The Role of Hypotonicity-Induced Myo-Inositol Efflux in Volume Regulation of Cultured Lens Epithelial Cells (HLE-B3)

Abstract

Abstract: : Purpose: We investigated some properties of the hypotonicity-induced myo-inositol (MI) efflux and its possible role in volume regulation. Methods: Lens epithelial cells (HLE-B3) were cultured to confluence and then equilibrated in media containing 5 µCi of ³H-MI for 3 days. MI efflux as function of time was determined by washing the cells repeatedly in 0.5 ml aliquots of Ringer solution for 5 minute intervals to establish the resting efflux. Cells were then exposed to test solutions made hypotonic by reducing the NaCl content or exposed to isosmotic test solutions in which a fraction of NaCl was substituted with urea or NH₄Cl. The radioactivity in the aliquots was counted in a scintillation counter. Protein content was determined by the Lowry method. Results: 1) MI efflux from cultured and in vitro lens epithelial cells occurs via a passive diffusion mechanism. The rate of efflux in cultured cells was approximately 0.11 % of total MI per minute or 20 nmol g^-1min^-1. MI efflux was stimulated in a dose-dependent manner by exposing the cells to hypotonic solutions. In contrast to other volume regulatory mechanisms, MI efflux remained elevated after the volume regulation was complete. 2) The resting and the stimulated efflux could be inhibited in a dose-dependent manner by the chloride channel inhibitors NPPB, DPC, and niflumic acid. 3) The total MI efflux in response to exposure to a hypotonic solution of 245 mOsm was approximately 0.85 µmol g^-1 during the 20 minute volume regulatory period, or 0.45 µmol g^-1 above the resting efflux. 4) Lastly, we exposed lens epithelial cells to isosmotic solutions in which 50mM NaCl was substituted with isosmotic urea or NH₄Cl to produce isosmotic swelling. MI efflux was stimulated in urea substituted solutions (low ionic strength), but was not stimulated in NH₄Cl substituted solutions of constant ionic strength. Conclusions: MI efflux occurs via putative chloride channels and can be stimulated in hypotonic solutions and by lowering the ionic strength. The stimulated efflux does not contribute significantly to acute volume regulation. Its possible role is to contribute to the adjustment of ionic strength during chronic exposure to hypotonicity.

Keywords: ion transporters • signal transduction • ion channels