|Budget Amount *help
¥6,600,000 (Direct Cost : ¥6,600,000)
Fiscal Year 1994 : ¥2,000,000 (Direct Cost : ¥2,000,000)
Fiscal Year 1993 : ¥4,600,000 (Direct Cost : ¥4,600,000)
Protein kinase C (PKC) is a family of enzymes, which are calcium-and phospholipid-dependent and are activated by diacylglycerol. In the retinal pigment epithelial cell (RPE), the role of PKC is uncertain, while in other tissues it functions in cell surface signal transduction and is believed to mediate many cellular functions. The purposes of this study are (1) to examine the ability of PKC inhibitors and activators to influence the healing of wound closure in cultured human RPE (2) to examine the effect of PKC inhibitors on phospholipase C (PLC) and phospholipase D (PLD) in RPE.
(1) Cells in tissue culture are advantageous for evaluating the effects of drugs and enzyme inhibitors/activators under defined conditions. We have therefore developed a tissue culture assay for wound closure using human RPE.RPE defects, 2 mm in diameter, were produced in confluent cultures by freezing. The time course of wound closure was measured from phase-contrast photomicrographs. The changes of wound-surr
ounding cells were examined using scanning electron microscopy. In control, the defect was 70% closed by one week, while in the presence of staurosporine (100 nM) or H-7 (100 uM) was minimal healing of the defect. Healing rates was similar to control RPE when the incubation media contained HA-1004 (100 uM), apotent inhibitor of c-AMP-and c-GMP-dependent protein kinases and a weak inhibitor of PKC that is structually similar to H-7. Following this results, we are going to examine the effect of a novel PKC inhibitor, myristoylated pseudosubstrate peptide. These results suggest that PKC activity is an important factor in regulating RPE migration/proliferation. (2) Muscarine receptors are present on human RPE,but the second messenger pathways elicited by activation of these receptors are incompletely understood. We examined the ability of acetylcholine to activate PLD in human RPE in vitro. Under physiological conditions, PLD catalyses hydrolysis of the terminal diester bond of phospholipids with the resultant formation of phosphatidic acid (PA). PA can then be hydrolyzed to 1,2-diacylglycerol, an important second messenger which activates PKC.We found that muscarine receptors are coupled to PLD in RPE in this study. Then, we heve a plan to examine the effect of PKC inhibitors on PLC and PLD coupled to muscarine receptors in cultured human RPE. Less