Oxidative stress and Ca2+ signalling in calf pulmonary artery endothelial cells

University College London (2003) J Physiol 547P, PC32

Poster Communications: Oxidative stress and Ca2+ signalling in calf pulmonary artery endothelial cells

Jenny A. Wilkinson and Ron Jacob

Centre for Cardiovascular Biology & Medicine, King's College London, GKT School of Biomedical Sciences, London SE1 1UL, UK

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We measured simultaneously cytosolic [Ca2+] ([Ca2+]i) and oxidative stress (OS) in ATP-stimulated calf pulmonary artery endothelial cells (obtained from The European Collection of Cell Cultures), using fura-2 and the fluorescin-based probe C-H2DCF, respectively. [Ca2+]i is reported as the fura-2 340/380 nm fluorescence ratio and OS in arbitrary units of 495 nm fluorescence. Values quoted are means ± S.E.M. P values are for one-tailed, paired t tests or two-tailed, two-way ANOVA.

Stimulation with 100 µM ATP stimulated Ca2+ release with a peak [Ca2+]i that was followed a few seconds later by a rapid increase in OS. In the continued presence of ATP, [Ca2+]i fell to a maintained plateau, while the level of OS usually continued to rise slowly. After wash-out of ATP, 100 µM H2O2 caused a further increase in OS that was 9 ± 1 times larger than the response to ATP (n = 25 coverslips, 5 cultures). Pretreatment of cells with the anti-oxidant vitamin C (100 µM for 20-24 h) attenuated both the ATP- and H2O2-induced increases in OS (0.24 ± 0.01 to 0.12 ± 0.02, P = 0.007 and 3.0 ± 0.2 to 1.7 ± 0.2, P < 0.001, t test, n = 3 cultures, 40 coverslips) indicating that the C-DCF signal reflects OS.

Low concentrations of H2O2 can increase the IP3 sensitivity of Ca2+ stores in permeabilised cells (Hu et al. 2000). Exposing cells to 5 µM H2O2 100 s prior to, and during a 10 min exposure to 100 µM ATP significantly enhanced the [Ca2+]i peak from 3.38 ± 0.16 to 5.80 ± 0.53 (n = 5 cell cultures, P < 0.001, ANOVA). The [Ca2+]i plateau was slightly but significantly increased. Both the initial increase in OS and that after 10 min ATP exposure were also significantly enhanced from 0.39 ± 0.09 to 0.61 ± 0.15 (P < 0.001) and from 0.65 ± 0.12 to 1.02 ± 0.19, respectively (n = 5 cell cultures, P < 0.001, ANOVA).

The potentiating effect of 5 µM H2O2 on the initial [Ca2+]i peak was mimicked by raising [Ca2+]o to 6 mM for 1 h prior to, and for the first 40 s of ATP exposure. This enhanced both the initial increase in OS (0.23 ± 0.06 to 0.61 ± 0.08; P = 0.002) and the level of OS observed after 10 min exposure to ATP (0.40 ± 0.08 to 0.64 ± 0.09; P = 0.04, unpaired, 1 culture, n = 11 coverslips), suggesting that the increased OS was a consequence of increased [Ca2+]i.

If the 5 µM H2O2 was removed before application of ATP the [Ca2+]i peak was still potentiated (4.00 ± 0.33 to 6.84 ± 0.85; n = 6 cell cultures, P < 0.001, ANOVA). The effect on the OS was variable in different cell cultures, but overall was also significantly increased (P < 0.001, ANOVA).

There is potential for complex interactions between [Ca2+]i and H2O2. Such interactions may shape physiological responses and also contribute to the endothelial dysfunction associated with many cardiovascular diseases, since oxidative stress is often observed in such conditions.

This work was funded by Guy’s and St Thomas’ Charitable Foundation.



Where applicable, experiments conform with Society ethical requirements.

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