Effect of aging on EGF-induced proliferative response in primary cultured periportal and perivenous hepatocytes

BACKGROUND/AIMS: Aging relates to declined proliferative capacity of the liver, but the molecular mechanism is not well understood. We examined whether functional changes of epidermal growth factor (EGF) receptor (EGFR) are involved in age-related decline in EGF-induced DNA synthesis using hepatocytes isolated in periportal and perivenous regions of the liver, which differ in the proliferative capacity. METHODS: Periportal hepatocytes (PPH) and perivenous hepatocytes (PVH) in 7-, 30-, and 90-week-old rats were isolated using the digitonin/collagenase perfusion technique. DNA synthesis was assessed by [methyl-(3)H]thymidine incorporation. EGFR binding affinity to EGF was analyzed by Scatchard analysis using [(125)I]EGF. EGFR dimerization and phosphorylation were determined by Western blot analysis. RESULTS: EGF-induced DNA synthesis was greater in PPH than in PVH from rats of 7 weeks, but the zonal difference disappeared with aging. [(125)I]EGF binding studies indicated that high-affinity EGFR in both subpopulations also disappeared with aging. Furthermore, EGF-induced dimerization in both subpopulations was down-regulated with aging, and the pattern of EGFR phosphorylation was parallel to that of dimerization. CONCLUSIONS: These data suggest that age-related decline in EGF-induced DNA synthesis of PPH and PVH is caused by down-regulation of EGFR dimerization through the decrease of high-affinity EGFR.     

The mitochondrial permeability transition contributes to acute ethanol-induced apoptosis in rat hepatocytes

Acute ethanol intoxication induces oxidative stress and apoptosis in primary cultured hepatocytes. Oxidative stress can trigger mitochondrial cytochrome c release initiating the mitochondrial pathway of apoptosis. Based on this information, we formulated the hypothesis that ethanol induced oxidative stress causes mitochondrial dysfunction resulting in apoptosis. In the present study, we found that the mitochondrial membrane permeability transition (MPT) is essential for induction of mitochondrial cytochrome c release and caspase activation of ethanol. The short-term incubation with ethanol (50 mmol/L) induced the MPT, cytochrome c release, caspase activation, and apoptosis of cultured rat hepatocytes. Hepatocyte apoptosis was prevented by caspase inhibitors (i.e., Z-VAD-fmk, DEVD-cho, and DMQD-cho). An MPT inhibitor, cyclosporin A, also prevented ethanol-induced cytochrome c release, caspase activation, and apoptosis, suggesting that acute ethanol-induced apoptosis is MPT dependent. Ethanol-induced MPT was also attenuated by N'N'-dimethylthiourea (DMTU, a scavenger of hydrogen peroxide, 10 mmol/L) and N-acetyl-cysteine (NAC, an antioxidant, 5 mmol/L). Preventing hepatocyte MPT by DMTU or NAC attenuated cytochrome c release as well as caspase activation, suggesting that ethanol-induced oxidative stress mediates the MPT. Thus, acute ethanol induces MPT via oxidative stress, and the MPT mediates mitochondrial pathway of apoptosis in hepatocytes exposed to acute ethanol.     

Separation of periportal and perivenous rat hepatocytes by fluorescence-activated cell sorting: confirmation with colloidal gold as an exogenous marker

Periportal and perivenous hepatocytes are known to display various functional differences. In this study we present a new method to separate periportal and perivenous cells: after selectively loading zone 1 or zone 3 with the fluorescent label acridine orange in an antegrade or retrograde perfusion, respectively, we separated the isolated hepatocytes on a fluorescence-activated cell sorter. The common way to check on proper separation is to estimate activities of enzymes known to exhibit a heterogeneous acinar distribution. Using enzyme histochemistry, however, we found that already on short collagenase perfusion, some enzymes displayed a more shallow gradient than in vivo, making enzyme activities less suitable as zonal markers. We therefore used colloidal gold granules (17 nm) injected intravenously (2.5 mg) into the rat 2 to 3 hr before cell isolation. The gold is taken up predominantly by perivenous hepatocytes, probably because of the efficient removal of gold granules in zone 1 by competing Kupffer cells. We compared acridine orange fluorescence, presence of gold particles and activities of six marker enzymes, three biochemically and three histochemically determined. Acridine orange and gold both pointed to a high enrichment of the fractions, whereas most enzyme activities were more randomly distributed among the cells as a result of the isolation procedure. Our separation procedure yielded fractions highly enriched in either viable periportal or perivenous cells, both from one liver. The use of colloidal gold as a marker to monitor separation is a valuable alternative to the more risky estimation of enzyme activities.     

Mechanism of azathioprine-induced injury to hepatocytes: roles of glutathione depletion and mitochondrial injury

BACKGROUND/AIMS: We sought evidence that azathioprine causes cell death through reduced glutathione (GSH) depletion and mitochondrial injury. METHODS: Studies were conducted in primary cultures of rat hepatocytes and cultured Hep G2 cells. RESULTS: Azathioprine toxicity to rat hepatocytes was preceded by depletion of GSH. Prior GSH depletion (by treatment with buthionine sulfoximine) enhanced toxicity whilst supplemental GSH or N-acetylcysteine was protective. In hepatocytes, GSH is consumed during metabolism of azathioprine to 6-mercaptopurine. 6-Mercaptopurine was not toxic to hepatocytes, suggesting that the later steps in azathioprine metabolism were not related to the pathogenic mechanism. In Hep G2 cells, azathioprine did not alter levels of GSH and was not toxic. Ultrastructural studies showed hepatocyte mitochondrial lesions after exposure to azathioprine, but no features of apoptosis. Azathioprine produced rapid and profound depletion of adenosine 5'-triphosphate (ATP). Cyclosporin A and glycine afforded protection against azathioprine toxicity, and Trolox and high-dose allopurinol also attenuated injury. CONCLUSIONS: The mechanism of azathioprine toxicity to hepatocytes involves depletion of GSH leading to mitochondrial injury with profound depletion of ATP and cell death by necrosis. Cell death was prevented by potent antioxidants, glycine and blocking the mitochondrial permeability transition pore.     

Selective isolation of intact periportal or perivenous hepatocytes by antero- or retrograde collagenase gradient perfusion

ABSTRACT— To study the role of functional heterogeneity in the pathogenesis of liver damage, a new technique for separation of intact periportal and perivenous parenchymal cells was developed. After a brief portal perfusion in situ, the upper vena cava was also cannulated and the medium was simultaneously and slowly pumped via the portal and hepatic veins so that the medium had to ooze through the liver capsule. For the isolation of periportal cells collagenase was added selectively to the medium perfused through the portal vein in order to concentrate the digestive effects of collagenase in the periportal parenchyma. Conversely, to obtain cells of perivenous origin, collagenase was added to the medium perfused via hepatic veins. After 6–7 min and a brief reoxygenation by portal perfusion the liver was rapidly sliced. The slices were shaken for 3 min in preoxygenated medium and free hepatocytes were isolated by centrifugation. Injured cells were eliminated by brief and gentle trypsin-DNase treatment terminated with trypsin inhibitor. With this procedure, 0.2–1 g packed cells with viability of over 90% could be routinely obtained. LDH-leakage during incubation of either hepatocyte preparation was small and similar to that of hepatocytes isolated by conventional collagenase perfusion. Histological HE-stained preparations of liver parenchyma after perfusion demonstrated selective loosening of either periportal or perivenous cellular subpopulations. Moreover, the activity of alanine aminotransferase was three times higher (p<0.001) in “periportal” than in “perivenous” cells. This difference is similar to that obtained by the microdissection technique. The isolated “perivenous” cells were significantly larger (+28%, p<0.001) than “periportal” cells, as measured from the packed cell volume. The results demonstrate that with this technique intact hepatocytes of either periportal or perivenous origin can be obtained in sufficient amounts to enable their further characterization by incubation or culture experiments.     

Different capacities for amino acid transport in periportal and perivenous hepatocytes isolated by digitonin/collagenase perfusion

Periportal and perivenous hepatocytes were isolated from rat liver by digitonin/collagenase perfusion for investigating the acinar heterogeneity of amino acid transport activities related to glutamine and ammonia metabolism. Immunocytochemical staining of the respective subpopulations for glutamine synthetase demonstrated that periportal subpopulations were essentially free of glutamine synthetase-positive cells, whereas perivenous subpopulations showed a 2- to 3-fold enrichment of glutamine synthetase-positive hepatocytes. The high perivenous/periportal ratio of 59 found for glutamine synthetase activity as well as the perivenous/periportal ratios of other marker enzymes further indicated the good separation of periportal and perivenous cells. alpha-Aminoisobutyric acid, histidine and glutamate were used to determine the distribution pattern of amino acid transport systems A, N and G-, as well as of the sodium-independent uptake of these compounds 1 hr after isolation and after maximal hormonal stimulation during primary culture. The strong heterogeneity of the sodium-independent transport of histidine, characterized by higher perivenous transport rates [perivenous/periportal ratio: 1.5 (1 hr) to 3.5 (48 hr)], suggests a significant role of facilitated diffusion, presumably in glutamine export. Conversely, the strong heterogeneity of the sodium-dependent glutamate transport (System G-) characterized by higher uptake rates in nonstimulated [perivenous/periportal ratio: 6.6 (1 hr)] and in hormonally treated perivenous hepatocytes (perivenous/periportal ratio: 2.2) reflects its possible significance with respect to the substrate availability for glutamine synthesis. The observed heterogeneities provide a basis for understanding how substrate fluxes related to glutamine metabolism might be established and regulated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective mitochondrial glutathione depletion by ethanol enhances acetaminophen toxicity in rat liver

Chronic alcohol consumption may potentiate acetaminophen (APAP) hepatotoxicity through enhanced formation of N-acetyl-p-benzoquinone imine (NAPQI) via induction of cytochrome P450 2E1 (CYP2E1). However, CYP2E1 induction appears to be insufficient to explain the claimed magnitude of the interaction. We assessed the role of selective depletion of liver mitochondrial glutathione (GSH) by chronic ethanol. Rats were fed the Lieber-DeCarli diet for 10 days or 6 weeks. APAP toxicity in liver slices (% glutathione-S-transferase alpha released to the medium, GST release) and NAPQI toxicity in isolated liver mitochondria (succinate dehydrogenase inactivation, SDH) from these rats were compared with pair-fed controls. Ethanol induced CYP2E1 in both the 10-day and 6-week groups by approximately 2-fold. APAP toxicity in liver slices was higher in the 6-week ethanol group than the 10-day ethanol group. Partial inhibition of NAPQI formation by CYP2E1 inhibitor diethyldithiocarbamate to that of pair-fed controls abolished APAP toxicity in the 10-day ethanol group only. Ethanol selectively depleted liver mitochondrial GSH only in the 6-week group (by 52%) without altering cytosolic GSH. Significantly greater GSH loss and APAP covalent binding were observed in liver slice mitochondria of the 6-week ethanol group. Isolated mitochondria of the 6-week ethanol group were approximately 50% more susceptible to NAPQI (25-165 micromol/L) induced SDH inactivation. This increased susceptibility was reproduced in pair-fed control mitochondria pretreated with diethylmaleate. In conclusion, 10-day ethanol feeding enhances APAP toxicity through CYP2E1 induction, whereas 6-week ethanol feeding potentiates APAP hepatotoxicity by inducing CYP2E1 and selectively depleting mitochondrial GSH.     

Impaired autophagy: A mechanism of mitochondrial dysfunction in anoxic rat hepatocytes

Autophagy selectively removes abnormal or damaged organelles such as dysfunctional mitochondria. The mitochondrial permeability transition (MPT) is a marker of impaired mitochondrial function that is evident in hepatic ischemia/reperfusion (I/R) injury. However, the relationship between mitochondrial dysfunction and autophagy in I/R injury is unknown. Cultured rat hepatocytes and mouse livers were exposed to anoxia/reoxygenation (A/R) and I/R, respectively. Expression of autophagy-related protein 7 (Atg7), Beclin-1, and Atg12, autophagy regulatory proteins, was analyzed by western blots. Some hepatocytes were incubated with calpain 2 inhibitors or infected with adenoviruses encoding green fluorescent protein (control), Atg7, and Beclin-1 to augment autophagy. To induce nutrient depletion, a condition stimulating autophagy, hepatocytes were incubated in an amino acid-free and serum-free medium for 3 hours prior to onset of anoxia. For confocal imaging, hepatocytes were coloaded with calcein and tetramethylrhodamine methyl ester to visualize onset of the MPT and mitochondrial depolarization, respectively. To further examine autophagy, hepatocytes were infected with an adenovirus expressing green fluorescent protein-microtubule-associated protein light chain 3 (GFP-LC3) and subjected to A/R. Calpain activity was fluorometrically determined with succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin. A/R markedly decreased Atg7 and Beclin-1 concomitantly with a progressive increase in calpain activity. I/R of livers also decreased both proteins. However, inhibition of calpain isoform 2, adenoviral overexpression, and nutrient depletion all substantially suppressed A/R-induced loss of autophagy proteins, prevented onset of the MPT, and decreased cell death after reoxygenation. Confocal imaging of GFP-LC3 confirmed A/R-induced depletion of autophagosomes, which was reversed by nutrient depletion and adenoviral overexpression. Conclusion: Calpain 2-mediated degradation of Atg7 and Beclin-1 impairs mitochondrial autophagy, and this subsequently leads to MPT-dependent hepatocyte death after A/R.     

Simvastatin attenuates oxidant-induced mitochondrial dysfunction in cardiac myocytes

3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors (statins) can exert beneficial effects independently of serum cholesterol reduction by increasing the bioavailability of nitric oxide. However, it is unclear whether statins can exert such effects directly on cardiac myocytes and whether mitochondria are potential targets. Neonatal rat cardiac myocytes were cultured and subjected to oxidant stress (1 hour of 100 micromol/L H2O2). Mitochondrial membrane potential, a key determinant of cardiomyocyte viability, was assessed by flow cytometric analysis of tetramethylrhodamine ethyl ester (TMRE)-loaded cells. Hydrogen peroxide significantly reduced mitochondrial membrane potential. Incubation of the cardiac myocytes in simvastatin (> or =1 micromol/L) 1 hour before peroxide exposure significantly attenuated the loss of TMRE fluorescence. This effect was inhibited by the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) or the ATP-sensitive mitochondrial potassium channel (mitoKATP) blocker 5-hydroxydecanoate. Simvastatin attenuates mitochondrial membrane depolarization after exposure to oxidant stress. These findings provide primary evidence that myocytes can act as triggers and effectors in the cardioprotective cascade of simvastatin therapy. These results bear implications of statin therapy as a potential clinical application of pharmacological preconditioning.     

Zonal differences in gallium-67 uptakes between perivenous versus periportal regions of rat liver following carbon tetrachloride treatment.

Gallium-67 ((67)Ga) has been used as a tumor or inflammation-imaging agent in nuclear medicine, although underlying mechanism has not been fully elucidated. To gain some insights into the mechanism of (67)Ga uptake by injured liver, we analyzed the difference between perivenous and periportal regions of rat liver in terms of (67)Ga uptake by hepatocytes at the site of inflammation caused by carbon tetrachloride (CCl(4))-treatment. Distribution of (67)Ga in rat liver sections was monitored with a BAS5000 system following hepatic injury by CCl(4)-treatment. Periportal hepatocytes (PPH) and perivenous hepatocytes (PVH) were prepared by modified digitonin-collagenase perfusion technique. Uptakes of (67)Ga in PVH region and PPH region reached to a maximum 2 days after CCl(4)-treatment, and the amount of maximum uptake of (67)Ga in PVH was twice as much as that in PPH. Liver damage as measured by lipid peroxidation and (45)Ca uptake occurred in PVH region within 1 day after CCl(4)-treatment. Incorporation of bromodeoxyuridine into hepatocytes reached to a maximum 2 days after CCl(4)-treatment, and peaked amount of DNA synthesis in PVH was twice as much as that in PPH. These results indicated that the uptake of (67)Ga by the PVH region was carried out during hepatic regeneration phase rather than hepatic damage period by CCl(4)-treatment.     

Cardiac mitochondrial dysfunction and DNA depletion in children with hypertrophic cardiomyopathy

Abnormalities in specific mitochondrial respiratory enzymes and DNA (mtDNA) have been reported in cardiomyopathy. In this study, we report 4 cases of severe hypertrophic cardiomyopathy (HCM) in which specific cardiac mitochondrial enzyme activity defects were found, including complex I (n = 2), complex III (n = 2), complex IV (n = 2) and complex V (n = 1). Other abnormalities were also noted including a marked depletion of mtDNA (n = 1) and decreased content of subunit 2 of cytochrome c oxidase (n = 1). None of the mtDNA point mutations and common deletions previously found in association with cardiomyopathy were detected in these patients. These data indicate that specific respiratory enzyme activity defects are frequently present in HCM. Also, our finding of a marked depletion of mtDNA in 1 patient suggests that cardiac mtDNA depletion, previously unreported in HCM, needs further examination in order to establish whether it plays a primary role in its pathogenesis.     

Differences in the lectin-binding patterns of the periportal and perivenous endothelial domains in the liver sinusoids

We have studied the distribution patterns of carbohydrate terminals on the endothelial surface of the mouse liver microvasculature. For this purpose, a wide battery of FITC lectins specific to glucose, mannose, galactose, fucose, N-acetyl-neuraminic acid, N-acetyl-galactosamine and N-acetyl-glucosamine residues were incubated on liver cryostat sections or intraportally perfused under physiological conditions. All the resulting hepatic sections were examined under fluorescent microscopy and confocal laser scanning microscopy. With the exception of N-acetyl-galactosamine- and fucose-binding lectins, all the perfused lectins specifically bound to the microvascular wall as confirmed by blocking methods using their corresponding sugars. A wide range of binding was, however, observed among the lectins, and the latter were classified into four groups according to their affinities for the different segments of the hepatic microvasculature: (a) equal affinity for all segments (concanavalin A); (b) different affinities depending on acinar zone (wheat germ agglutinin, Ricinus communis toxin, phytohemagglutinin E, Erythrina cristagalli agglutinin and Pisum sativum agglutinin); (c) preferential binding to the sinusoidal network (Lathyrus odoratus, phytohemagglutinin); and (d) lectins that fail to bind to the hepatic microvasculature (N-acetyl-galactosamine- and fucose-binding lectins). Sinusoidal segment walls in acinar zone 1 expressed a higher concentration of certain lectin-binding carbohydrate residues (N-acetyl-neuraminic acid, N-acetyl-galactosamine, galactose, mannose and glucose) than in acinar zone 3. The labeling patterns obtained through the incubation of liver sections or through in vivo perfusion with the different lectins did not always coincide. Only concanavalin A, wheat germ agglutinin and phytohemagglutinin E lectins proved to be concordant (i.e., they produced identical labeling patterns in both procedures).(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanisms for sensitization to TNF-induced apoptosis by acute glutathione depletion in murine hepatocytes

We previously reported that depletion of glutathione in murine hepatocytes by diethylmaleate (DEM) or acetaminophen (APAP) leads to oxidative stress-dependent necrosis and sensitizes to tumor necrosis factor (TNF)-induced apoptosis in an oxidative stress-independent fashion, which could not be explained by interference with nuclear factor kappaB (NF-kappaB) nuclear translocation. The present report explores the mechanisms of these effects. We observed that DEM led to necrosis when both mitochondrial and cytosol glutathione were depleted profoundly but sensitized to TNF-induced apoptosis when cytosol glutathione was depleted selectively. DEM and APAP lead to a significant decrease in reduced glutathione (GSH)/glutathione disulfide (GSSG) ratio. Glutathione depletion by DEM or APAP was associated with inhibition of TNF-induced NF-kappaB transactivation of anti-apoptotic genes, including inducible nitric oxide synthase (i-NOS). Provision of exogenous NO partially abrogated the sensitization to TNF in response to glutathione depletion. Glutathione depletion alone led to sustained increase in phospho-jun levels and c-Jun-N-terminal kinase (JNK) activity. JNK inhibitor partially blocked the sensitization to TNF-induced apoptosis accompanying glutathione depletion. In conclusion, these findings suggest that extramitochondrial glutathione depletion alters the thiol-disulfide redox state, leading to inhibition of NF-kappaB transactivation of survival genes and to sustained activation of JNK, both of which contribute to the sensitization to TNF-induced apoptosis.     

Feeding and fasting controls liver expression of a regulator of G protein signaling (Rgs16) in periportal hepatocytes

Background  

Heterotrimeric G protein signaling in liver helps maintain carbohydrate and lipid homeostasis. G protein signaling is activated by binding of extracellular ligands to G protein coupled receptors and inhibited inside cells by regulators of G protein signaling (RGS) proteins. RGS proteins are GTPase activating proteins, and thereby regulate Gi and/or Gq class G proteins. RGS gene expression can be induced by the ligands they feedback regulate, and RGS gene expression can be used to mark tissues and cell-types when and where Gi/q signaling occurs. We characterized the expression of mouse RGS genes in liver during fasting and refeeding to identify novel signaling pathways controlling changes in liver metabolism.     

Hepatic mitochondrial glutathione depletion and progression of experimental alcoholic liver disease in rats.

Long-term ethanol feeding has been shown to selectively reduce hepatic mitochondrial glutathione content by impairing mitochondrial uptake of this thiol. In this study, we assessed the role of this defect in evolution of alcoholic liver disease by examining the mitochondrial glutathione pool and lipid peroxidation during progression of experimental alcoholic liver disease to centrilobular liver necrosis and fibrosis. Male Wistar rats were intragastrically infused with a high-fat diet plus ethanol for 3, 6 or 16 wk (the duration that resulted in induction of liver steatosis, necrosis and fibrosis, respectively). During this feeding period, the cytosolic pool of glutathione remained unchanged in the ethanol-fed animals compared with that in pair-fed controls. In contrast, the mitochondrial pool of glutathione selectively and progressively decreased in rats infused with ethanol for 3, 6 or 16 wk, by 39%, 61% and 85%, respectively. Renal mitochondrial glutathione level remained unaffected throughout the experiment. Serum ALT levels increased significantly in the ethanol-fed rats at 6 wk and remained elevated at 16 wk. In the mitochondria with severely depleted glutathione levels at 16 wk, enhanced lipid peroxidation was evidenced by increased malondialdehyde levels. Thus a progressive and selective depletion of mitochondrial glutathione is demonstrated in the liver in this experimental model of alcoholic liver disease and associated with mitochondrial lipid peroxidation and progression of liver damage.     

Selective glutathione depletion of mitochondria by ethanol sensitizes hepatocytes to tumor necrosis factor

Background & Aims: Tumor necrosis factor (TNF)-α induces cell injury by generating oxidative stress from mitochondria. The purpose of this study was to determine the effect of ethanol on the sensitization of hepatocytes to TNF-α. Methods: Cultured hepatocytes from ethanol-fed (ethanol hepatocytes) or pair-fed (control hepatocytes) rats were exposed to TNF-α, and the extent of oxidative stress, gene expression, and viability were evaluated. Results: Ethanol hepatocytes, which develop a selective deficiency of mitochondrial glutathione (mGSH), showed marked susceptibility to TNF-α. The susceptibility to TNF-α, manifested as necrosis rather than apoptosis, was accompanied by a progressive increase in hydrogen peroxide that correlated inversely with cell survival. Nuclear factor κB activation by TNF-α was significantly greater in ethanol hepatocytes than in control hepatocytes, an effect paralleled by the expression of cytokine-induced neutrophil chemoattractant. Similar sensitization of normal hepatocytes to TNF-α was obtained by depleting the mitochondrial pool of GSH with 3-hydroxyl-4-pentenoate. Restoration of mGSH by S-adenosyl-L-methionine or by GSH–ethyl ester prevented the increased susceptibility of ethanol hepatocytes to TNF-α. Conclusions: These results indicate that mGSH controls the fate of hepatocytes in response to TNF-α. Its depletion caused by alcohol consumption amplifies the power of TNF-α to generate reactive oxygen species, compromising mitochondrial and cellular functions that culminate in cell death.GASTROENTEROLOGY 1998;115:1541-1551