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M. Nakamura, Y. Shichijo, A. Dota, K. Shinomiya, O. Katsuta, T. Ikuse; Stimulation of Mucin–Like Glycoprotein Secretion From Isolated Rabbit Conjunctival Tissue by Gefarnate in a Manner Dependent on Protein Kinase C . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4935.
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The effect of gefarnate on the secretion of mucin–like glycoproteins from isolated rabbit conjunctival tissue and the role of signaling by protein kinase C (PKC) in such secretion were investigated.
Pieces of rabbit conjunctival tissue (4 mm in diameter) isolated from the bulbar conjunctiva were cultured in normal bicarbonated Ringer’s solution. The concentration of mucin–like glycoproteins in culture supernatants was measured by enzyme–linked lectin assay with wheat germ agglutinin (WGA). Localization of WGA–binding glycoproteins in rabbit conjunctival tissue was examined by histochemical staining with WGA, and the size of these proteins in culture supernatants was evaluated by SDS–polyacrylamide gel electrophoresis and blot analysis with WGA.
Histochemical analysis revealed that mucous in the goblet cells and the apical part of the conjunctival epithelium showed positive reactivity for staining with WGA. Blot analysis showed that most WGA–binding glycoproteins in culture supernatants of conjunctival tissue were larger than 200 kDa. Exposure of conjunctival tissue to gefarnate resulted in a concentration– and time–dependent increase in the secretion of mucin–like glycoproteins. The PKC activator, phorbol 12–myristate 13–acetate (PMA) also stimulated mucin–like glycoprotein secretion in a concentration–dependent manner. Although the PKC inhibitor, calphostin C by itself did not affect mucin–like glycoprotein secretion at concentrations up to 100 nM, it inhibited the stimulatory effects of gefarnate and PMA on such secretion in a concentration–dependent manner.
These results suggest that gefarnate stimulates the secretion of mucin–like glycoproteins from rabbit conjunctival tissue in a manner dependent on signaling by PKC.
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