Periods of chronic hypoxia (CH) are well known to alter Ca²⁺-dependent mechanisms in a variety of cell types and such effects may contribute to disturbances of Ca²⁺ homeostasis associated with Alzheimer’s disease (e.g. Taylor et al. 1999; Smith et al. 2001). Here, we have investigated bradykinin-evoked changes of [Ca²⁺]_i in rat type 1 cortical astrocytes, and how these changes were modified by culturing cells in CH (24 h, 2.5 % O₂). [Ca²⁺]_i was monitored in fura-2-loaded cells as previously described (Smith et al. 2001). Transient rises of [Ca²⁺]_i above baseline were integrated and results are presented as means (± S.E.M.) ratio unit seconds (r.u.s.). Statistical significance was determined using Student’s unpaired t tests.

In Ca²⁺-free solution (replaced with 1 mM EGTA), 100 nM bradykinin evoked rises of [Ca²⁺]_i in control cells of 8.04 ± 0.92 r.u.s. (n = 14), which declined to basal levels with a t_1/2 of 43.6 ± 3.7s. Ca²⁺ transients in CH cells were significantly greater than in controls at 17.7 ± 1.5 r.u.s. (n = 13, P < 0.01) and declined with a t_1/2 of 61.7 ± 8.6 s (P < 0.04). Upon removal of external Na⁺ from the perfusate (replaced with N-methyl-D-glucamine), transient rises of Ca²⁺ in control cells (11.47 ± 0.93 r.u.s., n = 8) were significantly greater than in the presence of Na⁺ (P < 0.03). Under CH conditions, transient responses to bradykinin were not significantly altered by Na⁺ removal (23.56 ± 4.4 r.u.s., n = 8). Bath application of 10 mM FCCP/ 2.5 mg ml^-1 oligomycin evoked rises in [Ca²⁺]_i due to release from mitochondria which were significantly greater (P < 0.05) in CH-treated cells at 4.98 ± 0.82 r.u.s. (n = 8) than were observed in control cells (1.93 ± 0.62 r.u.s., n = 8). Subsequent application of 100 nM bradykinin in the continued presence of FCCP and oligomycin caused rises of [Ca²⁺]_i in control cells (19.6 ± 2.0 r.u.s., n = 13), which were not significantly different from those observed in CH cells (20.5 ± 2.4 r.u.s., n = 9).

Our results indicate that CH causes an increase in the Ca²⁺ content of mitochondria which in turn results in an apparent increase in responses to bradykinin due to a decreased ability of mitochondria to buffer Ca²⁺ released from the endoplasmic reticulum. In addition, Na⁺-Ca²⁺ exchange appears inhibited in CH cells, which may also be due to elevated mitochondrial Ca²⁺ levels (Opuni & Reeves, 2000).

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