Efficiency of enterosorbents and detoxication mechanisms in immature rats with simulated hepatitis

Different modifications of fibrous enterosorbent of the ACFM "Dnepr-MN" type exert a normalizing effect on the morphofunctional state of the liver of preadolescent rats with the model of toxic hepatitis. The distortion in the oxidation of lipids with peroxide and in the antioxidizing system protection removed, antitoxic liver function increases, which is confirmed by microsomal oxidation processes. A mediated effect of enterosorbents on the hepatocyte membrane structures is very significant for their detoxication effect.     

Activatory effect of regucalcin on hepatic plasma membrane (Ca2+-Mg2+)-ATPase is impaired by liver injury with carbon tetrachloride administration in rats

The alteration of the plasma membrane (Ca²⁺-Mg²⁺)-ATPase activity in the liver of rats administered orally carbon tetrachloride (CCl₄) solution was investigated. Rats received a single oral administration of CCl₄ (10, 25 and 50%, 1.0 ml/100 g body weight), and 3 or 24 h later they were sacrificed. CCl₄ administration caused a remarkable elevation of liver calcium content and a corresponding increase in liver plasma membrane (Ca²⁺-Mg²⁺)-ATPase activity, indicating that the increased Ca²⁺ pump activity is partly involved in calcium accumulation in liver cells. Moreover, the participation in regucalcin, which is an intracellular activating factor on the enzyme, was examined by using anti-regucalcin IgG. The plasma membrane (Ca²⁺-Mg²⁺)-ATPase activity increased by CCl₄ administration was not entirely inhibited by the presence of anti-regucalcin IgG (1.0 and 2.5 ug/ml) in the enzyme reaction mixture. However, the effect of regucalcin (0.25–1.0 uM) to activate (Ca²⁺-Mg²⁺)-ATPase in the liver plasma membranes of normal rats was not revealed in the liver plasma membranes obtained from CCl₄-administered rats. Also, the effect of regucalcin was not seen when the plasma membranes were washed with 1.0 mM EGTA, indicating that the disappearance of regucalcin effect is not dependent on calcium binding to the plasma membranes due to liver calcium accumulation. Now, the presence of dithiothreitol (5 mM) or heparin (20 ug/ml) caused a remarkable elevation of the plasma membrane (Ca²⁺-Mg²⁺)-ATPase activity in the liver obtained from CCl₄-administered rats. Thus, the regucalcin effect differed from that of dithiothreitol or heparin. The present study suggests that the impairment of regucalcin effect on Ca²⁺ pump activity in liver plasma membranes is partly contribute to hepatic calcium accumulation induced by liver injury with CCl₄ administration.     

Effect of magnesium deficiency on the metabolism of glycosamino-glycans in rats

Magnesium deficiency in rats has significant effect on the concentration of different glycosaminoglycans in the tissues, the nature of the change being different in different tissues. Total glycosaminoglycans, chondroitin-4-sulphate + chondroitin-6-sulphate and dermatan sulphate increased in the aorta while hyaluronic acid, heparan sulphate and heparin decreased. In the liver, total glycosaminoglycans, hyaluronic acid, chondroitin-4-sulphate + 6-sulphate and heparin decreased while total glycosamino-glycans and all the glycosaminoglycan fractions increased in the heart. In the kidney, total glycosaminoglycans showed no significant alteration, hyaluronic acid and heparin decreased while chondroitin-4-sulphate + 6-sulphate increased. Activity of biosynthetic enzymesviz. glucosamine-o-phosphate isomerase and UDPG-dehydrogenase showed decrease in the liver. The concentration of 3’-phosphoadenosine 5’-phosphosulphate, activity of sulphate activating system and sulphotransferase were also similarly altered in the liver in magnesium deficiency.     

Effect of heparin, spermidine and Be2+ ions on the phosphatase and RNAse activity of rat liver cell nuclei]

Effects of heparin, spermidine, and Be2+ ions on the ATPase and beta-glycerophosphatase and RNA-ase activities of the rat liver cell nuclei were studied. Be2+ was shown to inhibit the ATPase activity and, to a lesser extent, beta-glycerophosphatase activities. Physiological concentrations of heparin and spermidine also lowered the mentioned two activities, as well as the RNAase activity of the nuclei. Evidence is presented for the inhibitory effect of heparin and spermidine on endonucleases.     

Activity of lipoprotein lipase in thyroidectomized rats

Total plasma postheparin lipolytic activity as well as lipoprotein lipase activity in plasma was higher after heparin injection in thyroidectomized rats than in controls. In contrast, the activity of liver lipase was lower in thyroidectomized rats. Adipose tissue from thyroidectomized rats contained more lipoprotein lipase activity than adipose tissue from controls as measured both in extracts of tissue homogenates and medium from in vitro incubations of tissue pieces. There were no differences between control and hypothyroid rats in the disappearance of intravenously injected 125I-labeled lipoprotein lipase, but when a low dose of heparin was injected before the labeled enzyme, the disappearance of 125I-labeled lipoprotein lipase was more retarded in thyroidectomized rats. The elimination of heparin itself was slightly retarded by thyroidectomy.     

Effects of heparin administration on Trypanosoma brucei gambiense infection in rats

We examined whether heparin administration influences in vivo trypanosome proliferation in infected rats. Administration of heparin every 8 hr via cardiac catheter inhibited growth of Trypanosoma brucei gambiense and prolonged survival of treated rats. Heparin administration increased lipoprotein lipase activity, high-density lipoprotein (HDL) concentration in the blood, and haptoglobin messenger RNA content of the liver. The presence of heparin in culture media did not directly affect proliferation of trypanosomes in vitro. However, the addition of plasma from infected rats treated with heparin to culture media decreased the number of trypanosomes. This effect was decreased by incubating the trypanosomes with benzyl alcohol, a known inhibitor of receptor-mediated endocytosis of lipoprotein. These data suggested that heparin administration reduced the number of trypanosomes in infected rats. Trypanosome lytic factor, a HDL and haptoglobin-related protein, protects humans and some animals from infection by Trypanosoma brucei brucei. In rats, increases in HDL and haptoglobin may affect the proliferation of T. b. gambiense.     

Influence of heparin on the removal of serum lipoprotein lipase by the perfused liver of the rat

Isolated rat livers were perfused with whole rat blood containing postheparin lipoprotein lipase (LPL) activity. LPL activity disappeared rapidly from the perfusate; the extraction ratio (portal vein-hepatic vein difference) was 0.70 for all time periods studied. Control experiments established that the disappearance of LPL was not due to non-specific inactivation in the apparatus or to the release of an inhibitory by the liver. The addition of heparin to the perfusate in suitable concentration (4 units/ml) almost completely blocked the disappearance of LPL activity from the perfusate. In addition to the perfusion experiments, we studied the effect of heparin on LPL activity when added to the LPL assay system. When heparin was added to the assay system containing fresh postheparin serum from rats, it stimulated LPL activity by about 70%. When heparin was added to postheparin serum which had been perfused through the liver, it stimulated LPL activity over 200%, but it did not restore LPL to its preperfusion value. These observations are compatible with a two-step inactivation system for LPL by the liver. The first step may involve a dissociation of a heparin-apoenzyme complex followed by destruction of the heparin. The second step may involve the removal of the apoenzyme of LPL.     

Hepatotropic action of benzobamil in CCl4 poisoning

The administration of an antiepileptic drug--benzobamilum-to rats with CCl4-induced hepatitis prevents the development of liver parenchyma necrosis, promotes the retention of normal enzyme activity in hepatocytes, stimulates the excretory and antitoxic liver functions. The drug has antioxidant properties, inhibits the production of lysophosphatidylcholine and the reduction in phosphatidylcholine content in the liver homogenates but fails to intensify CCl4-induced steatosis.     

Studies on isolated cell components

1. The addition of heparin to rat liver, kidney, or brain nuclei has been found to bring about the release of a gel. Chemical analysis and histochemical studies on whole homogenates and isolated nuclei demonstrated that the material released by heparin contained desoxyribonucleic acid (DNA) and protein. The action of heparin on nuclei is interpreted as the result of a combination with the basic proteins of the nucleus with a consequent displacement of DNA. 2. The addition of heparin to a finely divided dilute liver homogenate prepared in a phosphate-sucrose solution at pH 7.1 brings about a marked increase in viscosity which reaches a maximum in 6 to 8 minutes at 23° and then declines. 3. The concentration threshold for the viscosity effect was 0.1 mg. per 100 mg. fresh rat liver, with further increases in viscosity at higher heparin concentrations. Over a period of several hours a marked decrease in response to heparin was observed in homogenates stored at 0°. 4. Fractionation of the homogenate demonstrated that the viscosity increase was due to the presence of the nuclei alone, other components showing no effect. Microscopic observation showed that the increase in viscosity was associated with the appearance of a clear gel around nuclei treated with heparin. 5. Heparin brought about the release of DNA from the nuclei of incubated rat liver, kidney, and brain homogenates. In some instances over half the DNA is found in the supernatant after high speed centrifugation (20 minutes, 21,000 x g). 6. No correlation was found between anticoagulant activity of heparin preparations and their effectiveness in causing an increase in the viscosity of liver homogenates. Desulfated heparin produced none of the results described here for heparin.     

Effect of hepatophyt on the choleretic function of the liver damaged by tetracycline]

In experimental injury of the liver in Wistar-line white rats induced by tetracycline the course therapeutic and prophylatic administration of the dry extract "Hepatophyt" in a dose of 0.1 g/kg inhibits the negative effect of tetracycline and promotes stimulation of choleretic and antitoxic functions of the liver. The dry extract was derived from the herbal mix of the same name, used in the practice of Tibetan medicine against liver diseases.     

Heparin and chondroitin sulfate inhibit adenine nucleotide hydrolysis in liver and kidney membrane enriched fractions

The inhibition of adenine nucleotide hydrolysis by heparin and chondroitin sulfate (sulfated polysaccharides) was studied in membrane preparations from liver and kidney of adult rats. Hydrolysis was measured by the activity of NTPDase and 5′-nucleotidase. The inhibition of NTPDase by heparin was observed at three different pH values (6.0, 8.0 and 10.0). In liver, the maximal inhibition observed for ATP and ADP hydrolysis was about 80% at pH 8.0 and 70% at pH 6.0 and 10.0. Similarly to the effect observed in liver, heparin caused inhibition of ATP and ADP hydrolysis that reached a maximum of 70% in kidney (pH 8.0). Na⁺, K⁺ and Rb⁺ changed the inhibitory potency of heparin, suggesting that its effects may be related to charge interaction. In addition to heparin, chondroitin sulfate also caused a dose-dependent inhibition in liver and kidney membranes. The maximal inhibition observed for ATP and ADP hydrolysis was about 60 and 50%, respectively. In addition, the hepatic and renal activity of 5′-nucleotidase was inhibited by heparin and chondroitin sulfate, except for kidney membranes where chondroitin sulfate did not alter AMP hydrolysis. On this basis, the findings indicate that glycosaminoglycans have a potential role as inhibitors of adenine nucleotide hydrolysis on the surface of liver and kidney cell membranes in vitro.     

Potentiation of the activity of mouse liver-derived HBGF-1-like growth factor by heparin and dithiothreitol: evidence for the involvement of a plasma factor.

The potentiation of mouse liver derived heparin binding growth factors 1 and 2 (HBGF-1, HBGF-2) activity has been investigated. It was found that both heparin and various sulfhydryl reagents (such as dithiothreitol, DTT) markedly potentiated HBGF-1 activity, but not HBGF-2 activity. Further studies with HBGF-1 indicated that the growth factor would interact with a plasma factor, in a temperature-dependent manner, to become inactive, and that sulfhydryl reagents would reverse this inactivation. Inactivation would not occur either in the presence of heparin or DTT, indicating that heparin and DTT can protect the growth factor from plasma inactivation. When assayed in the absence of plasma, both heparin and DTT were required to reactivate plasma inactivated HBGF-1-ML. A model is presented to explain these data. This model predicts that either DTT or heparin can block the plasma induced inactivation process, but that once inactivation has occurred only sulfhydryl reagents can restore activity. Furthermore, heparin is thought to activate growth factor activity in the absence of plasma by blocking non-productive growth factor binding to the extracellular matrix. The identification of a plasma inactivating factor for mouse liver derived HBGF-1 has important implications for understanding the regulation of extracellular growth factor activity.     

Effect of multiple parasitic infections on the tolerance to pollutant contamination

The horizontally-transmitted acanthocephalan parasite Polymorphus minutus and the vertically-transmitted microsporidian parasite Dictyocoela roeselum have both been shown to influence on the antitoxic responses of mono-infected Gammarus roeseli exposed to cadmium. The present study investigates the effect of this co-infection on the antitoxic defence responses of naturally infected females exposed to cadmium stress. Our results revealed that, depending on the cadmium dose, bi-infection induced only slight, significant increased cell damage in G. roeseli as compared to non-infection. In addition, the antitoxic defence pattern of cadmium-exposed bi-infected hosts was similar to the pattern of cadmium-exposed D. roeselum-infected hosts. Reduced glutathione concentrations, carotenoid levels and γ-glutamylcystein ligase activity decreased, while metallothionein concentrations increased. This similar pattern indicates that host physiology can be controlled to some extent by microsporidia under stress conditions. It supports the hypothesis of a disruption of acanthocephalan effects in the presence of microsporidia. However, the global negative effects of bi-infection on host condition should be tested on more biological models, since competition between parasites depends on life history trade-off.     

Effect of chronic heparin administration on serum lipolytic activity and some aspects of lipid metabolism

Chronic heparin administration to rats for periods up to 8 days by i.p. implantation of mini pumps, increased serum total lipolytic activity in a dose-dependent manner up to infusion rates of 10 U/h per 100 g body weight. This augmentation was predominantly due to lipoprotein lipase (LPL). Synchronously, heart muscle demonstrated a dose-dependent reduction in LPL activity and adipose tissue showed a biphasic response, LPL activity decreasing at low doses and rising towards control levels at higher doses. Lipolytic activities of skeletal muscle and liver were unaffected. Increased serum LPL could not be attributed to altered enzyme clearance from the circulation in chronically heparinised rats, but was accompanied by a reduced response to i.v. high-dose heparin indicating reduction in the pool of endothelial-bound enzyme. Fasting serum concentrations of triacylglycerol and glycerol were unaffected in chronically heparinised animals although accelerated clearance of exogenous 14C-labelled VLDL was demonstrated, together with enhanced uptake of the isotope by liver and heart. Since de novo synthesis of fatty acids and triacylglycerol from 3H2O was not increased by heparin, we suggest that serum triacylglycerol concentrations were maintained by enhanced re-esterification of preformed fatty acids taken up by the liver. Hepatic cholesterol synthesis from 3H2O was augmented by heparin; this observation is consistent with reported increases in serum total and HDL-cholesterol mediated by chronic heparin administration in man and dog.     

Lipolytic activity of whole isolated liver cells in aqueous suspension.

1. Liver contains a lipase which catalyzes in vitro the hydrolysis of esters of short-chain normal primary alcohols and fatty acids. It is shown that this enzymatic activity can be measured by using intact liver cells as source of enzyme. During short-term incubations of suspensions of cells isolated from rat liver, the lipase acts as a membrane-bound enzyme and readily attacks [3H] oleoylethanol added as an emulsion into the bathing medium. The lipolytic reaction proceeds linearly for at least 20 min at 37 degrees C, at the pH optimum of 8.5. [3H] Oleic acid, a reaction product, is mostly retained in the medium and is used to monitor the lipolytic process. 2. In the presence of heparin, the bound lipase is released in the medium in amounts representing one-third to one half the total activity contained in the cells. This release is very rapid and associated in all cases with a concomitant release of lactate dehydrogenase activity. Such effects are consistent with the interpretation that heparin, at concentrations comprised between 10 and 100 mug per ml, causes alterations of the plasma membrane of the isolated cells, resulting in the dispersion of membrane-bound and cytoplasmtic material. This action of heparin is totally blocked by protamine sulfate (1 mg/ml). No specific effect of heparin directed towards the selective release of lipase could be demonstrated under these conditions. 3. During incubations in the presence of heparin, it was observed that the release of monoester lipase was quantitatively related to a simultaneous decrease in membrane-bound as well as in total monoester lipase activity measureable in the cells after homogenization. This, along with the reappearance of membrane-bound activity immediately after heparin withdrawal, suggest that under the experimental conditions, the membrane-bound enzyme is replaced from inside the cell in proportion of its release by heparin.     

A gel filtration study of the fractional composition of tetanus antitoxic sera

The fraction composition of tetanus antitoxic sera has been studied by the method of gel filtration in columns packed with Sephadex G-200. Differences in the fraction composition of preparations manufactured by different enterprises have been revealed. The specific activity of antitoxic sera has been found to depend on the degree of their fragmentation.     

A comparison of the lipolytic activities in liver perfusates and liver plasma membranes from rats

We have undertaken a study to resolve the conflicting reports on the substrate specificity of the lipolytic enzyme(s) released by heparin from liver and report the following: (1) Heparin perfusates from liver contain an enzyme(s) capable of degrading triacylglycerol, diacylglycerophosphorylethanolamine and monoacylglycerol, whereas a heparin-solubilized fraction from liver plasma membranes hydrolyzes diacylglycerophosphorylethanolamine and monoacylglycerol only; (2) The lipolytic activities for the two sources behave differently on gel filtration but have the same behavior on heparin-Sepharose affinity chromatography; (3) Treatment of the preparation from the plasma membrane with Triton X-100 followed by heparin-Sepharose affinity chromatography produces forms of the enzyme(s) that now have activity on triacylglycerol This study suggests that the enzyme(s) from the two sources may be the same and that some change occurs when the enzyme is released from the intact liver.     

Heparin inhibits the activity of protein phosphatase-1

Heparin inhibited the dephosphorylation of rabbit skeletal muscle or liver phosphorylase a by protein phosphatase-1. Other glycosaminoglycans (chondroitin sulfates) and their constituents were found to be without effect. The chromatography of a partially purified phosphatase preparation on heparin-Sepharose CL-6B resulted in a fraction that did not bind to the matrix and its activity was not inhibited by heparin or inhibitor-1. The phosphatase bound to heparin-Sepharose was eluted by 0.2 M NaCl and was inhibited by heparin or inhibitor-1.     

Structural characterization of human liver heparan sulfate

The isolation, purification and structural characterization of human liver heparan sulfate are described. 1H-NMR spectroscopy demonstrates the purity of this glycosaminoglycan (GAG) and two-dimensional 1H-NMR confirmed that it was heparan sulfate. Enzymatic depolymerization of the isolated heparan sulfate, followed by gradient polyacrylamide gel, confirmed its heparin lyase sensitivity. The concentration of resulting unsaturated disaccharides was determined using reverse phase ion-pairing (RPIP) HPLC with post column derivatization and fluorescence detection. The results of this analysis clearly demonstrate that the isolated GAG was heparan sulfate, not heparin. Human liver heparan sulfate was similar to heparin in that it has a reduced content of unsulfated disaccharide and an elevated average sulfation level. The antithrombin-mediated anti-factor Xa activity of human liver heparan sulfate, however, was much lower than porcine intestinal (pharmaceutical) heparin but was comparable to standard porcine intestinal heparan sulfate. Moreover, human liver heparan sulfate shows higher degree of sulfation than heparan sulfate isolated from porcine liver or from the human hepatoma Hep 2G cell line.     

Influences of anionic polysaccharides on DNA synthesis in isolated nuclei and by DNA polymerase α: Correlation of observed effects with properties of the polysaccharides

The basis of the differential effect of anionic polysaccharides on replicative DNA synthesis in liver and hepatoma cell nuclei was investigated. The differential effect of heparin was lost when more than 40% of its sulfate was removed. DNA synthesis in liver nuclei was optimally stimulated by heparin of molecular weight 22 600 and sulfate to hexosamine ratio 2.42, but inhibited by heparin of molecular weight 4300 and sulfate to hexosamine ratio 2.35. A heparin fragment (molecular weight 2800 and sulfate to hexosamine ratio 1.81), prepared by partial nitrous acid treatment was a potent inhibitor of DNA synthesis in hepatoma nuclei. There was no significant difference in the rate of entry of heparin or its subfractions into either liver or hepatoma nuclei. In both cases less than 15% of added polysaccharide entered the nuclei and only about 4.5% was found associated with the chromatin. The influence of the anionic polysaccharides on DNA synthesis was correlated with their ability to complex with histones as determined by relative light scattering in a laser nephelometer. The relative light scattered on mixing with histones (H1, H2A + H3, H4) was high for DNA synthesis stimulators (heparin, dextran sulfate); medium for DNA synthesis inhibitors (chondroitin 4- and 6-sulfates, heparan sulfate) and low for non-effectors (keratan sulfate, hyaluronic acid). Heparin and chondroitin sulfate H, which at low concentrations stimulate DNA synthesis in liver nuclei, inhibited DNA synthesis by calf thymus DNA polymerase α at all concentrations. This inhibition was not simply due to electrostatic interactions.