Identification and preliminary validation of novel biomarkers of acute hepatic ischaemia/reperfusion injury using dual-platform proteomic/degradomic approaches.

Hepatic ischaemia/reperfusion (I/R), a major cause of liver damage associated with multiple trauma, haemorrhagic and septic shock, and liver transplantation, contributes significantly to multiple organ failure. Development of novel sensitive biomarkers that detect early stages of liver damage is vital for effective management and treatment of ischaemic liver injury. By using high-throughput immunoblotting and cation-anion exchange chromatography/reversed-phase liquid chromatography-tandem mass-spectrometry, we identified several hepatic proteins, including argininosuccinate synthase (ASS) and estrogen sulfotransferase (EST-1), which were degraded in the liver and rapidly released into circulation during I/R injury. ASS accumulated in serum within 10 min, reached a steady state at 30 min, and persisted up until 3 h after reperfusion following 30 min of total hepatic ischaemia. EST-1 appeared rapidly in blood and attained maximum within 1 hour followed by a decline at 3 h of reperfusion. No ASS or EST-1 protein was detected in serum of control or sham operated rats. ASS and EST-1 exhibited greater sensitivity and specificity toward I/R liver injury as compared with alanine aminotransferase (ALT), an established marker of hepatocellular necrosis. In contrast, serum ASS and EST-1 were undetectable in rats with chronic alcoholic liver disease, while the levels of ALT protein were significantly increased. In addition, ASS, but not EST-1 or ALT accumulated in blood only 6 h after treatment with hepatotoxic combination of lipopolysaccharide and D-galactosamine. These data demonstrate the utility of ASS and EST-1 as novel sensitive and specific biomarkers of acute liver ischaemic injury for prospective clinical studies.

Enhancement of survival by LPA via Erk1/Erk2 and PI 3-kinase/Akt pathways in a murine hepatocyte cell line.

First published September 5, 2001; 10.1152/ajpcell.00077.2001.-Protective mechanisms for lysophosphatidic acid (LPA) against cell death caused by Clostridium difficile toxin, or tumor necrosis factor-alpha (TNF-alpha) plus D-galactosamine, were investigated in a murine hepatocyte cell line AML12 expressing Edg2 LPA receptor. In these models of hepatocellular injury, LPA prevented hepatocyte damage, suppressed apoptosis, and enhanced cell survival in a dose-dependent fashion. The protective effects of LPA were abolished by wortmannin and LY-294002, specific inhibitors of phosphatidylinositol 3-phosphate kinase (PI 3-kinase), and by PD-98059 and U-0126, inhibitors of MEK1/MEK2. In nontreated hepatocytes, LPA elicited a gradual and sustained increase in phosphorylation of Erk1/Erk2 over 180 min of stimulation and downstream phosphorylation of p90RSK and transcription factor Elk-1. In C. difficile toxin-treated cells, LPA-induced phosphorylation of Erk1/Erk2 was rapid but transient, while p90RSK and Elk-1 phosphorylation did not change significantly. LPA stimulated phosphorylation of Akt in a time-dependent manner in both intact and toxin-treated AML12 hepatocytes. Wortmannin and LY-294002 abolished phosphorylation of Akt, further supporting activation of PI 3-kinase/Akt as a signaling pathway, which mediates hepatocyte protection by LPA. Taken together, these results demonstrate that LPA prevents cell apoptosis induced by C. difficile toxin and TNF-alpha/D-galactosamine in the AML12 murine hepatocyte cell line. Cell protection by LPA involves activation of the mitogen-activated protein kinase Erk1/Erk2 cascade and PI 3-kinase-dependent phosphorylation of Akt.

Endotoxin-stimulated macrophages decrease bile acid uptake in WIF-B cells, a rat hepatoma hybrid cell line.

Endotoxemia leads to cytokine-mediated alterations of the hepatocellular sodium-taurocholate-cotransporting polypeptide (ntcp). We hypothesized that stimulated macrophages are essential transducers for down-regulating hepatocellular bile salt uptake in response to endotoxin (lipopolysaccharide [LPS]) exposure. Using an in vitro model, we exposed mouse macrophages (IC-21 cell line) to LPS for 24 hours. Concentrations of cytokines tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1beta, and IL-6 increased 10.6-fold, 12.5-fold, and 444-fold, respectively, in LPS-conditioned IC-21 medium (CM) versus unconditioned IC-21 medium (UM). WIF-B rat hepatoma hybrid cells were incubated with either CM or UM or treated directly with medium containing recombinant TNF-alpha, IL-1beta, and IL-6. [(3)H]Taurocholate ([(3)H]TC) uptake decreased in WIF-B cells exposed to either TNF-alpha (54% of control), IL-1beta (78%), IL-6 (55%) as single additives, or in triple combination (TCC) (43%). A virtually identical decrease was observed after exposing WIF-B cells to CM (52%, P <.001). LPS had no direct effect on [(3)H]TC uptake. CM treatment did not decrease L-alanine transport in WIF-B cells. Blocking antibodies against TNF-alpha, IL-1beta, and IL-6 restored the diminished [(3)H]TC uptake in cells exposed to TCC and CM to 87% and 107% of controls, respectively. Northern blotting revealed that ntcp messenger RNA (mRNA) expression was significantly reduced in WIF-B cells after exposure to CM, and in primary rat hepatocytes exposed to CM or TNF-alpha (68%, 14%, and 29% of control, respectively). We conclude that macrophages and their ability to secrete the cytokines TNF-alpha, IL-1beta, and IL-6 may be essential in mediating the endotoxin-induced cholestatic effect of decreased hepatocellular bile salt uptake.

Hepatic regulation of platelet-activating factor acetylhydrolase and lecithin:cholesterol acyltransferase biliary and plasma output in rats exposed to bacterial lipopolysaccharide.

Normal rat bile contains secretory platelet-activating factor acetylhydrolase (PAF-AH), the enzyme capable of hydrolyzing the inflammatory mediator platelet-activating factor (PAF), and phospholipids containing oxidized truncated fatty acids. Because lecithin:cholesterol acyltransferase (LCAT) possesses intrinsic PAF-AH-like activity, it also may represent a potential anti-inflammatory enzyme. The behavior of PAF-AH and LCAT in hepatobiliary inflammatory responses in vivo has not been characterized. We therefore investigated the biliary and plasma secretion and pharmacological characteristics of these enzymes in rats subjected to intraportal bacterial endotoxin exposure (lipopolysaccharide [LPS], Escherichia coli, 055:B5). Portal vein LPS infusion (1 mg/kg, bolus) resulted in a maximal 4- to 5-fold increase in bile PAF-AH-specific activity with a gradual decline to baseline by 18 hours. Biliary PAF-AH hydrolyzed also the truncated sn-2-succinoyl and sn-2-glutaroyl analogs of PAF, indicating a broader activity of PAF-AH in bile toward byproducts of glycerophospholipid peroxidation. Plasma PAF-AH activity was not altered 5 hours after LPS injection compared with saline injection, but it was significantly elevated 18 hours after endotoxin exposure. The levels of LCAT in bile were low and declined to nearly undetectable values by 5 hours after cannulation in both control and LPS-exposed rats. Plasma LCAT activity was significantly increased after 5 hours and decreased 18 hours after LPS injection. In summary, hepatic exposure to endotoxin results in a rapid increase in biliary secretion of PAF-AH followed by elevation of LCAT and PAF-AH levels in plasma. We propose that biliary secretion of PAF-AH may be involved in the hepatic response to endotoxic insult by counteracting potential inflammatory damage in the biliary tree and gastrointestinal tract, whereas plasma increases in LCAT and PAF-AH may promote elimination of excess PAF and oxidized phospholipids in the circulation.

Decreased intraplatelet Ca2+ release and ATP secretion in pediatric nephrotic syndrome.

Platelets play an important role in the natural history of idiopathic nephrotic syndrome (NS). Although thromboembolic events are rare, the activation of circulating platelets is generally considered an important factor in the prethrombotic state in children with NS. Platelet-activating factor (PAF), a potent endogenous phospholipid mediator of inflammation, stimulates intracellular free calcium (Ca2+) mobilization, aggregation, and release reactions in platelets obtained from normal donors. Platelet-related effects of PAF in children with NS are unknown. We studied PAF-induced intracellular Ca2+ mobilization in washed platelets and ATP secretion in platelet-rich plasma in 34 children with idiopathic NS and in 7 healthy children. There was a significant decrease in ATP secretion: 0.021+/-0.011 microg/10(7) cells with 20 nM PAF and 0.089+/-0.017 microg/10(7) platelets with 200 nM PAF versus control values (0.195+/-0.004 microg/10(7) and 0.813+/-0.09 microg/10(7), respectively). Moreover, PAF-evoked increase in intracellular free Ca2+ concentration was twofold lower in NS patients than in control subjects (230.1+/-22.4 nM versus 455.6+/-15.3 nM). Also, thrombin-induced intracellular free Ca2+ mobilization was diminished in children with NS compared with the control group. Thus, contrary to expectations, a decrease of platelet reactivity in response to PAF in vitro was observed in children with idiopathic NS. We suggest that this decreased platelet reactivity may reflect a period refractory to PAF and may be considered as platelet desensitization to PAF released in vivo in children with NS.

Depressed plasma platelet-activating factor acetylhydrolase in patients presenting with acute myocardial infarction.

Cell membrane phospholipids, including platelet-activating factor (PAF), participate in the pathogenesis of acute myocardial infarction (AMI). The plasma level of PAF acetylhydrolase (AH) was determined in 18 patients at presentation with AMI before thrombolysis, and the administration of adjunctive therapy, and compared with 13 healthy controls. Plasma levels of PAF-AH were significantly lower in the AMI patients (23.15 +/- 1.75 nmol/min/ml) than in the controls (30.43 +/- 2.13 nmol/min/ml; p = 0.027). Considering normal plasma levels of PAF and lyso-PAF, and lack of evidence that anti-PAF antibodies are really beneficial in myocardial ischemia-reperfusion, it is reasonable to speculate that an inability of systemic PAF to 'turn on' PAF-AH enzymatic activity could contribute substantially to the observed events. Decreased PAF-AH activity in AMI patients may represent not a consequence, but rather, a risk factor for the development of acute coronary syndromes.

Secretory PAF-acetylhydrolase of the rat hepatobiliary system: characterization and partial purification.

Hepatocytes and Kupffer cells in primary culture both secrete plasma-type platelet-activating factor-acetylhydrolase (pPAF-AH) into serum-free culture medium. The rate of secretion of pPAF-AH by Kupffer cells was 20 to 25 times higher than from hepatocytes, and Kupffer cells expressed a higher level of pPAF-AH mRNA than did hepatocytes. Purified liver cell-secreted pPAF-AH exhibited a major protein band of 65-67 kDa on SDS-PAGE; this was the band predominantly labeled when the enzyme catalytic center was reacted with [3H]diisopropylfluorophosphate ([3H]DFP). Rat bile collected from cannulated bile ducts contained significant PAF-AH activity, and bile samples possessed a prominent band at 30-32 kDa, which was the exclusive target for [3H]DFP. Experiments using tunicamycin, an inhibitor of N-linked glycosylation, and endoglycosidase H suggested that pPAF-AH secreted constitutively by cultured hepatocytes and Kupffer cells is glycosylated. The present study supports the notion that hepatic secretion of pPAF-AH into the blood contributes to the regulation of PAF and oxidized phospholipid levels in the circulation, whereas secretion of PAF-AH into the bile may allow hepatic control of these phospholipid signaling molecules in the gastrointestinal tract.

Regulation of platelet-activating factor (PAF) biosynthesis via coenzyme A-independent transacylase in the macrophage cell line IC-21 stimulated with lipopolysaccharide.

The regulation of PAF synthesis by the macrophage cell line IC-21 challenged with bacterial endotoxin was investigated. The LPS-induced increase in cellular PAF levels was rapid, sustained and attained maximal levels within 30 min following LPS stimulation. PAF accumulation was accompanied by the activation of the CoA-independent transacylase and acetyl-CoA: lyso-PAF acetyltransferase, whereas the release of free [3H]arachidonic acid in prelabeled cells reflecting the activation of phospholipase A2, occurred primarily within the initial 1-5 min of treatment with LPS. Cell lysates from LPS-stimulated macrophages exhibited a markedly increased enzymatic activity that was capable of both acylation of 1-[3H]alkyl-2-lyso-GPC (lyso-PAF) and deacylation of 1-[3H]alkyl-2-acyl-GPC generating [3H]lyso-PAF via CoA-independent transacylation of exogenous lysoplasmenylethanolamine compared with extracts from resting macrophages. Pretreatment of the cells with LPS for 5 and 30 min enhanced significantly the transfer of [14C]arachidonic acid from 1-[3H]alkyl-2-[14C]arachidonoyl-GPC into plasmenylethanolamine in prelabeled cell homogenates following the addition of exogenous lysoplasmenylethanolamine. Taken together, these data suggest that the CoA-independent transacylase, but not phospholipase A2, is a key enzyme responsible for the prolonged generation of lyso-PAF and that the increased capability of CoA-independent transacylation followed by CoA-dependent acetylation of lyso-PAF can sustain the biosynthesis of PAF in LPS-stimulated IC-21 macrophages.

Signaling responses to alkyllysophosphatidic acid: the activation of phospholipases A2 and C and protein tyrosine phosphorylation in human platelets.

The profile of biochemical responses following stimulation of human platelets with 1-alkyl-2-lyso-sn-glycero-3-phosphate (ALPA), a derivative of platelet-activating factor (PAF), was investigated. In the presence of extracellular Ca2+, ALPA evoked a dose-dependent increase and sustained elevation of the intracellular free Ca2+ concentration and stimulated the formation of phosphatidic acid. Platelets released free [3H]arachidonic and [3H]oleic acid at maximal rates between 5 and 15 s following ALPA stimulation. However, in platelets labeled with myo-[3H]inositol, ALPA induced [3H]phosphoinositide breakdown and formation of [3H]inositol phosphates with slower kinetics. Intracellular Ca2+ mobilization and the release of free fatty acids and inositol phosphates were not inhibited by pretreatment of platelets with pertussis toxin (PTX) or the PAF receptor antagonist WEB 2086. Following platelet stimulation with ALPA, tyrosine phosphorylation of proteins with apparent molecular masses of 65-95, 110-130, and 145-170 kDa was increased in a time-dependent manner, while phosphorylation of 40- to 45-kDa proteins was decreased. One of the platelet proteins phosphorylated on tyrosine residues in response to ALPA was found to be PLC-gamma1. Exogenous [3H]ALPA was metabolized primarily to [1-3H]alkyl-2,3-diacylglycerol. The metabolic conversion of [3H]ALPA involved a dephosphorylation reaction, and the formation of the dephosphorylated product, [1-3H]alkyl-monoglycerol, was detected within 5 s. These data demonstrate that an ether-linked lysophosphatidic acid can activate human platelets by a PTX-insensitive mechanism which does not involve the PAF receptor. Upon stimulation of platelets, ALPA induces the activation of phospholipases A2 and C, and tyrosine phosphorylation of several cellular proteins including PLC-gamma1. These signal transduction responses in platelets are accompanied by rapid metabolism of ALPA.

Interaction of platelet-activating factor with rat hepatocytes: uptake, translocation, metabolism, and effects on PAF-acetylhydrolase secretion and protein tyrosine phosphorylation.

In the present study, the interaction of the phospholipid mediator platelet-activating factor (PAF) with rat hepatocytes in primary culture was examined. Following exposure to hepatocytes, exogenous [3H]alkyl-PAF was metabolized rapidly to [3H]lyso-PAF, the content of which was raised in the outer leaflet of the plasma membrane within the initial 5 min of incubation. Thereafter [3H]lyso-PAF was translocated into cells with concomitant reacylation to [3H]alkyl-acyl-glycerophosphocholine. A portion of untransformed [3H]PAF accumulated in the outer leaflet, and only a small amount of the [3H]PAF was translocated into the inner leaflet of the plasma membrane. Detectable levels of [3H]lyso-PAF were found in the medium of hepatocyte cultures at all times of incubation. These findings suggest that at least a portion of the cellular PAF-acetylhydrolase (PAF-AH) activity is located in the outer leaflet of the plasma membrane and can be secreted into the medium. Indeed, rat hepatocytes in culture released PAF-AH into the medium in a time-dependent fashion, Incubation of hepatocytes with exogenous PAF increased secretion of PAF-AH, whereas lyso-PAF and the nonhydrolyzable analog methylcarbamyl-PAF significantly reduced secretion. The structurally related PAF receptor antagonist CV 3988 markedly inhibited the activity of PAF-AH and also diminished its release by hepatocytes. In contrast, BN 50739 amd WEB 2170, thienotriazolodiazepine PAF receptor antagonists, did not affect the PAF-AH activity, but increased its secretion by the cells. A full-length 3.8-kb mRNA encoding the cell surface PAF receptor was absent in hepatocytes as indicated by Northern blot analysis using the rat PAF receptor cDNA, whereas PAF receptor mRNA was readily detected in Kupffer cells. Upon incubation with hepatocytes, PAF induced tyrosine phosphorylation of proteins with molecular masses of 120-130 and 160-180 kDa and dephosphorylation of 80-90-kDa proteins; these responses were not inhibited by WEB 2170 and BN 50739. The protein tyrosine kinase inhibitor genistein abolished the release of free arachidonic acid, suggesting a crucial role for tyrosine phosphorylation in PAF-induced phospholipase A2 activation in rat hepatocytes. Taken together, our data indicate that the interaction of PAF with rat hepatocytes is dependent upon its metabolism, involves protein tyrosine phosphorylation/dephosphorylation and arachidonic acid release, and does not involve the heteromeric G-protein-coupled PAF receptor which has been characterized in Kupffer cells. This metabolically regulated mechanism for PAF action on hepatocytes may be of potential biological importance in the liver under normal and pathological conditions.

The specific binding of the platelet-activating factor (PAF) receptor antagonist WEB 2086 and the benzodiazepine flunitrazepam to rat hepatocytes.

Thieno-triazolodiazepines WEB 2086 and BN 50739 have been described as the potent PAF receptor antagonists. Binding of radiolabeled [3H]WEB 2086 has been widely employed to characterize PAF receptors in different cells. In a search for a PAF receptor in isolated rat hepatocytes, we discovered that the binding of [3H]WEB to rat hepatocytes was highly specific but had a relatively low affinity with a Kd of 113 nM and Bmax of 0.65 pmol/10(6) cells in freshly isolated cell suspension and Kd of 1.65 muM and Bmax of 2.0 pmol/plate in cultured hepatocytes. No consistent specific binding of [3H]PAF itself was found in the same cell preparations. The binding of [3H]flunitrazepam in the presence of the peripheral type of benzodiazepine receptor antagonist Ro 5-4864 was saturated and exhibited a K(i) of 3.8 nM and Bmax of 3.5 pmol/plate. The central type of benzodiazepine receptor antagonist clonazepam was competed for the [3H]flunitrazepam binding, however with a much lower affinity. Various antagonists inhibited the binding of [3H]WEB 2086 with a rank order BN 50739>>Ro 5-4864 > or = clonazepam. Interestingly, bicuculline, specific antagonist of GABA(A) recognition sites, also significantly reduced the binding of [3H]WEB 2086. The binding of [3H]flunitrazepam was inhibited with a rank potency BN 50739>>WEB 2086. Taken together, these findings suggest that the specific binding of PAF receptor antagonists WEB 2086 and BN 50739 in rat hepatocytes does not involve PAF receptors and occurs via peripheral benzodiazepine and, possibly GABA(A) receptor sites.

[The effect of phospholipids on the adhesive properties of bacteria]. / Vliianie fosfolipidov na adgezivnye svoistva bakterii.

The present work shows that choline-containing phospholipids (lysophosphatidylcholine and lyso-1-alkyl-sn-glycerophosphocholine) inhibit the adhesion of some strains: Bacterium bifidum 1, B. adolescentis MC-42, B. longum B. 379M, Staphylococcus aureus P 209 and Klebsiella pneumoniae 52. Phosphatidylcholine produces no effect on the adhesiveness of these strains, while platelet activation factor stimulates adhesiveness only in strain S. aureus 209. The stimulating or inhibiting action of phospholipids on the adhesive process of microorganisms depends on the species of bacteria and on the concentration of reagents.

[A morphologic analysis of changes in the lungs and kidneys caused by acetyl glyceryl ether phosphorylcholine and limited by verapamil]. / Morfologicheskii analiz izmenenii v legkikh i pochkakh, vyzvannykh atsetilglitseril'nym éfirom fosforilkholina i ogranichennykh verapamilom.

Histological analysis of lung and kidney after multiple PAF i.v. injection into rabbits was performed. Under these conditions the effect of previously injected of verapamyl, calcium channel blocker, was studied. It was shown that RAF causes inflammatory reaction with cell infiltration, microcirculation disorders and thrombosis in lung and kidney, which may classify as interstitial nephritis, pneumonitis and vasculitis. Verapamyl inhibits vasospasm, abolishes fibrinogen but does not affect cell infiltration and oedema development. Thus, Ca channel-dependent mechanism of vasospasm and thrombogenesis induced by PAF in vivo is confirmed. Perhaps, another possibilities for realising PAF action in vivo may occur.

[Features of thrombophilia in nephrotic syndrome in children]. / Osobennosti trombofilii pri nefroticheskom sindrome v detskom vozraste.

A comprehensive study was made of the hemostatic system in 25 children with the nephrotic syndrome and combined forms of glomerulonephritis. Hypercoagulation and a number of factors preventing thrombosis formation were revealed, namely the presence of so-called thrombin-resistant blocked fibrinogen, a rise of the total antithrombin potential at the expense of proteins differing from antithrombin III, sufficient reserves of plasminogen, and a moderate increase of fibrin degradation products.