It is well established that signal transduction in several tissues involves phosphatidylinositol 4,5-biphosphate (PIP2) hydrolysis by phospholipase C (PLC) to generate myo-inositol trisphosphate (IP3)and diacylglycerol (DAG). While IP3 mobilizes Ca2+ from ER, DAG activates protein kinase C (PKC). Recent studies have demonstrated that phospholipase D (PLD), which hydrolyzes phosphatidylcholine (PC) into phosphatidic acid (PA), may also play an important role in the signal transduction cascade. The PA, thus produced, has been implicated in several cellular processes including cell proliferation. We have previously demonstrated that alpha1-adrenergic- and serotonergic-stimulation of the corneal epithelium results in PLC-mediated increased hydrolysis of PIP2 into IP3 and DAG. More recently, we have localized PLD activity, which is stimulated by elevated intracellular Ca2+ and PKC, in the corneal epithelium. We have little understanding of the process of signal transduction involving phosphoinositides in the corneal epithelium. At present, it is not clear which enzymes are involved, their mechanism of activation, or the specific functions of the putative second messengers such as IP3, DAG and PA in this tissue. EGF promotes reepithelization of the injured cornea, although the exact mechanism(s) underlying EGF effect remains unknown. Therefore, the overall objective of the present work is to investigate the role of phosphoinositides- and PC-derived second messengers in corneal function, in particular epithelial wound healing. There are five specific aims of the proposed research plan: (1) We will investigate the effects of EGF and other growth factors on PLC- and PLD-mediated hydrolysis of phosphoinositides and PC in bovine corneal epithelial cells, in vitro. (2) We will investigate the effects of EGF on PLC- and PLD-mediated phospholipid metabolism in alkali-induced corneal lesions undergoing healing process in rabbit. (3) We will determine subcellular distribution and substrate specificity of PLD in the corneal epithelium, and investigate the requirement for GTP-binding protein in PLD activation. (4) We will purify PLC from the soluble and microsomal fractions of the bovine corneal epithelium, and determine the isozymic nature of PLC-S by immunochemical and Western blotting procedures. The catalytic properties of PLC following phosphorylation with PKC and cAMP-- dependent protein kinase will also be investigated. (5) We will examine the effects of polyamines on properties of DAG-, PI-, PIP-kinase and PLC in bovine corneal epithelium. It is hoped that these studies will provide us with a greater insight into the role of phosphoinositide- and PC-derived second messengers in corneal epithelial functions. We believe that this information could be of considerable importance in clinical treatment(s) designed towards facilitating reepithelization process in the injured cornea.
| Akhtar, R A; Abdel-Latif, A A (1990) Specific binding of [3H]inositol 1,4,5-trisphosphate to bovine iris sphincter microsomal membranes. Curr Eye Res 9:387-92 |
