Hepatobiliary transport of diflunisal conjugates and taurocholate by the perfused rat liver: the effect of chronic exposure of rats to diflunisal

Acyl glucuronides are reactive electrophilic metabolites of carboxylate drugs which can form covalent adducts with endogenous macromolecules such as serum albumin and hepatic proteins. Such adducts have been suggested as initiating factors in certain immune and toxic responses to acidic drugs. In the present study, pretreatment of rats with high daily doses (50 mg/kg orally) of the non-steroidal anti-inflammatory drug (NSAID) diflunisal (DF) for 35 days, followed by perfusion of the isolated liver with 3 mg DF for 3 hr, resulted in appreciable concentrations of covalent adducts of DF with hepatic tissue (3.68 microg DF/g liver). Immunoblotting using a rabbit polyclonal DF antiserum showed the major DF-modified bands at about 110, 140 and 200 kDa. A vehicle-pretreated control group achieved adduct concentrations of only 0.37 microg DF/g liver, with the 200 kDa band not detectable in immunoblots. Elimination of DF from perfusate of the isolated perfused rat liver (IPRL) preparation was the same (t1/2 about 3.4 hr) in both DF- and vehicle-pretreated groups. Appearance of the sulfate (DS) conjugate, the major metabolite in perfusate, was also similar. However, higher concentrations of the acyl glucuronide (DAG) and phenolic glucuronide (DPG) conjugates were found in perfusate at later times, though a statistically significant difference in area under the concentration-time curve was found only in the case of DAG. At 3 hr, recoveries of dose as DAG and DPG were significantly higher in perfusate, but not in bile. No significant differences in uptake and biliary excretion of taurocholate were found between the two groups. The finding of higher perfusate concentrations of DAG and DPG could signal a minor compromise to biliary excretion processes for the glucuronides, though whether such a result is simply coincident with or attributable to DAG-derived covalent DF-protein adducts in liver remains indeterminate.     

Technetium-99m labelled LDL as a tracer for quantitative LDL scintigraphy. II. In vivo validation, LDL receptor-dependent and unspecific hepatic uptake and scintigraphic results

The purpose of this study was to determine whether the hepatic uptake of dialysed technetium-99m labelled low-density lipoprotein (99mTc-LDL) reflects the hepatic LDL receptor activity and to what extent the non-LDL receptor-dependent 99mTc-LDL uptake by non-parenchymal cells relates to the diagnostic utility of quantitative 99mTc-LDL scintigraphy of the liver. New Zealand White rabbits and Watanabe Heritable Hyperlipidaemic rabbits, which were sacrificed 24 h after simultaneous injection of 99mTc-LDL and iodine-125 labelled LDL, were clearly discriminated by their hepatic 99mTc-LDL uptake according to their genetically different hepatic LDL receptor activity. Yet the hepatic 99mTc-LDL uptake exceeded the 125I-LDL uptake in all animals. The different hepatic uptake of the tracers was elucidated in the isolated perfused rat liver and was due to rapid intracellular degradation and the release of low molecular catabolites of 125I-LDL. In contrast, 99mTc activity was trapped in the liver. Analysis of biliary 99mTc activity provided evidence for the excretion of 99mTc-labelled apolipoprotein B. The amount of biliary excreted protein-bound 99mTc was linked to total hepatic 99mTc-LDL uptake and presumably reflected LDL receptor-mediated apolipoprotein excretion. Collagenase liver perfusion in Sprague-Dawley rats 90 min following simultaneous injection of 99mTc- and 125I-LDL and subsequent cell separation by gradient centrifugation revealed that 99mTc-LDL and 125I-LDL had a comparably low uptake into non-parenchymal cells; thus its contribution can be neglected for scintigraphic purposes. Planar scintigraphy was performed in New Zealand White and Watanabe Heritable Hyperlipidaemic rabbits.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hepatic uptake and biliary excretion of d-tubocurarine and trimethyltubocurarine in the rat in vivo and in isolated perfused rat livers

The clearance from perfusion medium and the biliary excretion of d-tubocurarine (d-TC) and trimethyltubocurarine (tMeTC) was studied in isolated perfused rat livers. Despite the related structure, d-TC exhibited considerably higher lipophilicity and plasma protein binding than its trimethyl derivative. Significant differences in hepatic disposition of the two agents were found. The clearance constant of elimination from the perfusate for d-TC was 2.00 and 0.41 ml/min for tMeTC. Fifty-one percent of the administered d-TC was excreted in the bile during 2 hours of perfusion. For tMeTC this amounted to only 16%. Bile/plasma concentration ratios of d-TC were 10 times those of tMeTC. There was no evidence for biotransformation of the substances. The unequal biliary output cannot be explained by differences in subcellular distribution. After injection into rats in vivo, the major part of drug in the liver is confined to the particulate fractions. Subfractionation studies indicate binding to lysosomes. The hepatocyte cytosol concentrations of d-TC and tMeTC are in the same order and are lower than the concomitant plasma concentrations. Both bile/liver and liver/plasms concentration ratios were higher for d-TC. The results support the idea that the balance of hydrophilic and hydrophobic properties is an important factor determining hepatic transport of organic compounds.     

Role of a rat membrane inhibitor of complement in anti-basement membrane antibody-induced renal injury

In the kidneys of anti-glomerular basement membrane (anti-GBM) antibody disease, binding of antibodies to tubular basement membrane (TBM) is often observed. The present work was performed to explore the mechanisms of binding of anti-GBM antibodies to TBM in vivo with special reference to 5I2Ag, a rat membrane inhibitor of complement which regulates complement activation at C3 convertase level. To suppress functions of renal 5I2Ag, F(ab')2 fragment of 5I2 (a neutralizing mAb against 5I2Ag) was perfused in the left kidney and then blood circulation was restored. Mild proteinuria ( < 10 mg/16 hr) was observed during first several days. Five days later, there were tubulointerstitial injuries defined by tubular vimentin staining and leukocyte infiltration. Significant deposition of C3 was observed in the capillaries and in TBM. In rats intravenously injected with rabbit anti-rat GBM antibodies five minutes after kidney perfusion with 5I2, strong binding of rabbit IgG to TBM was observed at one and five days after injection. Although these rats showed mild proteinuria comparable to those perfused with 5I2 and those injected with normal rabbit serum, tubulointerstitial injury was significantly enhanced at Day 5. In contrast, rats perfused with irrelevant mAb and injected with anti-GBM antibodies did not show any significant binding of antibodies to TBM nor tubulointerstitial injury. Furthermore, rats which were made proteinuric by puromycin aminonucleoside and injected with anti-GBM antibodies did not show any significant binding of rabbit IgG to TBM. These results indicate that 5I2Ag, a rat membrane inhibitor of complement at the C3 convertase level, regulates vascular permeability in the living kidney, and that dysfunction or decreased expression of this molecule leads to increased accessibility of anti-GBM antibodies to TBM.     

Generation of coagulation factors V, XI, and XII by the isolated rat liver

When isolated rat livers were perfused with platelet-free erythrocytes suspended in Tyrode's solution containing 6% bovine serum albumin, the generation of factors V, XI, and XII was clearly demonstrable. On average, after 5 h of perfusion of a single liver, the concentrations in the perfusate (as a percentage of normal rat plasma) were about 6% for factor V, 8% for factor XI, and 5% for factor XII, compared with 20% for factor VII, which was used as the reference standard. When two livers were perfused, approximately twice these concentrations were achieved. When the properties of these factors in perfusate and plasma were compared, they agreed well except for differences in the celite adsorbability of factors XI and XII.     

Decreased accumulation as a mechanism of resistance to cis-diamminedichloroplatinum(II) in cervix carcinoma HeLa cells: relation to DNA repair

In vivo transport in plasma and in vitro transfer of ebselen to binding sites in the hepatocyte were studied. More than 90% of intravenously administered ebselen in mouse plasma is bound by selenium-sulfur bonds to reactive thiols in serum albumin. In in vitro experiments the uptake of [14C]-ebselen from a complex prepared with bovine serum albumin (BSA) was determined in isolated perfused rat liver. Radioactive ebselen metabolites were excreted into bile. In isolated hepatocytes, radioactivity was bound to all subcellular organelles. Ebselen is transferred from the BSA complex to membrane-associated proteins after reductive cleavage of the Se-S bond effected by endogenous protein thiols. In contrast, when proteins were separated by dialysis membranes, ebselen transfer from its BSA complex occurred only in the presence of externally added reductants. Among the physiological reductants tested, ebselen release from the BSA complex was highest with glutathione (75%) and lowest with ascorbic acid (less than 10%). Quantitative release of ebselen from its BSA complex was only achieved by the combined action of reductant, notably 2-mercaptoethanol, and guanidine thiocyanate, suggesting that ebselen interacts with proteins by covalent Se-S bonds as well as by ionic charge interactions.     

Isolated rat hepatocytes bind lactoferrins by the RHL-1 subunit of the asialoglycoprotein receptor in a galactose-independent manner

Isolated rat hepatocytes bind and internalize the iron-binding protein lactoferrin (Lf) by a set of high-affinity, recycling, Ca2+-dependent binding sites. We have purified a 45-kDa membrane protein (p45) from rat hepatocytes that exhibits Ca2+-dependent receptor activity. In this study, we found p45 to be identical to the major subunit (RHL-1) of the rat asialoglycoprotein receptor. Two tryptic fragments of p45 showed 100% identity with RHL-1 internal sequences (Leu121 --> Lys126 and Phe198 --> Lys220), and monospecific antisera against p45 and RHL-1 cross-reacted equally well with each protein. Molar excesses of anti-p45 IgG, anti-RHL-1 IgG, asialoorosomucoid, and asialofetuin competitively blocked the binding of 125I-Lf to isolated rat hepatocytes at 4 degrees C. Similarly, either excess anti-p45 or Lf blocked the binding of 125I-asialoorosomucoid to cells at 4 degrees C. We did not detect the minor subunits of the rat asialoglycoprotein receptor (RHL-2/3) in p45 preparations from Triton X-100 extracts of hepatocytes and 125I-Lf bound to purified RHL-1 but not to RHL-2/3 immobilized on nitrocellulose. Nonetheless, anti-RHL-2/3 IgG reduced the binding of 125I-Lf to hepatocytes at 4 degrees C. Exoglycosidases were used to remove terminally-exposed N-acetylneuraminyl, alpha- and beta-galactosyl, and N-acetylhexosaminyl sugars from human and bovine Lf glycans, and lectin blotting confirmed that glycosidase-treated Lfs lacked detectable terminal galactosyl sugars. Unexpectedly, these deglycosylated Lfs exhibited no loss in their ability to compete with unmodified Lfs for binding to isolated hepatocytes. In addition, molar excess of beta-lactose but not sucrose competitively blocked the binding of 125I-Lf to cells, indicating that Lf bound at or very near the carbohydrate-recognition domain of RHL-1. We conclude that RHL-1 is the Ca2+-dependent Lf receptor on hepatocytes and that it binds Lf at its carbohydrate-recognition domain yet in a galactose-independent manner.     

Reconstruction of hepatic organoid by rat small hepatocytes and hepatic nonparenchymal cells

Hepatic cells isolated from an adult rat liver, consisting of small hepatocytes (SHs), mature hepatocytes (MHs), liver epithelial cells (LECs), Kupffer cells, sinusoidal endothelial cells, and stellate cells, were cultured in a medium supplemented with 10% fetal bovine serum, 10 mmol/L nicotinamide, 1 mmol/L ascorbic acid 2-phosphate, 10 ng/mL epidermal growth factor, and 1% dimethyl sulfoxide. The SHs rapidly proliferated and formed a colony. About 10% of cytokeratin 8 (CK8)-positive cells formed SH colonies. All SHs at day 10 immunocytochemically showed positivity for albumin, transferrin, CK8, and CK18, which are markers for hepatocytes. In contrast, alpha-fetoprotein (AFP)-, CK14-, OC2-, and glutathione S-transferase placental type (GST-P)-positive cells, which are thought to be markers for hepatic immature cells, were rarely observed. At day 20 some cells in the colonies were positive for AFP, CK7, CK19, and GST-P. LECs and stellate cells proliferated and surrounded the colonies. About 2 weeks after plating, piled up cells were often observed on the SH colonies. In those colonies LECs and stellate cells invaded under the colonies. The invasion of the cells and gradual deposits of extracellular matrix (ECM) such as type I collagen, type IV collagen, and laminin induced alteration of the shape of the SHs from relatively flat to cuboidal or rectangular. With the cellular structural changes, the expression of albumin, connexin 32 (Cx32), and tryptophan 2,3-dioxygenase (TO) messenger RNAs increased. In addition, overlapping nonparenchymal cells (NPCs) on the piled up cells induced the formation of duct- or cyst-like structures consisting of MHs. In the present experiment we showed that SHs could differentiate to MHs by interacting with NPCs and ECM. Thus, SHs may be "committed progenitor cells" that can further differentiate into MHs.     

The effect of endothelin and its antagonist Bosentan on hemodynamics and microvascular exchange in cirrhotic rat liver

BACKGROUND/AIMS: To characterize the effects of endothelin-1 and of Bosentan, a mixed endothelin antagonist, on hepatic hemodynamics in cirrhotic animals in vivo and on hepatic microvascular exchange in the perfused rat liver. METHODS: Biliary cirrhosis was induced by bile duct ligation, and micronodular cirrhosis by chronic exposure to phenobarbital/CCl4 in male rats. Hepatic hemodynamics were studied under basal conditions and after administration of Bosentan (3-30 mg/kg) by the microsphere technique. Microvascular exchange was assessed in the in situ perfused rat liver by the multiple indicator dilution technique. RESULTS: Bosentan lowered portal pressure in a dose-dependent fashion; at the highest dose tested, this decrease averaged -29+/-11 and -26+/-8% in biliary and micronodular cirrhosis, respectively (p<0.01). This was achieved mainly via a marked decrease in hepatic arterial flow. In the perfused liver, endothelin-1 induced a dose-dependent vasoconstriction; up to 10(-9) mol/l; this was not associated with any effect on viability. At this dose, endothelin-1 markedly decreased extravascular albumin space in both controls and micronodular cirrhosis; this could be antagonized by Bosentan 10(-5) mol/l. CONCLUSIONS: Endothelin-1 affects hepatic microvascular exchange, presumably by a direct effect on hepatic sinusoidal endothelial cells. A mixed endothelin antagonist lowers portal pressure in vivo, presumably by acting on hepatic stellate cells, and counteracts the microvascular effects of endothelin-1 in vitro. These properties could prove useful in treatment of portal hypertension.     

Reversal of stereoselectivity in the hepatic availability of verapamil in isolated perfused rat livers. Role of protein binding

The effects of serum proteins on the stereoselective kinetics of the high clearance drug verapamil (VER) and its metabolite, norverapamil (NOR), were studied in isolated perfused rat livers (IPRLs). Livers were perfused, in a recirculating manner, with a solution containing human serum albumin (HSA), bovine serum albumin (BSA), or no serum albumin (N = 5 for each group). After presystemic administration of a single dose of racemic VER (2 mg), the concentrations of VER and NOR enantiomers in the perfusate were measured over 90 min. In addition, the fraction of the enantiomers bound to the plasma of perfusate was determined. Perfusate concentrations of both VER and NOR were stereoselective in all of the perfusates studied. However, the direction of stereoselectivity in the concentrations of VER enantiomers in the BSA perfusate (S-VER > R-VER) was opposite that in the HSA and albumin-free perfusates (R-VER > S-VER); this was associated with an opposite stereoselectivity in the concentrations of NOR in the BSA perfusate was higher than that in the HSA and albumin-free perfusates, an observation in agreement with the higher stereoselectivity in the binding of NOR to BSA. These data, along with other kinetic parameters such as apparent hepatic availability and intrinsic clearance, suggest that the apparent stereoselectivity in the presystemic elimination of VER by IPRLs is significantly influenced by the stereoselectivity in the protein binding of the drug.     

Hemodynamic characteristics of the intrahepatic portal vascular bed over an extended flow range: a study in the isolated perfused rat liver

The relationship between the perfusion pressure (P) and resistance (R) of the intrahepatic portal vascular bed was determined in isolated rat liver preparations perfused with fresh, heparinized rat blood (hematocrit 30%), with rat blood containing a vasodilator agent (sodium nitroprusside, 1 X 10(-3) mol/L), or with 2.5% bovine serum albumin in Krebs-Henseleit buffer (BSA-KH). Pressure-flow curves were constructed over an extended range of portal venous inflow (0 to 70 mL.min-1, corresponding to a flow rate per gram liver wet weight, Q, of approximately 0 to 7 mL.min-1.g-1). Subsequent analysis showed that two mathematical expressions adequately described the data over the full range of flow. Thus, the pressure-flow curve could be represented by (a) the sum of a linear plus a hyperbolic function, i.e., P = Q.R' + Pmax.Q/(Q + Km), where R', Pmax, and Km are constants, or (b) by the simple equation G = C.P, where G is the conductance (Q/P), and C is a conductivity constant. The values of R', Pmax, Km, and C were significantly different under each of the circumstances investigated, but the form of the curve was not altered. Hence, it is proposed that these parameters can be used to describe the fundamental hemodynamic properties of the portal vascular bed of the isolated rat liver. The results are discussed in terms of the microvascular recruitment and distensible resistance vessel models of the hepatic microcirculation.     

Specificity of Fcgamma receptors in human malignant tissue and normal lymphoreticular tissue

The specificity of the Fcgamma receptors in normal spleen and liver and in malignant tissues was studied using hemadsorption to cryostat sections. Indicator cells (EA) were sheep erythrocytes (E) sensitized with rabbit IgG antibody (A). The binding of EA to sections of normal and malignant tissues was inhibited by pooled IgG of human, rabbit, and guinea pig origin and by human IgG1, and IgG3, and IgG4 myeloma proteins. Heat-aggregated IgG inhibited the binding to sections of liver and some malignant tissues more effectively than monomeric IgG. The Fc fragments of IgG were also inhibitory, but not the F(ab')2, Fab', and Facb fragments. The inhibition obtained increased with decreasing amounts of A used for sensitization of E. The inhibitory activity of IgG was abolished after partial reduction and alkylation. No inhibition was obtained with IgG2, IgM, IgA, or albumin. E sensitized with Facb or F(ab')2 fragments of A did not bind to normal or malignant tissues. The specificity of the Fc receptors in normal spleen and liver and in malignant tissues is apparently very similar.     

Carbohydrate-mediated clearance of immune complexes from the circulation. A role for galactose residues in the hepatic uptake of IgG-antigen complexes

The purpose of these experiments was to evaluate the hypothesis that galactose residues on IgG mediate the clearance of IgG.antigen complexes from the circulation. After 28 days of immunization of rats with bovine serum albumin (BSA), approximately 90% of anti-BSA antibody was IgG; the circulating half-life of trace amounts of BSA antigen in immunized rats was 6 min, compared to 24 h in nonimmunized rats. Similarly, soluble IgG.125I-BSA complexes formed in vitro, under conditions of antibody excess, had a circulating half-life of 4 min in normal rats. For both antigen in immunized rats, or IgG.125I-BSA complexes in normal animals, clearance was markedly inhibited by pre- or co-injection of asialofetuin, but was insensitive to large doses of fetuin, ovalbumin, or mannan. Liver parenchymal cells were the major site of uptake of complexes formed in vivo or in vitro. In vitro binding of IgG.125I-BSA complexes by isolated hepatocytes was effectively competed by asialofetuin, asialo-orosomucoid, galactose, and N-acetylgalactosamine, but was unaffected by fetuin, orosomucoid, ovalbumin, mannan, or mannose. These data suggest that antigen-induced conformational changes in IgG result in both recognition of galactose residues on IgG and clearance of IgG-immune complexes from the circulation by the galactose-specific receptor in hepatic parenchymal cells.     

Comparative studies of rat IgG to further delineate the Fc:FcRn interaction site

Recent data have indicated that the MHC class I-related receptor, FcRn, regulates the half-lives of serum IgG in addition to its known role in transferring IgG from mother to young. In the current study, the activity of rat IgG (rIgG) isotypes in FcRn-mediated functions has been analyzed. The serum half-life and maternofetal transfer in mice decreased in the order rIgG2a > rIgG1 > rIgG2c > rIgG2b. This decrease in activity correlates well with reduced binding affinity for soluble mouse FcRn, and site-directed mutagenesis of a recombinant Fc-hinge fragment has been used to investigate the molecular basis for the differences in activities of the rIgG. Analysis of the serum half-lives of the mutated Fc-hinge fragments demonstrated that, in addition to Ile253, His310, His435 and His436 that were identified in earlier studies, amino acids at positions 257, 307 and 309 play a role in building the FcRn interaction site of IgG. The study also excludes the involvement of amino acids in a fourth loop located at the CH2-CH3 domain interface that encompasses residues 386-387 in FcRn binding. Sequence differences at positions 257, 307 and 309 between rIgG most likely account for the reduced affinity of rIgG2b and IgG2c relative to rIgG1 and rIgG2a for binding to FcRn.     

Improvement of perfusion-flow in the isolated rat liver, under the influence of streptase

Accumulation of radioactive material in the isolated rat liver was noted when 125I-fibrinogen was added to the perfusing medium. Owing to an enhanced production of fibrinogen and to a diminished fibrinolysis, fibrin deposits were found to occur in the capillaries of fhe isolated liver. This would cause a decrease of hepatic flow and an impairment of the nutrition of this organ which leads to subsequent necrosis. Administration of streptase removed the fibrin deposits and restored the perfusive flow in the isolated liver.     

Transport, binding, and metabolism of sulfate conjugates in the liver

Sulfate conjugates are a heterogeneous class of polar, anionic metabolites that result from the conjugation of endogenous and exogenous compounds. Sulfate conjugates exhibit a high degree of binding to albumin, the extent of which usually exceeds those of their parent compounds. Preponderant direct and indirect evidence suggests that sulfation activity is slightly higher in the periportal than in the perivenous (centrilobular) region of the liver, but recent immunohistochemical studies imply that specific isoforms of the sulfotransferases may also be preferentially localized in the perivenous region. Entry of sulfate conjugates into the liver cell is poor unless discrete carriers are present. Although known transport carriers exist for the sulfated bile acids, the specificity of the carriers for drug sulfate conjugates is presently unknown. The removal of sulfates is usually by way of biliary excretion while, on occasion, sulfates can be desulfated and participate in futile cycling with their parent compounds. The binding, transport, and hepatic elimination of various drug sulfate conjugates are examined. Non-recirculating studies carried out in the perfused rat liver with the multiple indicator dilution technique under varying input sulfate conjugate concentrations have provided essential information on the effects of vascular (red blood cells and plasma protein) binding on transport and removal of the conjugates. These studies clearly demonstrate the need to study protein binding, transmembrane transfer characteristics across the liver basolateral (sinusoidal) and canalicular membranes, and enzyme zonation in a distributed-in-space fashion in order to properly define the handling of sulfate conjugates in the liver.     

Immunoblot analysis of proteins associated with HEMA-MMA microcapsules: human serum proteins in vitro and rat proteins following implantation

Human serum proteins and their fragments, associated with hydroxyethyl methacrylate-methyl methacrylate (HEMA-MMA) copolymer microcapsules, were characterized using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis. Capsules were incubated with serum for 1 week in vitro and then dissolved in ethanol to also precipitate the adsorbed protein. The precipitate was dissolved in 2% (w/v) SDS (the 'capsule eluate') to be assayed by electrophoresis. The majority of proteins probed for in the immunoblots were detected in the capsule eluates. These included fibronectin, plasminogen, IgG, vitronectin, Factor B, Factor H, Factor I, C3, but not beta-lipoprotein, fibrinogen, HMWK, or IgM. Complement activation fragments were detected in both the immunoblots of the capsule eluates and the medium containing serum without capsules. Thus, the adsorption of these fragments, formed independent of capsule presence, may be partially or completely responsible for the complement fragments associated with capsules. The prevention of complement activation by the addition of 5.8 mM EDTA, at the beginning of the week-long incubation, resulted in fewer low-molecular-weight C3 fragments associated with capsules. Rat proteins were also detected in immunoblots of the eluate of 'free-floating' capsules from the rat peritoneal cavity following implantation for 1 day using anti-human antibodies. Detected proteins included HMWK, fibrinogen, antithrombin III, transferrin, alpha1-antitrypsin, fibronectin, albumin, alpha2-macroglobulin, vitronectin, beta2-microglobulin, Factor B and Factor I. Rat fibrinogen, IgG, and complement C3 fragments were also detected in these immunoblots, but with monoclonal antibodies against the rat proteins.     

Endotoxin modulates arachidonic acid-induced glycogenolysis in the perfused rat liver

Effects of endotoxin on arachidonic acid (AA)-induced hepatic glycogenolysis were examined in perfused rat liver. In normal rat liver, infusion of AA increased oxygen consumption and glucose production concurrently. In rats injected with lipopolysaccharide (LPS) 6 h before, AA increased glucose production but suppressed oxygen consumption. The changes in LPS-injected rat were abolished by a thromboxane (Tx) A2 receptor antagonist. The release of Tx B2 by AA increased after LPS-injection. These results suggest that priming of hepatic macrophage by endotoxin in vivo enhances Tx synthesis, resulting in modulating hepatic glycogenolysis.     

Effects of the nonsteroidal anti-inflammatory drug nimesulide on energy metabolism in livers from adjuvant-induced arthritic rats

The effects of nimesulide on energy metabolism and the hepatic metabolic alterations produced by adjuvant-induced arthritis were investigated in the perfused rat liver an in isolated liver mitochondria. Nimesulide, at therapeutic levels (20-50 microM), produced: (1) stimulation of oxygen consumption in the perfused rat liver and in isolated mitochondria, (2) inhibition of gluconeogenesis; (3) reduction of ADP/O ratio and the respiratory control ratio and stimulation of glycogenolysis in the livers from healthy rats, but not in livers from arthritic rats. These results indicate that nimesulide acts as a mitochondrial uncoupler. The main alterations produced by adjuvant-induced arthritis were: higher rates of oxygen consumption in both perfused livers and isolated mitochondria, with no decrease in the efficiency of mitochondrial energy transduction; (2) decreased gluconeogenesis and lack of glycogenolytic response to uncouplers, but not to alpha 1-agonists. These data allow to conclude that nimesulide-induced impairment of energy metabolism should worsen the hepatic disturbances that are already associated with the adjuvant disease.     

The disposition of nifurtimox in the rat isolated perfused liver: effect of dose size

The disposition of nifurtimox was studied in the rat isolated perfused liver using a recirculating system. The drug was administered as a bolus (5.0, 15.0 or 30.0 micrograms mL-1), and its disappearance was monitored by analysing perfusate samples. In all experiments perfusate disappearance was monoexponential, and no significant difference was found between the three doses for the elimination constant (0.016, 0.011 and 0.012 min-1, respectively), half-life (46.6, 65.8 and 66.8 min, respectively), extraction rate (0.128, 0.091 and 0.099, respectively) and distribution volume (41.1, 47.3 and 30.7 mL g-1, respectively). At 30 micrograms mL-1 the hepatic clearance was lower than the other concentrations of nifurtimox (0.66, 0.51 and 0.34 mL min-1 g-1, respectively). Relatively little parent drug was recovered from the liver at the end of the perfusions. In summary, nifurtimox is cleared slowly from the rat isolated perfused liver, is poorly extracted by hepatocyte cells and is completely metabolized from 2 to 4 h after perfusion.