okadaic-acid and Retinal-Degeneration

Reversible protein phosphorylation regulates the biological activities of many human proteins involved in crucial cellular processes, e.g., protein-protein interactions, cell signaling, gene transcription, cell growth, and death. A malfunction of cellular homeostasis in retinal pigment epithelial (RPE) cells is involved in the age-related retinal degeneration. In this study, we examined cytotoxicity in human RPE cells subjected to the protein phosphatase inhibitor, okadaic acid (OA). Moreover, the influence of Hsp90 inhibitor geldanamycin (GA), a benzoquinone ansamycin, in cytoprotection was assessed. Hsp70 protein levels were analyzed by Western blot. Cellular viability was determined by LDH and MTT assays. To study apoptotic cell death, caspase-3 enzyme activity was measured by assaying the cleavage of a fluorescent peptide substrate and Hoechst dye was used to visualize nuclear morphology. OA treatment caused morphological changes and induced cytotoxicity by caspase-3-independent manner in the RPE cells. No evidence of nuclear fragmentation was observed in response to OA. Interestingly, GA treatment accumulated Hsp70 protein and attenuated OA-induced cytotoxicity. This study suggests that Hsp70 and Hsp90 are closely related to cytoprotection of RPE cells in response to protein phosphatase inhibition.

Topics: Benzoquinones; Caspase 3; Caspases; Cell Death; Cell Line; Cell Nucleus; Cell Survival; Cysteine Proteinase Inhibitors; Cytoprotection; Enzyme Activation; Enzyme Inhibitors; Epithelial Cells; HSP70 Heat-Shock Proteins; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Okadaic Acid; Phosphoprotein Phosphatases; Phosphorylation; Pigment Epithelium of Eye; Quinones; Retinal Degeneration; Up-Regulation

The authors investigated intercellular communication among cultured rat retinal pigment epithelial (RPE) cells isolated from dystrophic Royal College of Surgeons (RCS) rats by studying the conduction of the [Ca2+]i wave elicited by mechanical stimulation. The effect of protein phosphorylation was measured by modulating the protein kinase C (PKC), protein kinase A (PKA), and tyrosine kinase activity.. Cultured RPE cells isolated from neonatal control Long-Evans (LE) and dystrophic RCS rats were analyzed using the fluorescent dye fluo-3 to measure the Ca2+-wave propagation on mechanical stimulation to investigate the intercellular communication.. Mechanical stimulation in LE-RPE cells resulted in a centrifugally spreading Ca2+ wave through the neighboring cells. When a mechanical stimulus was applied on RCS-RPE cells, a significantly reduced Ca2+-response was found in the neighboring cells compared with that of control RPE cells. Activation of PKC almost completed blocked the mechanically induced Ca2+ rise in the neighboring RCS-RPE cells. In contrast to LE-RPE cells, an activation of PKA also significantly decreased the Ca2+-wave propagation in RCS-RPE cells. Inhibition of PKA had no effect on the intercellular communication in LE- or RCS-RPE cells. In addition, when protein phosphatase activity or tyrosine kinase activity was inhibited, an increased Ca2+ rise in the neighboring cells on mechanical stimulation was measured, reaching levels currently found for LE-RPE cells.. In dystrophic RCS-RPE cells, a decreased intercellular Ca2+-wave propagation is found. This intercellular communication can be mediated by protein phosphorylation.

Topics: Aniline Compounds; Animals; Calcium; Calcium-Transporting ATPases; Cell Communication; Cells, Cultured; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Fluorescent Dyes; Okadaic Acid; Phosphorylation; Pigment Epithelium of Eye; Protein Kinase C; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Rats; Rats, Mutant Strains; Retinal Degeneration; Stress, Mechanical; Thapsigargin; Xanthenes