Ca2+ Mobilization Induced by Gliadin Figure 2. Effect of Ca2+-free and THP on [Ca2+]i increase induced by gliadin peptides in Caco-2 cells. (A) and (B) Superimposed single- cell traces representative for the effect of 20 mg/ml p31–43 and p57–68, respectively, in a Ca2+-free buffer, or in a Ca2+-free buffer plus 1 mM THP, on [Ca2+]i. Before peptide perfusion (black arrows), cells were preincubated with THP for 10 min (grey arrows), to deplete ER. (C) Quantification of the effect of the treatments reported in (A) and (B) on [Ca2+]i. Each bar represents the mean 6SEM of data obtained in three independent experimental sessions. *p,0.05 versus its respective control; **p,0.05 versus peptide alone. doi:10.1371/journal.pone.0045209.g002 were revealed using a chemiluminescence detection kit (Euroclone) according to the manufacturer’s instructions. Statistics Data regarding Ca2+ measurements were expressed as mean 6 SEM; statistical comparisons were performed using the one-way ANOVA, followed by Newman Keul’s test. Data regarding tTG transamidationg activity and tTG, GRP78, and CHOP expression were expressed as means 6 SD; statistical analysis was performed by using the Student’s t test. In all experiments, differences were considered to be statistically significant at p,0.05. Results Gliadin Peptides 31–43 and 57–68 Increase Intracellular Ca2+ Concentrations in Caco-2 Cells Using single-cell Fura-2 acetoxymethyl ester microfluorimetry, we first investigated whether p31–43 could alter the intracellular Ca2+ concentration ([Ca2+]i) of Caco-2 cells. At 20 mg/ml, p31–43 caused a rapid rise in [Ca2+]i when perfused in normal Krebs solution (Figure 1A). Similarly, also p57–68 (20 mg/ml) caused a rapid increase in [Ca2+]i when perfused in normal Krebs solution (Figure 1B). The effect of both peptides on [Ca2+]i was dose-dependent. However, p31–43 caused a higher [Ca2+]i increase than p57–68 (about 65% and 45%, respectively, with respect to the basal value) when tested at 20 mg/ml. At this concentration, pCTR did not affect [Ca2+]i. Interestingly, also at a low concentration, i.e. 0.2 mg/ml, p31–43 induced an increase in [Ca2+]i (45% of basal value), whereas at the same concentration, p57–68 was almost ineffective (Figure 1C and D). Consequently, the EC50 for p31–43 and p57–68 was 0.2 mg/ml and 0.9 mg/ml, respectively. We also evaluated whether p31–43 and p57–68 exert the same effects on [Ca2+]i when perfused in a Ca2+-free solution. The data reported in Figure 2 show that extracellular Ca2+ ions flowing through the plasma membrane did not contribute to the effect of the peptides on [Ca2+]i. This suggests that the peptide-induced [Ca2+]i increase was due to the release of Ca2+ from intracellular stores. Gliadin Peptides 31–43 and 57–68 mobilize Ca2+ from Different Intracellular Ca2+ Stores To identify the intracellular Ca2+ stores involved in the mechanism of Ca2+ release induced by p31–43 and p57–68, we first used THP to determine whether the ER was involved in the effect of the peptides. Treatment of cells with 1 mM THP significantly reduced the [Ca2+]i increase caused by p31–43 (20 mg/ml), but not that caused by p57–68 (20 mg/ml) (Figure 2A, B and C). This finding indicated that the ER was involved in the effect exerted by p31–43, but not by p57–68, on intracellular Ca2+ PLOS ONE | www.plosone.org 4 September 2012 | Volume 7 | Issue 9 | e45209