Ethacrynic acid disrupts steady state microtubules in vitro.

Published

Journal Article

PURPOSE:Ethacrynic acid (ECA) has been shown to increase facility of aqueous outflow in whole eyes and perfused anterior segments, to open up spaces between cells in the trabecular meshwork and inner wall of Schlemm's canal, and to cause separation and retraction of trabecular meshwork and endothelial cells in culture. One mechanism by which ECA has been proposed to act in cells is via disruption of microtubules, leading to cell retraction. Although it is known that ECA can inhibit de novo assembly of microtubules from tubulin subunits in vitro, we wanted to determine, as a better correlate to the proposed effect of ECA in cells, whether ECA could disrupt microtubule polymers that had reached steady state. We also wanted to determine whether calcium ion could enhance this process. METHODS:We therefore assembled purified and crude porcine brain tubulin to steady state at 37 degrees C and then added ECA and/or calcium. Reaction kinetics were followed spectrophotometrically. RESULTS:We found that ECA effectively disrupted assembled microtubules in vitro. Although 0.8-1.0 mM ECA was required to produce a half-maximal effect in pure tubulin microtubules and 0.2-0.3 mM ECA was necessary with crude microtubule protein, significant disassembly also occurred in the 0.01-0.2 mM range. Calcium had a greater maximal effect than ECA, and was more potent on a molar basis, showing half maximal effect between 2 and 12 microM free calcium ion. Combination experiments showed that ECA did not act synergistically with calcium to increase microtubule disassembly. CONCLUSIONS:Our results are consistent with the proposed disruptive action of ECA on the assembled microtubules of outflow pathway cells, but do not support a rise in intracellular calcium as being an added factor.

Full Text

Cited Authors

  • O'Brien, ET; Lee, RE; Epstein, DL

Published Date

  • September 1996

Published In

Volume / Issue

  • 15 / 9

Start / End Page

  • 985 - 990

PubMed ID

  • 8921220

Pubmed Central ID

  • 8921220

Electronic International Standard Serial Number (EISSN)

  • 1460-2202

International Standard Serial Number (ISSN)

  • 0271-3683

Digital Object Identifier (DOI)

  • 10.3109/02713689609017644

Language

  • eng