Activation of ESMCs by cross-linking of FcϵRI and PGE₂. (A) Phosphorylation of p42/44 ERK assayed by FACS using a phosphospecific antibody that recognizes phosphorylation of T202 and Y204. ESMCs (left panel) or CBMCs (right panel) were activated by FcϵRI cross-linking or by PGE₂ activation (black represents unactivated; green, PGE₂; red, anti-IgE; and blue, PGE₂ and anti-IgE). (B) Quantitative analysis (n = 6) of T202/Y204 phosphorylation in ESMCs stimulated by PGE₂ (white bar), FcϵRI (hatched bar), and FcϵRI and PGE₂ together (dark bar). (C) Intracellular calcium release in ESMCs (left panel) or CBMCs (right panel) and activated by FcϵRI cross-linking 3 μg/mL anti-IgE antibody (blue) or 1μM PGE₂ (red). Point of stimulation is shown by arrow. Calcium mobilization is expressed as the ratio of fluorescence of Fura-2 measured at 340 and 380 nm. Typical results from at least 2 experiments are shown. (D) Histamine release in ESMCs (left panel) or CBMCs (right panel) preincubated with hIgE and cross-linked with various concentrations of anti-IgE antibody alone or together with 10⁻⁶M PGE₂. Histamine release is presented as a percentage of total histamine assayed by lysis of cells by Triton X-100 (n = 3). *Significant difference from 0 μg/mL anti-IgE (P < .05). (E) Release of TNF-α was evaluated in ESMCs (left panel) and CBMCs (right panel) preincubated with hIgE and cross-linked with 3 μg/mL anti-IgE antibody alone or together with 10⁻⁶M PGE₂ (n = 3). (F) PGD₂ release from ESMCs (left panel) or CBMCs (right panel) measured using PGD₂ methoxime enzyme immunoassay kit (n = 3). *Significantly different from untreated cells (P < .05). Results shown in right panels in panels C and E were obtained under identical conditions to those in the left panels but on different days.