Glucocorticoids reduce intracellular calcium concentration and protects neurons against glutamate toxicity

Abstract

Glucocorticoids are steroid hormones which act through the glucocorticoid receptor. They regulate a wide variety of biological processes. Two glucocorticoids, the naturally occurring corticosterone and chemically produced dexamethasone, have been used to investigate the effect of glucocorticoids on Ca(2+)-signalling in cortical co-cultures of neurons and astrocytes. Dexamethasone and to a lesser degree corticosterone both induced a decrease in cytosolic Ca(2+) concentration in neurons and astrocytes. The effect of both compounds can be blocked by inhibition of the plasmamembrane ATPase, calmodulin and by application of a glucocorticoid receptor antagonist, while inhibition of NMDA receptors or the endoplasmic reticulum calcium pump had no effect. Glucocorticoid treatment further protects against detrimental calcium signalling and cell death by modulating the delayed calcium deregulation in response to glutamate toxicity. At the concentrations used dexamethasone and corticosterone did not show cell toxicity of their own. Thus, these results indicate that dexamethasone and corticosterone might be used for protection of the cells from calcium overload.

Fig. 1

The effect of glucocorticoids on [Ca²⁺]_c in co-cultured neurons and astrocytes. (A and B) Representative recording of fura-2 ratio from both cell types following exposure to DEX (1 μM; A and B) and corticosterone (1 μM; D). (C) DEX (1 μM) also reducing effect of [Ca²⁺]_c in fluo-4 loaded neurons. (E) Percentage of the cells in the experiment with calcium response to glucocorticoids.

Fig. 2

The effect of glucocorticoid receptor antagonist on glucocorticoid-induced Ca²⁺ decrease. Application of glucocorticoid receptor antagonist, inhibited the effect of DEX (A and B) and corticosterone-reduced (C and D) Ca²⁺ signal in primary co-culture neurons and astrocytes.

Fig. 3

Glutamate receptor or mitochondrial calcium uptake are not involved in the effect of DEX or CORT on [Ca²⁺]_c in neurons and astrocytes. Incubation cells with MK801 did not change the effect of DEX on [Ca²⁺]_c (A). (B) Changes in Δψ_m were measured using Rh123 in dequench mode (the loss of potential is seen as an increase in fluorescence). Corticosterone (1 μM) or DEX (1 μM) induced mild depolarisation of Δψ_m in both cell types. The Rh123 fluorescence signal in these traces is normalised between zero, representing the resting Rh123 fluorescence and 100, representing the maximal increase Rh123 fluorescence in response to complete mitochondrial depolarisation by 1 μM FCCP.

Fig. 4

Effect of Ca²⁺ pumps and calmodulin on the action of DEX and CORT. Depletion of the intracellular calcium pool with an inhibitor of the ER Ca²⁺ pump, thapsigargin (1 μM) (A) did not block the effect of DEX-reduced Ca²⁺ response in the cell (grey trace). Black trace represents the effect of 1 μM of thapsigargin without DEX. (B–D) Inhibitor of calcium ATPase – plasmalemmal Ca²⁺-ATPase – erythrosine B (10 μM) completely blocked the effect of DEX or corticosterone on the level of cytosolic calcium. Application of the inhibitor of calmodulin – calmidazolium (3 μM) blocked the effect of DEX (E) and CORT (F) on cytosolic calcium concentration of neurons and astrocytes. (G) Percentage of the neurons with response to DEX and CORT in the presence of inhibitors. **P < 0.001.

Fig. 5

The effect of glucocorticoids on glutamate-induced calcium signal in co-cultured neurons and astrocytes. (A) 5 μM glutamate induced typical [Ca²⁺]_c rise cortical neurons. Application of DEX (1 μM, B) or corticosterone (1 μM, C) significantly decreased the amplitude of calcium rise in response to 5 μM glutamate. (D) Differences in the amplitude (fura-2 ratio) of glutamate (5 μM)-induced calcium signal in the presence of DEX (1 μM) and corticosterone (1 μM) (*P < 0.05 and **P < 0.01).

Fig. 6

The effect of glucocorticoids on glutamate-induced calcium signal and mitochondrial membrane potential in co-cultured neurons and astrocytes. Simultaneous measurements of changes in [Ca²⁺]_c (fura-ff ratio) and Δψ_m (relative Rh123 fluorescence) were made from single neurons. Glutamate (100 μM) and glycine (10 μM) were applied in a Mg²⁺-free solution. An increase in Rh123 fluorescence reflects mitochondrial depolarisation. (A) The calcium response to glutamate shows initial phase followed by calcium deregulation and loss of mitochondrial membrane potential. (B and C) The differences in the dynamics of glutamate-induced changes in [Ca²⁺]_c and Δψ_m in the presence of 1 μM DEX or 1 μM corticosterone. The mitochondrial uncoupler FCCP (1 μM) was applied at the end of this and subsequent experiments to indicate the level of Rh123 fluorescence that reflected complete dissipation of Δψ_m. (D) Time (min) from application of glutamate and appearance of DCD; (E) differences in the amplitude (fura-ff ratio) of initial peak of glutamate (100 μM) induced calcium signal; (F) both glucocorticoids decrease the average of glutamate-induced Rh123 signal in neurons (*P < 0.05 and **P < 0.001).

Fig. 7

Glucocorticoids protects neurons against glutamate-induced cell death. The viability of neurons from rat cortex was measured 24 after 10 min exposure to 100 μM glutamate using PI fluorescence. Dead cells were counted with respect to the total number of cells present, identified by staining nuclei with Hoechst 33342. 1 μM DEX and 1 μM corticosterone were significantly protective, but caused significant toxicity when applied without glutamate. ***P < 0.0001 compared with control,^###P < 0.001 compared to glutamate. All data are expressed as mean ± S.E.M., n = 4.