Galactosamine decreases nitric oxide formation in cultured rat hepatocytes: mechanism of suppression

We have shown that nitric oxide production is dramatically decreased in rat primary hepatocyte cultures exposed to galactosamine. Cotreatment of the cells with uridine, which is known to prevent cytotoxicity, was found to also attenuate NO loss. In the present study, two possible mechanisms for the decreased nitric oxide production were examined. First, we examined the possibility that galactosamine could interfere with the uptake of extracellular arginine by the cultured hepatocytes. Cellular uptake of arginine was determined after addition of 14C-arginine at the time of hepatocyte attachment. Uptake of arginine was rapid in control cultures, and both the rate and level of uptake were unchanged by the addition of a cytotoxic concentration of galactosamine (4 mM). In addition, increased concentrations of arginine in the cell culture medium did not ameliorate the galactosamine-induced decrease in production of nitric oxide. Second, we determined whether the synthesis of inducible nitric oxide synthase in the hepatocyte cultures was inhibited by addition of galactosamine. Hepatocyte levels of inducible nitric oxide synthase were determined immunochemically at various times after the addition of galactosamine (4 mM). In control cultures, inducible nitric oxide synthase was detectable at 7 and 24 hours after attachment. In contrast, no nitric oxide synthase protein was detectable at any time in the galactosamine-treated cultures. Furthermore, addition of galactosamine after inducible nitric oxide synthase had already been synthesized (6.5 h after attachment) did not result in suppression of nitric oxide production in the hepatocyte cultures. The present studies suggest that galactosamine suppresses nitric oxide production in hepatocyte cultures by inhibiting synthesis of inducible nitric oxide synthase, rather than by interference in cellular uptake of arginine.     

Effect of bradykinin on nitric oxide production,urea synthesis and viability of rat hepatocyte cultures

Background  

It is well known that cytotoxic factors, such as lipopolysaccharides, derange nitrogen metabolism in hepatocytes and nitric oxide (NO) is involved among the other factors regulating this metabolic pathway. Hepatocytes have been shown to express large levels of NO following exposure to endotoxins, such as bacterial lipopolysaccharide and/or cytokines, such as tumour necrosis factor-α (TNFα), interleukin-1. The control role of arginine in both urea and NO biosynthesis is well known, when NO is synthesized from arginine, by the NOS reaction, citrulline is produced. Thus, the urea cycle is bypassed by the NOS reaction. Many authors demonstrated in other cellular types, like cardiomyocytes, that bradykinin caused the increase in reactive oxygen species (ROS) generation. The simultaneous increase of NO and ROS levels could cause peroxynitrite synthesis, inducing damage and reducing cell viability. The aim of this research is to study the effect of bradykinin, a proinflammatory mediator, on cell viability and on urea production in cultures of rat hepatocytes.     

The morphological and immunocytochemical evaluation of primary rat hepatocytes undergoing spontaneous cell death: modulation by the nitric oxide donor S-nitroso-N-acetylpenicillamine

Nitric oxide (NO) is one of the smallest molecules synthesised in the human body. It is produced by three distinct nitric oxide synthase isoenzymes (NOS) and plays a number of physiological functions in many organs and tissues. Among its numerous properties is the ability to influence programmed cell death. NO can either inhibit or induce apoptosis depending on the context of its production. In the liver, NO is produced in greater amounts especially during inflammation. The effect of NO in liver physiology and pathophysiology can be both beneficial and detrimental. Therefore, the aim of our study was to examine NO effect on cell viability and cell death in primary rat hepatocyte culture. By using NO donor, S-nitroso-N-acetylpenicillamine (SNAP), the potential of exogenously delivered NO to influence spontaneous cell death in culture was examined. The morphological approach was used in order to discriminate between apoptotic and necrotic cell death. The nitrite level, urea production and alanine aminotransferase leakage were determined in the culture medium. The immunocytochemical detection of three apoptotic markers: cleaved caspase-3, cleaved caspase-9 and lamin A, was performed. Immunocytochemical analysis of hepatocyte apoptosis revealed different labelling pattern for each method, while the detection of cleaved caspase-3 best correlated with defined phenotypical criteria. Our data showed that under present conditions NO improved the viability of primary rat hepatocytes compared to untreated cells. This was manifested by the increase of viable hepatocytes in contrast to the decrease of necrotic and apoptotic hepatocytes as assessed by the morphological examination of cell culture. The NO effect was dose-dependent in the range of SNAP concentration between 200-800 microM.     

A mechanobiological mathematical model of liver metabolism

The liver plays a complex role in metabolism and detoxification, and better tools are needed to understand its function and to develop liver-targeted therapies. In this study, we establish a mechanobiological model of liver transport and hepatocyte biology to elucidate the metabolism of urea and albumin, the production/detoxification of ammonia, and consumption of oxygen and nutrients. Since hepatocellular shear stress (SS) can influence the enzymatic activities of liver, the effect of SS on the urea and albumin synthesis are empirically modeled through the mechanotransduction mechanisms. The results demonstrate that the rheology and dynamics of the sinusoid flow can significantly affect liver metabolism. We show that perfusate rheology and blood hematocrit can affect urea and albumin production by changing hepatocyte mechanosensitive metabolism. The model can also simulate enzymatic diseases of the liver such as hyperammonemia I, hyperammonemia II, hyperarginemia, citrollinemia, and argininosuccinicaciduria, which disrupt the urea metabolism and ammonia detoxification. The model is also able to predict how aggregate cultures of hepatocytes differ from single cell cultures. We conclude that in vitro perfusable devices for the study of liver metabolism or personalized medicine should be designed with similar morphology and fluid dynamics as patient liver tissue. This robust model can be adapted to any type of hepatocyte culture to determine how hepatocyte viability, functionality, and metabolism are influenced by liver pathologies and environmental conditions.     

Loss of hepatocyte co-operative activity after inhibition of ganglioside GM1 synthesis and shedding

Pretreatment of hepatocyte cultures with 1 microM d-l-threo-1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol-HCL (PPPP) for 24 h decreased the ganglioside GM1 content of the cells by approximately 50% and that of the conditioned medium by 90%. No rhythm in the rate of protein synthesis was detected in dense cultures pretreated with PPPP, but was observed in control dense cultures. Conditioned medium from control dense cultures induced synchrony in sparse cultures, which were non-synchronous in their own medium. In contrast, conditioned medium from dense cultures pretreated with PPPP did not synchronize sparse cultures. Since protein synthesis rhythm is a marker of cell synchronization, i.e. their co-operative activity, then non-oscillatory behavior means loss of cell co-operation. The protein synthesis rhythm was restored 24 h after hepatocytes were transferred to PPPP-free medium. Restoration was more rapid when 0.9 microM gangliosides (standard mixture from bovine brain) were added to the medium just after the withdrawal of PPPP. These novel results concerning the loss of rhythm of protein synthesis in low GM1 ganglioside medium support the conclusion that ganglioside is implicated in the regulation of cell co-operative activity.     

The effects of cell density, attachment substratum and dexamethasone on spontaneous apoptosis of rat hepatocytes in primary culture

Summary The rates of spontaneous cell detachment, cell viability, and apoptosis in primary cultures of rat hepatocytes plated at high and low density were compared. Apoptosis was frequent in detached cells, and the rates of cell detachment and apoptosis were greater in high-density than in low-density cultures. Among attached cells, more cells had condensed or fragmented nuclei in high-density than in low-density cultures. Further, ladder-like DNA fragmentation was not seen in low-cell-density cultures but was clearly evident in high-density cultures. Bax was more highly expressed in cells cultured at high density, and on collagen vs. matrigel, whereas changes of Bcl-2 and Fas expression observed in culture appeared unrelated to the rate of apoptosis. The rate of hepatocyte apoptosis appeared to be identical in low-density cultures on collagen 1 and matrigel, but when cells were cultured at high density, matrigel suppressed apoptosis by more than 50% at 36 h. In hepatocytes cultured on collagen 1, dexamethasone (0.1 μM) suppressed apoptosis in both low- and high-density cultures; higher doses had no further effects. In high density cultures, aurintricarboxylic acid (10 μM) suppressed apoptosis and this improved cell attachment at 48 h. It is concluded that cell viability in primary cultures of rat hepatocytes grown on collagen I is dependent on optimal culture density and that the cell population is regulated, at least in part, by apoptosis. Corticosteroids suppress spontaneous apoptosis of cultured hepatocytes in a non-dose-dependent manner, whereas matrigel abolishes apoptosis induced by increasing cell density. Bax may be an important protein in the cell density and cell matrix-dependent regulation of apoptosis in cultured hepatocytes.     

Oxygen uptake rates and liver-specific functions of hepatocyte and 3T3 fibroblast co-cultures

Bioartificial liver (BAL) devices have been developed to treat patients undergoing acute liver failure. One of the most important parameters to consider in designing these devices is the oxygen consumption rate of the seeded hepatocytes which are known to have oxygen consumption rates 10 times higher than most other cell types. Hepatocytes in various culture configurations have been tested in BAL devices including those formats that involve co-culture of hepatocytes with other cell types. In this study, we investigated, for the first time, oxygen uptake rates (OUR)s of hepatocytes co-cultured with 3T3-J2 fibroblasts at various hepatocyte to fibroblast seeding ratios. OURs were determined by measuring the rate of oxygen disappearance using a ruthenium-coated optical probe after closing and sealing the culture dish. Albumin and urea production rates were measured to assess hepatocyte function. Lower hepatocyte density co-cultures demonstrated significantly higher OURs (2 to 3.5-fold) and liver- specific functions (1.6-fold for albumin and 4.5-fold for urea production) on a per cell basis than those seeded at higher densities. Increases in OUR correlated well with increased liver-specific functions. OURs (V(m)) were modeled by fitting Michaelis-Menten kinetics and the model predictions closely correlated with the experimental data. This study provides useful information for predicting BAL design parameters that will avoid oxygen limitations, as well as maximize metabolic functions.     

Feedback inhibition of bile acid synthesis in cultured pig hepatocytes

Bile acid synthesis by cultured pig hepatocytes, as measured by conversion of [14C]cholesterol to bile acids, increased during the second and third day of culture. This rise was inhibited after addition of various conjugated and unconjugated bile acids in a concentration of 100 microM. It could be completely prevented by cycloheximide, indicating that de novo protein synthesis is required for the increase in bile acid formation. No effect of exogenous bile salts on LDH release to the medium or on cellular ATP content was observed, demonstrating that hepatocyte viability was not affected. During the period in which bile acid synthesis was inhibited, pig hepatocytes were able to accumulate taurocholic acid (100 microM) up to 7-18 nmol per mg cell protein (decreasing during culture time). It is concluded that feedback regulation of bile acid synthesis is exerted by direct action of bile acids on the hepatocyte.     

Acetaminophen-induced hepatotoxicity of gel entrapped rat hepatocytes in hollow fibers

An important application of primary hepatocyte cultures is for hepatotoxicity research. In this paper, gel entrapment culture of rat hepatocytes in miniaturized BAL system were evaluated as a potential in vitro model for hepatotoxicity studies in comparison to monolayer cultures. After exposure for 24 and 48 h to acetaminophen (2.5 mM), gel entrapped hepatocytes were more severely damaged than hepatocyte monolayer detected by methyl thiazolyl tetrazolium (MTT) reduction, intracellular glutathione (GSH) content, reactive oxygen species (ROS) levels, urea genesis and albumin synthesis. CYP 2E1 activities detected by 4-nitrocatechol (4-NC) formation were higher in gel entrapped hepatocytes than in hepatocyte monolayers while the addition of CYP 2E1 inhibitor, diethyl-dithiocarbamate (DDC), more significantly reduced acetaminophen-induced toxicity in gel entrapped hepatocytes. In addition, protective effects of GSH, liquorice extract and glycyrrhizic acid against acetaminophen hepatotoxicity were clearly observed in gel entrapped hepatocytes but not in hepatocyte monolayer at an incubation time of 48 h. Overall, gel entrapped hepatocytes showed higher sensitivities to acetaminophen-induced hepatotoxicity than hepatocyte monolayer by a mechanism that higher CYP 2E1 activities of gel entrapped hepatocytes could induce more severe acetaminophen toxicity. This indicates that gel entrapped hepatocytes in hollow fiber system could be a promising model for toxicological study in vitro.     

Gas‐permeable membranes and co‐culture with fibroblasts enable high‐density hepatocyte culture as multilayered liver tissues

To engineer reliable in vitro liver tissue equivalents expressing differentiated hepatic functions at a high level and over a long period of time, it appears necessary to have liver cells organized into a three‐dimensional (3D) multicellular structure closely resembling in vivo liver cytoarchitecture and promoting both homotypic and heterotypic cell–cell contacts. In addition, such high density 3D hepatocyte cultures should be adequately supplied with nutrients and particularly with oxygen since it is one of the most limiting nutrients in hepatocyte cultures. Here we propose a novel but simple hepatocyte culture system in a microplate‐based format, enabling high density hepatocyte culture as a stable 3D‐multilayer. Multilayered co‐cultures of hepatocytes and 3T3 fibroblasts were engineered on collagen‐conjugated thin polydimethylsiloxane (PDMS) membranes which were assembled on bottomless frames to enable oxygen diffusion through the membrane. To achieve high density multilayered co‐cultures, primary rat hepatocytes were seeded in large excess what was rendered possible due to the removal of oxygen shortage generally encountered in microplate‐based hepatocyte cultures. Hepatocyte/3T3 fibroblasts multilayered co‐cultures were maintained for at least 1 week; the so‐cultured cells were normoxic and sustained differentiated metabolic functions like albumin and urea synthesis at higher levels than hepatocytes monocultures. Such a microplate‐based cell culture system appears suitable for engineering in vitro miniature liver tissues for implantation, bioartificial liver (BAL) development, or chemical/drug screening. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011.     

Suppression of liver cell apoptosis in vitro by the non-genotoxic hepatocarcinogen and peroxisome proliferator nafenopin

Suppression of apoptosis has been implicated as a mechanism for the hepatocarcinogenicity of the peroxisome proliferator class of non- genotoxic carcinogens. The ability of the peroxisome proliferator nafenopin to suppress or delay the onset of liver apoptosis was investigated using primary cultures of rat hepatocytes and the Reuber hepatoma cell line FaO. 50 microM nafenopin reversibly maintained the viability of primary rat hepatocyte cultures which otherwise degenerated within 8 d of establishment. The maintenance of viability of hepatocyte monolayers was associated with a significant decrease in the number of cells exhibiting chromatin condensation patterns typical of apoptosis. Apoptosis could be induced in hepatocytes by administration of 5 ng/ml TGF beta 1. Co-addition of 50 microM nafenopin significantly reduced TGF beta 1-induced apoptosis by 50-60%. TGF beta 1 (1-5 ng/ml) also induced apoptosis in the FaO rat hepatoma cell line. Cell death was accompanied by detachment of FaO cells from the monolayer and detached cells exhibited chromatin condensation and non-random DNA fragmentation patterns typical of apoptosis. Co-addition of 50 microM nafenopin to TGF beta 1-treated FaO cultures significantly reduced the number of apoptotic cells detaching from the monolayer at 24 h. In contrast, nafenopin had no significant effect on FaO apoptosis induced by the DNA damaging agents etoposide and hydroxyurea. We conclude that suppression of liver cell death by apoptosis may play a role in the hepatocarcinogenicity of the peroxisome proliferators, although the extent of this protection is dependent on the nature of the apoptotic stimulus.     

The differential effect of PGE(1) on d-galactosamine-induced nitrosative stress and cell death in primary culture of human hepatocytes

The pre-administration of PGE(1) reduced inducible nitric oxide synthase (NOS-2) expression and cell death induced by d-galactosamine (d-GalN) in cultured rat hepatocytes. The present study evaluated the role of nitric oxide (NO) during PGE(1) treatment in fully established d-GalN-induced cytotoxicity in cultured human hepatocytes. Human hepatocytes were isolated from liver resections by classic collagenase perfusion. PGE(1) (1 microM) was administered at 2 h before d-GalN (40 mM), or 2 or 10 h after d-GalN in cultured hepatocytes. The production of NO was inhibited by N-omega-nitroso-l-arginine methyl ester (l-NAME) (0.5 mM). Various parameters related to oxidative and nitrosative stress, mitochondrial dysfunction, NF-kappaB activation, NOS-2 expression and cell death were evaluated in hepatocytes. NO mediated mitochondrial disturbances, nitrosative stress and cell death in d-GalN-treated hepatocytes. The administration of PGE(1) 10 h after d-GalN enhanced NF-kappaB activation, NOS-2 expression and nitrosative stress. Although PGE(1) administered at 2 h before or 2h after d-GalN reduced apoptosis and necrosis, its administration 10 h after d-GalN had no beneficial effect on cell death. In conclusion, the administration of PGE(1) during advanced d-GalN cytotoxicity induced nitrosative stress and lost its cytoprotective properties in cultured human hepatocytes.     

Chitosan nanofiber scaffold enhances hepatocyte adhesion and function

To enhance cell attachment and promote liver functions of hepatocytes cultured in bioreactors, a chitosan nanofiber scaffold was designed and prepared via electrospinning. Effects of the scaffold on hepatocyte adhesion, viability and function were then investigated. Data showed that hepatocytes on chitosan nanofiber scaffold exhibited better viability and tighter cell-substrate contact than cells on regular chitosan film. In addition, urea synthesis, albumin secretion and cytochrome P450 activity of hepatocytes on chitosan nanofiber scaffold were all 1.5 to 2 folds higher than the controls. Glycogen synthesis was also increased as compared with the controls. These results suggested the potential application of this chitosan nanofiber scaffold as a suitable substratum for hepatocyte culturing in bioreactors.     

Ammonia removal using hepatoma cells in mammalian cell cultures

It was examined whether hepatocyte cell lines can be used for ammonia removal in mammalian cell cultures. It was found that there exists a critical ammonium concentration level for each hepatocyte cell to remove ammonia. Among the cells tested in this work, primary hepatocytes showed the strongest ammonia removal capability if ammonium concentration is higher than the critical level. However, primary hepatocytes lost the liver function gradually and finally died after 2-3 weeks. Because of this limitation, primary hepatocytes were not appropriate to be used for ammonia removal in long-term cultures. Hep G2 cells, which are immortal, also showed a strong ammonia removal activity. The ammonia removal activity of Hep G2 cells depended on the concentration of ammonium in the medium, as in the case of primary hepatocytes. However, urea could not be detected in the course of ammonia removal by Hep G2 cells. Instead of urea, Hep G2 cells secreted glutamine into the culture medium. The capacity for ammonia removal was higher in the absence than in the presence of glutamine. Thus we checked the activity of glutamine synthetase in the Hep G2 cells. The level of glutamine synthetase activity increased with the addition of ammonium chloride. This result accounts for the ammonium concentration dependency of Hep G2 cells in ammonia removal and glutamine synthesis. Furthermore Hep G2 cells could grow well in the absence of glutamine, which was necessarily required in mammalian cell cultures. These results prove that glutamine formation serves as the primary mechanism of detoxifying ammonia in hepatocyte cell lines as expected. In addition, it was demonstrated that ammonium level could be reduced 38% and that erythropoietin production increased 2-fold in the mixed culture of Hep G2 and recombinant CHO cells.     

Bidirectional concentration-dependent effects of glucagon and dibutyryl cyclic AMP on DNA synthesis in cultured adult rat hepatocytes

Glucagon and dibutyryl cyclic AMP exerted both stimulatory and inhibitory effects on hepatocyte DNA synthesis when added to primary monolayer cultures in the presence of serum, dexamethasone, insulin and epidermal growth factor. The stimulation occurred at low concentrations of glucagon (1 pM-1 nM) or dibutyryl cyclic AMP (1 nM-1 microM), while the agents inhibited DNA synthesis at higher concentrations (usually glucagon at over 10 nM or dibutyryl cyclic AMP at over 10 microM). The stimulatory effect was stronger at low cell densities (less than 20 X 10(3) hepatocytes/cm2). When the hepatocytes were cultured at higher densities, stimulatory effects were reduced or absent and the inhibition of (hormone-induced) DNA synthesis by a high concentration of glucagon was much more pronounced than at low cell densities. These results indicate dual, bidirectional, effects of cyclic AMP on hepatocyte DNA synthesis.     

Effect of tryptophan on isolated hepatocytes of rats

The addition of tryptophan to adult rat hepatocyte cultures stimulated DNA synthesis. The increase in DNA synthesis as measured by 3H-thymidine incorporation into DNA was observed on treatment of the cultures with tryptophan for 48 h but also as short as for 6 h in comparison with control cultures. An increase was also apparent at 30 h which was maintained for up to 48 h post treatment with tryptophan. The increase in DNA synthesis by tryptophan cannot be attributed to cell injury or to increased DNA degradation. Of the degradative enzymes added after harvesting the hepatocytes, only DNase decreased incorporation of 3H-thymidine. The observed effect was specific for tryptophan since treatment with kynurenine, isoleucine, methionine or serine failed to show a significant effect. Pretreatment of cultured hepatocytes with hydroxyurea prevented the tryptophan stimulated increase in DNA synthesis suggesting that the latter was due to replicative and not to reparative DNA synthesis. Experiments performed with the addition of diethylnitrosamine also alluded to tryptophan's role in replicative DNA synthesis. The mechanism of tryptophan-induced DNA synthesis is discussed.     

Absence of negative feedback control of bile acid biosynthesis in cultured rat hepatocytes

The effect of individual bile acids on bile acid synthesis was studied in primary hepatocyte cultures. Relative rates of bile acid synthesis were measured as the conversion of lipoprotein [4-14C]cholesterol into 4-14C-labeled bile acids. Additions to the culture media of cholate, taurocholate, glycocholate, chenodeoxycholate, taurochenodeoxycholate, glycochenodeoxycholate, deoxycholate, and taurodeoxycholate (10-200 microM) did not inhibit bile acid synthesis. The addition of cholate (100 microM) to the medium raised the intracellular level of cholate 10-fold, documenting effective uptake of added bile acid by cultured hepatocytes. The addition of 200 microM taurocholate to cultured hepatocytes prelabeled with [4-14C]cholesterol did not result in inhibition of bile acid synthesis. Taurocholate (10-200 microM) also failed to inhibit bile acid synthesis in suspensions of freshly isolated hepatocytes after 2, 4, and 6 h of incubation. Surprisingly, the addition of taurocholate and taurochenodeoxycholate (10-200 microM) stimulated taurocholate synthesis from [2-14C]mevalonate-labeled cholesterol (p less than 0.05). Neither taurocholate nor taurochenodeoxycholate directly inhibited cholesterol 7 alpha-hydroxylase activity in the microsomes prepared from cholestyramine-fed rats. By contrast, 7-ketocholesterol and 20 alpha-hydroxycholesterol strongly inhibited cholesterol 7 alpha-hydroxylase activity at low concentrations (10 microM). In conclusion, these data strongly suggest that bile acids, at the level of the hepatocyte, do not directly inhibit bile acid synthesis from exogenous or endogenous cholesterol even at concentrations 3-6-fold higher than those found in rat portal blood.     

Effects of oxygenation and flow on the viability and function of rat hepatocytes cocultured in a microchannel flat-plate bioreactor

The goal of this study was to investigate the viability and synthetic function of rat hepatocytes cocultured with 3T3-J2 fibroblasts in a small-scale microchannel flat-plate bioreactor with and without an internal membrane oxygenator under flow. Bioreactor channel heights ranged between 85 and 500 microm and medium flow rates ranged between 0.06 and 4.18 mL/min. The results showed that the bioreactor without the oxygenator resulted in significantly decreased viability and function of hepatocytes, whereas hepatocytes in the bioreactor with internal membrane oxygenator were able to maintain their viability and function. The shear stress calculations showed that, at lower wall shear stresses (0.01 to 0.33 dyn/cm(2)), hepatocyte functions, measured as albumin and urea synthesis rates, were as much as 2.6- and 1.9-fold greater, respectively, than those at higher wall shear stresses (5 to 21 dyn/cm(2)). Stable albumin and urea synthesis rates for 10 days of perfusion were also demonstrated in the bioreactor with internal membrane oxygenator. These results are relevant in the design of hepatocyte bioreactors and the eventual scaling-up to clinical devices.     

Opioid receptor-independent protection of ischemic rat hepatocytes by morphine

We studied the role of morphine in anoxia/reoxygenation injury to hepatocytes. Overnight cultured rat hepatocytes were incubated in anoxic buffer at pH 6.2 for 4h and reoxygenated at pH 7.4 for 2h to simulate anoxia/reoxygenation. Some hepatocytes were preincubated with 50 microM morphine for 10 min prior to onset of anoxia/reoxygenation. To study the effect of morphine on nitric oxide (NO), hepatocytes were loaded with 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM). Changes in NO concentration were assessed with a multi-well fluorescence reader and confocal microscopy. Morphine substantially improved cell viability after reoxygenation and increased NO generation, which was blocked by ATP-sensitive potassium channel blockers. Confocal images revealed that the increase in NO occurred mainly at the cytosol. However, treatment with opioid receptor antagonists did not reverse cytoprotection by morphine. These results indicate that morphine prevents anoxia/reoxygenation injury to hepatocytes. Protective mechanisms are associated with the potassium channels and NO, but are independent of opioid receptor-mediated signaling.