We have previously shown that chronic hypoxia (CH) potentiates evoked catecholamine secretion and augments Ca2+ current density in PC12 cells via a mechanism that appears to involve increased production of amyloid β peptides (AβPs; Taylor et al. 1999a; Green & Peers, 2001). CH and AβPs have been shown to increase reactive oxygen species (Mattson, 1997; Hohler et al. 1999). We have therefore investigated the ability of antioxidants to modulate Ca2+ current augmentation by both CH and AβP. Currents were recorded using the whole-cell patch-clamp technique (20 mM Ba2+ as charge carrier; see Taylor et al. 1999b, for solution details) using 200 ms ramps applied from -100 to +100 mV at 0.2 Hz (-80 mV inter-ramp holding potential). All reported values are taken from currents measured at +20 mV and statistical comparisons made using Student’s unpaired t tests.
In cells cultured normoxically, Ca2+ current density was -4.48 ± 0.67 pA pF-1 (n = 22 cells). This was significantly enhanced (P < 0.001) in CH cells (kept in 10 % O2 for 24 h) to -8.11 ± 0.77 pA pF-1 (n = 18) and in cells exposed to 100 nM AβP(1-40) (-13.73 ± 2.01 pA pF-1, n = 17, P < 0.0001). When cells were exposed to either CH or AβP(1-40) in the presence of melatonin (150 µM), current densities were not augmented (CH cells, -4.77 ± 1.43 pA pF-1, n = 6; AβP(1-40) treated cells -4.25 ± 0.62 pA pF-1, n = 7). Exposure of control cells to 150 µM melatonin was without significant effect (-4.03 ± 1.26 pA pF-1, n = 7). 24 h exposure of cells to ascorbic acid (200 µM) augmented currents in control cells (-7.76 ± 1.44 pA pF-1, n = 8, P < 0.02), but prevented further augmentation by CH (-8.05 ± 2.12 pA pF-1, n = 6) or by AβP(1-40) (-7.60 ± 2.70 pA pF-1, n = 9).
Our results suggest that both CH and AβPs augment Ca2+ current density in PC12 cells via a mechanism involving increased production of reactive oxygen species. This in turn lends further support to the idea that CH and AβPs exert their effects via a common pathway.
K.N.G. holds a MRC PhD Studentship.
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