Hepatocyte membrane water permeability measured by silicone layer filtering centrifugation

We previously found that hepatocytes are able to control their osmotic membrane water permeability (P_f) by regulating the number of surface aquaporin water channels. Hepatocyte P_f has been assessed by phase-contrast microscopy and cell image analysis, an established but relatively laborious procedure. We report here an alternative method to assess hepatocyte P_f based on a single silicone layer filtering centrifugation system. Isolated rat hepatocytes were incubated in hypotonic or isotonic buffers containing ³H₂O as a tracer and, then, were filtered by rapid centrifugation through a silicone layer down to a lysis layer. Osmotically driven radioactivity (i.e., ³H₂O) within hepatocytes was calculated as the difference between the dpm in lysis media measured under hypotonic and isotonic conditions. The P_f calculated from the initial slope of the radioactivity-versus-time curve was 18 μm/s at 4°C. Hepatocytes treated with dibutyryl cyclic AMP, to increase P_f through the plasma membrane insertion of aquaporins, showed an increased P_f value of 37 μm/s. The aquaporin blocker dimethyl sulfoxide selectively prevented the agonist-induced hepatocyte P_f. These data are in good agreement with the corresponding values determined by quantitative phase-contrast microscopy; thus, the method developed allows the rapid and reliable measurement of hepatocyte P_f.