Regulation of conjunctival goblet cell secretion by Ca2+ and protein kinase C

Conjunctival goblet cells secrete mucus in response to cholinergic (muscarinic) agonists, but the underlying signaling pathways activated in this tissue are not well understood. Cholinergic agonists usually activate phospholipase C to produce inositol 1,4,5 trisphosphate and diacylglycerol. Inositol 1,4,5 trisphosphate increases the intracellular Ca2+ concentration [Ca2+](i)) while diacylglycerol activates protein kinase C (PKC). PKC and Ca2+, either by itself or with calmodulin, activate cellular functions. Goblet cell glycoprotein secretion, our index of mucin secretion, was measured from pieces of rat conjunctiva with an enzyme-linked lectin assay using the lectin Ulex europaeus agglutinin 1 (UEA-I). UEA-I selectively recognizes high molecular weight glycoproteins secreted by the goblet cells. Increasing the [Ca+](i) with the Ca2+ ionophore ionomycin stimulated glycoprotein secretion from conjunctival goblet cells. Cholinergic agonist-induced secretion was completely blocked by chelation of extracellular Ca2+ and by the Ca2+/calmodulin-dependent protein kinase inhibitors KN93 and W7 as well as their inactive analogs KN92 and W5. Activation of classical and novel PKC isozymes by phorbol 12-myristate 13-acetate anti phorbol 12,13-dibutyrate stimulated goblet cell glycoprotein secretion. When ionomycin and PMA were added simultaneously secretion was additive. PKC isozymes were identified by Western blotting analyses with antibodies specific to nine of the 11 PKC isozymes (PKC gamma and zeta were not tested). All nine PKC isozymes were identified in the conjunctival epithelium. The cellular location of the PKC isozymes was determined by immunofluorescence microscopy. Goblet cells contained the classical PKC isozymes PKC alpha, -betaI and -beta II, the novel PKC isozymes PKC epsilon, -theta, and -mu and the atypical PI(C isozyme PKC zeta. We were unable to determine if PKC activation is required for cholinergic-agonist induced secretion because the PKC inhibitors chelerythrine and staurosporine alone greatly increased secretion. We conclude that Ca2+ plays a major role in cholinergic agonist-induced conjunctival goblet cell secretion, but this agonist appears not to use Ca2+/calmodulin-dependent protein kinases. We also conclude that activated PKC can stimulate goblet cell secretion and that seven different PKC isoforms are present in the goblet cells. (C) 2000 Academic Press.