Glucocorticoid modulation of Ca2+ homeostasis in human B lymphoblasts

Abstract

1. We determined the effect of cortisol (200 nM for 48 h) on the intracellular Ca2+ concentration ([Ca2+]i) and parameters of Ca2+i signalling in 19 lymphoblastoid cell lines (LCLs). 2. Using the fluorescent dye fura-2, the basal [Ca2+]i in Ca2+-containing medium was 63.5 +/- 2.4 nM in vehicle (ethanol)-treated LCLs and 55.7 +/- 2. 6 nM (mean +/- s.e.m.) in cortisol-treated LCLs. 3. Ca2+i signalling following platelet-activating factor (PAF, 100 nM) addition was enhanced by cortisol treatment, with LCLs having small PAF responses showing the largest percentage increase after cortisol treatment. Mean peak [Ca2+]i responses to PAF were enhanced 67.0% and 55.7% in Ca2+-free and Ca2+-containing medium, respectively. 4. The endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin (100 nM) caused a transient increase in [Ca2+]i in Ca2+-free medium in which the peak change was increased in cortisol-treated cells (98.5 +/- 5.8 vs. 79.8 +/- 4.5 nM). Peak changes in the freely exchangeable Ca2+ in response to 5 microM ionomycin were also enhanced in cortisol-treated cells (923.7 +/- 113.9 vs. 652.2 +/- 64.5 nM) and correlated to the PAF-evoked [Ca2+]i response. 5. Cortisol-treated LCLs exposed to thapsigargin to empty intracellular Ca2+ stores (10 min treatment in Ca2+-free medium) and exposed to CaCl2 or MnCl2 had a greater rate of Ca2+ entry (18.6 +/- 1.8 vs. 13.8 +/- 1.5 nM s-1) and higher rate constant for Mn2+ entry (0.0345 +/- 0.0029 vs. 0. 0217 +/- 0.0020) than vehicle-treated cells. Peak [Ca2+]i in cells exposed to CaCl2 was also enhanced (869.4 +/- 114.7 vs. 562.6 +/- 61.7 nM). Parameters of divalent cation influx were highly correlated to the peak [Ca2+]i elicited by thapsigargin or ionomycin. 6. Inclusion of RU 486 (a glucocorticoid antagonist) with cortisol prevented the decrease in basal [Ca2+]i and stimulatory actions of cortisol on all Ca2+i parameters. RU 486 alone had no apparent effects on basal [Ca2+]i or Ca2+i signalling. 7. Based on data obtained over a wide range of responses (in the presence and/or absence of cortisol or RU 486), the results show that cortisol stimulation of glucocorticoid receptors decreases basal [Ca2+]i and enhances PAF-evoked [Ca2+]i signalling, most probably through its effects on intracellular Ca2+ stores. In turn, the extent of Ca2+ entry via store-operated plasma membrane Ca2+ channels is closely linked to the size of the Ca2+ stores.

Figure 4. Cortisol treatment increases Mn²⁺ entry in TG-treated cells

A, representative experiments are depicted in which fura-2-loaded cells were centrifuged and pretreated with 500 nM TG (TG-labelled traces) or vehicle (D-labelled traces, for 0.5 % DMSO) in Ca²⁺-free medium for 10 min. Cells were then centrifuged and the pellet resuspended in 3 ml Ca²⁺-free medium; traces show the normalized response of fluorescence decline immediately after addition of 0.35 mM MnCl₂ (arrows). B, data used to calculate the rate constant for TG-sensitive quenching of fura-2 in vehicle (left panel) and cortisol-treated (right panel) LCLs (n= 18) as described in Methods and legend to Table 1. Shown are means ±s.e.m. of LCLs evaluated for Mn²⁺ entry; s.e.m.s smaller than the symbol are not shown. Mn²⁺ entry in vehicle- (□, DMSO) and TG-treated cells (○); TG-sensitive Mn²⁺ influx is given by (▵), and curve-fitted lines describing the exponential decay of fluorescence are given for each condition. C, results of weighted least-squares analysis of k₁ (rate constant, see also Table 1) indicated a P value < 0.01 between vehicle- and cortisol-treated LCLs.

Figure 1. Cortisol treatment enhances PAF-evoked Cai2+ signalling

A, example of ${\text{Ca}}_{\text{i}}^{\text{2+}}$ transients in an LCL exposed to 1, 10 or 100 nM PAF in HBS. 10 × 10⁶ cells were treated with vehicle (ethanol) or cortisol (200 nM) for 48 h and processed for ${\text{Ca}}_{\text{i}}^{\text{2+}}$ measurements as described in Methods. Basal [Ca²⁺]_i was determined as the mean [Ca²⁺]_i value immediately preceding addition of agonist, and the PAF-evoked [Ca²⁺]_i response was calculated as the difference between basal [Ca²⁺]_i and peak [Ca²⁺]_i after PAF addition. For this LCL, PAF addition (arrow) to cortisol-treated cells resulted in peak [Ca²⁺]_i increases of 123 % (1 nM), 163 % (10 nM) and 158 % (100 nM) compared with vehicle-treated cells. B and C, PAF-evoked [Ca²⁺]_i responses in LCLs suspended in Ca²⁺-free and Ca²⁺-containing HBS. Left panels: percentage stimulation of PAF-evoked [Ca²⁺]_i response in cortisol-treated cells (Δ[Ca²⁺]_i(C)) to vehicle-treated cells (Δ[Ca²⁺]_i(V)) as a function of control response. Right panels: mean changes in PAF-evoked [Ca²⁺]_i in cells treated for 48 h with vehicle, cortisol (200 nM), RU 486 (200 nM), or cortisol (200 nM) and RU 486 (200 nM) (n= 19). ANOVA indicated overall P values < 0.0001 for both Ca²⁺-free and Ca²⁺-containing HBS, with significant differences between cortisol and all other treatments.

Figure 2. Cortisol treatment enhances intracellular Ca²⁺ stores in LCLs

The left panels show representative examples of the TG-evoked [Ca²⁺]_i response (A) and the ionomycin-TG-evoked rise in [Ca²⁺]_i (FEC) (B) in LCLs treated as described in the legend to Fig. 1. Arrows indicate addition of 100 nM TG (A) or 5 μm ionomycin-100 nM TG (B) to cells suspended in Ca²⁺-free medium; TG was added to prevent reuptake of Ca²⁺ and diminution of the [Ca²⁺]_i transient. The peak [Ca²⁺]_i response minus the basal [Ca²⁺]_i was used to estimate TG-releasable Ca²⁺ pools defined by the SER and FEC. The right panels show the results from 19 (A) and 17 (B) LCLs. ANOVA indicated overall P values < 0.0001 (TG-evoked [Ca²⁺]_i) and < 0.0025 (ionomycin-TG-evoked [Ca²⁺]_i), with significant differences between cortisol and all other treatment conditions.

Figure 3. Cortisol enhancement of Ca²⁺ entry and peak [Ca²⁺]_i following Ca²⁺ addition to TG-treated cells

In representative experiments (A), cells challenged with 100 nM TG in Ca²⁺-free medium for 10 min (not shown) had 0.45 mM CaCl₂ added to the cuvette (final [Ca²⁺]_o= 0.15 mM). The first 10 s of the increase in [Ca²⁺]_i were used to estimate the rate of Ca²⁺ influx (nM s⁻¹, B), and peak changes in [Ca²⁺]_i were determined as the difference between [Ca²⁺]_i immediately prior to CaCl₂ addition and the maximal [Ca²⁺]_i (C). Control experiments with cells treated with DMSO (vehicle, 0.2 %) for 10 min had low (< 0.05 nM s⁻¹) rates of Ca²⁺ influx after CaCl₂ addition, hence no correction for non-TG-mediated increases in ${\text{Ca}}_{\text{i}}^{\text{2+}}$ was performed. ANOVA indicated overall P values < 0.0001 for both analyses, with significant differences between cortisol and all other treatment conditions.

Figure 5. Correlations between PAF-evoked [Ca²⁺]_i response and intracellular Ca²⁺ store size (A) and intracellular Ca²⁺ store size and parameters of SOCE (B and C)

A, correlation of PAF-evoked [Ca²⁺]_i responses (in Ca²⁺-free HBS) and FEC; parameters were obtained as described in legends to Figs 1 and 2. B, correlation of TG-evoked [Ca²⁺]_i and peak [Ca²⁺]_i levels achieved after 10 min TG treatment and readdition of CaCl_2. TG-evoked [Ca²⁺]_i was obtained as described in legend to Fig. 2, and peak [Ca²⁺]_i values were obtained as in legend to Fig. 3. C, intracellular Ca²⁺ store size (FEC) and TG-sensitive Mn²⁺ entry. FEC was determined as in the legend to Fig. 2, and Mn²⁺ entry was obtained from TG-sensitive decreases in fluorescence at 30 s after addition of MnCl₂ (cf. Fig. 4). Correlations were obtained with data from the four treatment conditions for 17-19 LCLs (for A, n= 72; for B, n= 71; for C, n= 67).