The long-term objective of the study is to determine the role of the cytoskeleton in retinal pigment epithelial (RPE) cell migration. By defining changes in the cytoskeletal configuration which occur during cell migration, the pathophysiologic mechanisms of proliferative vitreoretinopathy can be better understood, leading to more effective treatments of this condition.
The specific aim of this study is to define the effect of mediators of intracellular cyclic-3, '5'-AMP (cAMP) on the frequency and distribution of the contractile protein F-actin and the membrane-associated protein vinculin with respect to microfilament bundles which have been implicated in cell migration and chemotaxis. Mediators of intracellular cAMP have been demonstrated to inhibit migration and chemotaxis in human RPE cells, and have also been shown to promote formation and inhibit contraction of stress fibers (microfilament bundles consisting primarily of F-actin) in fibroblasts. Thus, these mediators which are both inhibitory of RPE migration and affect F-actin arrangements in fibroblasts are ideal agents to determine cytoskeletal changes associated with inhibition of cell migration of human RPE. Following adherence and spreading of human RPE cells in vitro, incubation with test reagents (dibutyryl-cAMP, forskolin, L- epinephrine, L-norepinephrine, L-isoproterenol, and caffeine) will be performed, followed by immunofluorescent labeling of vinculin using mouse monoclonal anti-chicken vinculin antibody and fluorescein-conjugated goat anti-mouse IgG antibody. Double labeling with the F-actin fluorescent probe, rhodamine-phalloidin, will then be performed. The frequency and distribution of actin and vinculin and their relationship to microfilament bundles will be documented by fluorescence microscopy and compared with controls. In the event that adrenergic agents induce cytoskeletal changes, pharmacologic blockade (timolol) will be used to determine the role of beta adrenergic receptors in the mediation of such changes.
