The Water Channel Aquaporin-8 Is Mainly Intracellular in Rat Hepatocytes, and Its Plasma Membrane Insertion Is Stimulated by Cyclic AMP

Abstract

We previously found that water transport across hepatocyte plasma membranes occurs mainly via a nonchannel 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 ϳ34 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.Bile is formed primarily by hepatocytes and subsequently delivered to the bile ducts where it is modified by cholangiocytes (i.e. the epithelial cells that line the bile ducts). Bile secretion by hepatocytes involves the active transport of solutes followed by the passive movement of water into the bile canaliculus in response to osmotic gradients created by these solutes (1, 2). Although a substantial amount of data have been published about the molecular identification of solute transporters and the mechanisms regulating solute transport by hepatocytes (3), little attention has been focused on the mechanistic and regulatory aspects involved in hepatocyte water transport.Water can cross cellular plasma membranes through the lipid portion of the bilayer by a diffusion mechanism or through aquaporin water channels. Aquaporins, a family of recently identified integral membrane proteins, increase cell membrane water permeability facilitating rapid movement of water in response to osmotic gradients (4, 5).We previously found based on biophysical and molecular biology studies that water transport across hepatocyte plasma membranes occurs mainly via a non-channel mediated pathway (6). As this observation seems to be in contradiction with the recent identification of transcript for the water channel aquaporin-8 (AQP8) 1 in hepatocytes (7-9), we further explored this issue by studying the protein expression, subcellular localization, and possible regulation of AQP8 water channels in isolated rat hepatocytes. MATERIALS AND METHODSIsolation and Incubation of ...

Rat Hepatocyte Aquaporin–8 Water Channels Are Down–Regulated in Extrahepatic Cholestasis

“…Exposure of liver slices from sham-operated rats to dibutyryl cAMP resulted in an increase of AQP8 in plasma membranes (97%; P Ͻ .05), in agreement with our previous observations in isolated hepatocytes. 5,10 Nevertheless, dibutyryl cAMP failed to increase plasma membrane AQP8 in liver slices from cholestatic rats, suggesting a defective translocation of AQP8 induced by 7 days of BDL.…”

“…Total liver homogenates were subjected to low-speed centrifugation to obtain postnuclear supernatants and then centrifuged at 200,000g for 60 minutes, yielding the total liver membrane fraction. 5 Fractions enriched in plasma or intracellular microsomal membranes were prepared from liver homogenates by differential centrifugation as previously described 5,10 by centrifugation at 200,000g for 60 minutes on a discontinuous 1.3 mol/L sucrose gradient. The plasma membrane band was removed and further purified on a 9% to 60% linear sucrose gradient, and the remainder of the gradient was sonicated, diluted to 0.3 mol/L, and centrifuged at 17,000g for 30 minutes.…”

“…[3][4][5][6][7][8][9][10] AQP8, which seems to be the most abundant AQP in hepatocytes, is predominantly localized in intracellular vesicles; its translocation to the canalicular plasma membrane is microtubule dependent and adenosine 3Ј,5Ј-cyclic monophosphate (cAMP) stimulated. 5,6,10 AQP9 resides exclusively on the sinusoidal plasma membranes of hepatocytes and thereby may facilitate the movement of water and certain small solutes. 4,10 AQP0 is localized intracellularly, and its significance in hepatocytes is currently unclear.…”

An emerging consensus on aquaporin translocation as a regulatory mechanism

“…AQPs may traffic within vesicles (Garcia et al, 2001), often via a microtubule network and sometimes with the involvement of the actin cytoskeleton, whereby rearrangement of the actin network assists in AQP membrane integration (Riethmuller et al, 2008). Once fully integrated into the membrane, newly synthesised AQPs exhibit certain chemical properties.…”

“…AQP8 expression may also be stimulated to increase at the cell surface by cAMP in hepatocytes; cells that normally exhibit low water permeability in their resting state due to intracellular sequestration of AQP8 (Garcia et al, 2001). Evidence for this was provided through labelling experiments, which showed an inverse correlation between AQP8 numbers located within microsomes and those on the plasma membrane, with an increase in the latter following stimulation by cAMP (Koyama et al, 1998).…”