005978:Glutathione / Q000494:pharmacology; D020287:In Situ Nick-End Labeling; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D019255:NADPH Oxidases / Q000378:metabolism; D009861:Onium Compounds / Q000494:pharmacology*; D046790:Pancreas, Exocrine / Q000166:cytology* / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D013482:Superoxide Dismutase / Q000494:pharmacology
D051033:Apoptosis Inducing Factor; D009861:Onium Compounds; D017382:Reactive Oxygen Species; C007517:diphenyleneiodonium; D002108:Ceruletide; D007770:L-Lactate Dehydrogenase; D013482:Superoxide Dismutase; D019255:NADPH Oxidases; D053148:Caspase 3; D005978:Glutathione; D002118:Calcium