A MECHANISM FOR TRABECULAR MESHWORK CELL RETRACTION - ETHACRYNIC-ACIDINITIATES THE DEPHOSPHORYLATION OF FOCAL ADHESION PROTEINS

Ethacrynic acid (ECA) increases aqueous humor outflow facility in huma n and animal model systems, and causes cellular retraction in cultured trabecular meshwork (TM) cells. ECA-induced retraction, a possible co rrelate to the opening of spaces in the outflow pathway in vivo, takes place coincident with disruption of cell-cell attachments and actin s tress fibers. Tyrosine phosphorylated proteins are located predominant ly where actin filaments terminate at sites of cell-to-cell and cell-t o-substrate adhesion, end are understood to regulate cellular adhesion s and filamentous (F) actin organization in many cell types. In the pr esent study we investigated whether ECA might affect cell adhesions an d F-actin in TM cells by altering levels of phosphotyrosine. We analys ed levels of phosphotyrosine in cultured human TM and calf pulmonary a rtery endothelial cells after exposure to ECA. Using immunoflourescenc e microscopy and antibodies to phosphotyrosinated proteins we found a rapid decrease in phosphotyrosine levels at the focal contacts of cell s treated with ECA. Immunoblots of whole cell extracts showed a decrea se in phosphotyrosine predominantly in a band running at about 120 kD, with a more subtle decrease in a band about 65 kD. Reprobing the blot with antibodies to pp120 focal adhesion kinase (FAK) or paxillin indi cated that the 120 kD band was FAK and the 65 kD band was likely paxil lin. Immunoprecipitation of FAK or paxillin and probing the resulting blot with antibodies to phosphotyrosine confirmed that these proteins were rapidly dephosphorylated after ECA addition. Loss of FAK and paxi llin proteins in cells was then confirmed using immunofluorescence mic roscopy. Dephosphorylation of these proteins was detected before the o nset of retraction, stress fiber disruption, or complete disruption of focal adhesions. A pure microtubule inhibitor (colchicine), did not c ause stress fiber disruption or decrease focal adhesion phosphorylatio n. We postulate that dephosphorylation of FAK and paxillin by ECA disr upts signaling pathways that normally maintain the stability of the ac tin cytoskeleton and cellular adhesions, and that this action leads bo th to cell shape change in culture, and to facility changes in vivo. ( C) 1997 Academic Press Limited.