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.