Induction of actin cytoskeleton rearrangement by methyl okadaate--comparison with okadaic acid.
Methyl okadaate is a derivative of the lipophilic polyether okadaic acid (OA), a well-known inducer of apoptosis. OA inhibits Ser/Thr protein phosphatases (PPs), among them types 1 and 2A (PP1 and PP2A), whereas methyl okadaate lacks PP1/PP2A inhibitory activity in vitro. As progressive loss of neuronal cytoarchitecture is a major event that precedes neuronal death, in this work we studied comparatively the effects of both toxins on actin cytoskeleton organization in human neuroblastoma cells by filamentous actin (F-actin) labeling with the specific dye Oregon Green 514 Phalloidin. Neither methyl okadaate nor OA modified the amount of F-actin per cell. However, confocal microscopy imaging showed that methyl okadaate induced reorganization of actin cytoskeleton, loss of the typical flattened morphology and adoption of a round shape, and a reduction in the number of neurites, with a consequent loss of cell attachment. These effects were identical to those induced by OA, although methyl okadaate potency was approximately 10-fold lower. In order to investigate the role of membrane potential and cytosolic Ca2+ concentration in morphological changes induced by these toxins, the cells were stained with bis-(1,3-dibutylbarbituric acid)-trimethine oxonol and fura-2. No toxin effect was detected on membrane potential or calcium influx, indicating that these two signals are not responsible for cytoskeletal/morphological change induction. Methyl okadaate induced an increase of Ser/Thr phosphorylation of cellular proteins detected by western blot, showing similar phosphorylation profiles to OA. Our data suggest that methyl okadaate is an active compound that shares a pharmacological target with OA that may be a Ser/Thr phosphatase, probably different from PP1 and PP2A.
Vilariño
Natalia
N
Departamento de Farmacología, Facultad de Veterinaria, Universidad de Santiago de Compostela, Lugo, Spain.
Ares
Isabel R
IR
Cagide
Eva
E
Louzao
M Carmen
MC
Vieytes
Mercedes R
MR
Yasumoto
Takeshi
T
Botana
Luis M
LM
D000199:Actins / Q000378:metabolism* / Q000648:ultrastructure; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D003599:Cytoskeleton / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D003600:Cytosol / Q000378:metabolism; D004988:Ethers, Cyclic / Q000494:pharmacology*; D006801:Humans; D008564:Membrane Potentials; D019319:Okadaic Acid / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects; D054645:Protein Phosphatase 1 / Q000037:antagonists & inhibitors*; D054648:Protein Phosphatase 2 / Q000037:antagonists & inhibitors*; D012694:Serine / Q000378:metabolism; D013912:Threonine / Q000378:metabolism
D000199:Actins; D004988:Ethers, Cyclic; D019319:Okadaic Acid; D013912:Threonine; D012694:Serine; C066239:methyl okadaate; D054645:Protein Phosphatase 1; D054648:Protein Phosphatase 2; D002118:Calcium