Volume-induced chloride transport in HT29 cells studied by X-ray microanalysis

Ion transport mediated by osmotically induced chloride channels in HT29 cells was investigated by X-ray microanalysis of cell cultures. The cells were cultured on cellulose nitrate filters, and exposed to isotonic, hypotonic, or hypertonic solutions. Then the NaCl-rich medium was removed by rinsing the cells with a washing fluid, the cells were frozen, freeze-dried, and analyzed. As washing solutions, distilled water, ammonium acetate, or mannitol (the latter two at different concentrations) were used. No major effects of the composition of the washing fluid was noted, except for mannitol, which left remnants that caused an apparent decrease in the intracellular elemental concentrations. In response to exposure to hypotonic solution (modified Krebs Ringer's buffer, KRB, with 90 mM NaCl), the cells lost Cl, K, and Na. On the other hand, exposure to hypertonic solution (modified KRB, 290 mM NaCl) caused an increase of the intracellular concentrations of Na and Cl and a decrease in K. In the presence of chloride channel blockers, chloride efflux induced by exposure to hypotonic medium was inhibited. This chloride efflux was also inhibited in the absence of extracellular calcium, indicating that the process is Ca2+ dependent.