Activation of the EGF Receptor by Histamine Receptor Subtypes Stimulates Mucin Secretion in Conjunctival Goblet Cells

PURPOSE: The purpose of this study was to determine if histamine receptors interact with the epidermal growth factor receptor (EGFR) in cultured rat conjunctival goblet cells. METHODS: Goblet cells from rat conjunctiva were grown in organ culture. First-passage goblet cells were used in all experiments. Phosphorylated (active) and total EGFR, AKT, and extracellular signal-regulated kinase (ERK)1/2 were measured by Western blot analysis. Cells were preincubated with the EGFR antagonist AG1478 for 30 minutes or small interfering RNA specific to the EGFR for 3 days prior to stimulation with histamine or agonists specific for histamine receptor subtypes for 2 hours. Goblet cell secretion was measured using an enzyme-linked lectin assay. Goblet cells were incubated for 1 hour with the calcium indicator molecule fura-2/AM, and intracellular [Ca(2+)] ([Ca(2+)](i)) was determined. Data were collected in real time and presented as the actual [Ca(2+)](i) with time and as the change in peak [Ca(2+)](i). RESULTS: Histamine increased the phosphorylation of the EGFR. Mucin secretion and increase in [Ca(2+)](i) stimulated by histamine, and agonists specific for each histamine receptor subtype were blocked by inhibition of the EGFR. Increase in [Ca(2+)](i) stimulated by histamine and specific agonists for each histamine receptor was also inhibited by TAPI-1, a matrix metalloproteinase (MMP) inhibitor. The histamine-stimulated increase in activation of AKT, but not ERK1/2, was blocked by AG1478. CONCLUSIONS: In conjunctival goblet cells, histamine, using all four receptor subtypes, transactivates the EGFR via an MMP. This in turn phosphorylates AKT to increase [Ca(2+)](i) and stimulate mucin secretion.