Chronic hypoxia (CH; 10 % O₂, 24 h) causes a dramatic enhancement of evoked catecholamine secretion and L-type Ca²⁺ channel activity in pheochromocytoma (PC12) cells, an effect that appears to involve increased production of Alzheimer’s amyloid β peptides (AβPs; Taylor et al. 1999; Green & Peers, 2001). AβPs are generated from amyloid precursor protein by sequential cleavage by β and γ secretases (e.g. Mattson, 1997). In order to evaluate the requirement for AβP production in mediating the enhancement of Ca²⁺ currents by CH, we investigated the effects of two novel γ secretase inhibitors. Ca²⁺ channel currents were recorded exactly as previously described (Green & Peers, 2001), and peak current density was measured at +10 mV test potential. Data are presented as means ± S.E.M. and statistical comparisons made using unpaired t tests.

Ca²⁺ current density in control (normoxically cultured) PC12 cells was -4.52 ± 1.00 pA pF^-1 (n = 8), and this was significantly enhanced (P < 0.001) to -8.89 ± 1.38 pA pF^-1 (n = 7) in CH cells, in good agreement with our previous studies (Green & Peers, 2001). When cells were cultured under the same hypoxic conditions, but in the additional presence of the γ secretase inhibitor 2-Napthyl-Val-Phe-CHO (NVP; 10 µM), current density (-5.41 ± 1.24 pA pF^-1, n = 12) was not significantly different from control levels, and significantly less (P < 0.02) than that observed in CH cells. Augmentation of Ca²⁺ current density by hypoxia was also inhibited by Boc-Gly-Val-Valinal (GVV), another γ secretase inhibitor. Ca²⁺ current density in cells cultured hypoxically for 24 h in the presence of 10 µM GVV was -4.09 ± 0.95 pA pF^-1 (n = 7; not significantly different from controls, but significantly (P = <0.01) reduced compared with CH cells).

NVP prevents production of both AβP_(1-40) and AβP_(1-42) by γ secretase (Sinha & Lieberberg, 1999), whereas GVV preferentially inhibits production of AβP_(1-40) (Murphy et al. 2000). Our findings indicate that AβP production is a necessary step in hypoxic augmentation of Ca²⁺ currents in PC12 cells, and that AβP_(1-40) may be the form of peptide required to mediate this action of hypoxia.

K.N.G. holds a MRC PhD Studentship.