Ethacrynic acid inhibition of microtubule assembly in vitro

Ethacrynic acid (ECA) is a sulfhydryl reactive diuretic drug. Recent studies show that ocular administration of ECA may have potential efficacy for treatment of glaucoma. ECA affects cell shape in cultured cells from the eye outflow pathway and the microtubule system is disrupted. We have studied the effect of ECA on microtubule protein (MTP) (tubulin and microtubule-associated proteins) and purified tubulin assembly. Fifty percent inhibition of MTP (1.8 mg/ml) assembly was found at 70 microM ECA in buffer and 410 microM ECA in 30% glycerol in buffer. If all sulfhydryl groups were attributed to tubulin, then approximately two sulfhydryls were blocked at 50% inhibition. Tubulin (2 mg/ml) assembly showed 50% inhibition at 175 microM ECA and approximately 2 sulfhydryl groups were lost. Increasing ECA preincubation times (0-60 min) with tubulin showed that the longer the preincubation time, the longer the lag time, and the slower the rate of assembly and that the percentage of inhibition was proportional to the ECA preincubation time. The number of blocked sulfhydryls also increased with preincubation time. Approximately two sulfhydryls were blocked at 50% inhibition of assembly. The critical concentration for assembly increased twofold when tubulin was preincubated with 0.1 mM ECA, suggesting a loss of active tubulin. Fifty percent inhibition of taxol-induced MTP and tubulin assembly occurred at 190 and 280 microM ECA, respectively, with 3.6 to 3.8 sulfhydryls blocked, respectively. Taxol protects microtubules from disassembly by ECA, suggesting that the ECA binding key sulfhydryls are blocked in the microtubule. These results suggest that ECA reacts slowly with tubulin and blocks sulfhydryl groups important for assembly. Microtubule-associated proteins and glycerol protect the sulfhydryls and so more ECA is necessary to inhibit assembly. Since the number of blocked sulfhydryls is greater at 50% inhibition for taxol-induced microtubules, sulfhydryl blocked tubulin incompetent to assemble under normal conditions may be induced to do so with taxol.