Hepatocyte Shrinkage Research Articles

Acute toxicity of palm oil mill effluent on zebrafish (Danio rerio Hamilton-Buchanan, 1822): Growth performance, behavioral responses and histopathological lesions

Evaluating the toxicity of Palm Oil Mill Effluent (POME) is critical as part of the effort to develop waste management regulations for the palm oil industry. In this study, we investigated the acute toxicity of POME on growth performance, behavioral response, and histopathology of gill and liver tissues of zebrafish (Danio rerio). In total, 550 adult male zebrafish were used for the toxicity experiment including range finding test, acute toxicity test, growth performance and behaviour test. Static non-renewal acute toxicity bioassays were conducted by exposing fish to POME (1.584–9.968 mL/L) for 96 h. Growth performance, behavior response, and histopathological lesions in untreated and POME treated (96-h LC50: 5.156 mL/L) fish were measured at 24, 48, 72 and 96 h. Time-dependent significant decline in body length and body weight of POME-exposed zebrafish was observed. Furthermore, several behavioral changes were recorded, including hyperactivity, loss of balance, excessive mucus secretion, and depigmentation. Decreasing operculum movement and oxygen consumption rate as well as alterations in gill tissues (i.e. hyperplasia, hypertrophy, hemorrhage, and necrosis) of POME-exposed zebrafish were observed, suggesting a dysfunction in respiratory performance. On the other hand, liver tissue alterations (congestion, hemorrhage, hyperplasia, shrinkage of hepatocytes, hydrophilic degeneration, and necrosis) indicated a disruption in detoxification performance. We conclude that exposure to POME at acute concentration caused histopathological lesions both in gill and liver tissue along with changes in fish behaviors which disrupted respiratory and detoxification performance, resulting in mortality and reduced growth of zebrafish. These findings might provide valuable information for guiding POME management and regulation.

Pathogenicity of Coagulase Negative Staphylococcus chicken isolates to 10 Days Old Broiler Chickens

One hundred and sixty, 1-day old broiler chicks were grouped into 4 equal groups, at the 10th day birds of groups 1- 3 were s.c inoculated with 0.5 ml containing y 1.5x 108 of S.xylosis, S. sciuri and S.lentus; respectively and group 4 was noninfected control. Clinical signs in infected groups started at 2-3dpi as general signs. Signs disappear in Ciprofloxacillin treated subgroups 24 hr post treatment and lasted to the 7th day in non treated. Average body weight gain in S.xylosis infected non treated was the highest (813.90 gm), followed by S. scuiri (778.50gm ) and 773.75 in S. lentus infected treated . FCR was the highest in control (1.69 treated and 1.74 non treated) followed by S. scuiri infected (1.81 non treated and 1.82 treated) and the lowest 1.94 was in S.lentus infected non treated. S. sciuri was reisolated from intestine and spleen (5th dpi) and from intestine (7 and 10 dpi). While S.lentuswas reisolated from intestine , liver and spleen (3rd dpi) ; from intestine and spleen (5th dpi) and intestine (10th dpi). Histopathological lesion was recorded in infected group as hemorrhages with sinusoidal dilation, focal areas of vacuolar degeneration , fatty degeneration and shrinkage of hepatocytes in liver, necrotic changes of lymphocytes and vacuolion of corpuscle in spleen. Leucocytic infiltration , degeneration and necrosis of epithelium surface and intraepithelial as well as submucosal leucoytic infiltration were seen in intestine. In conclusion the injected organisms induce mild subclinical disease with recording of histopathological lesions in liver, spleen and intestine. This area needs more investigation to explore pathogenicity of CoNS in chickens.

Regulation of Plasma Membrane Localization of the Na+-taurocholate cotransporting polypeptide (Ntcp) by Hyperosmolarity and Tauroursodeoxycholate

In perfused rat liver, hepatocyte shrinkage induces a Fyn-dependent retrieval of the bile salt export pump (Bsep) and multidrug resistance-associated protein 2 (Mrp2) from the canalicular membrane (Cantore, M., Reinehr, R., Sommerfeld, A., Becker, M., and Haussinger, D. (2011) J. Biol. Chem. 286, 45014-45029) leading to cholestasis. However little is known about the effects of hyperosmolarity on short term regulation of the Na(+)-taurocholate cotransporting polypeptide (Ntcp), the major bile salt uptake system at the sinusoidal membrane of hepatocytes. The aim of this study was to analyze hyperosmotic Ntcp regulation and the underlying signaling events. Hyperosmolarity induced a significant retrieval of Ntcp from the basolateral membrane, which was accompanied by an activating phosphorylation of the Src kinases Fyn and Yes but not of c-Src. Hyperosmotic internalization of Ntcp was sensitive to SU6656 and PP-2, suggesting that Fyn mediates Ntcp retrieval from the basolateral membrane. Hyperosmotic internalization of Ntcp was also found in livers from wild-type mice but not in p47(phox) knock-out mice. Tauroursodeoxycholate (TUDC) and cAMP reversed hyperosmolarity-induced Fyn activation and triggered re-insertion of the hyperosmotically retrieved Ntcp into the membrane. This was associated with dephosphorylation of the Ntcp on serine residues. Insertion of Ntcp by TUDC was sensitive to the integrin inhibitory hexapeptide GRGDSP and inhibition of protein kinase A. TUDC also reversed the hyperosmolarity-induced retrieval of bile salt export pump from the canalicular membrane. These findings suggest a coordinated and oxidative stress- and Fyn-dependent retrieval of sinusoidal and canalicular bile salt transport systems from the corresponding membranes. Ntcp insertion was also identified as a novel target of β1-integrin-dependent TUDC action, which is frequently used in the treatment of cholestatic liver disease.

Regulation of Plasma Membrane Localization of the Na+-Taurocholate Cotransporting Polypeptide (Ntcp) by Hyperosmolarity and Tauroursodeoxycholate

In perfused rat liver, hepatocyte shrinkage induces a Fyn-dependent retrieval of the bile salt export pump (Bsep) and multidrug resistance-associated protein 2 (Mrp2) from the canalicular membrane (Cantore, M., Reinehr, R., Sommerfeld, A., Becker, M., and Häussinger, D. (2011) J. Biol. Chem. 286, 45014-45029) leading to cholestasis. However little is known about the effects of hyperosmolarity on short term regulation of the Na(+)-taurocholate cotransporting polypeptide (Ntcp), the major bile salt uptake system at the sinusoidal membrane of hepatocytes. The aim of this study was to analyze hyperosmotic Ntcp regulation and the underlying signaling events. Hyperosmolarity induced a significant retrieval of Ntcp from the basolateral membrane, which was accompanied by an activating phosphorylation of the Src kinases Fyn and Yes but not of c-Src. Hyperosmotic internalization of Ntcp was sensitive to SU6656 and PP-2, suggesting that Fyn mediates Ntcp retrieval from the basolateral membrane. Hyperosmotic internalization of Ntcp was also found in livers from wild-type mice but not in p47(phox) knock-out mice. Tauroursodeoxycholate (TUDC) and cAMP reversed hyperosmolarity-induced Fyn activation and triggered re-insertion of the hyperosmotically retrieved Ntcp into the membrane. This was associated with dephosphorylation of the Ntcp on serine residues. Insertion of Ntcp by TUDC was sensitive to the integrin inhibitory hexapeptide GRGDSP and inhibition of protein kinase A. TUDC also reversed the hyperosmolarity-induced retrieval of bile salt export pump from the canalicular membrane. These findings suggest a coordinated and oxidative stress- and Fyn-dependent retrieval of sinusoidal and canalicular bile salt transport systems from the corresponding membranes. Ntcp insertion was also identified as a novel target of β1-integrin-dependent TUDC action, which is frequently used in the treatment of cholestatic liver disease.

The Src family kinases: distinct functions of c-Src, Yes, and Fyn in the liver.

The Src family kinases Yes, Fyn, and c-Src play a pivotal role in regulating diverse liver functions such as bile flow, proteolysis, apoptosis, and proliferation and are regulated by anisoosmotic cell volume changes, death receptor ligands, and bile acids. For example, cell swelling leads to an integrin-sensed and focal adhesion kinase-mediated activation of c-Src-triggering choleresis, proteolysis inhibition, regulatory volume decrease via p38MAPK and proliferation via the activation of the epidermal growth factor receptor and extracellular regulated kinases 1 and 2. In contrast, hepatocyte shrinkage generates an almost instantaneous oxidative stress response that triggers the activation of c-Jun N-terminal kinase and the Src family kinases Fyn and Yes. Whereas Fyn activation mediates cholestasis, Yes triggers CD95 activation and apoptosis. This review will discuss the role of Src family kinases in the regulation of liver function with emphasis on their role in osmo-signaling and bile acid signaling.

Arsenic induced toxicity and histopathological changes in gill and liver tissue of freshwater fish, tilapia (Oreochromis mossambicus)

Acute toxicity of arsenic to tilapia (Oreochromis mossambicus) and its histopathological impacts on gill and liver tissue were evaluated. The median lethal concentration (96 h; LC50) of arsenic (NaAsO₂) was calculated as 28.22 ppm in repeated semi static test method. Fish were exposed to 3 ppm, 28 ppm and 56 ppm concentrations of NaAsO₂ and gill and liver samples were collected after 48 h, 96 h and 192 h of exposure. The changes in gill were characterized by epithelial hyperplasia, epithelial lifting and oedema, lamellar fusion, aneurism, desquamation and necrosis, whereas, the liver tissue showed focal lymphocytic and macrophage infiltration, congestion, vacuolization and shrinkage of hepatocytes, dilation of sinusoids, cloudy swelling, vacuolar degeneration, focal necrosis and nuclear hypertrophy. The result showed that acute arsenic toxicity severely affects the normal behavior and vital organs which is deleterious for the exposed fish.

Observation on the Histopathological Alterations in the Liver of Cypermethrin Affected Fish,Cirrhinus mrigala(Hamilton)

The freshwater fish, Cirrhinus mrigala, was exposed to lethal and sub-lethal concentrations of cypermethrin (5.13 μg/l and 1.026 μg/l) and the histopathological changes were observed in liver tissues. Compared to the structure of control fish, exposed tissues were initially exhibited disarray of liver lobes, mild degree of degeneration of cytoplasm, occasional blood clots, congregation of nuclei, cloudy swelling of hepatocytes, granulisation of cytoplasm, hypertrophic, pyknotic nuclei, atrophic, hepatocytic nuclei, focal necrosis, vacuolation, shrinkage of hepatocytes, granular degeneration, rupture of blood vessels, necrosis dissolution of laminar structure and cytoplasmic disintegration in hepatocytes. In the sub-lethal concentration of cypermethrin, tissues initially exhibited few changes like slight disarray of liver lobes, mild degree of degeneration of cytoplasm, occasional blood clots, congregation of nuclei and cloudy swelling of hepatocytes, granulisation of cytoplasm, hypertrophic and pyknotic nuclei on 1 and 7 days. But on 14 and 21 day, certain degree of reorganisation in the structure of liver cords was observed.

Activation of Integrins by Urea in Perfused Rat Liver

High concentrations of urea were shown to induce a paradoxical regulatory volume decrease response with K(+) channel opening and subsequent hepatocyte shrinkage (Hallbrucker, C., vom Dahl, S., Ritter, M., Lang, F., and Häussinger, D. (1994) Pflügers Arch. 428, 552-560), although the hepatocyte plasma membrane is thought to be freely permeable to urea. The underlying mechanisms remained unclear. As shown in the present study, urea (100 mmol/liter) induced within 1 min an activation of β(1) integrins followed by an activation of focal adhesion kinase, c-Src, p38(MAPK), extracellular signal-regulated kinases, and c-Jun N-terminal kinase. Because α(5)β(1) integrin is known to act as a volume/osmosensor in hepatocytes, which becomes activated in response to hepatocyte swelling, the findings suggest that urea at high concentrations induces a nonosmotic activating perturbation of this osmosensor, thereby triggering a volume regulatory K(+) efflux. In line with this, similar to hypo-osmotic hepatocyte swelling, urea induced an inhibition of hepatic proteolysis, which was sensitive to p38(MAPK) inhibition. Molecular dynamics simulations of a three-dimensional model of the ectodomain of α(5)β(1) integrin in water, urea, or thiourea solutions revealed significant conformational changes of α(5)β(1) integrin in urea and thiourea solutions, in contrast to the simulation of α(5)β(1) in water. These changes lead to an unbending of the integrin structure around the genu, which may suggest activation, whereas the structures of single domains remained essentially unchanged. It is concluded that urea at high concentrations affects hepatic metabolism through direct activation of the α(5)β(1) integrin system.

Osmosensing and osmosignaling in the liver

Alterations of hepatocyte volume induced by either anisoosmotic environments or under the influence of hormones, concentrative amino acid uptake and oxidative stress are now recognized as an independent signal which contributes to the regulation of liver cell function and gene expression. Several long-known but mechanistically poorly understood effects of amino acids, which could not be related to their metabolism, such as the stimulation of glycogen synthesis or the inhibition of proteolysis are due to their effects on hepatocyte hydration, because they are quantitatively mimicked by swelling the cells in hypoosmotic media to the extent as the amino acids do. Likewise, transmembrane ion movements under the influence of hormones are an integral part of hormonal signal transduction mechanisms with alterations of cellular hydration acting as another "second messenger" of hormone action. Integrins act as osmosensors for hepatocyte swelling and trigger activation of mitogen- activated protein kinase systems as osmosignaling cascades towards choleresis and autophagy inhibition. On the contrary, hepatocyte shrinkage triggers endosomal acidification as a signal for a ceramide-dependent activation of NADPH oxidase isoenzymes, which results in an oxidative stress signal with proapoptotic effects. Disturbances of osmosignaling and osmosensing are involved in a variety of pathophysiological conditions such as insulin resistance, protein catabolic states and cholestatic liver injury. This article briefly summarizes some aspects of our own work on osmosignaling and osmosensing; for indepth surveys the reader is refered to recent reviews [1-6].

Effects of sediment sorbed linear alkylbenzene sulphonate on juveniles of the Senegal sole, Solea senegalensis: toxicity and histological indicators.

Many synthetic organic substances, including surfactants, tend to be sorbed on suspended solids and to accumulate finally on bottom sediments, where benthic communities may be exposed to them. Concentrations of Linear Alkylbenzene Sulphonates (LAS) have been detected in estuarine and coastal sediments, presenting wide concentration ranges depending on the presence of treatment facilities, hydrodynamic conditions, organic matter content etc. Senegal sole, Solea senegalensis, larvae (40 days posthatching; dph) were exposed to increasing concentrations of LAS spiked sediments, comprised between 0.37 and 880.78 mg LAS x kg(-1) during 30 days. The obtained results showed that survival of exposed larvae was not significantly affected at environmentally relevant concentrations, the LC50 value being obtained after 30 days 876.46 mg x kg(-1). However, the histological and histopathological analyses carried out in target organs revealed, that first alterations from the normal pattern were observed at concentrations of 222.66 mg x kg(-1), presenting effects such as blood extravasation and hyperplasy of the lamellar epithelium in gills, increase of inter-myotomal spaces of the skeletal musculature and edematous separation of the skin from epidermis. At the highest exposure concentrations (755.27 and 880.78 mg LAS x kg(-1)), shrinkage of hepatocytes, nuclear pycnosis and blood stagnation are observed in the liver, degeneration of pancreatic cells, reduction of hemocytopoietic tissue in the kidney and vacuolisation of intestinal enterocytes was observed at histological level, as well as severe separation of the epidermis from the underlying tissues. Simultaneously, a significant increase of the wet weight with exposure concentration was observed in the test organisms.

157. Volume regulation and mRNA expression of Aquaporine-8 in hepatocytes cold stored at 0 °C in preservation solutions

ID: mamprin Abstract No.: 158 Volume Regulation and mRNA Expression of Aaquaporine 8 in Hepatocytes Cold Stored at 0◦C in Preservation Solutions Maŕia Mamprin, Cristina Bellarosa, Silvana Petrocelli, Edgardo Guibert, Claudio Tiribelli, Joaquin Rodŕiguez a Ciencias Fisiologicas, Facultad de Ciencias Bioquimicas y Farmaceuticas. Universidad Nacional de Rosario. UNESCO Chair in Cryobiology, S2002LRK Rosario, Argentina b Ciencias Biologicas, Facultad de Ciencias Bioquimicas y Farmaceuticas. Universidad Nacional de Rosario. UNESCO Chair in Cryobiology, S2002LRK Rosario, Argentina d BBCM, Centro Studi Fegato, AREA Science Park, Basovizza SS 14 Km 136.5. University of Trieste, 34012 Trieste, Italy Different storage solutions, such as the University of Wisconsin solutions (UW) have been successfully used for the temporal maintenance of the liver of animals and humans. Many studies have demonstrated the effectiveness of cold storage of hepatocytes and successful application on Bioartificial liver devices and hepatocellular transplantation. The aim of the present study was to investigate the effect of cold storage time at 0◦C with two different preservation solutions: UW and a novel BES-based solution (BGS), on gene expression (at mRNA level for Aquaporine 8 (AQP8)) and water movements of isolated rat hepatocytes. Hepatocytes were isolated from male Wistar rats by collagenase perfusion and cold stored for 72 h at 0◦C in either UW or a 70 mM BES solution containing 50mM sucrose and 100mM potassium gluconate. Total RNA was isolated from hepatocytes taken at different time intervals. Real time RT-PCR was used to quantify the AQP8 mRNA in freshly isolated hepatocytes and after 72 h of cold storage and during a subsequent 2 h rewarming period; s-actin was used as housekeeping gene to normalize the expression of target genes. For the AQP8 (accession bank: NM 019158) we used Upper primer: 5-TTG GGG CTC ATC ATT GCT AC-3, position 333; and Lower primer: 5-AAG GCC TCC AAC CAG TGT GA-3, position 403. To evaluate the water content of cells during preservation and rewarming, the [wet weight dry weight] / dry weight of hepatocytes collected at different time intervals was determinate after 48 h desiccation at 90◦C. To monitor the evolution of hepatocyte water content (Vend / Vinitial), each volume (Vend) was normalized to the volume of hepatocyte at the corresponding time of control cells at 37◦C (Vinitial). Cold storage induced a significant hepatocyte shrinkage at 72 h: UW 0.56 ± 0.03; BCG: 0.87 ± 0.13 vs. Control 1.00 (n=10). On the contrary, no changes in expression of AQP8 at mRNA level was found During the rewarming, cells preserved in both solutions recover a volume similar to the control group (control: 1.15 ± 0.06; UW: 1.03 ± 0.09, BCG: 1.21 ± 0.12, n=10). From these data we conclude that BES-based solution is as effective as UW solution for hepatocyte preservation. The change in cell volume observed both during cold storage and after rewarming is not related to change in the mRNA levels of AQP8. Irrespective of the cell shrinkage observed at 0◦C, hepatocytes recover a normal cellular volume during the rewarming pointing to the possibility to safety preserve the cells in both solutions for further use. Conflict of interest: None declared Source of funding: Ministero degli Affari Esteri, Italy. Progetti di Grande Rilevanza Prot. 269/P/0114337. ANPCyT, Argentina, PICT 03-14492. UNESCO Chair in Cryobiology. Cryo 2006 www.cryo2006.org sputtek@uke.uni-hamburg.de

Hyperosmolarity triggers CD95 membrane trafficking and sensitizes rat hepatocytes toward CD95L-induced apoptosis

The effect of hyperosmolarity on CD95 membrane targeting and CD95 ligand (CD95L)–induced apoptosis was studied in rat hepatocytes. CD95 showed a predominant intracellular localization in normoosmotically exposed rat hepatocytes, whereas hyperosmotic exposure induced, within 1 hour, CD95 trafficking to the plasma membrane followed by activation of caspase-3 and -8. Hyperosmotic CD95 membrane targeting was sensitive to inhibition of c-Jun-N-terminal kinase (JNK), protein kinase C (PKC), and cyclic adenosine monophosphate, but not to inhibition of extracellular regulated kinases (Erks) or p38 mitogen activated protein kinase (p38 MAPK). Hyperosmotic CD95 targeting to the plasma membrane was dose-dependently diminished by glutamine or taurine, probably caused by an augmentation of volume regulatory increase. Despite CD95 trafficking to the plasma membrane and caspase activation, hyperosmolarity per se did not induce apoptosis. Hyperosmolarity, however, sensitized hepatocytes toward CD95L-induced apoptosis, as assessed by annexin V staining and terminal deoxynucleotidyl transferase-mediated X-dUTP nick-end labeling (TUNEL) assay. This sensitization was abolished when hyperosmotic CD95 membrane trafficking was prevented by cyclic adenosine monophosphate, PKC, or JNK inhibition, whereas these effectors had no effect on CD95L-induced apoptosis in normoosmotically exposed hepatocytes. CD95L addition under normoosmotic conditions caused CD95 membrane trafficking, which was sensitive to JNK inhibition, but not to cyclic adenosine monophosphate or inhibition of PKC, Erks, and p38 MAPK. In conclusion, multiple signaling pathways are involved in CD95 membrane trafficking. Hyperosmotic hepatocyte shrinkage induces CD95 trafficking to the plasma membrane, which involves JNK-, PKA-, and PKC-dependent mechanisms and sensitizes hepatocytes toward CD95L-mediated apoptosis. (H EPATOLOGY 2002;36:602-614.)

Involvement of Ca2+/calmodulin-dependent protein kinase II in the activation of carnitine palmitoyltransferase I by okadaic acid in rat hepatocytes.

The present work was undertaken to study the mechanism by which okadaic acid (OA), an inhibitor of protein phosphatases 1 and 2A, stimulates carnitine palmitoyltransferase I (CPT-I) in isolated rat hepatocytes [Guzmán, Kolodziej, Caldwell, Costorphine and Zammit (1994) Biochem. J. 300, 693-699]. The OA-induced stimulation of CPT-I was abolished by the general protein kinase inhibitor K-252a as well as by KN-62, a specific inhibitor of Ca2+/calmodulin-dependent protein kinase II (Ca2+/CM-PKII). However, neither the protein kinase C-specific inhibitor bisindolylmaleimide nor the protein kinase A/protein kinase C inhibitor H-7 was able to prevent the OA-induced stimulation of CPT-I. Hepatocyte-shrinkage-induced stimulation of CPT-I as well as OA-induced hepatocyte shrinkage was prevented by KN-62. KN-62 also antagonized the OA-enhanced release of lactate dehydrogenase from digitonin-permeabilized hepatocytes. Exposure of 32P-labelled hepatocytes to OA increased the degree of phosphorylation of Ca2+/CM-PKII, as immunoprecipitated by a monoclonal antibody raised against the alpha-subunit of rat brain kinase. This effect of OA was also antagonized by KN-62. The results thus indicate that the OA-dependent stimulation of CPT-I may be mediated (at least in part) by increased phosphorylation and subsequent activation of Ca2+/CM-PKII.

Are Cytoskeletal Components Involved in the Control of Hepatic Carnitine Palmitoyltransferase I Activity?

The present work was undertaken to test whether cytoskeletal components are involved in the control of rat-liver carnitine palmitoyltransferase I (CPT-I) activity by cellular effectors. The microtubule stabilizer taxol abolished the changes in CPT-I activity induced by the effectors tested. Taxol also prevented OA-induced shrinkage of hepatocytes as well as the enhanced release of lactate dehydrogenase from digitonin-permeabilized hepatocytes. On the basis of its relative sensitivity to tautomycin and OA, the modulation of CPT-I activity seemed to involve mostly protein phosphatase 1. These data suggest that the short-term control of hepatic CPT-I by cellular effectors may involve modulation of interactions between CPT-I and cytoskeletal components.

Evaluation of university of wisconsin cold-storage solution in warm hypoxic perfusion of rat liver: The addition of fructose reduces injury

Background : University of Wisconsin cold-storage solution (UW solution) has markedly improved organ preservation for liver transplantation. However, the efficacy of this solution in preserving hepatocyte viability during warm ischemia is undefined; hence, the effects of UW solution on warm hypoxic injury in the isolated perfused rat liver were examined. Methods : Livers were perfused using a modified protocol that included a period of hypoxic perfusion with isosmotic Krebs' solution at the end of each experiment. Hepatic injury was evaluated by aspartate aminotransferase (AST) release into the perfusate and the trypan blue perfusion technique. Results : Although UW solution appeared to decrease hepatic injury during hypoxic perfusion, as reflected by low AST release, perfusion with UW solution led to hepatocyte shrinkage and cessation of bile flow even under oxygenated conditions. UW solution did not protect against warm hypoxic injury, as assessed by AST release into the perfusate (182 ± 15 U/mL, mean ± SD) or trypan blue staining of the dead hepatocyte nuclei (56% ± 5%). However, the addition of fructose to UW solution resulted in a significant decrease in AST release (66 ± 15 U/mL) and parenchymal cell death (39% ± 7%). Conclusions : These data suggest that the addition of fructose or other gluconeogenic substrates may complement the overall hepatoprotective effects of UW solution, particularly during periods of warm hypoxia.

Changes in hepatocyte water volume affect protein synthesis.

Protein synthesis in isolated rat hepatocytes was determined from the incorporation of [3H]leucine (4 mM) into acid-precipitable material in the presence of amino acids at twice their physiological concentration. Protein synthesis increased linearly with time and incubated cell protein, and was inhibited by cycloheximide by more than 95%. In normo-osmotic incubations containing amino acids at twice the physiological concentrations the rate of [3H]leucine incorporation was 5.8 ± 0.2 nmol/h per mg cell protein (n = 26). Hyperosmotic cell shrinkage due to addition of 60 mM-NaCl or 120 mM-raffinose inhibited [3H]leucine incorporation into acid-precipitable material by 60% and 74%, respectively, whereas hypo-osmotic cell swelling was ineffective. Inhibition of protein synthesis by adding 120 mM-raffinose was largely counteracted by simultaneous lowering of the NaCl concentration by 60 mM. Glutamine (10 mM) had no effect on protein synthesis in normo-osmotic incubations (320 mosm), but stimulated protein synthesis in hyperosmotically (440 mosm) pre-shrunken cells almost to rates found in normo-osmotic (320 mosm) control incubations. Cyclic AMP and vasopressin inhibited protein synthesis by 23% and 8%, respectively, whereas insulin and phenylephrine were ineffective. However, inhibition of protein synthesis by cyclic AMP was about twice as strong in the presence of vasopressin or phenylephrine. When protein synthesis was preinhibited by cyclic AMP, [3H]leucine incorporation was stimulated by glutamine (10 mM), insulin or hypoosmotic exposure. There was a close relationship between the inhibition of protein synthesis and the extent of hepatocyte shrinkage induced by the abovementioned effectors, suggesting a role of cell volume in the regulation of hepatic protein synthesis.

Liver cell volume and protein synthesis.

Protein synthesis in isolated rat hepatocytes was determined from the incorporation of [3H]leucine (4 mM) into acid-precipitable material in the presence of amino acids at twice their physiological concentration. Protein synthesis increased linearly with time and incubated cell protein, and was inhibited by cycloheximide by more than 95%. In normo-osmotic incubations containing amino acids at twice the physiological concentration the rate of [3H]leucine incorporation was 5.8 +/- 0.2 nmol/h per mg of cell protein (n = 26). Hyperosmotic cell shrinkage due to addition of 60 mM-NaCl or 120 mM-raffinose inhibited [3H]leucine incorporation into acid-precipitable material by 60 and 74% respectively, whereas hypo-osmotic cell swelling was ineffective. Inhibition of protein synthesis by adding 120 mM-raffinose was largely counteracted by simultaneous lowering of the NaCl concentration by 60 mM. Glutamine (10 mM) had no effect on protein synthesis in normo-osmotic incubations (320 mosM), but stimulated protein synthesis in hyperosmotically (440 mosM) pre-shrunken cells almost to rates found in normo-osmotic (320 mosM) control incubations. Cyclic AMP and vasopressin inhibited protein synthesis by 23% and 8% respectively, whereas insulin and phenylephrine were ineffective. However, inhibition of protein synthesis by cyclic AMP was about twice as strong in the presence of vasopressin or phenylephrine. When protein synthesis was preinhibited by cyclic AMP, [3H]leucine incorporation was stimulated by glutamine (10 mM), insulin or hypo-osmotic exposure. There was a close relationship between the inhibition of protein synthesis and the extent of hepatocyte shrinkage induced by the above-mentioned effectors, suggesting a role of cell volume in the regulation of hepatic protein synthesis.

Cell surface changes and enzyme release during hypoxia and reoxygenation in the isolated, perfused rat liver.

We examined the effects of hypoxia and reoxygenation in isolated, perfused rat livers. Hypoxia induced by a low rate of perfusion led to near anoxia confined to centrilobular regions of the liver lobule. Periportal regions remained normoxic. Within 15 min, anoxic centrilobular hepatocytes developed surface blebs that projected into sinusoids through endothelial fenestrations. Periportal hepatocytes were unaffected. Both scanning and transmission electron microscopy suggested that blebs developed by transformation of preexisting microvilli. Upon reoxygenation by restoration of a high rate of perfusion, blebs disappeared. Other changes included marked shrinkage of hepatocytes, enlargement of sinusoids, and dilation of sinusoidal fenestrations. There was also an abrupt increase in the release of lactate dehydrogenase and protein after reoxygenation, and cytoplasmic fragments corresponding in size and shape to blebs were recovered by filtration of the effluent perfusate. We also studied phalloidin and cytochalasin D, agents that disrupt the cytoskeleton. Both substances at micromolar concentrations caused rapid and profound alterations of cell surface topography. We conclude that hepatic tissue is quite vulnerable to hypoxic injury. The morphological expression of hypoxic injury seems mediated by changes in the cortical cytoskeleton. Reoxygenation causes disappearance of blebs and paradoxically causes disruption of cellular volume control and release of blebs as cytoplasmic fragments. Such cytoplasmic shedding provides a mechanism for selective release of hepatic enzymes by injured liver tissue.