Thapsigargin blocks electromagnetic field-elicited intracellular Ca2+ increase in HEK 293 cells

Abstract

Biological effects of electromagnetic fields (EMFs) have previously been identified for cellular proliferation and changes in expression and conduction of diverse types of ion channels. The major effect elicited by EMFs seems to be directed toward Ca²⁺ homeostasis. This is particularly remarkable since Ca²⁺ acts as a central modulator in various signaling pathways, including, but not limited to, cell differentiation and survival. Despite this, the mechanisms underlying this modulation have yet to be unraveled. Here, we assessed the effect of EMFs on intracellular [Ca²⁺ ], by exposing HEK 293 cells to both radio-frequency electromagnetic fields (RF-EMFs) and static magnetic fields (SMFs). We detected a constant and significant increase in [Ca²⁺ ] subsequent to exposure to both types of fields. Strikingly, the increase was nulled by administration of 10 μM Thapsigargin, a blocker of sarco/endoplasmic reticulum Ca²⁺ -ATPases (SERCAs), indicating the involvement of the endoplasmic reticulum (ER) in EMF-related modulation of Ca²⁺ homeostasis.

Keywords: calcium; electromagnetic fields; endoplasmic reticulum; intracellular dynamics.

FIGURE 1

Flow chart of the experimental protocol. The impact on intracellular [Ca²⁺] of oscillating (RF‐EMFs) and static (SMFs) fields was initially assessed. To elucidate the origin of the detected increase, the RF‐EMFs experiments were then repeated by using different compounds. These were: 10 μM Nifedipine as Ca²⁺ channels blocker; 10 μM Thapsigargin as blocker of Ca²⁺‐dependent ATPases (SERCAs) inhibitor; 10 μM Dantrolene as antagonist of Ryanodine receptors of (RyRs)

FIGURE 2

Experimental arrangement of RF‐EMF and SMF experiments: plates were incubated for 1 h with Fluo‐4AM Ca²⁺ dye. A first measurement was used as baseline. For RF‐EMF exposure (a), a cell phone was used as source of radiation. Both plates were then placed on a heating plate at 38°C. The cell phone's antenna was directly placed on the charged wells (red dots in the figure) for maximum radiation. Sham group was subjected to the same condition of the exposed group, but for the presence of the phone. For SMFs (b), we used a custom generator composed of two parallel Helmholtz coils powered by a DC supply. Both plates were incubated at a monitored temperature of 38°C. Sham group was subjected to the same condition of the exposed group, but the generator was turned off. In both cases, plates were exposed for 30 min and fluorescence intensity, reflecting intracellular [Ca²⁺], measured after 15 and 30 min

FIGURE 3

SMF and RF‐EMFs increase intracellular [Ca²⁺] in HEK 293 cells. Data are displayed as relative increase of exposed group when compared to sham. (*) represent inter‐conditions significance while (#) display significance when compared to sham. (a) RF‐EMFs increase basal [Ca²⁺] The difference with sham group is significant at both T₁ and T₂. (b) SMFs lead to a similar increase as already found with RF‐EMFs. However, the increase is delayed when compared to what observed with RF‐EMFs, and only significant at T₂. (c) Comparison between RF‐EMF elicited increase in standard and Ca²⁺‐free conditions. The extracellular presence of Ca²⁺ makes no difference on the detected [Ca²⁺]_i, suggesting an intracellular origin for the elicited increase. N = 45 microplate wells from 3 independent experiments. p‐value < 0.001

FIGURE 4

Nifedipine administration confirms the presence of Nifedipine‐sensitive channels on the membrane of HEK 293 cells, as their involvement in the increase in intracellular Ca²⁺ elicited by RF‐EMFs. Data in (b) are displayed as relative increase of exposed group when compared to sham. (*) represent inter‐condition significance while (#) display significance when compared to sham. (a) The intracellular [Ca²⁺] is lowered by the addition of 10 μM Nifedipine. Response to Ca²⁺ chelator EGTA was observed to be minimal. (b) Administration of 10 μM Nifedipine is sufficient to null any difference between sham and exposed group at T₂ exposure. N = 45 microplate wells from 3 independent experiments. p‐value < 0.001

FIGURE 5

Blockage of ER replenishment impairs RF‐EMFs response. Data in (b) are displayed as relative increase of exposed group when compared to sham. (a) Baseline after incubation with 10 μM Dantrolene and 10 μM Thapsigargin. The block of SERCAs operated by Thapsigargin raise intracellular Ca²⁺, impairing ER replenishment. The effect of dantrolene is not significant in line with the poor functional expression of RyRs in HEK 293 cells. (b) Administration of Thapsigargin is sufficient to completely null any difference between sham and exposed group at both T₁ and T₂ Dantrolene treated group displays no significant difference when compared to control. N = 45 microplate wells from 3 independent experiments. p‐value < 0.001