JNK activation and translocation to mitochondria mediates mitochondrial dysfunction and cell death induced by VDAC opening and sorafenib in hepatocarcinoma cells.

The multikinase inhibitor sorafenib, and opening of voltage dependent anion channels (VDAC) by the erastin-like compound X1 promotes oxidative stress and mitochondrial dysfunction in hepatocarcinoma cells. Here, we hypothesized that X1 and sorafenib induce mitochondrial dysfunction by increasing reactive oxygen species (ROS) formation and activating c-Jun N-terminal kinases (JNKs), leading to translocation of activated JNK to mitochondria. Both X1 and sorafenib increased production of ROS and activated JNK. X1 and sorafenib caused a drop in mitochondrial membrane potential (ΔΨ), a readout of mitochondrial metabolism, after 60 min. Mitochondrial depolarization after X1 and sorafenib occurred in parallel with JNK activation, increased superoxide (O2•-) production, decreased basal and oligomycin sensitive respiration, and decreased maximal respiratory capacity. Increased production of O2•- after X1 or sorafenib was abrogated by JNK inhibition and antioxidants. S3QEL 2, a specific inhibitor of site IIIQo, at Complex III, prevented depolarization induced by X1. JNK inhibition by JNK inhibitors VIII and SP600125 also prevented mitochondrial depolarization. After X1, activated JNK translocated to mitochondria as assessed by proximity ligation assays. Tat-Sab KIM1, a peptide selectively preventing the binding of JNK to the outer mitochondrial membrane protein Sab, blocked the depolarization induced by X1 and sorafenib. X1 promoted cell death mostly by necroptosis that was partially prevented by JNK inhibition. These results indicate that JNK activation and translocation to mitochondria is a common mechanism of mitochondrial dysfunction induced by both VDAC opening and sorafenib.

Combined effect of G3139 and TSPO ligands on Ca(2+)-induced permeability transition in rat brain mitochondria.

Permeability of the mitochondrial outer membrane is determined by the activity of voltage-dependent anion channels (VDAC) which are regulated by many factors and proteins. One of the main partner-regulator of VDAC is the 18 kDa translocator protein (TSPO), whose role in the regulation of membrane permeability is not completely understood. We show that TSPO ligands, 1 µM PPIX and PK11195 at concentrations of 50 µM, accelerate opening of permeability transition pores (mPTP) in Ca(2+)-overloaded rat brain mitochondria (RBM). By contrast, PK11195 at 100 nM and anti-TSPO antibodies suppressed pore opening. Participation of VDAC in these processes was demonstrated by blocking VDAC with G3139, an 18-mer phosphorothioate oligonucleotides, which sensitized mitochondria to Ca(2+)-induced mPTP opening. Despite the inhibitory effect of 100 nM PK11195 and anti-TSPO antibodies alone, their combination with G3139 considerably stimulated the mPTP opening. Thus, 100 nM PK11195 and anti-TSPO antibody can modify permeability of the VDAC channel and mPTP. When VDAC channels are closed and TSPO is blocked, permeability of the VDAC for calcium seems to be the highest, which leads to accelerated pore opening.

Amphiregulin stimulates liver regeneration after small-for-size mouse liver transplantation.

This study investigated whether amphiregulin (AR), a ligand of the epidermal growth factor receptor (EGFR), improves liver regeneration after small-for-size liver transplantation. Livers of male C57BL/6 mice were reduced to ~50% and ~30% of original sizes and transplanted. After transplantation, AR and AR mRNA increased in 50% but not in 30% grafts. 5-Bromodeoxyuridine (BrdU) labeling, proliferating cell nuclear antigen (PCNA) expression and mitotic index increased substantially in 50% but not 30% grafts. Hyperbilirubinemia and hypoalbuminemia occurred and survival decreased after transplantation of 30% but not 50% grafts. AR neutralizing antibody blunted regeneration in 50% grafts whereas AR injection (5 µg/mouse, iv) stimulated liver regeneration, improved liver function and increased survival after transplantation of 30% grafts. Phosphorylation of EGFR and its downstream signaling molecules Akt, mTOR, p70S6K, ERK and JNK increased markedly in 50% but not 30% grafts. AR stimulated EGFR phosphorylation and its downstream signaling pathways. EGFR inhibitor PD153035 suppressed regeneration of 50% grafts and largely abrogated stimulation of regeneration of 30% grafts by AR. AR also increased cyclin D1 and cyclin E expression in 30% grafts. Together, liver regeneration is suppressed in small-for-size grafts, as least in part, due to decreased AR formation. AR supplementation could be a promising therapy to stimulate regeneration of partial liver grafts.

[The role of the voltage-dependent anion channels in the outer membrane of mitochondria in the regulation of cellular metabolism].

The role of the voltage-dependent anion channels (VDAC) harbored in the outer membrane of mitochondria in the regulation of cellular metabolism was investigated using an experimental model of ethanol toxicity in cultured hepatocytes. It was demonstrated that ethanol inhibits State 3 and uncoupled mitochondrial respirations, decreases the accessibility of mitochondrial adenylate kinase localized in the intermembrane space of mitochondria, and suppresses ureagenic respiration and synthesis of urea in cultured hepatocytes. Increasing the permeability of the outer mitochondrial membrane with closed VDAC with high concentrations of digitonin (> 80 microM), which creates pores in the membrane, allowing the alternative bypass of closed VDAC, and restores all reactions suppressed with ethanol. It is concluded that the effect of ethanol in hepatocytes leads to global loss of mitochondrial functions due to the closure of VDAC, which limits the free diffusion of metabolites into the intermembrane space of mitochondria. Our studies demonstrated that ethanol affects the main mitochondrial functions and revealed the role of VDAC channels in the outer mitochondrial membrane in the regulation of liver specific intracellular processes such as ureagenesis. The data obtained can be used for the development of pharmaceutical drugs that prevent the closure of VDAC in mitochondria of ethanol oxidizing liver, thus protecting liver tissue from the hepatotoxic action of alcohol.

C-Jun N-terminal kinase 2 promotes graft injury via the mitochondrial permeability transition after mouse liver transplantation.

The c-Jun N-terminal kinase (JNK) pathway enhances graft injury after liver transplantation (LT). We hypothesized that the JNK2 isoform promotes graft injury via the mitochondrial permeability transition (MPT). Livers of C57BL/6J (wild-type, WT) and JNK2 knockout (KO) mice were transplanted into WT recipients after 30 h of cold storage in UW solution. Injury after implantation was assessed by serum ALT, histological necrosis, TUNEL, Caspase 3 activity, 30-day survival, and cytochrome c and 4-hydroxynonenal immunostaining. Multiphoton microscopy after LT monitored mitochondrial membrane potential in vivo. After LT, ALT increased three times more in WT compared to KO (p < 0.05). Necrosis and TUNEL were more than two times greater in WT than KO (p < 0.05). Immunostaining showed a >80% decrease of mitochondrial cytochrome c release in KO compared to WT (p < 0.01). Lipid peroxidation was similarly decreased. Every KO graft but one survived longer than all WT grafts (p < 0.05, Kaplan-Meier). After LT, depolarization of mitochondria occurred in 73% of WT hepatocytes, which decreased to 28% in KO (p < 0.05). In conclusion, donor JNK2 promotes injury after mouse LT via the MPT. MPT inhibition using specific JNK2 inhibitors may be useful in protecting grafts against adverse outcomes from ischemia/reperfusion injury.

NIM811, a mitochondrial permeability transition inhibitor, prevents mitochondrial depolarization in small-for-size rat liver grafts.

ATP decreases markedly in small-for-size liver grafts. This study tested if the mitochondrial permeability transition (MPT) underlies dysfunction of small-for-size livers. Half-size livers were implanted into recipients of about twice the donor weight, resulting in quarter-size liver grafts. NIM811 (5 microM), a nonimmunosuppressive MPT inhibitor was added to the storage solutions. Mitochondrial polarization and cell death were assessed by confocal microscopy of rhodamine 123 (Rh123) and propidium iodide (PI), respectively. After quarter-size transplantation, alanine aminotransferase (ALT), serum bilirubin and necrosis all increased. NIM811 blocked these increases by >70%. After 38 h, BrdU labeling, a marker of cell proliferation and graft weight increase were 3% and 5%, respectively, which NIM811 increased to 30% and 42%. NIM811 also increased survival of quarter-size grafts. In sham-operated livers, hepatocytes exhibited punctate Rh123 fluorescence. By contrast, in quarter-size grafts at 18 h after implantation, mitochondria of most hepatocytes did not take up Rh123, indicating mitochondrial depolarization. Nearly all hepatocytes not taking up Rh123 continued to exclude PI at 18 h, indicating that depolarization preceded cell death. NIM811 and free radical-scavenging polyphenols strongly attenuated mitochondrial depolarization. In conclusion, mitochondria depolarized after quarter-size liver transplantation. NIM811 decreased injury and stimulated regeneration, probably by inhibiting free radical-dependent MPT onset.

Endothelial nitric oxide synthase protects transplanted mouse livers against storage/reperfusion injury: Role of vasodilatory and innate immunity pathways.

Endothelial nitric oxide synthase (eNOS) plays a role in microcirculatory and immunomodulatory responses after warm ischemia/reperfusion. We hypothesized that eNOS is essential to maintain microcirculation, attenuate macrophage infiltration and decrease graft injury after liver transplantation. Liver transplantation was performed after 18 hours of cold storage in University of Wisconsin (UW) solution from wildtype and eNOS-deficient (B6.129P2-Nos3(tm/Unc)/J) donor mice into wildtype mice. Serum ALT, necrosis by histology, apoptosis by TUNEL, and macrophage infiltration by immunostaining against F4/80 antigen were determined 2 to 8 hours after implantation. Hepatic microcirculation was investigated after 4 hours by intravital confocal microscopy following injection of fluorescein-labeled erythrocytes. After sham operation, livers of wildtype and eNOS-deficient mice were not different in ALT, necrosis, apoptosis, macrophage infiltration, and microcirculation. After transplantation, ALT increased >3 times more after transplantation of eNOS-deficient livers than wildtype livers. Necrosis was >4 times greater, and TUNEL and F4/80 immunostaining in nonnecrotic areas were 2 and 1.5 times greater in eNOS-deficient donor livers, respectively. Compared with wildtype and eNOS sham-operated mice, sinusoidal blood flow velocity increased 1.6-fold after wildtype transplantation, but sinusoidal diameter was not changed. After transplantation of eNOS-deficient livers, blood flow velocity and sinusoidal diameter decreased compared with transplanted wildtype livers. These results indicate that donor eNOS attenuates storage/reperfusion injury after mouse liver transplantation. Protection is associated with improved microcirculation and decreased macrophage infiltration. Thus, eNOS-dependent graft protection may involve both vasodilatory and innate immunity pathways.

Primary cirrhotic hepatocytes resist TGFbeta-induced apoptosis through a ROS-dependent mechanism.

BACKGROUND/AIMS: The cirrhotic liver manifests dysregulated hepatocyte growth by poor regenerative capacity, formation of regenerative nodules, and malignant transformation to hepatocellular carcinoma. The purpose of this study was to determine if dysregulated hepatocyte growth occurs through deficient apoptosis. METHODS: Hepatocytes were isolated from normal and CCl(4)-treated mice and treated with TGFbeta, TNFalpha, and UV-C, known apoptotic agents. RESULTS: Cirrhotic hepatocytes were less sensitive to TGFbeta- (45+/-5 vs. 15+/-3%; P<0.003), TNFalpha- (59+/-21 vs. 21+/-8%; P=0.02), and UV-C-induced (31+/-4 vs. 17+/-4%; P<0.03) apoptosis compared to normal hepatocytes. In normal hepatocytes, TGFbeta-induced apoptosis occurred through a ROS-, MPT-, and caspase-dependent pathway. Cirrhotic hepatocytes lacked caspase activation, had decreased procaspase-8 expression, failed to undergo the MPT, and had increased basal ROS activity compared to normal hepatocytes. After treatment with trolox, an antioxidant that reduced basal ROS activity, cirrhotic hepatocytes underwent apoptosis in response to TGFbeta treatment. CONCLUSIONS: These findings suggest that increased ROS activity in cirrhotic hepatocytes plays a critical role in mediating cirrhotic hepatocyte resistance to apoptosis. Cirrhotic hepatocyte resistance to TGFbeta-induced apoptosis is ROS-dependent and is a mechanism of dysregulated growth in the chronically inflamed liver.

Role of the mitochondrial permeability transition in apoptotic and necrotic death after ischemia/reperfusion injury to hepatocytes.

Reperfusion of ATP-depleted tissues after warm or cold ischemia causes pH-dependent necrotic and apoptotic cell death. In hepatocytes and other cell types as well, the mechanism underlying this reperfusion-induced cell death involves onset of the mitochondrial permeability transition (MPT). Opening of permeability transition (PT) pores in the mitochondrial inner membrane initiates the MPT, an event blocked by cyclosporin A (CsA) and pH less than 7.4. Thus, both acidotic pH and CsA prevent MPT-dependent reperfusion injury. Glycine also blocks reperfusion-induced necrosis but acts downstream of PT pore opening by stabilizing the plasma membrane. After the MPT, ATP availability from glycolysis or other source determines whether cell injury after reperfusion progresses to ATP depletion-dependent necrosis or ATP-requiring apoptosis. Thus, apoptosis and necrosis after reperfusion share a common pathway, the MPT. Cell injury progressing to either necrosis or apoptosis by shared pathways can be more aptly termed necrapoptosis.

Electrical properties and conduction in reperfused papillary muscle.

The reversibility of ischemia-induced changes of extracellular K(+) concentration ([K(+)](o)), resting membrane potential (E(M)), and passive cable-like properties, ie, extracellular resistance and cell-to-cell electrical coupling, and their relationship to recovery of conduction and contraction is described in 25 reperfused rabbit papillary muscles. No-flow ischemia caused extracellular K(+) accumulation, depolarization of E(M), an increase in whole-tissue (r(t)), external (r(o)), and internal (r(i)) longitudinal resistances, and failure of conduction and contraction. Muscles were reperfused 10 minutes after the onset of ischemia related cell-to-cell electrical uncoupling, ie, 26+/-1 minutes after arrest of perfusion. In 11 muscles, incomplete reflow occurred with only partial recovery of [K(+)](o) and r(t). In the remaining 14 muscles, reperfusion caused a rapid and parallel decrease in [K(+)](o), r(t), and r(o). When complete tissue reperfusion occurred, cell-to-cell electrical uncoupling was largely reversible. Thus, cell-to-cell electrical uncoupling did not indicate irreversible injury. Reperfusion induced a depolarizing current widening the difference between the K(+) equilibrium potential and the E(M). This difference decreased after longer periods of reperfusion. Conduction was restored and conduction velocity approached preischemic values as cell-to-cell electrical interaction was reestablished and E(M) recovered. The recovery of r(o) preceded r(i), decreasing the ratio of the extracellular to intracellular resistance early in reperfusion, an effect predicted to influence the amplitude of the extracellular voltage field and electrocardiographic ST segments during reperfusion.

The mitochondrial permeability transition initiates autophagy in rat hepatocytes.

Cells degrade excess and effete organelles by the process of autophagy. Autophagic stimulation of rat hepatocytes by serum deprivation and glucagon (1 M) caused a fivefold increase of spontaneously depolarizing mitochondria to about 1.5% of total mitochondria after 90 min. Cyclosporin A (CsA, 5 M), an immunosuppressant that blocks the mitochondrial permeability transition (MPT), prevented this depolarization. Depolarized mitochondria moved into acidic vacuoles labeled by LysoTracker Red. These autophagosomes also increased several-fold after autophagic stimulation. CsA blocked autophagosomal proliferation, whereas tacrolimus, an immunosuppressant that does not block the MPT, did not. In conclusion, the MPT initiates mitochondrial depolarization after autophagic stimulation and the subsequent sequestration of mitochondria into autophagosomes.

Glycine blocks opening of a death channel in cultured hepatic sinusoidal endothelial cells during chemical hypoxia.

Using confocal microscopy, we investigated mechanisms underlying loss of plasma membrane integrity during necrotic death of cultured hepatic sinusoidal endothelial cells exposed to 2.5 mM potassium cyanide (chemical hypoxia). After 2-3 h, the anionic fluorophore calcein abruptly began to enter the cytosol, and nuclei labeled with cationic propidium after another 2-5 min. As calcein permeated, growth of blebs on the plasma membrane accelerated. Lucifer yellow, another anionic fluorophore, entered identically to calcein, whereas high molecular weight dextrans (40-2000 kDa) entered like propidium. Glycine slowed, but did not prevent calcein entry, whereas permeation of propidium and high molecular weight dextrans was blocked completely by glycine. These findings suggest that opening of a glycine-sensitive organic anion channel, or death channel, precipitates a metastable state characterized by rapid cell swelling and bleb growth. This metastable state culminates in non-specific breakdown of the plasma membrane permeability barrier and irreversible cell death.

Screening assays for the mitochondrial permeability transition using a fluorescence multiwell plate reader.

Opening of permeability transition (PT) pores in the mitochondrial inner membrane causes the mitochondrial permeability transition (MPT) and leads to mitochondrial swelling, membrane depolarization, and release of intramitochondrial solutes. Here, our aim was to develop high-throughput assays using a fluorescence plate reader to screen potential inducers and blockers of the MPT. Isolated rat liver mitochondria (0.5 mg/ml) were incubated in multiwell plates with tetramethylrhodamine methyl ester (TMRM, 1 microM), a potential-indicating fluorophore, and Fluo-5N (1 microM), a low-affinity Ca(2+) indicator. Incubation led to mitochondrial polarization, as indicated by uncoupler-sensitive quenching of the red TMRM fluorescence. CaCl(2) (100 microM) addition led to ruthenium red-sensitive mitochondrial Ca(2+) uptake, as indicated by green Fluo-5N fluorescence. After Ca(2+) accumulation, mitochondria depolarized, released Ca(2+) into the medium, and began to swell. This swelling was monitored as a decrease in light absorbance at 620 nm. Swelling, depolarization, and Ca(2+) release were prevented by cyclosporin A (1 microM), confirming that these events represented the MPT. Measurements of Ca(2+), mitochondrial membrane potential, and swelling could be made independently from the same wells without cross interference, and all three signals could be read from every well of a 48-well plate in about 1 min. In other experiments, mitochondria were ester-loaded with carboxydichlorofluorescein (carboxy-DCF) during the isolation procedure. Release of carboxy-DCF after PT pore opening led to an unquenching of green carboxy-DCF fluorescence occurring simultaneously with swelling. By combining measurements of carboxy-DCF release, Ca(2+) uptake, membrane potential, and swelling, MPT inducers and blockers can be distinguished from uncouplers, respiratory inhibitors, and blockers of Ca(2+) uptake. This high-throughput multiwell assay is amenable for screening panels of compounds for their ability to promote or block the MPT.

Activated Kupffer cells cause a hypermetabolic state after gentle in situ manipulation of liver in rats.

Harvesting trauma to the graft dramatically decreases survival after liver transplantation. Since activated Kupffer cells play a role in primary nonfunction, the purpose of this study was to test the hypothesis that organ manipulation activates Kupffer cells. To mimic what occurs with donor hepatectomy, livers from Sprague-Dawley rats underwent dissection with or without gentle organ manipulation in a standardized manner in situ. Perfused livers exhibited normal values for O(2) uptake (105 +/- 5 micromol. g(-1). h(-1)) measured polarigraphically; however, 2 h after organ manipulation, values increased significantly to 160 +/- 8 micromol. g(-1). h(-1) and binding of pimonidazole, a hypoxia marker, increased about threefold (P < 0.05). Moreover, Kupffer cells from manipulated livers produced three- to fourfold more tumor necrosis factor-alpha and PGE(2), whereas intracellular calcium concentration increased twofold after lipopolysaccharide compared with unmanipulated controls (P < 0.05). Gadolinium chloride and glycine prevented both activation of Kupffer cells and effects of organ manipulation. Furthermore, indomethacin given 1 h before manipulation prevented the hypermetabolic state, hypoxia, depletion of glycogen, and release of PGE(2) from Kupffer cells. These data indicate that gentle organ manipulation during surgery activates Kupffer cells, leading to metabolic changes dependent on PGE(2) from Kupffer cells, which most likely impairs liver function. Thus modulation of Kupffer cell function before organ harvest could be beneficial in human liver transplantation and surgery.

Alteration in Kupffer cell function after mild hemorrhagic shock.

Functional changes in Kupffer cells occur after profound hemorrhagic shock. This study was performed to demonstrate if Kupffer cell changes also occur after mild hemorrhagic shock. Sprague-Dawley rats were bled to a systolic blood pressure of 60 to 70 mmHg and resuscitated with Lactated Ringers solution (twice the shed blood volume) after 30 min. Resuscitation produced immediate recovery of blood pressure and allowed long-term recovery of the animals. Sham animals received anesthesia and monitoring only. Thirty minutes after resuscitation, Kupffer cells were isolated by centrifugal elutriation and cultured for 48 h. In Kupffer cells isolated from shocked animals, phorbol ester-stimulated superoxide production increased 7-fold and lipopolysaccharide- (LPS) stimulated prostaglandin E2 (PGE2) production increased 4-fold. Tumor necrosis factor-alpha (TNFalpha) production, on the other hand, was decreased by 50%. A non-significant trend toward increased phagocytosis was also observed, whereas LPS-stimulated nitric oxide production was unchanged. In conclusion, mild hemorrhagic shock produced increases in superoxide and PGE2 production, and decreases in TNFalpha production by Kupffer cells, changes that may be appropriate to defend against the infectious challenges that often follows trauma and hemorrhage.

Ischemic preconditioning of rat livers against cold storage-reperfusion injury: role of nonparenchymal cells and the phenomenon of heterologous preconditioning.

Brief periods of ischemia followed by reperfusion render tissues resistant against subsequent prolonged ischemia, a phenomenon called ischemic preconditioning. The effect of ischemic preconditioning on liver transplantation was investigated in relation to sinusoidal endothelial cell injury and Kupffer-cell activation, which are prominent features of storage and reperfusion injury leading to liver graft failure. Rat livers were preconditioned by 5 or 10 minutes of ischemia and 5 minutes of reperfusion and stored in University of Wisconsin (UW) solution for 30 hours. Livers were then reperfused for 15 minutes with physiological buffer containing trypan blue. Under these conditions, injury occurs predominantly to sinusoidal endothelial cells, reflected by trypan blue staining of nonparenchymal cells in histological sections. Ischemic preconditioning decreased nonparenchymal cell killing by more than 50%. When half the liver was preconditioned, sinusoidal endothelial cells were also protected in the contralateral half. Other stored livers were reperfused with nitroblue tetrazolium, which is converted to insoluble formazan by superoxide radicals. Ischemic preconditioning decreased the intensity of formazan deposition over Kupffer cells. Finally, stored livers were transplanted into nontreated rats. Ischemic preconditioning improved recipient long-term survival after 30 hours of cold ischemic storage in UW solution from 30% to 80% and decreased serum tumor necrosis factor-alpha levels in posthepatic blood 4 hours postoperatively from 98 to 54 pg/mL. In conclusion, ischemic preconditioning protects sinusoidal endothelial cells and suppresses Kupffer-cell activation after storage and reperfusion. As a result, graft survival improves after liver transplantation. Moreover, ischemia to half the liver confers protection to the other half. Such heterologous preconditioning provides a new means to protect liver tissue against ischemia-reperfusion injury without imposing ischemia on the target tissue.

NF-kappaB stimulates inducible nitric oxide synthase to protect mouse hepatocytes from TNF-alpha- and Fas-mediated apoptosis.

BACKGROUND AND AIMS: Hepatocyte apoptosis is induced by tumor necrosis factor alpha (TNF-alpha) and Fas ligand. Although nuclear factor-kappaB (NF-kappaB) activation protects hepatocytes from TNF-alpha-mediated apoptosis, the NF-kappaB responsive genes that protect hepatocytes are unknown. Our aim was to study the role of NF-kappaB activation and inducible nitric oxide synthases (iNOSs) in TNF-alpha- and Fas-mediated apoptosis in hepatocytes. METHODS: Primary cultures of hepatocytes from wild-type and iNOS knockout mice were treated with TNF-alpha, the Fas agonistic antibody Jo2, a nitric oxide (NO) donor (S-nitroso-N-acetylpenicillamine), an NO inhibitor (N(G)-methyl-L-arginine acetate), and/or adenovirus-expressing NF-kappaB inhibitors. RESULTS: The IkappaB superrepressor and a dominant-negative form of IkappaB kinase beta (IKKbeta) inhibited NF-kappaB binding activity by TNF-alpha or Jo2 and sensitized hepatocytes to TNF-alpha- and Jo2-mediated apoptosis. TNF-alpha and Jo2 induced iNOS messenger RNA and protein levels through the induction of NF-kappaB. S-nitroso-N-acetylpenicillamine inhibited Bid cleavage, the mitochondrial permeability transition, cytochrome c release, and caspase-8 and -3 activity, and reduced TNF-alpha- and Fas-mediated death in hepatocytes expressing IkappaB superrepressor. N(G)-methyl-L-arginine acetate partially sensitized hepatocytes to TNF-alpha- and Fas-mediated cell killing. TNF-alpha alone or Jo2 alone induced moderate cell death in hepatocytes from iNOS(-)/(-) mice. CONCLUSIONS: NO protects hepatocytes from TNF-alpha- and Fas-mediated apoptosis. Endogenous iNOS, which is activated by NF-kappaB via IKKbeta, provides partial protection from apoptosis.

Proinflammatory kupffer cell alterations after femur fracture trauma and sepsis in rats.

This study examined effects of trauma and sepsis on Kupffer cell function. When CBA/J mice had femur fracture (FFx), no deaths occurred. After cecal ligation and puncture (CLP), 44% died. Following combined injuries (FFx + CLP), mortality increased to 60%, suggesting a deleterious effect between FFx + CLP. Kupffer cell ablation with GdCI3 decreased mortality to 13% after CLP and 5% after FFx + CLP. After FFx, CLP, and FFx + CLP, Kupffer cells isolated from Sprague-Dawley rats produced 720%, 1,100%, and 2,130% more O2. than sham, respectively. Phagocytosis increased 320%, 610%, and 150%. Kupffer cell PGE2 production also increased 300%, 510%, and 300% over sham. After FFx alone, TNF-alpha production decreased 40%. By contrast, CLP and FFx + CLP increased TNF-alpha release 25% and 100%, respectively. After FFx, NO. production decreased 44%, whereas NO increased 280% and 260% after CLP and FFx + CLP. These findings indicate that Kupffer cells mediate mortality after CLP and FFx + CLP. Increased mortality is associated with a more proinflammatory and less antimicrobial Kupffer cell phenotype.

Kupffer cells mediate increased anoxic hepatocellular killing from hyperosmolarity by an oxygen- and prostaglandin-independent mechanism.

The purpose of this study was to evaluate the roles of Kupffer cells, prostaglandin biosynthesis, and glycolytic metabolism in accelerated anoxic cell killing by hyperosmolar stress. Isolated rat livers were perfused with anoxic normosmolar Krebs-Heinseleit bicarbonate buffer (KHB) or anoxic hyperosmolar KHB (+40 mM NaCl). Hyperosmolar KHB accelerated LDH release during anoxia in livers from both fed and fasted rats by as much as 3.7-fold. GdCl(3) pretreatment to inactivate Kupffer cells substantially delayed anoxic LDH release during normosmolar perfusions and blocked entirely the hyperosmolarity-induced acceleration of LDH release. Cyclooxygenase inhibition with indomethacin failed to alter LDH release during anoxia in hyperosmolar KHB. Neither GdCl(3) nor hyperosmolarity changed glycolytic flux during hypoxia, and hyperosmolarity did not change basal oxygen uptake. We conclude that accelerated cell killing in hyperosmolar buffer is a Kupffer cell-dependent event that is independent of oxygen-requiring prostaglandin synthesis, changes of glycolytic flux, and activation of cellular ATP demand. Another as yet unidentified Kupffer cell product appears to mediate the effect of hyperosmolarity of anoxic hepatocellular injury.