Glucocorticoid modulation of Ca2+ homeostasis in human B lymphoblasts

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.