Structural reaction pattern of hepatocytes following exposure to hypotonicity

Cell swelling stimulates phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) in hepatocytes, and the PI3K signaling pathway is involved in cAMP-mediated translocation of sinusoidal Na ؉ /taurocholate (TC) cotransporter (Ntcp) to the plasma membrane. We determined whether cell swelling also stimulates TC uptake and Ntcp translocation via the PI3K and/or MAPK signaling pathway. All studies were conducted in isolated rat hepatocytes. Hepatocyte swelling induced by hypotonic media resulted in: 1) time-and medium osmolarity-dependent increases in TC uptake, 2) an increase in the V max of Na ؉ /TC cotransport, and 3) wortmannin-sensitive increases in TC uptake and plasma membrane Ntcp mass. Hepatocyte swelling also induced wortmannin-sensitive activation of PI3K, protein kinase B, and p70 S6K . Rapamycin, an inhibitor of p70 S6K , inhibited cell swelling-induced activation of p70 S6K but failed to inhibit cell swelling-induced stimulation of TC uptake. Because PD98095, an inhibitor of MAPK, did not inhibit cell swelling-induced increases in TC uptake, it is unlikely that the effect of cell swelling on TC uptake is mediated via the MAPK signaling pathway. Taken together, these results indicate that 1) cell swelling stimulates TC uptake by translocating Ntcp to the plasma membrane, 2) this effect is mediated via the PI3K, but not MAPK, signaling pathway, and 3) protein kinase B, but not p70 S6K

Section: Discussionsupporting

confidence: 62%

Section: Discussionmentioning

confidence: 99%

Cell Swelling-induced Translocation of Rat Liver Na+/Taurocholate Cotransport Polypeptide Is Mediated via the Phosphoinositide 3-Kinase Signaling Pathway

Webster

Blanch

Phillips

et al. 2000

“…Swelling-Osmotic stress induces dramatic cell shape changes in a number of cell types (39), including rapid formation of membrane protrusions in fibroblasts upon hypotonic challenge (4). Because we previously had observed irregular cell morphologies in connection with swelling-induced plasma membrane PLC␥ localization in HTC hepatoma cells (3), we investigated whether hypotonic exposure of these cells would elicit plasma membrane protrusions analogous to those in fibroblasts, and how the formation of swelling-induced membrane protrusions temporally correlated with volume recovery.…”

Section: Transient Membrane Protrusions Form In Response To Cellmentioning

confidence: 99%

Src Regulates Distinct Pathways for Cell Volume Control through Vav andPhospholipaseCγ

Barfod

Moore²,

Melnick³

et al. 2005

Cell volume recovery in response to swelling requires reorganization of the cytoskeleton and fluid efflux. We have previously shown that electrolyte and fluid efflux via K ؉ and Cl ؊ channels is controlled by swelling-induced activation of phospholipase C␥ (PLC␥). Recently, integrin engagement has been suggested to trigger responses to swelling through activation of Rho family GTPases and Src kinases. Because both PLC␥ and Rho GTPases can be regulated by Src during integrin-mediated cytoskeletal reorganization, we sought to identify swelling-induced Src effectors. Upon hypotonic challenge, Src was rapidly activated in transient plasma membrane protrusions, where it colocalized with Vav, an activator of Rho GTPases. Inhibition of Src with PP2 attenuated phosphorylation of Vav. PP2 also attenuated phosphorylation of PLC␥, and inhibited swelling-mediated activation of K ؉ and Cl ؊ channels and cell volume recovery. These findings suggest that swelling-induced Src regulates cytoskeletal dynamics, through Vav, and fluid efflux, through PLC␥, and thus can coordinate structural reorganization with fluid balance to maintain cellular integrity.

“…Calcein was selected for the following reasons: (a) calcein labels exclusively cytosol (16) and, during hypotonic swelling, hepatocyte cytosol shows identical changes to those of the whole cell (19); (b) calcein emits pH insensitive fluorescence; (c) calcein causes no effect on osmotic hepatocyte swelling as judged by quantitative phase-contrast microscopy of hepatocytes with and without calcein loading (data not shown); and (d) spontaneous and swelling-induced calcein leakage from hepatocytes during the time of the experiments was negligible (less than 2%).…”

Section: Osmotic Water Permeability Studiesmentioning

confidence: 99%

Rat Hepatocytes Transport Water Mainly via a Non-channel-mediated Pathway

Yano

Marinelli

Roberts

et al. 1996

During bile formation by the liver, large volumes of water are transported across two epithelial barriers consisting of hepatocytes and cholangiocytes (i.e. intrahepatic bile duct epithelial cells). We recently reported that a water channel, aquaporin-channel-forming integral protein of 28 kDa, is present in cholangiocytes and suggested that it plays a major role in water transport by these cells. Since the mechanisms of water transport across hepatocytes remain obscure, we performed physiological, molecular, and biochemical studies on hepatocytes to determine if they also contain water channels. Water permeability was studied by exposing isolated rat hepatocytes to buffers of different osmolarity and measuring cell volume by quantitative phase contrast, fluorescence and laser scanning confocal microscopy. Using this method, hepatocytes exposed to hypotonic buffers at 23°C increased their cell volume in a time and osmolarity-dependent manner with an osmotic water permeability coefficient of 66.4 ؋ 10 ؊4 cm/s. In studies done at 10°C, the osmotic water permeability coefficient decreased by 55% (p < 0.001, at 23°C; t test). The derived activation energy from these studies was 12.8 kcal/mol. After incubation of hepatocytes with amphotericin B at 10°C, the osmotic water permeability coefficient increased by 198% (p < 0.001) and the activation energy value decreased to 3.6 kcal/mol, consistent with the insertion of artificial water channels into the hepatocyte plasma membrane. Reverse transcriptase polymerase chain reaction with hepatocyte RNA as template did not produce cDNAs for three of the known water channels. Both the cholesterol content and the cholesterol/phospholipid ratio of hepatocyte plasma membranes were significantly (p < 0.005) less than those of cholangiocytes; membrane fluidity of hepatocytes estimated by measuring steady-state anisotropy was higher than that of cholangiocytes. Our data suggests that the osmotic flow of water across hepatocyte membranes occurs mainly by diffusion via the lipid bilayer (not by permeation through water channels as in cholangiocytes).Bile formation by the liver involves two phases: secretion of primary bile by hepatocytes at the canalicular domain and delivery to a network of interconnecting ducts where bile is modified by cholangiocytes via the secretion of ions and water.Bile secretion by hepatocytes involves the active transport of both organic and inorganic solutes, followed by the passive movement of water into bile canaliculi in response to osmotic gradients established by these solutes (1). While a substantial amount of recent data have permitted a clearer understanding of the cellular mechanisms regulating solute transport by hepatocytes (2, 3), the mechanisms regulating water movement across hepatocytes remain obscure (2, 3).Theoretically, water may move across the epithelial barrier of hepatocytes by two pathways: a paracellular pathway between adjacent cells, or a transcellular pathway across the cell (4). Further, transcellular water movement may occur throu...