Study of cytoskeletal changes induced by okadaic acid in BE(2)-M17 cells by means of a quantitative fluorimetric microplate assay.
The diarrhogenic activity of the marine toxin okadaic acid (OA) has been associated to its actin-disrupting effect, which could reflect the loosening of tight junctions in vivo. In this report, we present results obtained using a fluorimetric microplate assay for quantitative measurements of OA-induced changes on F-actin pools in BE(2)-M17 cells. The proposed method shows important advantages over classical methods in terms of rapidity, sensitivity (less than 5000 cells per well) and reproducibility, thus providing a very useful tool for studying F-actin levels in living cells. Results obtained demonstrate a time- and dose-dependent decrease of F-actin pools (IC(50)=100 nM at 1 h) in OA-treated cells, which was partly counteracted by TPA, H89, forskolin, wortmannin, ionomycin and orthovanadate at early stages, but remained unaffected after 24 h of incubation. Cells exposed for 1 h to 1 nM OA showed a slight increase of F-actin pools (1.5-fold), which was blocked by genistein and lavendustin A, thus suggesting a role for tyrosine kinases-dependent pathways in OA-induced polymerization at low concentrations. These results suggest direct interactions of Ser/Thr protein phosphatases with actin-binding proteins in the regulation of actin polymerization, thus indicating that disruption of cytoskeletal structure may be a key mechanism of OA-induced diarrhea.
Leira
F
F
ANFACO-CECOPESCA, Campus Universitario de Vigo, 36310 Vigo, Spain. fleira@anfaco.cesga.es
Alvarez
C
C
Vieites
J M
JM
Vieytes
M R
MR
Botana
L M
LM
D000199:Actins / Q000187:drug effects / Q000378:metabolism; D000730:Androstadienes / Q000494:pharmacology; D005576:Colforsin / Q000494:pharmacology; D003592:Cytophotometry / Q000379:methods*; D003599:Cytoskeleton / Q000187:drug effects* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D019833:Genistein / Q000494:pharmacology; D006801:Humans; D015759:Ionomycin / Q000494:pharmacology; D007546:Isoquinolines / Q000494:pharmacology; D009447:Neuroblastoma; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism; D019319:Okadaic Acid / Q000494:pharmacology*; D010636:Phenols / Q000494:pharmacology; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013449:Sulfonamides; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D014638:Vanadates / Q000494:pharmacology; D000077191:Wortmannin
D000199:Actins; D000730:Androstadienes; D004791:Enzyme Inhibitors; D007546:Isoquinolines; D010636:Phenols; D013449:Sulfonamides; D005576:Colforsin; D019319:Okadaic Acid; D014638:Vanadates; C062898:lavendustin A; D015759:Ionomycin; D019833:Genistein; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide; D013755:Tetradecanoylphorbol Acetate; D000077191:Wortmannin