Phospholipid-Ca2+ dependent, diacylglycerol modulated protein phosphorylation (PKC), as well as the metabolism of phosphatidylinositol, phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate, will be studied in the rabbit cornea to test the hypothesis that the inositol lipid cycle and protein phosphorylation reactions are interrelated at the post- receptor activation stage in the cornea, and are involved as intracellular growth control pathways during corneal wound healing. Also, it may be that growth factors modulate corneal wound healing by controlling the inositol lipid cycle and protein phosphorylation in the cornea. Histone H1 will be used as an exogenous substrate to assay protein kinase C. Also, endogenous phosphorylation, including tyrosine phosphorylation, will be evaluated for various time periods after wounding by assessing calcium-phospholipid sensitivity and tumor promoter-phospholipid activity in both the soluble and particulate fractions. This design will enable us to follow the time course of soluble and particulate protein phosphorylation during wound healing and to correlate these events with the inositol lipid cycle. Two models will be used, a mechanical model in which the epithelium is selectively wounded, and a cryogenic model in which the three layers of the cornea are affected. In both models, protein and lipid phosphorylation will be followed during the process of cell migration and profileration in the epithelium, stroma and endothelium. The first model will allow us to study biochemical alterations in the other layers when the epithelium is wounded, and the extent to which these alterations affect the healing of the epithelium. The second model will allow us to study the healing of the stroma and endothelium as well as the epithelium. Correlation with cell movement and proliferation will be made by histology, DNA and radius of the wound measurement. Biochemical techniques such as high performance liquid chromatography, capillary gas liquid chromatography, and low- and high-voltage electrophoresis will be used. The results of this proposal will provide a better understanding of the biochemistry of inositol lipids and protein phosphorylation in the cornea and will be useful in the management of corneal wound healing- by generating information on new postreceptor pathways of cell signaling, where drugs may have the potential to modulate the breakdown of cell-to-cell communication in cornea cells after wounding.
