Male Reproductive Tract

Indazole Inhibition of Cystic Fibrosis Transmembrane Conductance Regulator Cl^- Channels in Rat Epididymal Epithelial Cells

Abstract

Previous studies have shown that two indazole compounds, lonidamine [1-(2,4-dichlorobenzyl)-indazole-3-carboxylic acid] and its analog AF2785 [(1-(2,4-dichlorobenzyl)-indazol-3-acrylic acid] suppress fertility in male rats. We also found they inhibit the cystic fibrosis transmembrane conductance regulator-chloride (CFTR-Cl^-) current in epididymal epithelial cells. To further investigate how lonidamine and AF2785 inhibit CFTR, we used a spectral analysis protocol to study whole-cell CFTR current variance. Application of lonidamine or AF2785 to the extracellular membrane of rat epididymal epithelial cells introduced a new component of the whole-cell current variance. Spectral analysis of this variance suggested a blocker on rate of 3.68 µmoles^-1s^-1, an off rate of 69.01 s^-1 for lonidamine; and a blocker on rate of 3.27 µmoles^-1s^-1, an off rate of 108 s^-1 for AF2785. Single CFTR Cl^- channel activity studied using excised inside-out membrane patches from rat epididymal epithelial cells revealed that addition of lonidamine to the intracellular solution caused a flickery block (a reduction in channel open time) at lower concentration (10 µM) without any effect on open channel probability or single-channel current amplitude. At higher concentrations (50 and 100 µM), lonidamine not only showed a flickery block but also caused a decrease in open channel probability. The flickery block by lonidamine was both voltage-dependent and concentration-dependent. These results suggest that lonidamine and AF2785 which are open channel blockers of CFTR at low concentrations also affect CFTR gating at high concentrations. It is concluded that these indazole compounds provide new pharmacological tools for the investigation of CFTR. By virtue of their interference with reproductive processes, these drugs have the potential for development into novel male contraceptives.