We have previously shown that chronic hypoxia (CH) potentiates Ca²⁺ influx in catecholamine-secreting cells via a mechanism that involves increased production of amyloid β peptides (AβPs), and can be mimicked by AβP application (Taylor et al. 1999). Recent studies have indicated that presenilins – probably enzymes central to formation of AβPs – modulate Ca²⁺ mobilization from intracellular stores and subsequent capacitative Ca²⁺ entry (CCE; Leissring et al. 2000; Yoo et al. 2000). For this reason, we have investigated muscarine-evoked changes of [Ca²⁺]_i in human neuroblastoma SH-SY5Y cells. [Ca²⁺]_i was monitored in fura-2-loaded cells under conditions previously described (McDonald et al. 1997). Results are presented as mean (± S.E.M.) changes in ratio units (r.u.) above baseline levels. Statistical significance was determined using Student’s unpaired t tests.

Bath application of muscarine (100 µM) caused a peak rise of [Ca²⁺]_i of 0.38 ± 0.03 r.u. (n = 14 recordings), which declined towards a plateau level of 0.15 ± 0.03 r.u. after 180 s. The peak rise of [Ca²⁺]_i was significantly greater (P < 0.01) in cells exposed to CH (2.5 % O₂, 24 h), being 0.50 ± 0.02 r.u. (n = 14), and was also significantly greater (P < 0.01) in cells exposed to 1 µM AβP_(1-40) for 24 h (0.52 ± 0.02 r.u., n = 14). In Ca²⁺-free solution (replaced with 1 mM EGTA), 100 µM muscarine evoked transient rises of [Ca²⁺]_i in control cells of 0.34 ± 0.03 r.u. (n = 17), which declined to basal levels with a t _1/2 of 25.3 ± 1.7 s. Peak rises of [Ca²⁺]_i in CH and AβP-treated cells were significantly greater than controls at 0.52 ± 0.03 r.u. (n = 18, P < 0.01) and 0.56 ± 0.03 r.u. (n = 18, P < 0.01), respectively. The rates of decline of [Ca²⁺]_i were similar in AβP-treated cells (t _1/2 26.4 ± 1.2 s), but significantly slowed (P < 0.01) in CH cells (t _1/2 42.8 ± 4.4 s). Following exposure of cells to Ca²⁺-free solution containing 1 µM thapsigargin (to deplete intracellular Ca²⁺ stores), exposure of cells to 2.5 mM Ca²⁺ induced rises of [Ca²⁺]_i of 0.22 ± 0.01 r.u. in control cells (n = 8), and a similar rise (0.25 ± 0.02 r.u.) in AβP-treated cells (n = 9). In CH cells, however, the rise of [Ca²⁺]_i was significantly (P < 0.01) smaller at 0.16 ± 0.01 r.u (n = 9).

Our results indicate that CH modulates Ca²⁺ stores and CCE in a manner that is distinct from exposure of cells to amyloid peptides.

This work was supported by Pfizer Central Research.

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Taylor, S.C., Batten, T.F. & Peers, C. (1999). J. Biol. Chem. 274, 31217-31222.

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