Mechanisms Transducing Cell Shrinkage in Xenopus Oocytes † 176

Hypertonicity shrinks cells, which restore their volume by osmolyte uptake. The transduction mechanism leading to osmolyte uptake is not known. Our aim was to determine whether cells transduce H₂O loss via distortion of the membrane/cytoskeleton or via increasing concentrations of intracellular ions or macromolecules. We previously showed, in Xenopus oocytes, that hypertonicity induces Ca²⁺-dependent Cl^- currents (I_hi). Both the rate and extent of I_hi are increased by expressing mRNA for the water channel protein aquaporin-1 to speed shrinkage. In order to correlate I_hi activation and cell volume changes, we simultaneously measured I_hi using the 2-electrode voltage clamp, and cell diameter using a video caliper in AQP1-expressing oocytes under three conditions: hypertonic shrinkage, hypertonic exposure at constant cell volume, and isotonic withdrawal of cytoplasm. Cell volume was calculated from cell diameter assuming a spherical shape. Volume was manipulated directly by injecting H₂O or withdrawing cytoplasm via a computerized microsyringe connected to a third intracellular micropipette. Increasing bath osmolality (200 to 400 mosm/l) shrunk cells from 2.0 to 1.8 μl and activated I_hi (-55±12 nA at -65 mV). Moreover, hypertonicity activated I_hi before volume changed significantly. Indeed, I_hi activated even when volume was held constant (±0.3%) using feedback controlled injection of H₂O. During this “volume clamp”, we observed decreased water flux with time without any change in the apparent water permeability judged from the time elapsed before cell rupture when the bath solution was replaced with distilled water. This suggested an osmotic gradient was established within the cell and is consistent with activation of I_hi by an increase in the concentration of some critical intracellular molecule. Withdrawing 120 nl of cytoplasm did not activate I_hi (n=7). This suggests that membrane deformation alone is not sufficient to activate the current. We conclude that increased ionic/macromolecular concentration beneath the membrane, and not mechanical distortion, activates I_hi.