Numerous studies have indicated that acute hypoxia modulates plasmalemmal ion channel activity, thereby regulating Ca²⁺ entry in a variety of cell types (Lopez-Barneo et al. 2001). Hypoxic modulation of intracellular Ca²⁺ stores may also be involved in the overall cellular response to hypoxia in some (Dipp et al. 2001) but not all (Vicario et al. 2000) tissue types. Here, we report the effects of acute hypoxia (PO₂ ²Dgr³{special} 25 mmHg) on Ca²⁺ stores in rat type I cortical astrocytes isolated from animals that had been humanely killed.

[Ca²⁺]_i was monitored in Fura-2 loaded cells (Smith et al. 2001). Transient rises of [Ca²⁺]_i above baseline were integrated and results are presented as mean (± S.E.M.) ratio unit seconds (r.u.s.). Statistical significance was determined using Student’s unpaired t tests. All experiments were conducted in a Ca²⁺-free perfusate containing 1 mM EGTA (Smith et al. 2001).

Bath application of 100 nM bradykinin (BK) evoked transient rises of [Ca²⁺]_i (mean 9.84 ± 0.66 r.u.s., n = 35 cells). Exposure of cells to acute hypoxia evoked smaller rises, of 2.7 ± 0.39 r.u.s. (n = 20 cells, P < 0.01), and following such challenges, subsequent exposure to 100 nM BK evoked significantly smaller responses (mean 4.7 ± 0.5 r.u.s., n = 20 cells, P < 0.01) than were seen in the absence of hypoxia. Hypoxia failed to elicit rises of [Ca²⁺]_i following exposure to BK (n = 12). Pretreatment of cells for 20 min with 1 mM thapsigargin prevented any rises of [Ca²⁺]_i when cells were exposed either to hypoxia (n = 12) or 100 nM BK (n = 12). When cells were not exposed to either hypoxia or BK, but Ca²⁺ (2.5 mM) was readmitted to the perfusate in place of EGTA, no rises of [Ca²⁺]_i were observed (n = 12). However, when Ca²⁺ was readmitted following BK application, a rise of [Ca²⁺]_i was consistently observed (mean plateau level 0.15 ± 0.01 r.u., n = 13). Similarly, following exposure to hypoxia, readmission of Ca²⁺ to the perfusate evoked rises of [Ca²⁺]_i (mean plateau level 0.12 ± 0.02 r.u., n = 11).

Our results indicate that acute hypoxia can evoke Ca²⁺ release from intracellular store(s) in type I cortical astrocytes. This store can also be liberated by BK, and is depleted by prior treatment with thapsigargin. Furthermore, hypoxic mobilization of Ca²⁺ from intracellular stores appears sufficient to trigger capacitative Ca²⁺ entry. These data imply a possible role for altered calcium signalling in the pathology of central ischaemia.

This work was supported by The Wellcome Trust, The Medical Research Council and Pfizer Central Research.