The water channel aquaporin-8 is mainly intracellular in rat hepatocytes, and its plasma membrane insertion is stimulated by cyclic AMP.
We previously found that water transport across hepatocyte plasma membranes occurs mainly via a non-channel mediated pathway. Recently, it has been reported that mRNA for the water channel, aquaporin-8 (AQP8), is present in hepatocytes. To further explore this issue, we studied protein expression, subcellular localization, and regulation of AQP8 in rat hepatocytes. By subcellular fractionation and immunoblot analysis, we detected an N-glycosylated band of approximately 34 kDa corresponding to AQP8 in hepatocyte plasma and intracellular microsomal membranes. Confocal immunofluorescence microscopy for AQP8 in cultured hepatocytes showed a predominant intracellular vesicular localization. Dibutyryl cAMP (Bt(2)cAMP) stimulated the redistribution of AQP8 to plasma membranes. Bt(2)cAMP also significantly increased hepatocyte membrane water permeability, an effect that was prevented by the water channel blocker dimethyl sulfoxide. The microtubule blocker colchicine but not its inactive analog lumicolchicine inhibited the Bt(2)cAMP effect on both AQP8 redistribution to cell surface and hepatocyte membrane water permeability. Our data suggest that in rat hepatocytes AQP8 is localized largely in intracellular vesicles and can be redistributed to plasma membranes via a microtubule-depending, cAMP-stimulated mechanism. These studies also suggest that aquaporins contribute to water transport in cAMP-stimulated hepatocytes, a process that could be relevant to regulated hepatocyte bile secretion.
Instituto de Fisiologia Experimental, Consejo Nacional de Investigaciones Cientificas y Técnicas (CONICET), Universidad Nacional de Rosario, Rosario, Santa Fe, Argentina 2000.
F
F
García
A
A
Kierbel
M C
MC
Larocca
S A
SA
Gradilone
P
P
Splinter
N F
NF
LaRusso
R A
RA
Marinelli
D000818:Animals; D020346:Aquaporins / Q000378:metabolism*; D003994:Bucladesine / Q000494:pharmacology*; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D003078:Colchicine / Q000494:pharmacology; D022781:Hepatocytes / Q000187:drug effects* / Q000378:metabolism; D007473:Ion Channels; D008297:Male; D051381:Rats; D017208:Rats, Wistar; D013347:Subcellular Fractions / Q000378:metabolism
D020346:Aquaporins; D007473:Ion Channels; C108330:aquaporin 8; D003994:Bucladesine; D003078:Colchicine