Effect of MgCl2 and GdCl3 on ORAI1 Expression and Store-Operated Ca2+ Entry in Megakaryocytes.

In chronic kidney disease, hyperphosphatemia upregulates the Ca2+ channel ORAI and its activating Ca2+ sensor STIM in megakaryocytes and platelets. ORAI1 and STIM1 accomplish store-operated Ca2+ entry (SOCE) and play a key role in platelet activation. Signaling linking phosphate to upregulation of ORAI1 and STIM1 includes transcription factor NFAT5 and serum and glucocorticoid-inducible kinase SGK1. In vascular smooth muscle cells, the effect of hyperphosphatemia on ORAI1/STIM1 expression and SOCE is suppressed by Mg2+ and the calcium-sensing receptor (CaSR) agonist Gd3+. The present study explored whether sustained exposure to Mg2+ or Gd3+ interferes with the phosphate-induced upregulation of NFAT5, SGK1, ORAI1,2,3, STIM1,2 and SOCE in megakaryocytes. To this end, human megakaryocytic Meg-01 cells were treated with 2 mM ß-glycerophosphate for 24 h in the absence and presence of either 1.5 mM MgCl2 or 50 µM GdCl3. Transcript levels were estimated utilizing q-RT-PCR, protein abundance by Western blotting, cytosolic Ca2+ concentration ([Ca2+]i) by Fura-2 fluorescence and SOCE from the increase in [Ca2+]i following re-addition of extracellular Ca2+ after store depletion with thapsigargin (1 µM). As a result, Mg2+ and Gd3+ upregulated CaSR and blunted or virtually abolished the phosphate-induced upregulation of NFAT5, SGK1, ORAI1,2,3, STIM1,2 and SOCE in megakaryocytes. In conclusion, Mg2+ and the CaSR agonist Gd3+ interfere with phosphate-induced dysregulation of [Ca2+]i in megakaryocytes.

1. Introduction

The impairment of renal phosphate excretion in chronic kidney disease (CKD) increases the plasma phosphate concentration with subsequent osteogenic reprogramming of vascular smooth muscle cells (VSMCs) [1,2,3,4] and vascular calcification [5,6,7,8]. Underlying signaling includes nuclear factor of activated T cells 5 (NFAT5) [9,10,11,12], serum and glucocorticoid-inducible kinase 1 (SGK1) [13,14] and Ca2+ channel ORAI with its activator stromal interaction molecule (STIM) [15,16,17]. Opening of ORAI by STIM upon intracellular Ca2+ depletion results in store-operated Ca2+ entry (SOCE) [15], which contributes to the orchestration of vascular calcification [16,17,18] as well as mineralization of bone [19] and enamel [20,21].

NFAT5, SGK1 and ORAI1/STIM1 similarly participate in activation of blood platelets [22,23,24,25,26,27] and thus contribute to the development of thrombosis and thrombo-occlusive events [23,26,27]. Platelets are generated by megakaryocytes, and protein abundance in platelets depends on megakaryocytic transcript and protein expression [28,29]. Megakaryocytes express ORAI1, ORAI2 and ORAI3 [30], thus accomplishing SOCE [31,32,33,34]. ORAI expression and SOCE are upregulated by the phosphate donor ß-glycerophosphate [30]. The stimulation of vascular calcification by phosphate has been shown to be reversed by Mg2+ and the calcium-sensing receptor (CaSR) agonist Gd3+ [17,35].

Expression of the calcium-sensing receptor has previously been shown in both megakaryocytes and platelets [36]. CaSR activation has been shown to counteract activation of blood platelets in hyperhomocysteinemia [37]. However, to the best of our knowledge, a functional role of CaSR in megakaryocytes and a role of CaSR in the regulation of ORAI1/STIM1 abundance and activity of megakaryocytes and platelets have never been reported before. The present paper thus explored whether Mg2+ or Gd3+ modifies NFAT5, SGK1, ORAI1,2,3 and STIM1,2 expression as well as SOCE in megakaryocytes without or with prior exposure to phosphate donor ß-glycerophosphate.

2. Results

In the first set of experiments, RT-PCR was employed to quantify transcript levels encoding the calcium-sensing receptor (CaSR), the transcription factor NFAT5, the NFAT5-regulated SGK1, the SGK1-sensitive Ca2+ release-activated ion channels ORAI1, ORAI2 and ORAI3 and the ORAI-activating Ca2+ sensor isoforms STIM1 and STIM2. According to RT-PCR (Figure S1), ORAI1 was, by far, the predominant ORAI isoform and STIM1 the prevailing STIM isoform. As shown in Figure 1, MgCl2 treatment significantly upregulated CaSR expression with or without prior exposure to ß-glycerophosphate. In agreement with earlier observations [30], a 24-h pretreatment of human megakaryocytes with the phosphate donor ß-glycerophosphate upregulated the transcript levels of NFAT5, SGK1, ORAI1, ORAI2, ORAI3, STIM1 and STIM2. All those effects were significantly blunted or virtually abrogated by additional exposure to 1.5 mM MgCl2. In the absence of ß-glycerophosphate, MgCl2 did not significantly modify the transcript levels of NFAT5, SGK1, ORAI1, ORAI2, ORAI3, STIM1 and STIM2 (Figure 1).

Figure 1

Upregulation of CaSR transcription by MgCl2 and reversal of ß-glycerophosphate-induced NFAT5, SGK1, ORAI1, ORAI2, ORAI3, STIM1 and STIM2 transcription in megakaryocytes by MgCl2. (A–H) Arithmetic means (±SEM, n = 6) of CaSR (A), NFAT5 (B), SGK1 (C), ORAI1 (D), ORAI2 (E), ORAI3 (F), STIM1 (G) and STIM2 (H) transcript levels in megakaryocytes without (CTR) and with prior 24-h exposure to 1.5 mM MgCl2 alone (MgCl2), 2 mM ß-glycerophosphate alone (Pi) or 2 mM ß-glycerophosphate and 1.5 mM MgCl2 (Pi+MgCl2). * (p < 0.05), ** (p < 0.01), *** (p < 0.001) indicate statistically significant differences compared to CTR; # (p < 0.05), ## (p < 0.01) indicate statistically significant differences compared to Pi alone (ANOVA). CaSR, calcium-sensing receptor; CTR, control.

Western blots were performed to test whether the observed alterations in ORAI1 and STIM1 transcript levels are paralleled by the respective changes in ORAI1 and STIM1 protein abundance. As illustrated in Figure 2, ß-glycerophosphate upregulated the ORAI1 and STIM1 protein abundance, an effect significantly blunted by additional exposure to 1.5 mM MgCl2. Again, in the absence of ß-glycerophosphate, MgCl2 did not significantly modify the ORAI1 and STIM1 protein abundance (Figure 2).

Figure 2

Reversal of ß-glycerophosphate-induced ORAI1 and STIM1 protein expression in megakaryocytes by MgCl2. (A) Original Western blots of ORAI1, STIM1 and GAPDH protein abundance in megakaryocytes without (CTR) and with prior 24-h exposure to 1.5 mM MgCl2 alone (MgCl2), 2 mM ß-glycerophosphate alone (Pi) or 2 mM ß-glycerophosphate and 1.5 mM MgCl2 (Pi+MgCl2). (B,C) Arithmetic means ± SEM (n = 6) of ORAI1 (B) and STIM1 (C) protein abundance in megakaryocytes without (CTR) and with prior 24-h exposure to 1.5 mM MgCl2 alone (MgCl2), 2 mM ß-glycerophosphate alone (Pi) or 2 mM ß-glycerophosphate and 1.5 mM MgCl2 (Pi+MgCl2). ** (p < 0.01), *** (p < 0.001) indicate statistically significant differences compared to CTR; # (p < 0.05), ## (p < 0.01) indicate statistically significant differences compared to Pi alone (ANOVA). CTR, control.

Cytosolic Ca2+ activity ([Ca2+]i) was estimated utilizing Fura-2 fluorescence to test whether the enhanced expression of ORAI and STIM is followed by the respective alterations in store-operated Ca2+ entry (SOCE). The 340 nm/380 nm ratio reflecting [Ca2+]i was, prior to store depletion, similar in ß-glycerophosphate-treated (1.298 ± 0.086, n = 6) and untreated (1.202 ± 0.094, n = 6) megakaryocytes. For determination of SOCE, cells were exposed to thapsigargin (1 µM), a sarco/endoplasmic reticulum Ca2+/ATPase (SERCA) inhibitor, and Ca2+-free solutions to deplete intracellular Ca2+ stores. In the following, extracellular Ca2+ was re-added in the continued presence of thapsigargin to quantify SOCE from the increase in [Ca2+]i. As illustrated in Figure 3, ß-glycerophosphate pretreatment significantly increased both the peak and the slope of SOCE, an effect significantly blunted by additional exposure to 1.5 mM MgCl2. Again, in the absence of ß-glycerophosphate, MgCl2 did not significantly modify SOCE. Neither ß-glycerophosphate nor MgCl2 significantly modified Ca2+ release.

Figure 3

Reversal of ß-glycerophosphate-induced increase in store-operated Ca2+ entry (SOCE) in megakaryocytes by MgCl2. (A) Representative tracings of Fura-2 fluorescence ratio in fluorescence spectrometry before and following extracellular Ca2+ removal and addition of thapsigargin (1 µM) as well as re-addition of extracellular Ca2+ in megakaryocytes without (CTR) or with prior 24-h exposure to 1.5 mM MgCl2 alone (MgCl2), 2 mM ß-glycerophosphate alone (Pi) or 2 mM ß-glycerophosphate and 1.5 mM MgCl2 (Pi+MgCl2). (B,C) Arithmetic means (±SEM, n = 6) of peak (B) and slope (C) increase in Fura-2 fluorescence ratio following addition of thapsigargin (1 µM) in megakaryocytes without (CTR) or with prior 24-h exposure to 1.5 mM MgCl2 alone (MgCl2), 2 mM ß-glycerophosphate alone (Pi) or 2 mM ß-glycerophosphate and 1.5 mM MgCl2 (Pi+MgCl2). (D,E) Arithmetic means (±SEM, n = 6) of peak (D) and slope (E) increase in Fura-2 fluorescence ratio following re-addition of extracellular Ca2+ in megakaryocytes without (CTR) or with prior 24-h exposure to 1.5 mM MgCl2 alone (MgCl2), 2 mM ß-glycerophosphate alone (Pi) or 2 mM ß-glycerophosphate and 1.5 mM MgCl2 (Pi+MgCl2). *** (p < 0.001) indicates statistically significant difference compared to CTR; # (p < 0.05), ## (p < 0.01) indicate statistically significant differences compared to Pi alone (ANOVA). CTR, control.

Additional experiments were performed to test whether the effects of MgCl2 were mimicked by calcium-sensing receptor agonist Gd3+. As illustrated in Figure 4, CaSR transcription was significantly upregulated by the exposure to 50 µM GdCl3 both in the absence and presence of ß-glycerophosphate. The upregulation of NFAT5, SGK1, ORAI1, ORAI2, ORAI3, STIM1 and STIM2 transcript levels by 2 mM ß-glycerophosphate was significantly blunted by additional exposure to 50 µM GdCl3. In the absence of ß-glycerophosphate, 50 µM GdCl3 did not significantly modify the transcript levels of NFAT5, SGK1, ORAI1, ORAI2, ORAI3, STIM1 and STIM2 (Figure 4).

Figure 4

Upregulation of CaSR transcription by GdCl3 and reversal of ß-glycerophosphate-induced NFAT5, SGK1, ORAI1, ORAI2, ORAI3, STIM1 and STIM2 transcription in megakaryocytes by GdCl3. (A–H) Arithmetic means (±SEM, n = 6) of CaSR (A), NFAT5 (B), SGK1 (C), ORAI1 (D), ORAI2 (E), ORAI3 (F), STIM1 (G) and STIM2 (H) transcript levels in megakaryocytes without (CTR) and with prior 24-h exposure to 50 µM GdCl3 alone (GdCl3), 2 mM ß-glycerophosphate alone (Pi) or 2 mM ß-glycerophosphate and 50 µM GdCl3 (Pi+GdCl3). * (p < 0.05), ** (p < 0.01), *** (p < 0.001) indicate statistically significant differences compared to CTR; # (p < 0.05), ## (p < 0.01) indicate statistically significant differences compared to Pi alone (ANOVA). CaSR, calcium-sensing receptor; CTR, control.

As shown in Figure 5, 50 µM GdCl3 further significantly blunted the ß-glycerophosphate-induced upregulation of ORAI1 and STIM1 protein abundance (Figure 5). Again, 50 µM GdCl3 did not significantly modify the ORAI1 and STIM1 protein abundance in the absence of ß-glycerophosphate (Figure 5).

Figure 5

Reversal of ß-glycerophosphate-induced ORAI1 and STIM1 protein expression in megakaryocytes by GdCl3. (A) Original Western blots of ORAI1, STIM1 and GAPDH protein abundance in megakaryocytes without (CTR) and with prior 24-h exposure to 50 µM GdCl3 alone (GdCl3), 2 mM ß-glycerophosphate alone (Pi) or 2 mM ß-glycerophosphate and 50 µM GdCl3 (Pi+GdCl3). (B,C) Arithmetic means ± SEM (n = 6) of ORAI1 (B) and STIM1 (C) protein abundance in megakaryocytes without (CTR) and with prior 24-h exposure to 50 µM GdCl3 alone (GdCl3), 2 mM ß-glycerophosphate alone (Pi) or 2 mM ß-glycerophosphate and 50 µM GdCl3 (Pi+GdCl3). *** (p < 0.001) indicates statistically significant difference compared to CTR; # (p < 0.05), ## (p < 0.01) indicate statistically significant differences compared to Pi alone (ANOVA). CTR, control.

As illustrated in Figure 6, 50 µM GdCl3 significantly blunted the ß-glycerophosphate-induced upregulation of the SOCE peak and slope without significantly modifying Ca2+ release. Again, in the absence of ß-glycerophosphate, 50 µM GdCl3 did not significantly modify SOCE.

Figure 6

Reversal of ß-glycerophosphate-induced increase in store-operated Ca2+ entry (SOCE) in megakaryocytes by GdCl3. (A) Representative tracings of Fura-2 fluorescence ratio in fluorescence spectrometry before and following extracellular Ca2+ removal and addition of thapsigargin (1 µM) as well as re-addition of extracellular Ca2+ in megakaryocytes without (CTR) or with prior 24-h exposure to 50 µM GdCl3 alone (GdCl3), 2 mM ß-glycerophosphate alone (Pi) or 2 mM ß-glycerophosphate and 50 µM GdCl3 (Pi+GdCl3). (B,C) Arithmetic means (±SEM, n = 6) of peak (B) and slope (C) increase in Fura-2 fluorescence ratio following addition of thapsigargin (1 µM) in megakaryocytes without (CTR) or with prior 24-h exposure to 50 µM GdCl3 alone (GdCl3), 2 mM ß-glycerophosphate alone (Pi) or 2 mM ß-glycerophosphate and 50 µM GdCl3 (Pi+GdCl3). (D,E) Arithmetic means (±SEM, n = 6) of peak (D) and slope (E) increase in Fura-2 fluorescence ratio following re-addition of extracellular Ca2+ in megakaryocytes without (CTR) or with prior 24-h exposure to 50 µM mM GdCl3 alone (GdCl3), 2 mM ß-glycerophosphate alone (Pi) or 2 mM ß-glycerophosphate and 50 µM GdCl3 (Pi+GdCl3). ** (p < 0.01), *** (p < 0.001) indicate statistically significant differences compared to CTR; # (p < 0.05), ## (p < 0.01) indicate statistically significant differences compared to Pi alone (ANOVA). CTR, control.

3. Discussion

The present study confirms previous observations [30] demonstrating the upregulation of NFAT5, ORAI1, ORAI2, ORAI3, STIM1 and STIM2 expression as well as SOCE by the phosphate donor ß-glycerophosphate in megakaryocytes.

More importantly, the present study reveals that all those effects were blunted or virtually abolished by 1.5 mM MgCl2 and by 50 µM GdCl3. MgCl2 and GdCl3 are, at least in part, effective by suppression of NFAT5 expression. NFAT5 upregulates the expression of SGK1 [13], which fosters degradation of the inhibitor protein IκBα and subsequent nuclear translocation of the transcription factor NFκB [15]. NFκB is a powerful stimulator of ORAI1 expression [15]. The present observations do not, however, exclude further signaling contributing to the upregulation of ORAI/STIM by ß-glycerophosphate and its reversal by MgCl2 or GdCl3 in megakaryocytes. It should be pointed out that the signaling shown here is triggered by sustained exposure to MgCl2 or GdCl3. Different signaling elements may prevail following acute stimulation with MgCl2 or GdCl3, such as G protein-dependent activation of phospholipase C and inositol trisphosphate formation, as shown in other cell types [38,39,40,41,42,43].

Without ß-glycerophosphate treatment, the addition of 50 µM GdCl3 did not significantly modify SOCE (Figure 6). The possibility should be considered that CaSR activation disrupts the upregulation of ORAI by ß-glycerophosphate/NFAT5/NFκB but does not modify basal ORAI expression. However, the scatter of the data does not allow exclusion of minor effects.

ORAI and STIM participate in the orchestration of platelet activation [44]. At least in theory, inhibition of ORAI and STIM expression and SOCE could contribute to the previously observed inhibition of platelet activity by CaSR activation in hyperhomocysteinemia [37]. Moreover, in view of the present observations and the known role of platelets in the pathophysiology of cardiac infarction and stroke [45], CaSR activation could counteract upregulation of platelet activity by phosphate and thus reduce the risk of cardiac infarction and stroke in CKD patients [46,47]. Clearly, additional experimental effort and clinical studies are required to define the potentially protective effect of CaSR agonists on pathological platelet activity in CKD.

In conclusion, the activation of CaSR by MgCl2 and GdCl3 reverses the upregulation of NFAT5, SGK1, ORAI1, ORAI2, ORAI3, STIM1 and STIM2 expression as well as SOCE by the phosphate donor ß-glycerophosphate in megakaryocytes (Figure 7). The effect may decrease the cardiovascular risk in hyperphosphatemic chronic kidney disease patients.

Figure 7

Mg2+ and Gd3+ interfere with phosphate-induced SOCE upregulation by the activation of CaSR in megakaryocytes—a schematic representation. In megakaryocytes, the phosphate donor ß-glycerophosphate upregulates NFAT5 and SGK1 expression, which leads to the degradation of the NFκB inhibitory protein IκB with subsequent nuclear translocation of NFκB and NFκB-dependent transcription of ORAI1 and STIM1. CaSR can be activated by cations such as Mg2+ and Gd3+ and inhibits phosphate-induced SOCE enhancement, at least partially, via the downregulation of the signaling pathway. CaSR, calcium-sensing receptor; ER, endoplasmic reticulum.

4. Materials and Methods

4.1. Cell Culture

Human megakaryocytic cells (Meg-01) from American Type Culture Collection (ATCC, Manassas, VA, USA) were cultured in RPMI 1640 (Roswell Park Memorial Institute medium, Gibco, Paisley, UK) containing 10% fetal bovine serum (FBS, Gibco, Paisley, United Kingdom) and 1% Penicillin/Streptomycin in a humidified incubator at 37ºC and 5% CO2. Where indicated, the cells were exposed to 2 mM ß-glycerophosphate (Sigma, Steinheim, Germany) for 24 h in the absence and presence of 1.5 mM MgCl2 or 50 µM GdCl3 (Sigma, Steinheim, Germany). In analysis of vascular calcification, phosphate donor ß-glycerophosphate is widely used as a substitute for phosphate and a well-established stimulator of tissue calcification [48,49,50].

4.2. Quantitative PCR

To determine transcript levels of CaSR, NFAT5, SGK1, ORAI1, ORAI2, ORAI3, STIM1 and STIM2, total RNA was extracted according to the manufacturer’s instructions with TriFast (Peqlab, Erlangen, Germany) [16,51,52,53,54]. DNAse digestion was performed to avoid DNA contamination and was followed by reverse transcription using Oligo(dT)15 primers (Promega, Mannheim, Germany) and the GoScript reverse transcription system (Promega, Mannheim, Germany). Real-time polymerase chain reaction (RT-PCR) amplification of the respective genes was set up in a total volume of 15 µL using 100 ng of cDNA, 500 nM forward and reverse primers and 2× GoTaq® qPCR Master Mix (Promega, Hilden, Germany) following the manufacturer’s protocol. Cycling conditions were as follows: initial denaturation at 95 °C for 3 min, followed by 40 cycles of 95 °C for 15 s, 60 °C for 30 s and 72 °C for 30 s. The primers used for amplification in this study are given in Table 1 (Invitrogen, Darmstadt, Germany).

Table 1

List of the primer sequences for RT-PCR.

Name	Orientation	Sequence
GAPDH	Forward	5′-TCAAGGCTGAGAACGGGAAG-3′
	Reverse	5’-TGGACTCCACGACGTACTCA-3′
CaSR	Forward	5’-ATGCCAAGAAGGGAGAAAGACTCTT-3′
	Reverse	5′-TCAGGACACTCCACACACTCAAAG-3′
NFAT5	Forward	5′-GGGTCAAACGACGAGATTGTG-3′
	Reverse	5′-GTCCGTGGTAAGCTGAGAAAG-3′
SGK1	Forward	5′-AGGAGGATGGGTCTGAACGA-3′
	Reverse	5′-GGGCCAAGGTTGATTTGCTG-3′
ORAI1	Forward	5′-CACCTGTTTGCGCTCATGAT-3′
	Reverse	5′-GGGACTCCTTGACCGAGTTG-3′
ORAI2	Forward	5′-CAGCTCCGGGAAGGAACGTC-3′
	Reverse	5′-CTCCATCCCATCTCCTTGCG-3′
ORAI3	Forward	5′-CTTCCAATCTCCCACGGTCC-3′
	Reverse	5′-GTTCCTGCTTGTAGCGGTCT-3′
STIM1	Forward	5′-AAGAAGGCATTACTGGCGCT-3′
	Reverse	5′-GATGGTGTGTCTGGGTCTGG-3′
STIM2	Forward	5′-AGGGGATTCGCCTGTAACTG-3′
	Reverse	5′-GGTTTACTGTCGTTGCCAGC-3′

Melting curves were analyzed to confirm PCR product specificity. The CFX96 Real-Time System (BioRad, Munich, Germany) was used to perform real-time PCR amplifications. All experiments were conducted in duplicate. Relative mRNA expression was calculated by the 2−ΔΔCT method using the housekeeping gene Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as internal reference, normalized to the control group.

4.3. Western Blotting

Protein abundance of ORAI1, STIM1 and GAPDH was determined by Western blotting [16,51,52,53,54]. Megakaryocyte suspensions were centrifuged for 5 min at 300× g and 4 °C. The pellet was washed with ice-cold PBS and suspended in 40 μL ice-cold RIPA lysis buffer (Cell Signaling Technology, Danvers, MA, USA) containing Protease Inhibitor Cocktail (Thermo-Fisher Scientific, Waltham, MA, USA). After centrifugation (20,000× g, 4 °C for 20 min), the supernatant was taken to determine protein concentration using the Bradford assay (BioRad, Munich, Germany). For Western blotting, 30 µg of proteins was electro-transferred onto a poly-vinylidene difluoride (PVDF) membrane after electrophoresis using 10% SDS-PAGE and blocked with 5% milk in TBST at room temperature for 1 h. The membranes were incubated with primary anti-ORAI1 antibody (1:1000, Proteintech, Chicago, IL, USA), anti-STIM1 antibody (1:1000, Cell Signaling Technology, Danvers, MA, USA) and anti-GAPDH antibody (1:2000, Cell Signaling Technology, Danvers, MA, USA) at 4 °C overnight. After washing (TBST), the blots were incubated with secondary anti-rabbit antibody conjugated with horseradish peroxidase (1:2000, Cell Signaling Technology, Danvers, MA, USA) for 2 h at room temperature. Protein bands were detected after additional washes (TBST) with an ECL detection reagent (Thermo-Fisher Scientific, Waltham, MA, USA). For densitometry image analysis, Western blots were scanned and analyzed by ImageJ software (Version 1.52, NIH, Bethesda, MD, USA). The results are shown as the ratio of total protein to GAPDH. Protein-Marker (Thermo-Fisher Scientific, Waltham, MA, USA) was used as reference to assign the right protein size.

4.4. Cytosolic Calcium Measurements

To determine the cytosolic Ca2+ concentration ([Ca2+]i), Fura-2 fluorescence was utilized [16,51,52,53,54,55]. Cells were preincubated for 30–45 min with Fura-2/AM (2 µM, Invitrogen, Goettingen, Germany) at 37 °C and excited alternatively at 340 nm and 380 nm in an inverted phase-contrast microscope (Axiovert 100, Zeiss, Oberkochen, Germany) through an objective (Fluor 40×/1.30 oil). At 505 nm, the emitted fluorescence intensity was recorded. Data (6/minute) were acquired using computer software Metafluor (Version 7.5, Universal Imaging, Downingtown, PA, USA). To estimate cytosolic Ca2+ activity, ratiometer (340 nm/380 nm)-based analysis was employed. SOCE was determined following extracellular Ca2+ removal causing store depletion and subsequent Ca2+ re-addition in constant presence of SERCA inhibitor thapsigargin (1 µM, Invitrogen, Goettingen, Germany). For quantification of Ca2+ entry, the slope (delta ratio/s) and peak (delta ratio) were determined following re-addition of Ca2+. Experiments were performed with HEPES solution containing (in mM): 125 NaCl, 5 KCl, 1.2 MgSO4, 2 Na2HPO4, 32 HEPES, 5 glucose, 1 CaCl2, pH 7.4. Ca2+-free conditions were achieved by using Ca2+-free HEPES solution containing (in mM): 125 NaCl, 5 KCl, 1.2 MgSO4, 2 Na2HPO4, 32 HEPES, 5 glucose, 0.5 EGTA, pH 7.4.

4.5. Statistical Analysis

Statistical analysis was conducted using SPSS software (Version 25.0, SPSS Inc., Chicago, IL, USA). Data are provided as means ± SEM, and n represents the number of independent experiments (i.e., in fluorescence experiments, the number of dishes measured). All data were tested for significance using Student’s t test or ANOVA. Results with p < 0.05 were considered statistically significant.