Hypoxia potentiates exocytosis and Ca2+ channels in PC12 cells via increased amyloid beta peptide formation and reactive oxygen species generation.
Exposure of PC12 cells to chronic hypoxia (CH; 10 % O(2), 24 h) augments catecholamine secretion via formation of a Cd2+-resistant Ca2+ influx pathway, and up-regulates native L-type Ca2+ channels. These effects are mimicked by exposure of cells to Alzheimer's disease-associated amyloid beta peptides (AbetaPs). Since pathological effects of AbetaPs have been associated with increased levels of reactive oxygen species (ROS), the involvement of ROS in hypoxia-mediated up-regulation of exocytosis and Ca2+ channel activity was examined. Both melatonin and ascorbic acid (two structurally unrelated antioxidants) fully blocked the enhancement of catecholamine secretion caused by CH (as determined amperometrically). Enhanced immunofluorescence, observed in chronically hypoxic cells using a primary monoclonal antibody raised against the N-terminus of AbetaP, was also suppressed by melatonin. Ascorbic acid, melatonin and ebselen (an additional antioxidant) also fully prevented augmentation of whole-cell Ca2+ currents caused by CH (as monitored using whole-cell patch-clamp recordings). Exposure of normoxic cells to H(2)O(2) (40 microM, 24 h), like hypoxia, caused Ca2+ channel up-regulation. Importantly, AbetaP formation appeared to be an absolute requirement for the effects of hypoxia, since the ability of CH to augment exocytosis and Ca2+ channel activity was blocked by two novel inhibitors of gamma secretase, an enzyme complex required for AbetaP formation. Our results indicate that the effects of hypoxia require ROS generation from AbetaPs, and suggest that elevated levels of ROS mediate hypoxic and AbetaP-mediated pathological remodelling of Ca2+ homeostasis.
Green
Kim N
KN
Institute for Cardiovascular Research, University of Leeds, UK.
Boyle
John P
JP
Peers
Chris
C
D016229:Amyloid beta-Peptides / Q000096:biosynthesis*; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D001205:Ascorbic Acid / Q000494:pharmacology; D002104:Cadmium / Q000494:pharmacology; D015220:Calcium Channels / Q000502:physiology*; D004594:Electrophysiology; D005089:Exocytosis / Q000502:physiology*; D000860:Hypoxia / Q000378:metabolism / Q000503:physiopathology*; D007150:Immunohistochemistry; D008550:Melatonin / Q000494:pharmacology; D008564:Membrane Potentials / Q000502:physiology; D008839:Microelectrodes; D018384:Oxidative Stress / Q000502:physiology; D016716:PC12 Cells; D018408:Patch-Clamp Techniques; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism*
D016229:Amyloid beta-Peptides; D000975:Antioxidants; D015220:Calcium Channels; D017382:Reactive Oxygen Species; D002104:Cadmium; D008550:Melatonin; D001205:Ascorbic Acid