Et. Obrien et al., A MECHANISM FOR TRABECULAR MESHWORK CELL RETRACTION - ETHACRYNIC-ACIDINITIATES THE DEPHOSPHORYLATION OF FOCAL ADHESION PROTEINS, Experimental Eye Research, 65(4), 1997, pp. 471-483
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.