Products of the phospholipase A(2) pathway mediate the dihydrorhodamine fluorescence response evoked by endogenous and exogenous peroxynitrite in PC12 cells.
A short-term exposure of PC12 cells to tert-butylhydroperoxide promotes a rapid oxidation of dihydrorhodamine sensitive to nitric oxide synthase inhibitors and peroxynitrite scavengers. This response was not directly caused by peroxynitrite, but rather appeared to be mediated by peroxynitrite-dependent activation of phospholipase A(2). The following lines of evidence support this inference: (i) the peroxynitrite-dependent dihydrorhodamine fluorescence response was blunted by low concentrations of two structurally unrelated phospholipase A(2) inhibitors; (ii) under similar conditions, the phospholipase A(2) inhibitors prevented release of arachidonic acid; (iii) low levels of arachidonic acid restored the dihydrorhodamine fluorescence response in nitric oxide synthase- as well as phospholipase A(2)-inhibited cells; (iv) the dihydrorhodamine fluorescence response induced by authentic peroxynitrite was also blunted by phospholipase A(2) inhibitors and restored upon addition of reagent arachidonic acid. We conclude that endogenous, or exogenous, peroxynitrite does not directly oxidize dihydrorhodamine in intact cells. Rather, peroxynitrite appears to act as a signalling molecule promoting release of arachidonic acid, which in turn leads to formation of species causing the dihydrorhodamine fluorescence response.
Palomba
L
L
Istituto di Farmacologia e Farmacognosia, Università di Urbino, Urbino, Italy.
Sestili
P
P
Guidarelli
A
A
Sciorati
C
C
Clementi
E
E
Fiorani
M
M
Cantoni
O
O
D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes; D018613:Microscopy, Confocal; D009566:Nitrates / Q000378:metabolism* / Q000633:toxicity*; D016877:Oxidants / Q000378:metabolism / Q000633:toxicity; D016716:PC12 Cells; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D012235:Rhodamines; D020122:tert-Butylhydroperoxide / Q000633:toxicity
D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D009566:Nitrates; D016877:Oxidants; D017382:Reactive Oxygen Species; D012235:Rhodamines; C058319:dihydrorhodamine 123; C039642:peroxynitric acid; D016718:Arachidonic Acid; D020122:tert-Butylhydroperoxide; D010741:Phospholipases A