Ginkgolide B Maintains Calcium Homeostasis in Hypoxic Hippocampal Neurons by Inhibiting Calcium Influx and Intracellular Calcium Release

Abstract

Ginkgolide B (GB), a terpene lactone and active ingredient of Ginkgo biloba, shows protective effects in neuronal cells subjected to hypoxia. We investigated whether GB might protect neurons from hypoxic injury through regulation of neuronal Ca²⁺ homeostasis. Primary hippocampal neurons subjected to chemical hypoxia (0.7 mM CoCl₂) in vitro exhibited an increase in cytoplasmic Ca²⁺ (measured from the fluorescence of fluo-4), but this effect was significantly diminished by pre-treatment with 0.4 mM GB. Electrophysiological recordings from the brain slices of rats exposed to hypoxia in vivo revealed increases in spontaneous discharge frequency, action potential frequency and calcium current magnitude, and all these effects of hypoxia were suppressed by pre-treatment with 12 mg/kg GB. Western blot analysis demonstrated that hypoxia was associated with enhanced mRNA and protein expressions of Ca_v1.2 (a voltage-gated Ca²⁺ channel), STIM1 (a regulator of store-operated Ca²⁺ entry) and RyR2 (isoforms of Ryanodine Receptor which mediates sarcoplasmic reticulum Ca²⁺ release), and these actions of hypoxia were suppressed by GB. Taken together, our in vitro and in vivo data suggest that GB might protect neurons from hypoxia, in part, by regulating Ca²⁺ influx and intracellular Ca²⁺ release to maintain Ca²⁺ homeostasis.

Keywords: cytoplasmic calcium; ginkgolide B; homeostasis; hypoxia; neuron.

Figure 1

GB increased the cellular metabolic activity of cultured neurons under hypoxic conditions. Chemical hypoxia was induced in cultured primary hippocampal neurons by treatment with 0.7 mM CoCl₂ for 12 h. 0.4 mM GB was administered for 24 h before and during exposure to CoCl₂. Data are shown as the mean ± SEM. **P < 0.01 vs. control group; ^#P < 0.05 vs. hypoxia group (Student's t-test).

Figure 2

GB decreased the [Ca²⁺]_i of cultured neurons under hypoxic conditions. (A) Representative images showing fura-4 fluorescence (green; used as a measure of [Ca²⁺]_i) in cultured primary hippocampal neurons from each of the four experimental groups. Scale bar: 10 μm. (B) Quantification of the fluorescence intensity in the control, GB, hypoxia and hypoxia+GB groups. Data are expressed as the mean ± SEM (n = 8). **P < 0.01 vs. control group; ^#P < 0.05 vs. hypoxia group (Student's t-test).

Figure 3

GB inhibited the post-synaptic excitability of hippocampal neurons exposed to hypoxia in vivo. (A) Representative traces showing spontaneous discharges reflecting spontaneous excitatory post-synaptic potentials (sEPSCs) recorded from hippocampal slices obtained from the control, GB, hypoxia and hypoxia+GB groups. (B,C) Summary results for the effects of GB on sEPSCs. Data in (C) are expressed as the mean ± SEM (n = 9). **P < 0.01 vs. control group; ^#P < 0.05 vs. hypoxia group (Student's t-test).

Figure 4

GB reduced calcium currents in hippocampal neurons exposed to hypoxia in vivo. (A) Representative traces showing action potentials recorded from hippocampal neurons during the injection of a 40-pA depolarizing current for 400 ms. (B) Summary data for the number of action potentials (APs) recorded for each injected current amplitude for neurons in the control, GB, hypoxia and hypoxia+GB groups. (C) Representative traces showing calcium currents recorded from neurons in the control, GB, hypoxia and hypoxia+GB groups. (D) Current-voltage (I-V) curves obtained for neurons in the control, GB, hypoxia and hypoxia+GB groups. Data in (C) and (D) are expressed as the mean ± SEM (n = 9). *P < 0.05, **P < 0.01 for hypoxia group vs. control group; ^#P < 0.05, ^##P < 0.01 for hypoxia+GB group vs. hypoxia group (one-way ANOVA and Bonferroni post-hoc test).

Figure 5

GB reduced the expressions of proteins involved in intracellular calcium homeostasis in neurons exposed to hypoxia. (A) Representative western blots showing the expressions of Ca_v1.2, STIM1 and RyR2 proteins in hippocampal tissue from the various groups. (B) Quantification of the western blot data. (C) qPCR analysis of the mRNA levels of Ca_v1.2, STIM1 and RyR2. Datafor (B) and (C) are expressed as the mean ± SEM (n = 3). **P < 0.01 for hypoxia group vs. control group; ^#P < 0.05, ^##P < 0.01 for hypoxia-GB group vs. hypoxia group (Student's t-test).