Figure 6.

1-Heptanol inhibits mast cell degranulation and calcium mobilization in activated cells. (A) IgE-sensitized BMMCs pretreated for 15 min with vehicle (PBS) alone (Control) or 2.5 mM 1-heptanol were non-activated (time 0 min; n = 8) or activated for 15 min with antigen (TNP-BSA; 0.5 µg/mL; n = 8). The level of β-glucuronidase released into the supernatant was determined using 4-methylumbelliferyl-β-D-glucuronide hydrate as a substrate. (B) In the experiment with reversibility of 1-heptanol action, the cells were treated as in A, but 1-heptanol was washed out before antigen activation. (C) Intracellular calcium mobilization in IgE-sensitized BMMCs in the presence of extracellular calcium and vehicle (Control) or 1-heptanol. BMMCs were loaded for 15 min in the presence of 1.8 mM calcium with Fura 2-AM alone (Control; black line; n = 5) or with 2.5 mM 1-heptanol (red line; n = 5). (D) Alternatively, the cells were loaded for 15 min with Fura-2-AM in the Ca²⁺-free BSS-BSA in the absence (Control–Ca²⁺; black line; n = 5) or presence of 2.5 mM 1-heptanol (red line; n = 5). The cells were activated with antigen (Ag, the arrow, TNP-BSA; 0.5 µg/mL) followed by the addition of calcium (final concentration 1.8 mM) at the indicated time interval (blue arrow Ca²⁺). (E) Intracellular calcium mobilization was analyzed as in C, except that thapsigargin (Th) at a final concentration of 1 µM was used for triggering (n = 4). The extent of calcium mobilization in (C,D) was monitored by measuring the fluorescence ratios. The bars in (A,B) indicate statistically significant intergroup differences. The bars above the curves in (C,D) indicate statistically significant differences between controls and 1-heptanol-treated groups. Values indicate means ± SEM, calculated from n, which show numbers of biological replicates; * p < 0.05, range of *** p < 0.001 **** p < 0.0001.