Role of the cytoskeleton in stimulation of Na+ channels in A6 cells by changes in osmolality.

Permeable supports with A6 cell monolayers were mounted in an Ussing chamber and bilaterally bathed with Ringer solution at room temperature. Short-circuit current (Isc) was recorded continuously, and noise analysis revealed microscopic channel current characteristics. Our investigation focuses on the stimulation of apical Na+ entry caused by exposing the serosal surface of the A6 cell monolayers to hyposmotic Ringer solution. To evaluate the possible role of the cytoskeleton in the regulation of Na+ channels in response to a change in osmolality we used four different experimental approaches. In the control group, which were not exposed to any cytoskeleton-influencing drugs, there was a 1.5-fold increase in Isc and in the number of open Na+ channels after osmotic stimulation. For the second group cytochalasin D (0.1 microg/ml) was present on the serosal side during the experiments. Neither Isc nor the number of open Na+ channels increased after osmotic stimulation. In the third group colchicine (0.2 mM) or nocodazole (20 microM) was present on the serosal side, which resulted in 1.8-fold and 1.5-fold increases in Isc as well as 3-fold and 2-fold increases in the number of Na+ channels, respectively. In the fourth experimental group erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride (EHNA, 0.5 mM), a dynein inhibitor, was present on the serosal side. In this group Isc decreased to about 0.4 microA/cm2, and subsequent application of amiloride abolished Isc completely. Under hyposmolar conditions EHNA abolished entirely the sensitivity of Isc to the osmotic challenge. Because of the EHNA-induced down-regulation of Isc, the density of apical Na+ channels in this experimental group could not be determined. These results show that the cytoskeleton is dominantly involved in osmotic channel regulation at the apical membrane, and that actin filaments, microtubules and molecular motors are involved in the recruitment of additional Na+ channels.