Presence And Location Of Protein Kinase C Isoforms In Cultured Rat Conjunctival Goblet Cells

Abstract

Abstract: : Purpose: Multiple protein kinase C (PKC) isoforms are present in stratified squamous and goblet cells of intact conjunctiva. Activation of PKC stimulates conjunctival goblet cell secretion from pieces of conjunctiva. To determine which PKC isoforms are present in these cells and their cellular location, we used rat goblet cells in culture Methods:Conjunctiva was removed from male Sprague Dawley rats and pieces were placed in culture in RPMI medium to propagate goblet cells. Cultured cells were identified as goblet cells by the presence of the lectin UEA–1. Cultured goblet cells were homogenized in buffer containing 30 mm Tris–HCL, pH–7.5, 10 mm EGTA, 5mm EDTA, 1 mm DTT and 250 mm sucrose and proteins were separated by SDS–PAGE. The presence of PKC isoforms was evaluated using antibodies to specific isoforms by Western Blot analysis. Goblet cells were also grown on cover slips and fixed in 4% formaldehyde in phosphate–buffered saline. The cellular localization of the specific PKC isoforms was determined by immunofluorescence microscopy. Results: PKC isoforms α , ß 1, δ , ε , ι , µ , and ζ were detected in goblet cells from three separate animals by western blotting. PKC isoforms ß Π, &#952, ζ, γ and η were not detected. By immunofluorescence microscopy staining PKCα , ß I and δ were visualized in cytoplasm. PKC µ , ε and ι were found in filamentous structures in the cytosol. Conclusions: We conclude that multiple isoforms of PKC are present in rat goblet cells in primary culture. These PKC isoforms could support multiple functions in goblet cells.

Keywords: conjunctiva • microscopy: light/fluorescence/immunohistochemistry