We previously found that hepatocytes are able to control their osmotic membrane water permeability (Pf) by regulating the number of surface aquaporin water channels. Hepatocyte Pf 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 Pf 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 Pf 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 Pf through the plasma membrane insertion of aquaporins, showed an increased Pf value of 37 μm/s. The aquaporin blocker dimethyl sulfoxide selectively prevented the agonist-induced hepatocyte Pf. 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 Pf.
Bibliographical noteFunding Information:
1This work was supported by Grant PICT 05-03589 (to R. A. Marinelli) from Agencia Nacional de Promoción Científica y Tecno-lógica and by Consejo Nacional de Investigaciones Científicas y Téc-nicas (CONICET).