Review

The use of isolated rat hepatocyte couplets in hepatobiliary physiology

Most cholestatic disorders are caused by defects in cholangiocytes. The type 3 isoform of the inositol 1,4,5-trisphosphate receptor (ITPR3) is the most abundant intracellular calcium release channel in cholangiocytes. ITPR3 is required for bicarbonate secretion by bile ducts, and its expression is reduced in intrahepatic bile ducts of patients with cholestatic disorders. We investigated whether the nuclear factor, erythroid 2-like 2 (NFE2L2 or NRF2), which is sensitive to oxidative stress, regulates expression of ITPR3.

The activity of the ITPR3 promoter was measured in normal human cholangiocyte (NHC) cells and primary mouse cholangiocytes. Levels of ITPR3 protein and messenger RNA were examined by immunoblot and polymerase chain reaction analyses, respectively. ITPR3 activity was determined by measuring calcium signaling in normal human cholangiocyte cells and secretion in isolated bile duct units. Levels of NRF2 were measured in liver tissues from rats with cholestasis (induced by administration of α-napthylisothiocyanate) and from patients with biliary diseases.

We identified a musculo-aponeurotic fibrosarcoma recognition element in the promoter of ITPR3 that bound NRF2 directly in NHC cells and mouse cholangiocytes. Increasing binding of NRF2 at this site resulted in chromatin remodeling that reduced promoter activity. Mutant forms of the musculo-aponeurotic fibrosarcoma recognition element did not bind NRF2. Activation of NRF2 with quercetin or by oxidative stress reduced expression of ITPR3 and calcium signaling in NHC cells; quercetin also reduced secretion by bile duct units isolated from rats. Knockdown of NRF2 with small interfering RNAs restored expression and function of ITPR3 in NHC cells incubated with quercetin. Bile ducts from rats with cholestasis and patients with cholangiopathic disorders expressed higher levels of NRF2 and lower levels of ITPR3 than ducts from control rats or patients with other liver disorders.

The transcription factor NRF2 binds to the promoter of ITPR3 to inhibit its expression in cholangiocytes, leading to reduced calcium signaling and bile duct secretion. This could be a mechanism by which oxidative stress inhibits these processes and contributes to cholangiopathies.

Although bile formation requires that large volumes of water be rapidly transported across liver epithelia, including hepatocytes, the molecular mechanisms by which water is secreted into bile are obscure. The aquaporins are a family of 10 channel-forming, integral membrane proteins of ∼28 kDa numbered 0–9 that allow water to rapidly traverse epithelial barriers in several organs including kidney, eye, and brain. We found transcripts of three of 10 aquaporins in hepatocytes (aquaporin 8 ≫ aquaporin 9 > aquaporin 0) by reverse transcription-polymerase chain reaction and quantitative ribonuclease protection assays; immunohistochemistry confirmed the presence of these three proteins in liver. Immunoblots of subcellular fractions of hepatocytes showed enrichment of aquaporins 0 and 8 in microsomes and canalicular plasma membranes; aquaporin 9 was enriched only in basolateral plasma membranes. Immunofluorescence of hepatocyte couplets confirmed the intracellular/canalicular localization of aquaporins 0 and 8 and the basolateral localization of aquaporin 9. Upon exposure of couplets to a choleretic stimulus (i.e. dibutyryl cAMP), aquaporin 8 redistributed to the canalicular plasma membrane; the subcellular distributions of aquaporins 0 and 9 were unaffected. In addition, exposure of couplets to dibutyryl cAMP caused an increase in canalicular water transport in the presence and absence of an osmotic gradient, an effect that was blocked by aquaporin inhibitors. These results provide evidence that aquaporins are present in hepatocytes and that aquaporins are involved in agonist-stimulated canalicular bile secretion.

Hepatocytes constitutively express CD95 (also called Fas/APO-1) and are therefore potential targets for CD95-ligand (CD95L)-mediated injury. To study this mechanism of cell death in hepatocytes we developed an in vitro model of liver cell apoptosis using membrane-bound CD95L as the inducing agent. Primary mouse hepatocytes were cocultured with NIH 3T3 fibroblasts, stably transfected with mouse CD95L (F^CD95L+). Fibroblasts stably transfected with vector only (F^CD95L−) served as controls. Hepatocytes from mice expressing low levels of CD95 (Fas^lpr mice) served as controls for effects unrelated to CD95. Morphologic and biochemical studies indicate that CD95 is expressed in cultured mouse hepatocytes. Membrane-bound CD95 from transfected fibroblasts destroyed all cocultured hepatocytes within 24 hours in the absence of protein synthesis inhibitors. Characteristic features of apoptosis were observed in dying hepatocytes and occurred in the following sequence: formation of cytoplasmic blebs and nuclear condensation after 3 hours; nuclear fragmentation and DNA strand breaks after 4 hours. These changes were observed only when normal hepatocytes were cocultured with F^CD95L+ and were not observed with F^CD95L− or in hepatocytes from Fas^lpr mice. Anti-CD95 antibody (Jo2) evoked similar changes in hepatocytes, although to a much lesser extent. We conclude that coculture of mouse hepatocytes with F^CD95L+ is a useful in vitro model for CD95-mediated apoptosis induced by CD95L. The high incidence of apoptosis caused by membrane-bound CD95L differs from the much smaller effects induced by the Jo2 antibody. In view of the high sensitivity of hepatocytes towards CD95L we speculate that CD95L-induced liver damage in vivo may be minimized by restricting exposure of hepatocytes to CD95L.

(Hepatology 2000;32:779-785.)

High density lipoprotein (HDL) can protect low density lipoprotein (LDL) against oxidation. Oxidized cholesterol esters from LDL can be transferred to HDL and efficiently and selectively removed from the blood circulation by the liver and adrenalin vivo. In the present study, we investigated whether scavenger receptor BI (SR-BI) is responsible for this process. At 30 min after injection, the selective uptake of oxidized cholesterol esters from HDL for liver and adrenal was 2.3- and 2.6-fold higher, respectively, than for native cholesterol esters, whereas other tissues showed no significant difference. The selective uptake of oxidized cholesterol esters from HDL by isolated liver parenchymal cells could be blocked for 75% by oxidized LDL and for 50% by phosphatidylserine liposomes, both of which are known substrates of SR-BI.

In vivo uptake of oxidized cholesterol esters from HDL by parenchymal cells decreased by 64 and 81% when rats were treated with estradiol and a high cholesterol diet, respectively, whereas Kupffer cells showed 660 and 475% increases, respectively. These contrasting changes in oxidized cholesterol ester uptake were accompanied by similar contrasting changes in SR-BI expression of parenchymal and Kupffer cells. The rates of SR-BI-mediated selective uptake of oxidized and native cholesterol esters were analyzed in SR-BI-transfected Chinese hamster ovary cells. SR-BI-mediated selective uptake was 3.4-fold higher for oxidized than for native cholesterol esters (30 min of incubation). It is concluded that in addition to the selective uptake of native cholesterol esters, SR-BI is responsible for the highly efficient selective uptake of oxidized cholesterol esters from HDL and thus forms an essential mediator in the HDL-associated protection system for atherogenic oxidized cholesterol esters.

The organic hydroperoxide, tertiary-butylhydroperoxide (tBOOH), causes oxidative damage in a number of cell types. It is used here in an isolated rat hepatocyte couplet preparation to study adverse hepatobiliary effects of peroxidative damagein vitro.At subcytotoxic concentrations (as determined by lactate dehydrogenase release and maintenance of cytoplasmic ATP concentrations) tBOOH caused decreased accumulation of a fluorescent bile acid analogue, cholyl-lysyl-fluorescein (CLF), in the canalicular vacuole of couplets (a hepatobiliary effect; cholestasis). This was dose dependent in the range 100–200 μM. At the same concentrations it brought about release of preaccumulated CLF, suggesting that its effect was more likely to be on sealing properties of the vacuole than processes of uptake, transcytosis, and secretion. Pretreatment of tBOOH-treated couplets with the antioxidants deferoxamine mesylate (iron chelator) and dimethyl sulfoxide (free radical scavenger) resulted in the prevention of both canalicular vacuolar accumulation (cVA, which assesses canalicular function) and canalicular vacuolar retention (cVR, which assesses the retaining ability of couplets) depression at 100 μM tBOOH but not at higher concentrations. This indicates that the cholestatic effect of tBOOH has a preventable and nonpreventable phase and that free radicals are involved in these processes. By selectively generating the two types of tBOOH radical, peroxyl (tBOO·) and alkoxyl (tBO·), using suitable catalysts, we were able to determine that the peroxyl radical was most probably involved in tBOOH-induced cholestasis. This was further supported by experiments employing specific peroxyl and alkoxyl radical scavengers; only the peroxyl scavenger reduced the effect of tBOOH upon canalicular function under the conditions studied.