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 (3)H(2)O as a tracer and, then, were filtered by rapid centrifugation through a silicone layer down to a lysis layer. Osmotically driven radioactivity (i.e., (3)H(2)O) 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 microm/s at 4 degrees 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 microm/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).
Gradilone
Sergio A
SA
Instituto de Fisiología Experimental, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Universidad Nacional de Rosario, Suipacha 570, Santa Fe, Rosario, 2000, Argentina.
Ochoa
J Elena
JE
García
Fabiana
F
Larocca
M Cecilia
MC
Pellegrino
José M
JM
Marinelli
Raúl A
RA
D000818:Animals; D020346:Aquaporins; D002462:Cell Membrane / Q000378:metabolism*; D002463:Cell Membrane Permeability; D002498:Centrifugation / Q000379:methods; D017666:Deuterium Oxide; D005374:Filtration; D022781:Hepatocytes / Q000378:metabolism* / Q000648:ultrastructure; D008297:Male; D051381:Rats; D017208:Rats, Wistar; D012828:Silicones; D014867:Water / Q000378:metabolism*
D020346:Aquaporins; D012828:Silicones; D014867:Water; D017666:Deuterium Oxide