## 3 Results and Discussion
Thus, the suppressed extracellular release of \(\beta\)-hexosaminidaseases by L-glutamic or L-aspartic acid-containing dipeptides is based on degranulation inhibition rather than on cytotoxicity. Effect of an L-Glutamic Acid-Containing Dipeptide His-Glu on the Antigen-Induced Elevation of Cytosolic Calcium Ion Concentration in RBL-2H3 Cells  
Next, we attempted to elucidate the mechanism by which acidic amino acid residue-containing dipeptides suppress degranulation. Antigen-induced degranulation firstly causes an elevation in cytosolic calcium ion concentration accompanied by a calcium ion influx from outside of the cell [17]. We thus investigated the effect of acidic amino acid residue-containing dipeptides on the elevation of cytosolic calcium ion concentration during degranulation. Two millimolar His-Glu was used in the following experiments as a representative of acidic amino acid residue-containing dipeptides, at which concentration there is no cytotoxicity. As can be seen in Figure 1B, the relative fluorescence intensity representing cytosolic calcium ion concentration rapidly increased upon stimulation with an antigen. Additionally, in cells exposed to His-Glu, cytosolic calcium ion concentration quickly increased, as well as in the control cells. The calcium ion influx tended to decrease when treating the cells with His-Glu; however, no statistically significant difference was observed between the His-Glu-treated and control cells. Since previous studies have shown that some dipeptides possess a chelating property [18; 19], the inhibitory effect of acidic amino acid residue-containing dipeptides on the degranulation of RBL-2H3 cells was suspected due to the chelating effect, which blocks the influx of calcium ion into cells. However, it was suggested that the degranulation-inhibitory effect of acidic amino acid residue-containing dipeptides is not mainly caused by the chelating effect, because the  
Figure 1: Effect of an acidic amino acid residue-containing dipeptide on antigen-induced degranulation. Anti-DNP IgE-sensitized RBL-2H3 cells were treated with His-Glu and subsequently stimulated with antigen to induce degranulation. (**A**) Relative degranulation was calculated based on the enzymatic activity of \(\beta\)-hexosaminidase, used as a marker of degranulation. (**B**) The elevation in cytosolic calcium ion concentration in RBL-2H3 cells was monitored using Fluo-3. The relative fluorescence intensity of Fluo-3 reflects the cytosolic calcium ion concentration. Closed circle, RBL-2H3 cells untreated with His-Glu and stimulated with antigen; gray circle, the cells treated with 2.0 mM His-Glu and stimulated with antigen; open circle, the cells not treated with His-Glu and not stimulated with antigen. Data are shown as mean \(\pm\) SEM (\(n=3\)). ***** \(p\) < 0.0001 against the control by Dunnett’s test. increase in cytosolic calcium ion concentration accompanied by an influx from outside the cell tended to be downregulated, but not statistically significant, in cells treated with His-Glu. Thus, we consider that the degranulation-inhibitory effect of His-Glu was not due to inhibition of the increase in cytosolic calcium ion concentration, but to another mechanism, although a weak chelating effect of His-Glu may have occurred.