BACKGROUND: Muscarinic cholinoceptors are distributed widely in both the central and peripheral nervous system. The presence of muscarinic cholinoceptors in corneal tissue is well established. Previous reports have shown that corneal muscarinic cholinoceptors are of the m2 or m4 subtype. However, recent studies have indicated the presence of the m5 muscarinic cholinoceptor subtype in human corneal epithelium and endothelium. The aim of the study was to confirm the presence of the m5 cholinoceptor subtype in bovine corneal epithelium and endothelium and the activation of phosphatidyl inositol pathway by its stimulation. METHODS: Muscarinic m5 cholinoceptor sites, phosphatidyl inositol 4,5-biphosphate, inositol 1,4,5-triphosphate and protein kinase C, were studied using immunocytochemistry and immunofluorescence. Activation of protein kinase C after stimulation of the m5 muscarinic cholinoceptor subtype was measured using the HTS protein kinase C assay kit. RESULTS: Immunocytochemistry/immunofluorescence revealed the presence of the m5 muscarinic cholinoceptor subtype, phosphatidyl inositol 4,5-biphosphate and protein kinase C in bovine corneal epithelial and endothelial cells. In bovine corneal epithelium and endothelium, protein kinase C activity was stimulated by acetylcholine in a dose-dependent manner (P<0.0001). CONCLUSIONS: Our findings indicate that acetylcholine-induced stimulation of muscarinic m5 cholinoceptors activates the phosphatidyl inositol pathway in corneal epithelial and endothelial cells, resulting in increased protein kinase C activity. Further work will be needed to clear the physiologic role of this signaling pathway in corneal epithelium and endothelium.
BACKGROUND: Muscarinic cholinoceptors are distributed widely in both the central and peripheral nervous system. The presence of muscarinic cholinoceptors in corneal tissue is well established. Previous reports have shown that corneal muscarinic cholinoceptors are of the m2 or m4 subtype. However, recent studies have indicated the presence of the m5 muscarinic cholinoceptor subtype in human corneal epithelium and endothelium. The aim of the study was to confirm the presence of the m5 cholinoceptor subtype in bovine corneal epithelium and endothelium and the activation of phosphatidyl inositol pathway by its stimulation. METHODS: Muscarinic m5 cholinoceptor sites, phosphatidyl inositol 4,5-biphosphate, inositol 1,4,5-triphosphate and protein kinase C, were studied using immunocytochemistry and immunofluorescence. Activation of protein kinase C after stimulation of the m5 muscarinic cholinoceptor subtype was measured using the HTS protein kinase C assay kit. RESULTS: Immunocytochemistry/immunofluorescence revealed the presence of the m5 muscarinic cholinoceptor subtype, phosphatidyl inositol 4,5-biphosphate and protein kinase C in bovine corneal epithelial and endothelial cells. In bovine corneal epithelium and endothelium, protein kinase C activity was stimulated by acetylcholine in a dose-dependent manner (P<0.0001). CONCLUSIONS: Our findings indicate that acetylcholine-induced stimulation of muscarinic m5 cholinoceptors activates the phosphatidyl inositol pathway in corneal epithelial and endothelial cells, resulting in increased protein kinase C activity. Further work will be needed to clear the physiologic role of this signaling pathway in corneal epithelium and endothelium.